A landscape of genetic heterogeneity in germline predisposition to familial chronic lymphocytic leukemia

A landscape of genetic heterogeneity in germline predisposition to familial chronic lymphocytic leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A landscape of genetic heterogeneity in germline predisposition to familial chronic lymphocytic leukemia Luca Laurenti, Eugenio Sangiorgi, Idanna Innocenti, Alberto Fresa, and 34 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7313089/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study provides a comprehensive analysis of the germline landscape in 81 families with chronic lymphocytic leukemia (CLL). We uncovered key genetic pathways associated with CLL predisposition, including telomere maintenance, DNA double-strand break (DSB) repair, and immune regulation. Notably, pathogenic variants were found in POT1 and TINF2 , suggesting defects in telomere stability, while variants in CHEK2 , MRE11 , CDKN1B , RAD51D , ERCC2 and RAD50 indicate defects in DSB repair mechanisms. Variants in immune-related genes such as RUNX1 and TNFRSF13B were also identified, with TNFRSF13B mutations occurring in 4 different families and 4 of the 57 (7%) investigated sporadic cases, further highlighting their potential role in disease susceptibility. Other novel candidates included RASA2 , involved in MAPK/ERK signaling, and NCOR2, a nuclear co-repressor involved in regulation of B cell development and maintenance of genomic integrity. These findings expand the understanding of genetic heterogeneity in familial CLL, supporting a model in which low-penetrance tumor suppressor genes and complex genetic interactions drive disease development. Our results underscore the importance of integrating genetic studies to unravel the hereditary basis of CLL and to further characterize the mechanisms that drive the development of the disease. Biological sciences/Cancer/Haematological cancer/Leukaemia/Chronic lymphocytic leukaemia Biological sciences/Cancer/Cancer genetics Figures Figure 1 Figure 2 Introduction Chronic lymphocytic leukemia (CLL) is the most common form of leukemia worldwide. Prevalence rates vary among different populations, with the highest incidence observed in Western countries, while it is relatively rare in Asia 1 . In the majority of patients, the disease follows a relatively indolent course and can be monitored without immediate intervention 2 . Among all B cell malignancies, CLL is the disorder with the strongest familial predisposition. Having a first-degree relative with the disease is the best-recognized risk factor, and no other environmental risk factors have been identified with the same level of significance 3 . According to studies conducted in different regions of the world, the prevalence of familial cases ranges from 7–13% of all CLL cases 4 . Unlike other tumor-predisposing conditions, most CLL families present with only two affected individuals. Although the age of onset is earlier in familial than in sporadic cases, the average onset typically remains above fifty years 5 . In both familial and sporadic cases, other family members may manifest a benign condition called monoclonal B lymphocytosis (MBL), which is common in the general population. However, in familial CLL, this condition is more often characterized by a high lymphocyte count, and its progression to full-blown CLL has been described 6 . In families with two CLL cases, the risk of having two affected siblings or a parent and child affected is the same, suggesting that single dominant mutations, rather than recessive variants, account for most familial cases 7 . A dominant inheritance with penetrance defect has been observed in families with more than two affected individuals 8 . The search for the elusive gene(s) responsible for familial CLL began in the 1990s, during the era of gene hunting. Linkage analysis and the candidate gene approach yielded results with limited impact. Three major linkage studies using microsatellites, involving over 300 families and employing both parametric and non-parametric approaches, identified several loci on chromosomes 1, 2, 3, 5, 6, 10, 11, 12, 13, 14, 17, and 18, but none reached statistical significance 9 – 11 . The advent of HapMap and next-generation sequencing expanded genetic studies aimed at uncovering the genetic predisposition to familial CLL. Several large genome-wide association studies (GWAS) identified numerous single nucleotide polymorphisms (SNPs) in various genes, each carrying a small incremental risk, that predispose individuals to CLL in different cohorts 12 – 19 . The emergence of exome sequencing eventually led to the identification of the first strong candidate gene for familial CLL. POT1 , along with its direct interactors, was the first tumor suppressor gene found to be mutated in approximately 10% of familial CLL cases 20 . POT1 had already been implicated in melanoma predisposition, and it was later discovered that germline mutations in POT1 can lead to clonal hematopoietic expansion through telomere elongation 21 . Despite this wealth of data and extensive research efforts, the genetic causes of most familial and sporadic CLL cases remain elusive. In this study, we present the results of an analysis of 81 families and 131 individuals with CLL through whole exome sequencing. We identified rare variants in genes involved in telomere biology, DNA repair, B lymphocyte signal transduction, and immune system function, and recurrent mutations in newly identified genes. This study highlights the broad heterogeneous contribution to the genetic landscape of familial CLL with many genes identified here for the first time in familial CLL. Materials and Methods Patients The study was approved by the Bioethics Committee of Fondazione Policlinico Gemelli on April 21, 2022 (Protocol ID: 4858). 81 families and 131 CLL patients were recruited via a national call involving multiple hematological centers. All participants had a confirmed diagnosis of CLL according to the iwCLL criteria 22 and at least one first-degree relative affected by CLL. Written informed consent was obtained from all participants. Bilateral buccal swabs were taken to obtain constitutional DNA from all available CLL patients in each family for exome sequencing. For each patient, clinical and family histories were recorded, and standard and molecular blood tests were conducted as part of routine medical assessments. Detailed clinical and molecular characterization of this cohort is presented in a separate publication 23 . As part of the study, blood samples were collected to isolate CLL DNA at enrollment. To extend the molecular findings from the familial exomes, a cohort of 57 sporadic patients with CLL was recruited exclusively from the Haematology Unit of the Fondazione Policlinico Gemelli. Specific genes or variants were analyzed in this cohort of patients by Sanger sequencing. For those patients the same procedures of the familial cases were followed. DNA extraction from blood and buccal samples was performed using the Qiagen QIAamp DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The purified DNA was quantified using standard spectrophotometric techniques. Exome sequencing was performed on DNA from the buccal swabs of all available family members with CLL. Variants that did not segregate with the CLL phenotype, when at least two affected individuals were present in the same family, were excluded from the final dataset. In families with only one CLL patient for analysis all variants contributed to the whole dataset. Whole Exome Sequencing and bioinformatics analyses Whole exome sequencing (WES) was conducted on service by Dantelabs SRL (L’Aquila, Italy) using Illumina technology. DNA samples extracted from buccal mucosa were sequenced from a targeted library covering 41.5 megabases of coding nucleotides. Each sample generated approximately 50 gigabases of reads, with an average coverage of 100X in coding regions. The following bioinformatics analysis was done in house. Paired-end FASTQ files were processed on the Galaxy platform 24 ( https://usegalaxy.org ) using standard pipelines. Reads were trimmed with Trimmomatic 25 and mapped with BWA-MEM 26 . High-quality, non-duplicated reads were selected, and all indel variants were left-aligned. Variants were identified using FreeBayes 27 and merged into a multisample file for each family. Variants common to all affected individuals within a family were retained, while a separate file of unique variants for each individual was generated. All variants were annotated using the wANNOVAR platform 28 ( https://wannovar.wglab.org ). Filters included only exonic or splicing variants while excluding synonymous variants. Variants with a minor allele frequency (MAF) below 0.001 in the gnomAD database ( https://gnomad.broadinstitute.org/ ; v4.1.0) were considered for analysis. Genes associated with olfactory receptors, taste receptors, collagen, and keratin genes were excluded from further evaluation. All variants were annotated according to their MANE Select transcript. Variant Prioritization and In Silico Analysis ClinVar classification was used to prioritize all submitted variants 29 . ClinVar is a public database of human variations that provides a platform to access data regarding genetic variants associated with diseases, their clinical significance, and evidence supporting these associations. To identify genes with a potential role in CLL biology, all genes were prioritized using VarElect 30 . This tool provides automated assessments of the clinical significance of genomic variants by analyzing multiple data sources, including scientific literature, previous clinical reports, and population databases. Genes receive a relevance score depending (in part) on the weight of each query term that appears in relation to a given gene. The weight of a term is determined by the frequency it appears in association with a gene (term frequency) compared to all genes (inverse document frequency). If a term appears more often in the annotations associated with a given gene, and less often in all genes, the weight of that term for the given gene increases. The following relevant keywords were used: “CLL,” “shelterin complex,” “ POT1 ,” “chronic lymphocytic leukemia,” “hematopoietic system,” “autoimmunity,” “lymphoproliferative disorder,” “lymphoma,” “ TP53 ,” “Bruton tyrosine kinase,” “tumor suppressor gene,” and “immunodeficiency.” Based on relevance, two gene lists were generated: a high-priority list for directly related genes and a secondary list for indirectly related genes; each gene had their scores related to the above mentioned keywords. Based on the evaluation from gnomAD, ClinVar and VarElect, top variants were selected according to the presence of the following parameters: - Rare variants in gnomAD (MAF < 0.0001). This allelic frequency was chosen to identify rare variants with a potential stronger effect. - Variants highly associated with tumorigenesis or CLL biology (genes with a score above 4 in VarElect). - Variants classified as pathogenic/likely pathogenic/uncertain significance/conflicting interpretation of pathogenicity or without ClinVar submissions. - Variants segregating among affected individuals within a family when this was possible. Across 131 exomes, approximately 40,000 variants were identified after filtering, among which 1,434 variants were selected based on the above criteria. Variants in this group were further analyzed using tools such as CADD 31 , REVEL 32 , and the Cancer Genome Interpreter (CGI) 33 , which evaluate the potential functional impact and predict the pathogenicity of individual variants. Clustering of pathogenic variants and identification of common pathways involved in CLL predisposition was done using the STRING database 34 ( https://string-db.org/ ). Sanger Sequencing Primers (available upon request) were designed to confirm the presence of certain variants or exons in CLL DNA, to assess loss-of-heterozygosity and conduct segregation analysis. PRF1 , TNFRSF13B and RUNX1 genes were also sequenced by Sanger in our cohort of sporadic CLL patients. Purified PCR products were sequenced using Big Dye chemistry on an ABI Prism sequencer, and sequence analysis was conducted using SeqScape Studio. Results Hematology units across Italy contributed data from 81 families and 131 individuals to this study. An in-depth clinical description of these families is provided in a separate paper 23 . Exome sequencing was performed on all available individuals with CLL from each family. In 34 families, only one individual was available for analysis (Table 1 ). In 44 families, exome sequencing was performed on two CLL-affected individuals. Although the segregation analysis has limited value, it helped eliminate many variants that did not segregate among the affected individuals. Variants that did not segregate with the phenotype within the same family were classified and evaluated separately but did not contribute to the primary findings of this report. Here, we describe the results of the 1434 variants relevant to CLL. Table 1 Table describing the number of affected individuals in each family and the individuals available for the exome analysis CLL cases Exomes CLL cases Exomes CLL cases Exomes PED1 2 1 PED28 2 2 PED55 3 1 PED2 4 4 PED29 2 1 PED56 2 1 PED3 2 2 PED30 2 2 PED57 2 2 PED4 3 2 PED31 3 2 PED58 2 1 PED5 4 2 PED32 2 1 PED59 2 2 PED6 2 2 PED33 2 2 PED60 2 2 PED7 2 2 PED34 3 1 PED61 4 1 PED8 3 2 PED35 2 2 PED62 2 1 PED9 2 2 PED36 5 1 PED63 2 2 PED10 2 2 PED37 2 1 PED64 2 1 PED11 2 2 PED38 2 1 PED65 2 2 PED12 3 2 PED39 2 1 PED66 2 1 PED13 3 2 PED40 2 1 PED67 2 2 PED14 2 2 PED41 2 2 PED68 2 2 PED15 2 2 PED42 2 1 PED69 2 1 PED16 2 2 PED43 2 1 PED70 2 1 PED17 2 2 PED44 2 1 PED71 2 1 PED18 5 2 PED45 2 1 PED72 2 1 PED19 2 2 PED46 2 2 PED73 3 2 PED20 2 2 PED47 2 1 PED74 2 2 PED21 2 2 PED48 2 2 PED75 2 2 PED22 2 2 PED49 2 2 PED76 2 2 PED23 2 2 PED50 2 1 PED77 2 1 PED24 3 1 PED51 3 1 PED78 2 1 PED25 4 2 PED52 2 1 PED79 2 2 PED26 2 2 PED53 2 1 PED80 2 1 PED27 3 3 PED54 2 1 PED81 2 2 Given the previously established role of telomere biology in prior studies 21 , we initially focused our analysis on the POT1 pathway. Two pathogenic variants were identified in two separate families (PED54 and PED59): one in POT1 and another in TINF2 (Table 2 ). Based on in silico analyses predicting their effects on protein function and ClinVar annotations, these variants were deemed to play a primary role in disease pathogenesis within their respective families. Although the TINF2 gene has not been previously reported in familial CLL, its known association with telomere biology disorders and cancer predisposition 35 , along with the presence of a stop codon mutation, strongly suggests a pathogenic role in CLL predisposition. Table 2 Description of variants in POT1 and its interactors. Variants are ordered top to bottom with the most pathogenic ones on top. