Molecular Characterization of Mismatch Repair Deficient Tumors In Young Jordanian Patients

Molecular Characterization of Mismatch Repair Deficient Tumors In Young Jordanian Patients | 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 Case Report Molecular Characterization of Mismatch Repair Deficient Tumors In Young Jordanian Patients Olfat Ahmad, Iyad Sultan, Maysa Hussaini, Osama Smadi, Christian Sutter, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9017459/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract In Jordan, approximately 19% of colorectal carcinomas (CRCs) in patients younger than 45 years show mismatch repair deficiency (MMRD) by immunohistochemistry (IHC). MMRD in pediatric high-grade gliomas (HGG) and CRC in Jordan appears substantially more frequent than global estimates (39% and 44%, respectively), likely reflecting high consanguinity rates and an increased contribution of constitutional MMRD (CMMRD). However, molecular characterization of MMRD-associated tumors in the Middle East remains limited. We analyzed 14 Jordanian patients (< 45 years) from 12 families with clinical or IHC evidence of MMRD, including four brain tumors and 11 CRCs; five patients were children. Pathogenic or likely pathogenic MMR variants were identified in nine families, including one affected sibling pair, comprising five frameshift variants, three single nucleotide variants, and one large deletion. A variant of uncertain significance in MSH6 was detected in another sibling pair. Apparent large deletions suggestive of sequencing artifacts were observed in six patients, underscoring the need for confirmatory copy number analysis by multiplex ligation-dependent probe amplification (MLPA). Three adult cases lacked germline MMR variants, MLH1 hypermethylation, or alterations in other cancer predisposition genes and were considered likely somatic. Tumor mutational burden (TMB) ranged from 2 to 352 variants/Mb (median: 23). Microsatellite instability (MSI) was low in seven tumors, likely due to degraded DNA from older Formalin-Fixed Paraffin-Embedded (FFPE) samples. Among tumors with low TMB and MSI, only one lacked an identifiable germline MMR variant. All tumors showed overrepresentation of C > T transitions, consistent with MMRD-associated mutational signature 6. This study represents the first comprehensive molecular characterization of MMRD-associated tumors in Jordan. Mismatch repair deficiency (MMRD) Constitutional mismatch repair deficiency (CMMRD) Lynch syndrome Colorectal cancer High-grade glioma Jordan / Middle East Figures Figure 1 Figure 2 Introduction Lynch syndrome (LS) is one of the most common adult-onset hereditary cancer predisposition syndromes (CPSs), caused by heterozygous pathogenic (P) or likely pathogenic (LP) variants in the DNA mismatch repair (MMR) genes MLH1 , MSH2 , MSH6 , and PMS2 , together with monoallelic deletions of the EPCAM gene vicinal to MSH2 [ 1 ]. On the other hand, constitutional mismatch repair deficiency (CMMRD) syndrome, a more severe manifestation of MMR deficiency (MMRD) due to biallelic P/LP variants in the MMR genes, typically leads to pediatric onset of cancers [ 2 ]. Similar to global figures, the rate of suspected LS in young adult Jordanians with colorectal carcinoma (CRC) has been estimated to be 19% by immunohistochemistry (IHC) [ 6 ]. Notably, CMMRD is more commonly reported in the Near and Middle East compared to other regions, primarily due to higher rates of consanguinity, which increases the likelihood of inheriting biallelic MMR mutations [ 3 , 4 ]. For example, while the consanguinity rate in Jordan is estimated to be around 30%, and up to 70% in other Middle and Near Eastern countries, it is less than 4% in most other regions worldwide [ 5 ]. Not surprisingly therefore, previous reports from Jordan showed high frequencies of pediatric MMRD CRC (44%) and high-grade glioma (HGG) (39%) [ 3 , 4 ]. In both LS and CMMRD, the defective MMR system results in microsatellite instability (MSI) and increased predisposition to tumors with high tumor mutational burden (TMB). While there are