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter Pedigree Gene Variant CADD REVEL gnomAD ClinVar CGI VarElect 54 POT1 c.410G > A:p.R137H 28.2 0.848 5/1613924 1P 1VUS driver 118 59 TINF2 c.331C > T:p.Q111X 35 - - - - 51 78 POT1 c.1367A > C:p.E456A 32 0.234 3/1590226 2VUS passenger 118 19 TERF1 c.811A > G:p.T271A 14.41 0.042 . - passenger 33 26 TERF2 c.973A > G:p.I325V 11.86 0.03 4/1612512 - passenger 31 62 TEP1 c.1280G > A:p.G427D 3.73 0.02 1218/1614144 1VUS passenger 4 15 TEP1 c.6319C > T:p.R2107C 32 0.394 21/1613558 - passenger 4 17 TEP1 c.6287C > G:p.P2096R 24 0.346 . - passenger 4 66 TEP1 c.3167G > A:p.R1056Q 21.7 0.08 50/1613894 1VUS passenger 4 67 TEP1 c.152G > A:p.C51Y 22.9 0.64 2/1613674 - passenger 4 Additional variants were identified in POT1 or other genes within the shelterin complex, including one variant each in TERF1 and TERF2 . However, due to conflicting deleterious prediction scores in CADD and REVEL (i.e., high CADD and low REVEL scores), the significance of these variants remains uncertain (Table 2 ). Additionally, five variants were found in TEP1 , another gene involved in telomere biology but not previously associated with any specific condition. At this time, the role of TEP1 variants in CLL pathogenesis remains unclear. A pathogenic variant in CHEK2 (c.514dupA; p.T172Nfs14*) was identified in PED67 (Table 3 ), present in two affected sisters. One sister had CLL and myelodysplasia with a 5q- deletion, while the other, in addition to CLL, had papillary thyroid cancer, haemolytic anaemia, and glomerulonephritis. Their mother, who passed away at 84, had a history of colon cancer, but no CLL. Six additional CHEK2 missense variants were identified across six families (Table 3 ). In order to classify them we used published functional studies assessing kinase activity 36 of the mutated variants, gnomAD frequencies, and i n silico evaluations. From all different evaluations two missense variants (identified in PED77 and PED31) can be considered potentially relevant to CLL pathogenesis, while the other four are considered less likely to play a significant role due to their normal results in the kinase assays in vitro . Table 3 Description of variants in CHEK2 . Variants are ordered top to bottom with the most pathogenic ones on top. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter. CHK2 assay and KAP1assay are functional assays evaluating the activity of the mutated protein. ID, impaired; IM, intermediate Pedigree Gene Variant CADD REVEL gnomAD CHK2assay KAP1assay ClinVar CGI VarElect 66 CHEK2 c.514dupA:p.T172Nfs*14 . . . . . Pathogenic driver 67 77 CHEK2 c.G549C:p.L183F 24.2 0.858 14/1613880 ID ID 4LP 8VUS driver 67 31 CHEK2 c.A980G:p.Y327C 27.6 0.684 29/1613830 IM IM 1LP 16VUS driver 67 26 CHEK2 c.C1216T:p.R406C 24.9 0.281 103/1613698 IM ID 11 VUS 2B driver 67 16 CHEK2 c.C1067T:p.S356L 29.7 0.214 7/1612796 WT IM 10VUS driver 67 76 CHEK2 c.C1283T:p.S428F 27.9 0.323 444/1610148 WT WT 17P 10LP 1VUS passenger 67 7 CHEK2 c.G1312T:p.D438Y 28.3 0.337 606/1613498 WT WT 14 VUS/9B driver 67 Since pathogenic variants were identified in genes involved in telomere maintenance and in CHEK2 , a protein involved in double-strand break (DSB) DNA repair, we conducted a search for additional potentially pathogenic variants in this pathway. Potentially pathogenic variants were identified in the MRE11 , CDKN1B , RAD51B , and ERCC2 genes in one family each, while variants in RAD50 were observed in two families (Table 4 ). These known tumor suppressors are associated with low-penetrance breast cancer predisposition and are reported here for the first time in association with familial CLL. Table 4 Description of variants in genes involved in DSB DNA repair. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter Pedigree Gene Variant CADD REVEL gnomAD ClinVar CGI VarElect 70 MRE11 c.1726C > T:p.R576X 36 . 61/1613702 7P - 32 73 CDKN1B c.31C > T:p.P11S 26.7 0.566 67/1613724 1LP 4VUS 1LB driver 52 31 RAD50 c.3716G > A:p.R1239Q 32 0.438 34/1614148 5VUS driver 38 8 RAD50 c.3857T > C:p.F1286S 31 0.701 4/1614036 3VUS driver 38 26 RAD51D c.493C > T:p.R165W 32 0.29 111/1612350 10VUS driver 9 67 ERCC2 c.335G > A:p.R112H 29.3 0.887 40/1613406 7 Pathogenic driver 30 Additional pathogenic or damaging variants were found in genes involved in immune regulation. A pathogenic variant and a variant of uncertain significance were identified in the RUNX1 gene in PED34 and PED61, respectively (Table 5 ). The pathogenic variant (c.602G > A:p.R201Q), previously reported in families with myeloid malignancies and thrombocytopenia 37 , was likely inherited from the proband’s father, who had CLL. DNA was not available from the father for analysis, but the proband’s mother, unaffected siblings, and brother did not carry the variant. This variant was confirmed in blood DNA, with no additional variants or loss of heterozygosity detected. To assess whether RUNX1 could be implicated in sporadic CLL, we screened 57 sporadic cases, but identified no additional RUNX1 lesions. The second RUNX1 variant (PED61) was considered of uncertain significance because of conflicting evaluations from the CADD and REVEL tools and ClinVar reports (Table 5 ). Table 5 Description of variants in genes involved syndromes causing immunedeficiency. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter. Pedigree Gene Variant CADD REVEL gnomAD ClinVar VarElect 34 RUNX1 c.602G > A:p.R201Q 24.3 0.94 1/1613142 Pathogenic 50 61 RUNX1 c.967A > G:p.T323A 17.8 0.103 12/1614060 2VUS 50 45 TNFRSF13B c.431C > A:p.S144X 34 - 61/1613948 8P 84 25 TNFRSF13B c.260T > A:p.I87N 24 0.623 771/1614204 15P 84 35 TNFRSF13B c.542C > A:p.A181E 8.97 0.607 8694/1614040 19P/LP 1VUS 84 65 TNFRSF13B c.G779A:p.G260E 9.12 0.505 79/1612466 4VUS 84 Sporadic patients Sp TNFRSF13B c.310T > C:p.C104R 25.8 0.919 8783/1614226 24P 4VUS 2B 84 Fo TNFRSF13B c.431C > A:p.S144X 34 - 61/1613948 8P 84 Fo TNFRSF13B c.577T > C:p.C193R 1.71 0.338 129/1613764 4VUS 84 Ca TNFRSF13B c.605G > A:p.R202H 13.2 0.554 1563/1613944 4VUS 84 37 PRF1 c.853_855del:p.K285del 9.41 - 77/ 1614030 Pathogenic 79 64 PRF1 c.1122G > A:p.W374X 41 - 25/ 1612474 Pathogenic 79 39 UNC13D c.2346_2349del:p.R782Sfs*12 26.3 - 241/1613448 Pathogenic 37 25 PRF1 c.1310C > T:p.A437V 25 0.498 1743/ 1614020 1LP 7VUS 79 64 PRF1 c.1070G > A:p.R357Q 23 0.452 390/ 1613648 5VUS 1LB 79 3 PRF1 c.755A > G:p.N252S 12.9 0.213 10357/ 1614236 1VUS 9B 79 63 UNC13D c.1015G > A:p.A339T 23.7 0.565 43/1613790 1VUS 37 60 UNC13D c.929C > T:p.S310F 25.6 0.335 48/1613502 3VUS 37 Pathogenic variants in TNFRSF13B , which encodes the TACI receptor, were identified in four families (Table 5 ). This gene plays a critical role in B lymphocyte signal transduction 38 and is commonly mutated in common variable immunodeficiency disease (CVID) 39 . None of the four families had a history of immunodeficiency before their CLL diagnoses. Given the complexity of TNFRSF13B biology, including incomplete penetrance among mutation carriers, high-frequency pathogenic variants that are common in the general population, and variable expressivity within the same family, segregation analysis was performed only among CLL patients. It would be impossible to assign a specific CLL risk to a healthy individual carrying a pathogenic variant in TNFRSF13B . In these families, the entire gene was sequenced in affected individuals using CLL DNA to identify additional variants or loss of heterozygosity, but none were found. Screening of 57 sporadic CLL cases revealed four additional variants, including one patient (#fo) who carried two distinct variants in trans, one of which (p.S144Ter) was also present in family PED45. At the time of this report, this patient remains the only affected individual in his family. Additional heterozygous pathogenic variants were found in genes associated with recessive immune system dysregulation phenotype. In three families, heterozygous pathogenic variants were identified in genes linked to familial hemophagocytic lymphohistiocytosis (FHLH), including PRF1 and UNC13D . Several studies suggest that heterozygous variants in the perforin pathway predispose individuals to non-Hodgkin lymphoma and other lymphoproliferative disorders 40 – 42 . Variants in PRF1 were found in two families (PED37 and PED64), while a single UNC13D variant was identified in PED37 (Table 5 ). Screening of sporadic cases revealed no additional PRF1 variants. Additional variants of uncertain significance were identified in three families in PRF1 and UNC13D . PED2 was the family with the highest number of available affected individuals (four). One individual had both CLL and mycosis fungoides, while another had mycosis fungoides without CLL, but was still considered affected for this study, and his constitutional DNA was sequenced (Fig. 1 ). By selecting variants shared among all four affected members, we reduced the number of shared variants to fewer than fifty, with only one Group 1 variant in the RASA2 gene. This variant (c.1618C > T:p.L540F), absent from gnomAD and predicted to be damaging by in silico tools, resides in a functional protein domain and has been annotated as a driver mutation in the Cancer Genome Interpreter database (Table 6 ). RASA2 is involved in B lymphocyte signal transduction as an inhibitor of the MAPK/ERK pathway 43 . A second family (PED52) carried a missense variant in a nearby codon (c.1621A > C:p.I541L), exhibiting similar features, suggesting a potential mutational hotspot in RASA2 . Screening of sporadic CLL patients for these hotspot mutations yielded no additional variants. Table 6 Description of variants in RASA2. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation. P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter Pedigree Gene Variant CADD REVEL gnomAD ClinVar CGI VarElect 2 RASA2 c.1618C > T:p.L540F 29.4 0.54 - - driver 5 52 RASA2 c. 1621A > C:p.I541L 23.6 0.211 2/1609670 - driver 5 Finally, using VarElect to prioritize genes recurrently mutated in multiple families, we identified several potential candidates. Among them, NCOR2 , PRR2CA , ITGB4 , and DOCK8 emerged as the top four most frequently mutated genes, none of which have been previously associated with CLL. While these genes do not have a clearly established pathogenic role, they remain potential candidates for future replication and functional studies (Supplementary Table 1). Additional variants were found in well-known tumor suppressor genes, but none were pathogenic based on Clinvar submissions, nor did patients exhibit phenotypes consistent with mutations in those specific genes (Supplementary Table 2). A notable mention goes to the ATM gene, a key somatic driver in CLL, which has also been implicated in both familial and sporadic cases 44 , 45 . In this cohort, six variants segregating with CLL were identified within families, along with three variants found in single affected individuals. Based on in silico evaluations, gnomAD frequencies, and ATM-specific pathogenicity criteria 46 , none of these variants could be classified as pathogenic (Supplementary Table 3). To identify common pathways involved in CLL predisposition, genes harboring pathogenic variants were clustered using the STRING database. Two main clusters emerged: one related to telomere biology and DSB repair machinery, and the other involving genes and proteins associated with immune system deficiencies. These two clusters encompassed also most of the recurrently mutated genes that had not been classified as pathogenic, suggesting that the majority of variants identified in this study are linked to these pathways (Fig. 2 ). Discussion In this study, we provide a comprehensive overview of the germline landscape of familial CLL. Our analysis focused on pathogenic variants that segregated with the phenotype. This stringent approach allowed us to identify new genes that may contribute to the disease. Supplementary Table 4 summarizes the key findings of this study. The first column lists the most probable causative gene and its variant for each family in which a direct role in CLL could be established. Other columns include genes whose role and/or variant significance remains uncertain. We identified two pathogenic variants in the POT1 pathway: one in POT1 itself, previously discovered in a familial melanoma case 47 , and another in TINF2 in PED59. TINF2 encodes a protein within the shelterin complex and is responsible for an autosomal form of dyskeratosis congenita. In heterozygosity, it is considered a low-penetrance tumour suppressor gene 35 . In PED59, the only malignant disease detected in the family was CLL in father and son. The second telomere-related pathway involved DSB repair, with pathogenic variants identified in CHEK2 , MRE11 , CDKN1B , RAD51B , ERCC2 , and RAD50 . These genes have been implicated in genetic predisposition to breast cancer, though they have also been reported in families with other tumors 41 , 48 – 50 . At least regarding breast cancer, the risk alleles are considered to have moderate penetrance. In all affected families in our study, CLL was the predominant cancer, and none of these families were initially suspected of having a hereditary cancer predisposition. Another major pathway implicated in familial CLL predisposition involves immune regulation. RUNX1 has previously been associated only with myeloid malignancies and thrombocytopenia. In our cohort, we identified a single well-described pathogenic RUNX1 variant in one of 81 families, and none in 57 sporadic cases, suggesting that germline RUNX1 mutations are rare in CLL predisposition. The most frequently mutated gene in our cohort was TNFRSF13B , which encodes the TACI receptor. It was mutated in both familial and sporadic cases. TNFRSF13B has a complex genotype-phenotype correlation. In recessive cases, it is commonly associated with common variable immunodeficiency disease (CVID), while in heterozygosity, it is linked to various degrees of antibody deficiency, autoimmune manifestations and lymphoproliferative disorders with low/moderate penetrance 39 . The complexity of TACI biology is further compounded by the presence of some common variants in the general population. However, all pathogenic alleles identified in our familial and sporadic cases were rare, suggesting that CLL predisposition may be mediated only by rare variants. We also investigated whether a second variant could be present in CLL DNA that might explain tumorigenesis, but Sanger sequencing did not detect additional mutations. The most likely pathogenic role of TNFRSF13B mutations is through a loss-of-function mechanism, as highlighted by functional studies on missense variants, the presence of stop codons, and studies with knockout mice exhibiting spontaneous B cell lymphoproliferation and a lethal autoimmune syndrome 51 . Additional pathogenic variants were identified in genes involved in the perforin pathway, which has previously been linked to lymphoma predisposition. A disputed common variant, p.A91V in PRF1 , has been suggested as a predisposing factor. More recently, heterozygous rare variants in this pathway have been found enriched in lymphoma and other lymphoproliferative disorders 40 – 42 , 52 . In the three families with pathogenic variants in PRF1 and UNC13D , the only malignancy-related phenotype was CLL. RASA2 was identified in PED2 after a stringent segregation analysis of rare variants across all four affected individuals. This was the only gene relevant to CLL biology in that family. RASA2 is mutated in up to 5% of melanomas 53 and acts as an inhibitor of the MAPK/ERK pathway. Its role in melanoma is linked to a loss-of-function mechanism, likely leading to increased MAPK/ERK signal transduction. RASA2 was also found to be disrupted by a translocation in a case of Sézary leukemia 54 , and its inactivation in CAR-T cells enhanced their self-renewal capacity 55 , underscoring its importance in both pathological and physiological hematopoietic contexts. Interestingly, in two different families, one in which CLL co-segregated with mycosis fungoides and another with two cases of CLL, a mutational hotspot was observed, suggesting a shared mechanism. The final analysis focused on recurrently mutated genes with a VarElect score above 4, which could indicate a role in CLL biology or pathology. The top recurrently mutated gene was NCOR2 , a transcriptional corepressor in B or T lymphocytes. Experimental studies found that tissue-specific knockout of NCOR1 and NCOR2 causes leukemia in mice, while constitutional knockout results in early embryonic lethality 56 . This gene as well as the other genes recurrently mutated could represent novel tumor suppressor genes causing CLL. In this study, a pathogenic variant potentially responsible for CLL was identified in 20 out of 81 families (Supplementary Table 4). In the remaining families, the primary mutation may be associated with one of the variants of uncertain significance described above or with genes not yet implicated in CLL biology, which were not considered in this analysis. This study expands our understanding of the genetic heterogeneity underlying CLL predisposition. The two main pathways that emerged were related to telomere and DSB repair and immune deficiency. All the tumor suppressor genes identified are considered low-penetrance genes, as reflected by the limited number of affected individuals in each family. Interestingly, in some families, more than one mutated gene was present in the same affected individuals, with both variants being rare. This finding supports a potential digenic/oligogenic inheritance model, which could explain the limited familial recurrence and significant genetic heterogeneity observed. In many cases, cancer development requires multiple mutational events in a multistep process. The two-hit hypothesis suggests that a second mutation in the same locus, affecting the remaining wild-type allele, is necessary for full transformation. At least for the genes we tested, the second hit hypothesis was not confirmed. In CLL, an oligogenic or digenic inheritance of rare alleles may be required for leukemia to develop. Even within the same families, only specific combinations of two or more rare variants, varying among individuals, seem capable of triggering B-lymphocyte transformation. Declarations Acknowledgments We would like to thank all the patients enrolled in this study, their family members and caregivers. MoH-Ricerca Corrente 2024. Authorship Contributions ES, GR, MB and MR performed research. AF, II, AT, CV, AS, AMF, AV, AAR, PS, AG, FP, RM, FRM, AM, FM, RL, AG, IA, EC, RM, GFDA, RP, FA, JO, LS, GB, AC, MIDP, AT, MC, and DG collected data. ES, GR, MB and MR performed data analysis. ES and LL wrote the manuscript. DGE, ES and LL supervised the study. All authors reviewed the manuscript. Conflict of Interest Disclosures Fresa: AstraZeneca, BeiGene, Abbvie: Honoraria. Vitale: AstraZeneca: Honoraria, Other: support for attending meetings; Takeda: Other: support for attending meetings; AbbVie: Honoraria; Johnson & Johnson: Honoraria. Frustaci: AbbVie, BeiGene: Other: Travel, accommodations, expenses; AbbVie, BeiGene, AstraZeneca, Janssen: Consultancy. Visentin: Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy, Research Funding, Speakers Bureau; J&J: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; AstraZenca SpA: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Sportoletti: Janssen; AstraZeneca, Abbvie; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mauro:AstraZenca SpA: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Morelli: BeiGene: Current Employment. Autore: BeiGene, AstraZeneca, AbbVie, Janssen: Honoraria. Tedeschi: AstraZeneca, AbbVie, BeiGene, Janssen, Lilly: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Coscia: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: support for attending meetings; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: support for attending meetings; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: support for attending meetings. Laurenti: AstraZeneca, AbbVie: Research Funding; AstraZeneva, AbbVie, Johnson and Johnson, BeiGene, Lilly: Honoraria; AstraZeneca, AbbVie, Johnson and Johnson, BeiGene, Lilly: Membership on an entity's Board of Directors or advisory committees. Data Availability Statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Goldin L, Slager S. Familial CLL: genes and environment. Hematology American Society of Hematology Education Program 2007. doi:10.1182/asheducation-2007.1.339. 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Nature 2022; 609 : 174–182. Lee RD, Knutson TP, Munro SA, Miller JT, Heltemes-Harris LM, Mullighan CG et al. Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation. Nat Immunol 2022; 23 : 1763–1776. Additional Declarations Yes there is potential conflict of interest. Supplementary Files manuscriptCLLrev23072025ESLeukemia2025supplementarytable1.docx Supplementary table 1 manuscriptCLLrev23072025ESLeukemia2025supplementarytable2.docx Supplementary table 2 manuscriptCLLrev23072025ESLeukemia2025supplementarytables3.docx Supplementary table 3 manuscriptCLLrev23072025ESLeukemia2025supplementarytable4.docx Supplementary table 4 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Businco, ARNAS“G. Brotzu","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Murru","suffix":""},{"id":500718746,"identity":"3e35aa89-d2e6-4df7-9afc-52b1eb1b164f","order_by":17,"name":"Francesca Mauro","email":"","orcid":"https://orcid.org/0000-0003-2425-9474","institution":"Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Mauro","suffix":""},{"id":500718747,"identity":"6ef0a72a-9937-4116-9b64-ea67c1fe6eeb","order_by":18,"name":"Antonio Mosca","email":"","orcid":"","institution":"Catholic University of Sacred Heart","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Mosca","suffix":""},{"id":500718748,"identity":"34469eb7-3ffd-4497-aa89-42ed83d1f9ae","order_by":19,"name":"Francesca Morelli","email":"","orcid":"","institution":"Università degli Studi di Firenze","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Morelli","suffix":""},{"id":500718749,"identity":"2c3dcf5a-4a2d-45d7-910f-910660a45de9","order_by":20,"name":"Roberta Laureana","email":"","orcid":"","institution":"University Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Laureana","suffix":""},{"id":500718750,"identity":"18607bc7-88fb-4015-9237-78f87b7f2c5f","order_by":21,"name":"Andrea Galitzia","email":"","orcid":"https://orcid.org/0000-0002-9122-4258","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Galitzia","suffix":""},{"id":500718751,"identity":"5ce8f111-27f6-4da6-9072-1d38c967a307","order_by":22,"name":"Ilaria Angeletti","email":"","orcid":"","institution":"azienda ospedaliera cosenza","correspondingAuthor":false,"prefix":"","firstName":"Ilaria","middleName":"","lastName":"Angeletti","suffix":""},{"id":500718752,"identity":"c3830931-b0ad-4ec8-b1ce-e158c4abf20a","order_by":23,"name":"esmeralda conte","email":"","orcid":"","institution":"Haematology Unit, Department of Clinical and Molecular Medicine, Sant'Andrea University Hospital, Sapienza University, Rome, Italy","correspondingAuthor":false,"prefix":"","firstName":"esmeralda","middleName":"","lastName":"conte","suffix":""},{"id":500718753,"identity":"6ee2c611-7be6-494c-a6a6-dfb2d91bfb6b","order_by":24,"name":"Riccardo Moia","email":"","orcid":"https://orcid.org/0000-0001-7393-1138","institution":"Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale, Novara, Italy","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Moia","suffix":""},{"id":500718754,"identity":"aa094a4e-9736-4efb-a0b3-d3098c9a966c","order_by":25,"name":"Giovanni D'Arena","email":"","orcid":"","institution":"University of Modena and Reggio Emilia","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"","lastName":"D'Arena","suffix":""},{"id":500718755,"identity":"b68bfb6a-ebbe-47ff-b40f-a2f747e8c177","order_by":26,"name":"Raffaella Pasquale","email":"","orcid":"","institution":"Azienda Sanitaria Universitaria Friuli Centrale (ASU FC), SOC Clinica Ematologia, Udine, Italy","correspondingAuthor":false,"prefix":"","firstName":"Raffaella","middleName":"","lastName":"Pasquale","suffix":""},{"id":500718756,"identity":"8fa0a057-26da-4628-9886-643cf8b52ff3","order_by":27,"name":"Francesco Autore","email":"","orcid":"https://orcid.org/0000-0002-7868-7469","institution":"Fondazione