some differences in the tumor spectrum caused by the two syndromes, both predispose mainly to CRC and brain tumors, among other malignancies. Despite the significant contribution of MMRD to CRC and HGG in Jordanian pediatric and young adult populations, molecular characterization of MMRD-associated tumors from Jordan and the broader Middle East remains limited [ 7 ]. In this case series, we aim to provide insights into molecular characterization of young Jordanian patients (< 45 years) with MMRD-associated HGG and/or CRC. For cases in which no pathogenic MMR variant was identified, an extended hereditary CPS gene panel was additionally analyzed. Material and Methods Patients Cohort This is a retrospective analysis of Jordanian patients conducted as a collaboration between King Hussein Cancer Center (KHCC), the German Cancer Research Center (DKFZ) and the Institute of Human Genetics Heidelberg, Germany. Patients younger than 45 years, clinically diagnosed with a biopsy-proven CRC or brain tumor at KHCC from January 1991 until October 2021 were recruited and shortlisted according to their MMR status as suggested by IHC of the corresponding four MMR proteins (MLH1, MSH2, MSH6 and PMS2), or if they scored ≥ 3 on CMMRD criteria regardless of staining pattern [ 8 ]. Institutional Review Board (IRB) approval from KHCC was obtained for contacting living patients, collecting their pedigrees, and obtaining blood samples for germline analysis at DKFZ. In addition, DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) sections and shipped to DKFZ for molecular analysis. Pathology Review At KHCC, pathology reports and tumor blocks were reviewed for MMRD by IHC of the corresponding four MMR proteins (PMS2, MSH6, MLH1, and MSH2). Ventana Benchmark © automated immunostainer © was used to run the IHC stains according to the manufacturer’s instructions. Positive and negative controls were applied on each slide. A tumor was considered MMR proficient if nuclear staining of all of the four MMR proteins was detected in the studied tumor cells, regardless of the intensity of staining or the percentage of positive cell nuclei. MMR-deficient tumors exhibited an absence of any detectable nuclear signal in either one or two MMR proteins in the tumor cells by definition; the absence of nuclear MMR staining of both tumor and normal cells is a hallmark of CMMRD, while intact nuclear staining of normal cells suggested LS. Molecular Analysis Next generation sequencing was performed on leukocyte DNA using a NovaSeq 6000 PE 150 S4 platform employing Iow coverage whole genome sequencing (lcWGS) and whole exome sequencing (WES) (Illumina, San Diego, CA, USA). Data were processed and aligned to the GRCh37/hg19 reference human genome assembly using the Biomedical Genomics Workbench (Qiagen, Hilden, Germany), and assessed using Ingenuity Variant Analysis (Qiagen) and DKFZ in-house NGS data analysis workflow. To predict the pathogenicity/impairment of genetic variants, we utilized several computational tools, including SIFT ( http://sift.jcvi.org/ ) and PolyPhen-2 ( http://genetics.bwh.harvard.edu/pph2/ ). Additionally, we referred to Varsome ( https://landing.varsome.com/varsome ), ClinVar ( https://www.ncbi.nlm.nih.gov/clinvar/ ), and gnomAD ( https://gnomad.broadinstitute.org/ ) databases to gather further insights into the clinical significance and pathogenicity of the identified variants. To assess copy number variants (CNVs) we initially implemented CNVkit v2.1.0 [ 9 ], to identify the variants and generate B allele frequency (BAF) plots. For the six patients who showed a CNV in MSH2 or MLH1 by rough computational mapping of potentially deleted regions identified by CNVkit, Multiplex Ligation-dependent Probe Amplification (MLPA) analysis was performed for confirmation. For QC of complete hybridization and ligation in the MLPA process all samples were evaluated for correct ratio and peak height of the three-quality control (QC) peaks. Using the probe set P003-D1* (MRC-Holland) for CNV detection within MLH1/MSH2 (including EPCAM exon 9). A 50% intensity- reduced signal for the probe matching MSH2 exon 7 was obtained. In addition, an alternative MLPA kit (P248-B1) containing a specific probe matching a region in intron 7 (51 nt downstream of exon 7) likewise showed a 50% intensity-reduced signal that confirmed this deletion. This also allowed a crude mapping of the extent of the MSH2 deletion, with limitations due to this methodology. No other sample showed deletions/duplications detectable by MLPA analysis. For the cases in which no single nucleotide variant (SNV), indel, or CNV was identified in the MMR genes (n = 3) or cases that has a VUS variant of MMR gene (n = 2), MLH1 promoter methylation status was evaluated using the Illumina EPIC methylation array. To visualize methylation patterns, a heatmap of MLH1 -associated CpG sites was generated in RStudio (v.4.2.0), enabling assessment of methylation levels across the most relevant CpG sites cg23658326, cg11600697, cg21490561, and cg00893636 [ 10 ]. In addition, a CPS panel comprising 213 cancer predisposition genes (listed in Supplementary Table 1). TMB was calculated based on WES data, as the number of high-quality (quality score ≥ 8) somatic functional SNVs and Indels in exonic regions divided by the total size of the exonic regions in the genome (total targeted regions overlapping annotated exons), multiplied by 1 million (TMB = mutations/ genome × 1,000,000). Target regions were derived from the Agilent 7 (without Untranslated Region (UTRs)) exome enrichment kit, and annotations were based on Gencode version 19. Mutational signatures were generated utilizing paired normal/tumor WES data via COSMIC signature assignment tool https://cancer.sanger.ac.uk/signatures/assignment/ . MSI was assessed on the basis of NGS data of WES utilizing MSIsensor https://github.com/ding-lab/msisensor [ 11 ]. We used a cut-off MSIsensor score ≥ 10 to define MSI high (MSI-H) [ 12 ]. Results Fourteen Jordanian patients, all aged younger than 45 years, from 12 families, were enrolled in the study. They presented with CRCs (n = 11) and/or brain tumors (n = 4) associated with suspected MMRD by IHC staining or clinical features, with one patient diagnosed with both CRC and a HGG (patient #12, Table 1). The cohort included five children: two siblings with CRCs (ages 13, and 15) and three with brain tumors (ages 9, 13, and 17 years). Nine patients exhibited IHC staining patterns compatible with LS. Three patients had IHC staining pattern consistent with CMMRD, and two patients with normal IHC staining but suggestive overall CMMRD score, according to the recommendations from the international consensus working group [8]. Table abbreviations : Number (No), Identifier (ID), Microsatellite Instability (MSI), and Tumor Mutational Burden (TMB), Immunohistochemistry (IHC), Colorectal Cancer (CRC), Lynch Syndrome (LS), Male (M), High (H), Chromosome (Chr), Large (lg), Deletion (Del), Genome Reference Consortium Human Build 37 (GRCh37), Not available (NA), Kilo base (kb), Codon (c.), Protein (p.), MLPA (Multiplex Ligation-dependent Probe Amplification), Duplication (Dup), Frameshift (Fs), Insertion (ins), Low (L), Likely Pathogenic (LP), Missense Mutation (ms), Untranslated region (UTR), Pathogenic (P), Constitutional Mismatch Repair Deficiency (CMMRD). Pedigrees’ Key: Identified Germline Variants of MMR Genes Five distinct frameshift (fs) indels of MMR genes were identified in five unrelated patients, including 3 MLH1 variants (patients # 5, 8 and 9), one MSH2 variant (patient #4) and another PMS2 variant (patient #14). Patient #4 (Table 1) is a 33-year-old male with CRC whose brother has also developed CRC. He carries MSH2 variant NM_000251.3:c.1321dup (p.Thr441fs), which is fs variant classified as P variant in ClinVar. Patient # 5 (Table 1) is a 28-year-old male with CRC and a family history of various tumors including CRC, gastric, esophageal, endometrial, brain, and breast cancers. He has a fs variant in MLH1 gene ENST00000231790.2:c.1164_1165insGAAT(p.Ser388fs), which is classified as P variant in Clinvar. Patient #8 (Table 1) is a 37-year-old male with a family history notable for multiple relatives affected predominantly by CRC and two cases of breast cancer. He carries the MLH1 variant NM_000249.4:c.901del (p.