Policlinico Universitario A. Gemelli IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Autore","suffix":""},{"id":500718757,"identity":"e5fce403-8366-4cba-aeb6-315155c3a6e0","order_by":28,"name":"Jacopo Olivieri","email":"","orcid":"","institution":"Azienda Ospedaliera Universitaria S. Maria Misericordia, Udine, Italy","correspondingAuthor":false,"prefix":"","firstName":"Jacopo","middleName":"","lastName":"Olivieri","suffix":""},{"id":500718758,"identity":"023370ad-804c-41e1-9581-3172e4ba5d5e","order_by":29,"name":"Luca Stirparo","email":"","orcid":"","institution":"Catholic University of Sacred Heart","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Stirparo","suffix":""},{"id":500718759,"identity":"541bcbae-9c37-440b-80da-9da89d18363e","order_by":30,"name":"Chiara Maria Rapolla","email":"","orcid":"","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"Maria","lastName":"Rapolla","suffix":""},{"id":500718760,"identity":"8167eaa8-e4d9-4eb0-9688-27aee3033675","order_by":31,"name":"Giulia Benintende","email":"","orcid":"","institution":"Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Universitätsklinikum","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Benintende","suffix":""},{"id":500718761,"identity":"bf4d371c-7249-4fe5-966d-78e4863d48b5","order_by":32,"name":"Andrea Corbingi","email":"","orcid":"","institution":"S.M. Goretti Hospital","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Corbingi","suffix":""},{"id":500718762,"identity":"f4d0aa35-2355-4cad-91c1-8e25ff449ee9","order_by":33,"name":"Maria Ilaria Del Principe","email":"","orcid":"","institution":"University Tor Vergata, Policlinico Tor Vergata","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Ilaria Del","lastName":"Principe","suffix":""},{"id":500718763,"identity":"9b37f26f-48cd-4e5e-a229-f2e944150645","order_by":34,"name":"diana giannarelli","email":"","orcid":"","institution":"Biostatistic Unit, Scientific Directorate, Fondazione Policlinico Universitario A. Gemelli, IRCCS","correspondingAuthor":false,"prefix":"","firstName":"diana","middleName":"","lastName":"giannarelli","suffix":""},{"id":500718764,"identity":"1cd739a3-3def-439a-8f4b-71e0fe964d90","order_by":35,"name":"Alessandra Tedeschi","email":"","orcid":"","institution":"Department of Hematology, Niguarda Cancer Center, ASST Grande Ospedale Metropolitano Niguarda","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Tedeschi","suffix":""},{"id":500718765,"identity":"3b35c425-5094-4fa5-84d5-88254e0d9cd5","order_by":36,"name":"Marta Coscia","email":"","orcid":"","institution":"University of Insubria","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Coscia","suffix":""},{"id":500718766,"identity":"167e5c79-5a74-4f6e-9f61-23e36b0931e2","order_by":37,"name":"Dimitar Efremov","email":"","orcid":"https://orcid.org/0000-0001-9081-5462","institution":"International Centre for Genetic Engineering \u0026 Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Dimitar","middleName":"","lastName":"Efremov","suffix":""}],"badges":[],"createdAt":"2025-08-06 21:40:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7313089/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7313089/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90284654,"identity":"275fcc38-e61c-47b4-9707-81e41c542d47","added_by":"auto","created_at":"2025-09-01 05:53:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29103,"visible":true,"origin":"","legend":"\u003cp\u003ePedigree of PED2. The DNA from individuals with CLL and mycosis fungoides was exome sequenced\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7313089/v1/e5249e5b8f41d89d25bfe778.png"},{"id":90284659,"identity":"d6bcfa77-7a71-4b07-b82d-f63cec3b039f","added_by":"auto","created_at":"2025-09-01 05:53:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":180611,"visible":true,"origin":"","legend":"\u003cp\u003eCluster analysis in STRING database of genes found mutated in familial CLL cases. In this analysis were included genes with pathogenic mutations and genes recurrently mutated in multiple families. Two clusters emerged one involving proteins in the telomere biology and DNA repair machinery (top left), the second cluster has proteins involved in the immunodeficiency (bottom right). Each circle is a node, the strength of each connection between node(s) is highlighted by the number of interconnecting lines.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7313089/v1/db634fea795c7ac92d383ccf.png"},{"id":90325900,"identity":"71ad270a-43e2-4389-a6f3-ecaac7a509d6","added_by":"auto","created_at":"2025-09-01 12:00:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1784751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7313089/v1/3a970008-d879-4436-92a3-8557caf656a1.pdf"},{"id":90284658,"identity":"a28fc11d-522b-4db0-9a13-96d78241e069","added_by":"auto","created_at":"2025-09-01 05:53:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24656,"visible":true,"origin":"","legend":"Supplementary table 1","description":"","filename":"manuscriptCLLrev23072025ESLeukemia2025supplementarytable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7313089/v1/ddef417fb74620ffba4af0cc.docx"},{"id":90284665,"identity":"bf2a397f-c7c3-4b49-b576-2c25d2aafdc1","added_by":"auto","created_at":"2025-09-01 05:53:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33558,"visible":true,"origin":"","legend":"Supplementary table 2","description":"","filename":"manuscriptCLLrev23072025ESLeukemia2025supplementarytable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7313089/v1/d03d3c370adf998b865f7178.docx"},{"id":90285404,"identity":"c5c5ff18-f86a-420a-8c09-d79cd07682e3","added_by":"auto","created_at":"2025-09-01 06:01:04","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":26466,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table 3\u003c/p\u003e","description":"","filename":"manuscriptCLLrev23072025ESLeukemia2025supplementarytables3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7313089/v1/972b24121de9caccb7f358c0.docx"},{"id":90284671,"identity":"e62004b9-0aaf-4f98-b4c0-426a8336afb9","added_by":"auto","created_at":"2025-09-01 05:53:05","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":33521,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table 4\u003c/p\u003e","description":"","filename":"manuscriptCLLrev23072025ESLeukemia2025supplementarytable4.docx","url":"https://assets-eu.researchsquare.com/files/rs-7313089/v1/0d898160d5a8585b12e5f96b.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential conflict of interest.","formattedTitle":"A landscape of genetic heterogeneity in germline predisposition to familial chronic lymphocytic leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic lymphocytic leukemia (CLL) is the most common form of leukemia worldwide. Prevalence rates vary among different populations, with the highest incidence observed in Western countries, while it is relatively rare in Asia\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In the majority of patients, the disease follows a relatively indolent course and can be monitored without immediate intervention\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAmong all B cell malignancies, CLL is the disorder with the strongest familial predisposition. Having a first-degree relative with the disease is the best-recognized risk factor, and no other environmental risk factors have been identified with the same level of significance\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. According to studies conducted in different regions of the world, the prevalence of familial cases ranges from 7\u0026ndash;13% of all CLL cases\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Unlike other tumor-predisposing conditions, most CLL families present with only two affected individuals. Although the age of onset is earlier in familial than in sporadic cases, the average onset typically remains above fifty years\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In both familial and sporadic cases, other family members may manifest a benign condition called monoclonal B lymphocytosis (MBL), which is common in the general population. However, in familial CLL, this condition is more often characterized by a high lymphocyte count, and its progression to full-blown CLL has been described\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In families with two CLL cases, the risk of having two affected siblings or a parent and child affected is the same, suggesting that single dominant mutations, rather than recessive variants, account for most familial cases\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. A dominant inheritance with penetrance defect has been observed in families with more than two affected individuals\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe search for the elusive gene(s) responsible for familial CLL began in the 1990s, during the era of gene hunting. Linkage analysis and the candidate gene approach yielded results with limited impact. Three major linkage studies using microsatellites, involving over 300 families and employing both parametric and non-parametric approaches, identified several loci on chromosomes 1, 2, 3, 5, 6, 10, 11, 12, 13, 14, 17, and 18, but none reached statistical significance\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe advent of HapMap and next-generation sequencing expanded genetic studies aimed at uncovering the genetic predisposition to familial CLL. Several large genome-wide association studies (GWAS) identified numerous single nucleotide polymorphisms (SNPs) in various genes, each carrying a small incremental risk, that predispose individuals to CLL in different cohorts\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe emergence of exome sequencing eventually led to the identification of the first strong candidate gene for familial CLL. \u003cem\u003ePOT1\u003c/em\u003e, along with its direct interactors, was the first tumor suppressor gene found to be mutated in approximately 10% of familial CLL cases\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePOT1\u003c/em\u003e had already been implicated in melanoma predisposition, and it was later discovered that germline mutations in \u003cem\u003ePOT1\u003c/em\u003e can lead to clonal hematopoietic expansion through telomere elongation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite this wealth of data and extensive research efforts, the genetic causes of most familial and sporadic CLL cases remain elusive. In this study, we present the results of an analysis of 81 families and 131 individuals with CLL through whole exome sequencing. We identified rare variants in genes involved in telomere biology, DNA repair, B lymphocyte signal transduction, and immune system function, and recurrent mutations in newly identified genes. This study highlights the broad heterogeneous contribution to the genetic landscape of familial CLL with many genes identified here for the first time in familial CLL.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePatients\u003c/h2\u003e\n \u003cp\u003eThe study was approved by the Bioethics Committee of Fondazione Policlinico Gemelli on April 21, 2022 (Protocol ID: 4858). 81 families and 131 CLL patients were recruited via a national call involving multiple hematological centers. All participants had a confirmed diagnosis of CLL according to the iwCLL criteria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and at least one first-degree relative affected by CLL. Written informed consent was obtained from all participants. Bilateral buccal swabs were taken to obtain constitutional DNA from all available CLL patients in each family for exome sequencing.\u003c/p\u003e\n \u003cp\u003eFor each patient, clinical and family histories were recorded, and standard and molecular blood tests were conducted as part of routine medical assessments. Detailed clinical and molecular characterization of this cohort is presented in a separate publication\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. As part of the study, blood samples were collected to isolate CLL DNA at enrollment. To extend the molecular findings from the familial exomes, a cohort of 57 sporadic patients with CLL was recruited exclusively from the Haematology Unit of the Fondazione Policlinico Gemelli. Specific genes or variants were analyzed in this cohort of patients by Sanger sequencing. For those patients the same procedures of the familial cases were followed.\u003c/p\u003e\n \u003cp\u003eDNA extraction from blood and buccal samples was performed using the Qiagen QIAamp DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer\u0026rsquo;s protocol. The purified DNA was quantified using standard spectrophotometric techniques.\u003c/p\u003e\n \u003cp\u003eExome sequencing was performed on DNA from the buccal swabs of all available family members with CLL. Variants that did not segregate with the CLL phenotype, when at least two affected individuals were present in the same family, were excluded from the final dataset. In families with only one CLL patient for analysis all variants contributed to the whole dataset.