(Gln301ArgfsTer66)), a fs alteration confirmed to be P variant in Clinvar. Patient #9 (Table 1) is a 43-year-old female with a family history of various gastrointestinal malignancies, together with breast, lung and parathyroid tumors. She has MLH1 variant NM_000249.4:c.1836_1839del (p.(Val612del)), which is a fs deletion, confirmed by expert panel to be LP in Clinvar. Patient #14 (Table 1) is a 9-year-old with a HGG and a family history of CRC, uterine cancer in the mother and thyroid tumors. She has a PMS2 variant ENST00000441476.2:c.1269dup (p.(Gln424AlafsTer12)). As endometrial cancer is an integrate tumor of LS, it is likely that the mother is a carrier of this variant. Three missense (ms) variants were identified across three families, including two families with affected sibling pairs (five patients in total). These included a P MSH6 variant, a LP PMS2 variant, and a VUS in MSH6 : Patient #11 (Table 1) is a 17-year-old girl who presented with a HGG, carries a pathogenic MSH6 variant ENST00000234420.5:c.2314C > T (p.(Arg772Trp)), which is confirmed as P variant in Clinvar. Her parents have no history of cancer; however, the family is consanguineous. Notably, the maternal grandmother was diagnosed with uterine cancer at age 60, and the paternal grandfather developed colorectal cancer at age 40. This background highlights the need for genetic screening of both currently healthy parents. Patients #6 and #7 (Table 1) are sisters aging 13 and 15 years when presented with CRC, who are identified to carry an ms variant of PMS2 gene NM_000535.7:c.903G > T (p.Lys301Asn), classified as LP in Clinvar. The 15-year-old girl had a pilomatrixoma as well, and they had a family history of leukemia, uterine, pancreatic and skin malignancy. Similar to patient #11, the parents must have been yet undetected carriers of one of the PMS2 variants. Although the children’s parents were not tested yet it is conceivable that both of them could be carrier of this PMS2 missense variant. Patients #12 and #13 (Table 1) are siblings: a 22-year-old brother diagnosed with CRC and HGG, and his 13-year-old sister diagnosed with HGG. Their mother died from a brain tumor, and two maternal uncles were also diagnosed with brain tumors in their twenties. Based on this strong family history, the siblings were enrolled in the study despite negative IHC results for MMRD. Germline analysis came back positive only for a shared VUS in MSH6 gene, ENST00000234420.10: c.1217G > A (p.Cys406Tyr), which makes it worth reporting. Patient #2 (Table 1) is a 30-year-old female, who is diagnosed with CRC. er family history is highly suggestive of a hereditary CPS with several family members affected with gastrointestinal, uterine, sarcomas and other malignancies. No SNVs or indels were identified in her sample by sequencing. A large deletion of approximately 74 kb in MSH2 was suggested by lcWGS (see sequencing artifacts section below), and MLPA analysis subsequently confirmed a heterozygous deletion involving exon 7 only (Supplementary Information (SI)). This deletion represents a known pathogenic copy number variant predicted to cause a frameshift and is associated with LS [13–15]. Additional Testing for Patients with No Validated MMR Variants For the two siblings (Patients #12 and #13) who carried only a VUS in MSH6 (ENST00000234420.10:c.1217G > A; p.Cys406Tyr), as well as for Patients #1, #3, and #10 (Table 1), in whom no SNVs, indels, or CNVs were identified, a CPS gene panel (specified in the methods section) was performed and did not reveal any P/LP variants. For the adult patients with CRC in this group (Patients #1, #3, and #10), MLH1 promoter hypermethylation analysis was performed (Fig. 1) and yielded negative results in all cases. Taken together, although not proven by functional tests, these findings suggest a possible contribution of the MSH6 VUS in the two siblings (Patients #12 and #13). In contrast, in Patients #1, #3, and #10—who were enrolled based solely on young age at diagnosis (39, 44, and 38 years, respectively) and loss of MMR protein expression by IHC, but without strong clinical suspicion—the observed IHC pattern is more likely attributable to a somatic event. Figure 1. MLH1 promoter methylation analysis for patients #1, #3, and #10 showing absence of methylation at four relevant CpG sites (cg23658326, cg11600697, cg21490561, and cg00893636), indicated by the red box at the left side of the graph. Apparent large deletions likely representing sequencing artifacts In addition to the identified sequence variants, the next generation sequencing (NGS) data suggested large deletions in six patients: three involving MSH2 and three involving MLH1 . However, MLPA analysis confirmed only part of one deletion in a single patient (patient #2, Table 1), suggesting that most of these findings may represent sequencing artifacts rather than true constitutional deletions. Furthermore, the three cases with presumed MSH2 deletion have overlapping segment of 74.6 kb (chr2:47,635,736–47,710,309, GRCh37) (Fig. 2-A), and the three patients with presumed MLH1 deletion have an overlapping area spanning approximately 53 kb (chr3:37,037,285–37,090,106, GRCh37) (Fig. 2-B), originally calculated from NGS based BAF plots. The recurrence of highly overlapping genomic regions across unrelated patients further supports the interpretation of these events as technical artifacts. This conclusion is also consistent with the overall clinical and molecular characterization of the tumors, as discussed below on a case-by-case basis. Figure 2-B Apparent overlapping deletions of MLH1 gene detected in three unrelated Jordanian patients as visualized on UCSC genomic browser, likely representing artifacts. The following cases showed putative artifacts in MSH2. BAF plots for all patients are available in SI : Patient #1 (Table 1) exhibited a deletion spanning approximately 390 kb (chr2:47,641,377–48,033,551, GRCh37), resulting in loss of MSH2 from intron 4 onward as well as deletion of the first eight exons of MSH6 . As explained in previous paragraph, no additional molecular events were identified: specifically, no SNVs or indels in the MMR genes, no pathogenic variants in other CPS genes, and no MLH1 promoter hypermethylation. The absence of strong clinical suspicion and patient’s enrolment based solely on young age (39 years), the IHC staining pattern could be attributable to a somatic event. Patient #2 (Table 1) showed a 74 kb deletion (chr2:47,635,736–47,710,309, GRCh37), which encompasses most of the gene from intron 2 to parts of the 3’UTR of MSH2 and overlaps with the first deletion. MLPA analysis carried out with two different probe kits only confirmed a large deletion of exon 7 and of 51nt of DNA downstream of exon 7 exclusively, as explained above. Patient #10 (Table 1) presented with a 112 kb deletion (chr2:47,635,736–47,748,646, GRCh37) encompassing intron 2 of MSH2 and extending into the KCNK12 gene, overlapping part of the deleted region observed in Patients #1 and #2. As explained in previous paragraph, and similar to patient #1, no additional molecular germline events were identified. Apart from his age at diagnosis (38 years), this patient did not exhibit strong clinical suspicion for a hereditary CPS. His tumor mutational burden was low (TMB 2.1), and MSI status was also low (0.13%). The loss of MMR protein expression on IHC may therefore reflect a somatic rather than germline event. The following cases showed putative artifacts in MLH1. BAF plots for all patients are available in SI : Patient #3 (Table 1) exhibited a deletion spanning approximately 53 kb (chr3:37,037,285–37,090,106, GRCh37), affecting exons 2–17 (parts of intron 1 to parts of intron 17). As explained in previous paragraph, and similar to patients #1 and 10, no additional molecular germline events were identified. Given the absence of strong clinical suspicion and his enrolment based solely on young age (44 years), the IHC staining pattern could be attributable to a somatic event. Patient #13 (Table 1) showed an approximately 132 kb (chr3:37,038,204–37,170,640, GRCh37), encompassing most parts of intron 2 far into the 3' untranslated region (UTR) and the noncoding UBE2F pseudogene 1 (UBE2FP1). However, the patient’s sibling (patient #12, Table 1) did not show the same deletion. Both siblings had normal IHC despite the suggestive clinical picture of CMMRD. Indeed, both siblings showed a VUS in MSH6 gene ENST00000234420.10: c.1217G > A (p.