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eWhole Exome Sequencing and bioinformatics analyses\u003c/h3\u003e\n\u003cp\u003eWhole exome sequencing (WES) was conducted on service by Dantelabs SRL (L\u0026rsquo;Aquila, Italy) using Illumina technology. DNA samples extracted from buccal mucosa were sequenced from a targeted library covering 41.5 megabases of coding nucleotides. Each sample generated approximately 50 gigabases of reads, with an average coverage of 100X in coding regions. The following bioinformatics analysis was done in house. Paired-end FASTQ files were processed on the Galaxy platform\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://usegalaxy.org\u003c/span\u003e\u003c/span\u003e) using standard pipelines. Reads were trimmed with Trimmomatic\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and mapped with BWA-MEM\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. High-quality, non-duplicated reads were selected, and all indel variants were left-aligned. Variants were identified using FreeBayes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and merged into a multisample file for each family. Variants common to all affected individuals within a family were retained, while a separate file of unique variants for each individual was generated.\u003c/p\u003e\n\u003cp\u003eAll variants were annotated using the wANNOVAR platform\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wannovar.wglab.org\u003c/span\u003e\u003c/span\u003e). Filters included only exonic or splicing variants while excluding synonymous variants. Variants with a minor allele frequency (MAF) below 0.001 in the gnomAD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/\u003c/span\u003e\u003c/span\u003e; v4.1.0) were considered for analysis. Genes associated with olfactory receptors, taste receptors, collagen, and keratin genes were excluded from further evaluation. All variants were annotated according to their MANE Select transcript.\u003c/p\u003e\n\u003ch3\u003eVariant Prioritization and In Silico Analysis\u003c/h3\u003e\n\u003cp\u003eClinVar classification was used to prioritize all submitted variants\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. ClinVar is a public database of human variations that provides a platform to access data regarding genetic variants associated with diseases, their clinical significance, and evidence supporting these associations.\u003c/p\u003e\n\u003cp\u003eTo identify genes with a potential role in CLL biology, all genes were prioritized using VarElect\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. This tool provides automated assessments of the clinical significance of genomic variants by analyzing multiple data sources, including scientific literature, previous clinical reports, and population databases. Genes receive a relevance score depending (in part) on the \u003cem\u003eweight\u003c/em\u003e of each query term that appears in relation to a given gene. The weight of a term is determined by the frequency it appears in association with a gene (term frequency) compared to all genes (inverse document frequency). If a term appears more often in the annotations associated with a given gene, and less often in all genes, the weight of that term for the given gene increases. The following relevant keywords were used: \u0026ldquo;CLL,\u0026rdquo; \u0026ldquo;shelterin complex,\u0026rdquo; \u0026ldquo;\u003cem\u003ePOT1\u003c/em\u003e,\u0026rdquo; \u0026ldquo;chronic lymphocytic leukemia,\u0026rdquo; \u0026ldquo;hematopoietic system,\u0026rdquo; \u0026ldquo;autoimmunity,\u0026rdquo; \u0026ldquo;lymphoproliferative disorder,\u0026rdquo; \u0026ldquo;lymphoma,\u0026rdquo; \u0026ldquo;\u003cem\u003eTP53\u003c/em\u003e,\u0026rdquo; \u0026ldquo;Bruton tyrosine kinase,\u0026rdquo; \u0026ldquo;tumor suppressor gene,\u0026rdquo; and \u0026ldquo;immunodeficiency.\u0026rdquo; Based on relevance, two gene lists were generated: a high-priority list for directly related genes and a secondary list for indirectly related genes; each gene had their scores related to the above mentioned keywords.\u003c/p\u003e\n\u003cp\u003eBased on the evaluation from gnomAD, ClinVar and VarElect, top variants were selected according to the presence of the following parameters:\u003c/p\u003e\n\u003cp\u003e- Rare variants in gnomAD (MAF\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This allelic frequency was chosen to identify rare variants with a potential stronger effect.\u003c/p\u003e\n\u003cp\u003e- Variants highly associated with tumorigenesis or CLL biology (genes with a score above 4 in VarElect).\u003c/p\u003e\n\u003cp\u003e- Variants classified as pathogenic/likely pathogenic/uncertain significance/conflicting interpretation of pathogenicity or without ClinVar submissions.\u003c/p\u003e\n\u003cp\u003e- Variants segregating among affected individuals within a family when this was possible.\u003c/p\u003e\n\u003cp\u003eAcross 131 exomes, approximately 40,000 variants were identified after filtering, among which 1,434 variants were selected based on the above criteria. Variants in this group were further analyzed using tools such as CADD\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, REVEL\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, and the Cancer Genome Interpreter (CGI)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, which evaluate the potential functional impact and predict the pathogenicity of individual variants. Clustering of pathogenic variants and identification of common pathways involved in CLL predisposition was done using the STRING database\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSanger Sequencing\u003c/h3\u003e\n\u003cp\u003ePrimers (available upon request) were designed to confirm the presence of certain variants or exons in CLL DNA, to assess loss-of-heterozygosity and conduct segregation analysis. \u003cem\u003ePRF1\u003c/em\u003e, \u003cem\u003eTNFRSF13B\u003c/em\u003e and \u003cem\u003eRUNX1\u003c/em\u003e genes were also sequenced by Sanger in our cohort of sporadic CLL patients. Purified PCR products were sequenced using Big Dye chemistry on an ABI Prism sequencer, and sequence analysis was conducted using SeqScape Studio.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eHematology units across Italy contributed data from 81 families and 131 individuals to this study. An in-depth clinical description of these families is provided in a separate paper\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Exome sequencing was performed on all available individuals with CLL from each family. In 34 families, only one individual was available for analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In 44 families, exome sequencing was performed on two CLL-affected individuals. Although the segregation analysis has limited value, it helped eliminate many variants that did not segregate among the affected individuals. Variants that did not segregate with the phenotype within the same family were classified and evaluated separately but did not contribute to the primary findings of this report. Here, we describe the results of the 1434 variants relevant to CLL.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTable describing the number of affected individuals in each family and the individuals available for the exome analysis\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCLL cases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExomes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCLL cases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eExomes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCLL cases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eExomes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED28\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED29\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED56\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED30\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED57\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED31\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED58\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED32\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED59\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED34\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED61\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED62\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED36\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED63\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED37\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED64\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED38\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED39\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED66\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED40\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED41\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED68\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED42\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED69\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED43\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED70\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED44\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED71\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED18\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED72\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED46\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED73\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED20\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED47\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED74\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED21\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED48\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED75\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED22\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED49\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED76\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED23\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED77\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED24\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED51\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED78\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED52\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED79\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED26\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED53\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED80\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePED27\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePED54\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePED81\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eGiven the previously established role of telomere biology in prior studies\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we initially focused our analysis on the POT1 pathway. Two pathogenic variants were identified in two separate families (PED54 and PED59): one in \u003cem\u003ePOT1\u003c/em\u003e and another in \u003cem\u003eTINF2\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on \u003cem\u003ein silico\u003c/em\u003e analyses predicting their effects on protein function and ClinVar annotations, these variants were deemed to play a primary role in disease pathogenesis within their respective families. Although the \u003cem\u003eTINF2\u003c/em\u003e gene has not been previously reported in familial CLL, its known association with telomere biology disorders and cancer predisposition\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, along with the presence of a stop codon mutation, strongly suggests a pathogenic role in CLL predisposition.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescription of variants in \u003cem\u003ePOT1\u003c/em\u003e and its interactors. Variants are ordered top to bottom with the most pathogenic ones on top. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePedigree\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCADD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eREVEL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003egnomAD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eClinVar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCGI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eVarElect\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e54\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePOT1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.410G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R137H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.848\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5/1613924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1P 1VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e118\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e59\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTINF2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.331C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.Q111X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e78\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePOT1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1367A\u0026thinsp;\u0026gt;\u0026thinsp;C:p.E456A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.234\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3/1590226\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e118\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTERF1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.811A\u0026thinsp;\u0026gt;\u0026thinsp;G:p.T271A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.042\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e26\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTERF2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.973A\u0026thinsp;\u0026gt;\u0026thinsp;G:p.I325V\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4/1612512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e62\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTEP1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1280G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.G427D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1218/1614144\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTEP1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.6319C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.R2107C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.394\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e21/1613558\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTEP1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.6287C\u0026thinsp;\u0026gt;\u0026thinsp;G:p.P2096R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.346\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e66\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTEP1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.3167G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R1056Q\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e50/1613894\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTEP1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.152G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.C51Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2/1613674\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAdditional variants were identified in POT1 or other genes within the shelterin complex, including one variant each in \u003cem\u003eTERF1\u003c/em\u003e and \u003cem\u003eTERF2\u003c/em\u003e. However, due to conflicting deleterious prediction scores in CADD and REVEL (i.e., high CADD and low REVEL scores), the significance of these variants remains uncertain (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, five variants were found in \u003cem\u003eTEP1\u003c/em\u003e, another gene involved in telomere biology but not previously associated with any specific condition. At this time, the role of \u003cem\u003eTEP1\u003c/em\u003e variants in CLL pathogenesis remains unclear.\u003c/p\u003e\u003cp\u003eA pathogenic variant in \u003cem\u003eCHEK2\u003c/em\u003e (c.514dupA; p.T172Nfs14*) was identified in PED67 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), present in two affected sisters. One sister had CLL and myelodysplasia with a 5q- deletion, while the other, in addition to CLL, had papillary thyroid cancer, haemolytic anaemia, and glomerulonephritis. Their mother, who passed away at 84, had a history of colon cancer, but no CLL. Six additional \u003cem\u003eCHEK2\u003c/em\u003e missense variants were identified across six families (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In order to classify them we used published functional studies assessing kinase activity\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e of the mutated variants, gnomAD frequencies, and i\u003cem\u003en silico\u003c/em\u003e evaluations. From all different evaluations two missense variants (identified in PED77 and PED31) can be considered potentially relevant to CLL pathogenesis, while the other four are considered less likely to play a significant role due to their normal results in the kinase assays \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescription of variants in \u003cem\u003eCHEK2\u003c/em\u003e. Variants are ordered top to bottom with the most pathogenic ones on top. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter. CHK2 assay and KAP1assay are functional assays evaluating the activity of the mutated protein. ID, impaired; IM, intermediate\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePedigree\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCADD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eREVEL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003egnomAD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCHK2assay\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eKAP1assay\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eClinVar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eCGI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eVarElect\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e66\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.514dupA:p.T172Nfs*14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePathogenic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e77\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.G549C:p.L183F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.858\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14/1613880\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4LP 8VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.A980G:p.Y327C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.684\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e29/1613830\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eIM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eIM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1LP 16VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e26\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.C1216T:p.R406C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.281\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e103/1613698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eIM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e11 VUS 2B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.C1067T:p.S356L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.214\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7/1612796\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eIM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e76\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.C1283T:p.S428F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e444/1610148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e17P 10LP 1VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003epassenger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCHEK2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.G1312T:p.D438Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.337\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e606/1613498\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e14 VUS/9B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSince pathogenic variants were identified in genes involved in telomere maintenance and in \u003cem\u003eCHEK2\u003c/em\u003e, a protein involved in double-strand break (DSB) DNA repair, we conducted a search for additional potentially pathogenic variants in this pathway. Potentially pathogenic variants were identified in the \u003cem\u003eMRE11\u003c/em\u003e, \u003cem\u003eCDKN1B\u003c/em\u003e, \u003cem\u003eRAD51B\u003c/em\u003e, and \u003cem\u003eERCC2\u003c/em\u003e genes in one family each, while variants in \u003cem\u003eRAD50\u003c/em\u003e were observed in two families (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These known tumor suppressors are associated with low-penetrance breast cancer predisposition and are reported here for the first time in association with familial CLL.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescription of variants in genes involved in DSB DNA repair. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePedigree\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCADD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eREVEL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003egnomAD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eClinVar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCGI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eVarElect\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e70\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMRE11\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1726C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.R576X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e61/1613702\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e73\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCDKN1B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.31C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.P11S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.566\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e67/1613724\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1LP 4VUS 1LB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRAD50\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.3716G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R1239Q\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.438\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e34/1614148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRAD50\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.3857T\u0026thinsp;\u0026gt;\u0026thinsp;C:p.F1286S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.701\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4/1614036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e26\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRAD51D\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.493C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.R165W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e111/1612350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eERCC2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.335G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R112H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.887\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40/1613406\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7 Pathogenic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAdditional pathogenic or damaging variants were found in genes involved in immune regulation. A pathogenic variant and a variant of uncertain significance were identified in the \u003cem\u003eRUNX1\u003c/em\u003e gene in PED34 and PED61, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The pathogenic variant (c.602G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R201Q), previously reported in families with myeloid malignancies and thrombocytopenia\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, was likely inherited from the proband\u0026rsquo;s father, who had CLL. DNA was not available from the father for analysis, but the proband\u0026rsquo;s mother, unaffected siblings, and brother did not carry the variant. This variant was confirmed in blood DNA, with no additional variants or loss of heterozygosity detected. To assess whether \u003cem\u003eRUNX1\u003c/em\u003e could be implicated in sporadic CLL, we screened 57 sporadic cases, but identified no additional \u003cem\u003eRUNX1\u003c/em\u003e lesions. The second \u003cem\u003eRUNX1\u003c/em\u003e variant (PED61) was considered of uncertain significance because of conflicting evaluations from the CADD and REVEL tools and ClinVar reports (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescription of variants in genes involved syndromes causing immunedeficiency. CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePedigree\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCADD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eREVEL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003egnomAD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eClinVar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eVarElect\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eRUNX1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.602G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R201Q\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1/1613142\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePathogenic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e61\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eRUNX1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.967A\u0026thinsp;\u0026gt;\u0026thinsp;G:p.T323A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12/1614060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.431C\u0026thinsp;\u0026gt;\u0026thinsp;A:p.S144X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e61/1613948\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.260T\u0026thinsp;\u0026gt;\u0026thinsp;A:p.I87N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.623\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e771/1614204\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e15P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.542C\u0026thinsp;\u0026gt;\u0026thinsp;A:p.A181E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.607\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8694/1614040\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19P/LP 1VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.G779A:p.G260E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.505\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e79/1612466\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eSporadic patients\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSp\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.310T\u0026thinsp;\u0026gt;\u0026thinsp;C:p.C104R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.919\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8783/1614226\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e24P 4VUS 2B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFo\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.431C\u0026thinsp;\u0026gt;\u0026thinsp;A:p.S144X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e61/1613948\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFo\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.577T\u0026thinsp;\u0026gt;\u0026thinsp;C:p.C193R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.338\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e129/1613764\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTNFRSF13B\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.605G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R202H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.554\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1563/1613944\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e37\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePRF1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.853_855del:p.K285del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e77/ 1614030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePathogenic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e64\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePRF1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.1122G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.W374X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e25/ 1612474\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePathogenic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e39\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eUNC13D\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.2346_2349del:p.R782Sfs*12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e241/1613448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePathogenic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePRF1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.1310C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.A437V\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.498\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1743/ 1614020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1LP 7VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e64\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePRF1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.1070G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.R357Q\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e390/ 1613648\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5VUS 1LB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePRF1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.755A\u0026thinsp;\u0026gt;\u0026thinsp;G:p.N252S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.213\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10357/ 1614236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1VUS 9B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e63\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eUNC13D\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.1015G\u0026thinsp;\u0026gt;\u0026thinsp;A:p.A339T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.565\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e43/1613790\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eUNC13D\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec.929C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.S310F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.335\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e48/1613502\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3VUS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePathogenic variants in \u003cem\u003eTNFRSF13B\u003c/em\u003e, which encodes the TACI receptor, were identified in four families (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This gene plays a critical role in B lymphocyte signal transduction\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and is commonly mutated in common variable immunodeficiency disease (CVID)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. None of the four families had a history of immunodeficiency before their CLL diagnoses. Given the complexity of \u003cem\u003eTNFRSF13B\u003c/em\u003e biology, including incomplete penetrance among mutation carriers, high-frequency pathogenic variants that are common in the general population, and variable expressivity within the same family, segregation analysis was performed only among CLL patients. It would be impossible to assign a specific CLL risk to a healthy individual carrying a pathogenic variant in \u003cem\u003eTNFRSF13B\u003c/em\u003e. In these families, the entire gene was sequenced in affected individuals using CLL DNA to identify additional variants or loss of heterozygosity, but none were found. Screening of 57 sporadic CLL cases revealed four additional variants, including one patient (#fo) who carried two distinct variants in trans, one of which (p.S144Ter) was also present in family PED45. At the time of this report, this patient remains the only affected individual in his family.\u003c/p\u003e\u003cp\u003eAdditional heterozygous pathogenic variants were found in genes associated with recessive immune system dysregulation phenotype. In three families, heterozygous pathogenic variants were identified in genes linked to familial hemophagocytic lymphohistiocytosis (FHLH), including \u003cem\u003ePRF1\u003c/em\u003e and \u003cem\u003eUNC13D\u003c/em\u003e. Several studies suggest that heterozygous variants in the perforin pathway predispose individuals to non-Hodgkin lymphoma and other lymphoproliferative disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Variants in \u003cem\u003ePRF1\u003c/em\u003e were found in two families (PED37 and PED64), while a single \u003cem\u003eUNC13D\u003c/em\u003e variant was identified in PED37 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Screening of sporadic cases revealed no additional \u003cem\u003ePRF1\u003c/em\u003e variants. Additional variants of uncertain significance were identified in three families in \u003cem\u003ePRF1\u003c/em\u003e and \u003cem\u003eUNC13D\u003c/em\u003e.\u003c/p\u003e\u003cp\u003ePED2 was the family with the highest number of available affected individuals (four). One individual had both CLL and mycosis fungoides, while another had mycosis fungoides without CLL, but was still considered affected for this study, and his constitutional DNA was sequenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By selecting variants shared among all four affected members, we reduced the number of shared variants to fewer than fifty, with only one Group 1 variant in the \u003cem\u003eRASA2\u003c/em\u003e gene. This variant (c.1618C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.L540F), absent from gnomAD and predicted to be damaging by \u003cem\u003ein silico\u003c/em\u003e tools, resides in a functional protein domain and has been annotated as a driver mutation in the Cancer Genome Interpreter database (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). RASA2 is involved in B lymphocyte signal transduction as an inhibitor of the MAPK/ERK pathway\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. A second family (PED52) carried a missense variant in a nearby codon (c.1621A\u0026thinsp;\u0026gt;\u0026thinsp;C:p.I541L), exhibiting similar features, suggesting a potential mutational hotspot in \u003cem\u003eRASA2\u003c/em\u003e. Screening of sporadic CLL patients for these hotspot mutations yielded no additional variants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescription of variants in \u003cem\u003eRASA2.\u003c/em\u003e CADD and REVEL scores are considered significant above 20 and 0.7 respectively. In ClinVar column are indicated, if present, the number of submissions and their evaluation. P stands for Pathogenic, VUS for variant of undetermined significance. CGI Cancer Genome Interpreter\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePedigree\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCADD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eREVEL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003egnomAD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eClinVar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCGI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eVarElect\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRASA2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1618C\u0026thinsp;\u0026gt;\u0026thinsp;T:p.L540F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e52\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRASA2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec. 1621A\u0026thinsp;\u0026gt;\u0026thinsp;C:p.I541L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2/1609670\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003edriver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFinally, using VarElect to prioritize genes recurrently mutated in multiple families, we identified several potential candidates. Among them, \u003cem\u003eNCOR2\u003c/em\u003e, \u003cem\u003ePRR2CA\u003c/em\u003e, \u003cem\u003eITGB4\u003c/em\u003e, and \u003cem\u003eDOCK8\u003c/em\u003e emerged as the top four most frequently mutated genes, none of which have been previously associated with CLL. While these genes do not have a clearly established pathogenic role, they remain potential candidates for future replication and functional studies (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eAdditional variants were found in well-known tumor suppressor genes, but none were pathogenic based on Clinvar submissions, nor did patients exhibit phenotypes consistent with mutations in those specific genes (Supplementary Table\u0026nbsp;2). A notable mention goes to the ATM gene, a key somatic driver in CLL, which has also been implicated in both familial and sporadic cases\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In this cohort, six variants segregating with CLL were identified within families, along with three variants found in single affected individuals. Based on in silico evaluations, gnomAD frequencies, and ATM-specific pathogenicity criteria\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, none of these variants could be classified as pathogenic (Supplementary Table\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eTo identify common pathways involved in CLL predisposition, genes harboring pathogenic variants were clustered using the STRING database. Two main clusters emerged: one related to telomere biology and DSB repair machinery, and the other involving genes and proteins associated with immune system deficiencies. These two clusters encompassed also most of the recurrently mutated genes that had not been classified as pathogenic, suggesting that the majority of variants identified in this study are linked to these pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we provide a comprehensive overview of the germline landscape of familial CLL. Our analysis focused on pathogenic variants that segregated with the phenotype. This stringent approach allowed us to identify new genes that may contribute to the disease.\u003c/p\u003e\u003cp\u003eSupplementary Table\u0026nbsp;4 summarizes the key findings of this study. The first column lists the most probable causative gene and its variant for each family in which a direct role in CLL could be established. Other columns include genes whose role and/or variant significance remains uncertain.\u003c/p\u003e\u003cp\u003eWe identified two pathogenic variants in the POT1 pathway: one in \u003cem\u003ePOT1\u003c/em\u003e itself, previously discovered in a familial melanoma case\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, and another in \u003cem\u003eTINF2\u003c/em\u003e in PED59. \u003cem\u003eTINF2\u003c/em\u003e encodes a protein within the shelterin complex and is responsible for an autosomal form of dyskeratosis congenita. In heterozygosity, it is considered a low-penetrance tumour suppressor gene\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In PED59, the only malignant disease detected in the family was CLL in father and son.