(Cys406Tyr)), which might be causative of their clinical picture. Patient #11 (Table 1) showed an approximately 54 kb in size (chr3: 37,038,204–37,093,204, GRCh37), affecting most parts of intron 2 to the 3' UTR. This variant cannot explain the IHC loss of MSH2 , MSH6 . In addition, this patient had a P variant of MSH6 gene ENST00000234420.10: c.2314C > T (p.(Arg772Trp)), which clearly explains the patient’s clinical picture including her diagnosis with HGG at the age of 17 years among other features. Additional Molecular Findings TMB varied significantly among patients, ranging from 2.1 to 352 variant/Mb, with a median of 23 variant/Mb and a mode of 19 variant/Mb. Notably, four tumors exhibited a TMB of less than 10 variant/Mb, while one had a TMB exceeding 100 variant/Mb. MSI on the basis of NGS data of WES was low in eight of 14 patients, including six of the proved 11 MMRD tumors, with scores ranging from 0.13% to 26% (mean: 10.6%). This limitation might be related to FFPE material as explained in the discussion below. Mutation signatures in all the tumor cases showed an overrepresentation of C > T transitions (Table 1), similar to signature 6 which has been associated with mismatch repair-deficient tumor tissue [16]. Discussion Nine of the enrolled fourteen patients have a P/LP variant in an MMR gene. In addition, a pair of enrolled siblings have a VUS of MSH6 gene. Three patients (patient #1, #3 and #10, Table 1 ) have not shown any SNV, indel, or CNV of MMR genes and were suggested to be somatic cases of MMR deficiency. Indeed, a substantial proportion of patients who meet the clinical criteria of LS or CMMRD, do not show germline mutations in any of the respective genes. Two studies of 28 and 38 cases of suspected CMMRD tumors reported 25% and 26% of the cases to have no identified P or LP variants [ 8 ]. Similarly, it is estimated that only 53–70% of suspected LS cases show germline variants, with the higher end of the range relating to cases with IHC loss of MSH2 / MSH6 [ 17 ]. Other cancer predisposition genes explained some cases [ 17 ]. In our cases series, all the three patients for whom no variants were identified, have weak clinical suspicion and were enrolled because of their age, and suggestive IHC results. Nevertheless, we examined them for other CPS and negative results were obtained. In addition, none of those cases showed evidence of MLH1 promoter hypermethylation. The two siblings (patients #12 and #13) exhibit strong clinical suspicion for CMMRD despite negative IHC stains of MMR genes, with no SNV, indel or CNV of MMR genes identified. The fact that both of them carry the same VUS variant of MSH6 gene ENST00000234420.10:c.1217G > A (p.(Cys406Tyr)) makes it worth reporting. Functional genomic testing for this variant can be helpful to confirm the diagnosis [ 18 ]. Apparent large deletions likely representing sequencing artifacts were identified in 6 patients, 3 showing overlapping deletions of MLH1 and 3 showing overlapping deletions in MSH2 . At first, the deletions looked similar to each other which raised our suspicion for artifacts (BAF plots, SI). Moreover, two patients already carry other germline MMR events that could explain their situation (patients #11 and #13). Accordingly, MLPA[ 19 ], which is a sensitive technique for detecting large CNVs through quantitative analysis of specific regions of the MMR genes was conducted. Only one patient of the six proved to have part of the identified sequencing deletion of MSH2 confirmed by MLPA, leaving three cases (patient #1, #3 and #10, Table 1 ) suspected to be somatic MMRD given the absence of other germline events and the weak clinical suspicion. TMB in our cohort exhibited a wide