\u003c/p\u003e\u003cp\u003eThe second telomere-related pathway involved DSB repair, with pathogenic variants identified in \u003cem\u003eCHEK2\u003c/em\u003e, \u003cem\u003eMRE11\u003c/em\u003e, \u003cem\u003eCDKN1B\u003c/em\u003e, \u003cem\u003eRAD51B\u003c/em\u003e, \u003cem\u003eERCC2\u003c/em\u003e, and \u003cem\u003eRAD50\u003c/em\u003e. These genes have been implicated in genetic predisposition to breast cancer, though they have also been reported in families with other tumors\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. At least regarding breast cancer, the risk alleles are considered to have moderate penetrance. In all affected families in our study, CLL was the predominant cancer, and none of these families were initially suspected of having a hereditary cancer predisposition.\u003c/p\u003e\u003cp\u003eAnother major pathway implicated in familial CLL predisposition involves immune regulation. \u003cem\u003eRUNX1\u003c/em\u003e has previously been associated only with myeloid malignancies and thrombocytopenia. In our cohort, we identified a single well-described pathogenic \u003cem\u003eRUNX1\u003c/em\u003e variant in one of 81 families, and none in 57 sporadic cases, suggesting that germline \u003cem\u003eRUNX1\u003c/em\u003e mutations are rare in CLL predisposition.\u003c/p\u003e\u003cp\u003eThe most frequently mutated gene in our cohort was \u003cem\u003eTNFRSF13B\u003c/em\u003e, which encodes the TACI receptor. It was mutated in both familial and sporadic cases. \u003cem\u003eTNFRSF13B\u003c/em\u003e has a complex genotype-phenotype correlation. In recessive cases, it is commonly associated with common variable immunodeficiency disease (CVID), while in heterozygosity, it is linked to various degrees of antibody deficiency, autoimmune manifestations and lymphoproliferative disorders with low/moderate penetrance\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The complexity of TACI biology is further compounded by the presence of some common variants in the general population. However, all pathogenic alleles identified in our familial and sporadic cases were rare, suggesting that CLL predisposition may be mediated only by rare variants. We also investigated whether a second variant could be present in CLL DNA that might explain tumorigenesis, but Sanger sequencing did not detect additional mutations. The most likely pathogenic role of \u003cem\u003eTNFRSF13B\u003c/em\u003e mutations is through a loss-of-function mechanism, as highlighted by functional studies on missense variants, the presence of stop codons, and studies with knockout mice exhibiting spontaneous B cell lymphoproliferation and a lethal autoimmune syndrome\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdditional pathogenic variants were identified in genes involved in the perforin pathway, which has previously been linked to lymphoma predisposition. A disputed common variant, p.A91V in \u003cem\u003ePRF1\u003c/em\u003e, has been suggested as a predisposing factor. More recently, heterozygous rare variants in this pathway have been found enriched in lymphoma and other lymphoproliferative disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In the three families with pathogenic variants in \u003cem\u003ePRF1\u003c/em\u003e and \u003cem\u003eUNC13D\u003c/em\u003e, the only malignancy-related phenotype was CLL.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRASA2\u003c/em\u003e was identified in PED2 after a stringent segregation analysis of rare variants across all four affected individuals. This was the only gene relevant to CLL biology in that family. \u003cem\u003eRASA2\u003c/em\u003e is mutated in up to 5% of melanomas\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and acts as an inhibitor of the MAPK/ERK pathway. Its role in melanoma is linked to a loss-of-function mechanism, likely leading to increased MAPK/ERK signal transduction. \u003cem\u003eRASA2\u003c/em\u003e was also found to be disrupted by a translocation in a case of S\u0026eacute;zary leukemia\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, and its inactivation in CAR-T cells enhanced their self-renewal capacity\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, underscoring its importance in both pathological and physiological hematopoietic contexts. Interestingly, in two different families, one in which CLL co-segregated with mycosis fungoides and another with two cases of CLL, a mutational hotspot was observed, suggesting a shared mechanism.\u003c/p\u003e\u003cp\u003eThe final analysis focused on recurrently mutated genes with a VarElect score above 4, which could indicate a role in CLL biology or pathology. The top recurrently mutated gene was \u003cem\u003eNCOR2\u003c/em\u003e, a transcriptional corepressor in B or T lymphocytes. Experimental studies found that tissue-specific knockout of \u003cem\u003eNCOR1\u003c/em\u003e and \u003cem\u003eNCOR2\u003c/em\u003e causes leukemia in mice, while constitutional knockout results in early embryonic lethality\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. This gene as well as the other genes recurrently mutated could represent novel tumor suppressor genes causing CLL.\u003c/p\u003e\u003cp\u003eIn this study, a pathogenic variant potentially responsible for CLL was identified in 20 out of 81 families (Supplementary Table\u0026nbsp;4). In the remaining families, the primary mutation may be associated with one of the variants of uncertain significance described above or with genes not yet implicated in CLL biology, which were not considered in this analysis.\u003c/p\u003e\u003cp\u003eThis study expands our understanding of the genetic heterogeneity underlying CLL predisposition. The two main pathways that emerged were related to telomere and DSB repair and immune deficiency. All the tumor suppressor genes identified are considered low-penetrance genes, as reflected by the limited number of affected individuals in each family.\u003c/p\u003e\u003cp\u003eInterestingly, in some families, more than one mutated gene was present in the same affected individuals, with both variants being rare. This finding supports a potential digenic/oligogenic inheritance model, which could explain the limited familial recurrence and significant genetic heterogeneity observed. In many cases, cancer development requires multiple mutational events in a multistep process. The two-hit hypothesis suggests that a second mutation in the same locus, affecting the remaining wild-type allele, is necessary for full transformation. At least for the genes we tested, the second hit hypothesis was not confirmed. In CLL, an oligogenic or digenic inheritance of rare alleles may be required for leukemia to develop. Even within the same families, only specific combinations of two or more rare variants, varying among individuals, seem capable of triggering B-lymphocyte transformation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the patients enrolled in this study, their family members and caregivers. MoH-Ricerca Corrente 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eES, GR, MB and MR performed research. AF, II, AT, CV, AS, AMF, AV, AAR, PS, AG, FP, RM, FRM, AM, FM, RL, AG, IA, EC, RM, GFDA, RP, FA, JO, LS, GB, AC, MIDP, AT, MC, and DG collected data. ES, GR, MB and MR performed data analysis. ES and LL wrote the manuscript. DGE, ES and LL supervised the study. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Disclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresa: AstraZeneca, BeiGene, Abbvie: Honoraria. Vitale: AstraZeneca: Honoraria, Other: support for attending meetings; Takeda: Other: support for attending meetings; AbbVie: Honoraria; Johnson \u0026amp; Johnson: Honoraria. Frustaci: AbbVie, BeiGene: Other: Travel, accommodations, expenses; AbbVie, BeiGene, AstraZeneca, Janssen: Consultancy. Visentin: Abbvie: Membership on an entity\u0026apos;s Board of Directors or advisory committees, Research Funding; Beigene: Consultancy, Research Funding, Speakers Bureau; J\u0026amp;J: Membership on an entity\u0026apos;s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Membership on an entity\u0026apos;s Board of Directors or advisory committees; AstraZenca SpA: Consultancy, Membership on an entity\u0026apos;s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Sportoletti: Janssen; AstraZeneca, Abbvie; BeiGene: Honoraria, Membership on an entity\u0026apos;s Board of Directors or advisory committees. Mauro:AstraZenca SpA: Consultancy, Membership on an entity\u0026apos;s Board of Directors or advisory committees, Speakers Bureau. Morelli: BeiGene: Current Employment. Autore: BeiGene, AstraZeneca, AbbVie, Janssen: Honoraria. Tedeschi: AstraZeneca, AbbVie, BeiGene, Janssen, Lilly: Membership on an entity\u0026apos;s Board of Directors or advisory committees, Speakers Bureau. Coscia: AbbVie: Honoraria, Membership on an entity\u0026apos;s Board of Directors or advisory committees, Other: support for attending meetings; AstraZeneca: Honoraria, Membership on an entity\u0026apos;s Board of Directors or advisory committees, Other: support for attending meetings; Janssen: Honoraria, Membership on an entity\u0026apos;s Board of Directors or advisory committees, Other: support for attending meetings. Laurenti: AstraZeneca, AbbVie: Research Funding; AstraZeneva, AbbVie, Johnson and Johnson, BeiGene, Lilly: Honoraria; AstraZeneca, AbbVie, Johnson and Johnson, BeiGene, Lilly: Membership on an entity\u0026apos;s Board of Directors or advisory committees.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGoldin L, Slager S. Familial CLL: genes and environment. \u003cem\u003eHematology American Society of Hematology Education Program\u003c/em\u003e 2007. doi:10.1182/asheducation-2007.1.339.\u003c/li\u003e\n \u003cli\u003eChiorazzi N, Chen S-S, Rai KR. 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\u003cstrong\u003e609\u003c/strong\u003e: 174\u0026ndash;182.\u003c/li\u003e\n \u003cli\u003eLee RD, Knutson TP, Munro SA, Miller JT, Heltemes-Harris LM, Mullighan CG \u003cem\u003eet al.\u003c/em\u003e Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation. \u003cem\u003eNat Immunol\u003c/em\u003e 2022; \u003cstrong\u003e23\u003c/strong\u003e: 1763\u0026ndash;1776.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7313089/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7313089/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study provides a comprehensive analysis of the germline landscape in 81 families with chronic lymphocytic leukemia (CLL). We uncovered key genetic pathways associated with CLL predisposition, including telomere maintenance, DNA double-strand break (DSB) repair, and immune regulation. Notably, pathogenic variants were found in \u003cem\u003ePOT1\u003c/em\u003e and \u003cem\u003eTINF2\u003c/em\u003e, suggesting defects in telomere stability, while variants in \u003cem\u003eCHEK2\u003c/em\u003e, \u003cem\u003eMRE11\u003c/em\u003e, \u003cem\u003eCDKN1B\u003c/em\u003e, \u003cem\u003eRAD51D\u003c/em\u003e, \u003cem\u003eERCC2\u003c/em\u003e and \u003cem\u003eRAD50 \u003c/em\u003eindicate defects in DSB repair mechanisms. Variants in immune-related genes such as \u003cem\u003eRUNX1\u003c/em\u003e and \u003cem\u003eTNFRSF13B\u003c/em\u003e were also identified, with \u003cem\u003eTNFRSF13B\u003c/em\u003e mutations occurring in 4 different families and 4 of the 57 (7%) investigated sporadic cases, further highlighting their potential role in disease susceptibility. Other novel candidates included \u003cem\u003eRASA2\u003c/em\u003e, involved in MAPK/ERK signaling, and \u003cem\u003eNCOR2, \u003c/em\u003ea nuclear co-repressor involved in regulation of B cell development and maintenance of genomic integrity. These findings expand the understanding of genetic heterogeneity in familial CLL, supporting a model in which low-penetrance tumor suppressor genes and complex genetic interactions drive disease development. Our results underscore the importance of integrating genetic studies to unravel the hereditary basis of CLL and to further characterize the mechanisms that drive the development of the disease.\u003c/p\u003e","manuscriptTitle":"A landscape of genetic heterogeneity in germline predisposition to familial chronic lymphocytic leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 05:52:59","doi":"10.21203/rs.3.rs-7313089/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"642cc39d-29b2-417b-90e1-166284333b04","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53196535,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Chronic lymphocytic leukaemia"},{"id":53196536,"name":"Biological sciences/Cancer/Cancer genetics"}],"tags":[],"updatedAt":"2025-09-01T11:52:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-01 05:52:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7313089","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7313089","identity":"rs-7313089","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}