range, with four tumors having normal TMB unexpectedly, with only one of which suspected to be a somatic case of CRC (patient #10). The majority displayed hypermutation (> 10 variants/Mb) and one was classified as ultra-hypermutated (> 100 variants/Mb). Hypermutation and ultra-hypermutation have been strongly linked to MMRD, which are characteristic of LS and CMMRD, with the latter being more likely to demonstrate extreme mutational loads [ 8 , 20 ]. Unexpectedly, six of the proved MMRD tumors of 11 patients exhibited low MSI. This might be related to the quality of DNA extracted from old FFPE blocks dating back to 1991 for the oldest block (patient #5, Table 1 ). According to Qingli Guo et al. “Application of MSIsensor to an unrepaired CRC sample could lead to miscalling of MSI status for the tumour” [ 21 ]. Additionally, tumor purity (percentage of tumor cells per slide) is a prerequisite for successful MSI analysis[ 22 ]. Utilizing the more sensitive Low-Pass Genomic Instability Characterisation Assay (LOGIC) would be helpful in such scenario [ 23 ]. Finally, this study highlights the importance of surveillance in affected Jordanian families. Notably, the presence of a germline MMR variant in affected children may serve as an indicator of cancer risk in their often asymptomatic parents. This is illustrated by patients #6, #7 and #11, who developed tumors while parents were still clinically unaffected. In addition, the study underscores the limitations of using FFPE material for molecular characterization of MMRD tumors and highlights the need to confirm sequencing-based CNVs by MLPA analysis. Another limitation is the unavailability of parental DNA samples, which precluded discrimination between biallelic and monoallelic inheritance. Abbreviations LS Lynch syndrome CPSs Cancer predisposition syndromes P Pathogenic LP Likely pathogenic MMR Mismatch repair CMMRD Constitutional mismatch repair deficiency MMRD Mismatch repair deficiency CRC Colorectal carcinoma IHC Immunohistochemistry HGG High grade glioma MSI Microsatellite instability TMB Tumor mutational burden KHCC King Hussein Cancer Center DKFZ German Cancer Research Center IRB Institutional Review Board FFPE Formalin-Fixed Paraffin-Embedded lcWGS Low coverage whole genome sequencing WES Whole exome sequencing UBE2FP1 UBE2F pseudogene 1 CNV Copy number variant BAF B allele frequency MLPA Multiplex ligation-dependent probe amplification QC Quality control SNV Single nucleotide variant UTR Untranslated region MSI-H Microsatellite instability- high fs Frameshift ms Missense SI Supplementary Information NGS Next generation sequencing Declarations Conflict of Interest None Ethics Statement Patients were recruited from King Hussein Cancer Center (KHCC). The study was approved by the Institutional Review Board of KHCC (ethics approval number: 140F-2021) and was conducted in accordance with the ethical standards of the institutional research committee and the principles of the Declaration of Helsinki. Freely given, informed consent to participate in the study and to publish the results was obtained from all participants prior to inclusion. For participants under the age of 16, informed consent was obtained from a parent or legal guardian. Written informed consent for publication of pseudonymized clinical and molecular data was obtained from all participants or their legal guardians, where applicable. Participants were informed about the purpose of the study and the intended publication of their data in a scientific journal. Consent to Publish As indicated in the ethics statement, informed consent was obtained from participants and/or their parents including consent to participate and to publish their results. Funding Statement This research was partially supported by King Hussein Cancer Center (KHCC), Amman, Jordan, where patients were recruited, pathology blocks were cut and reviewed and DNA was extracted. Molecular analysis was supported by the German Cancer Research Center (DKFZ), Heidelberg, Germany, as part of the fellowship project of O.A. Author Contribution O. A. Conceptualized the work, was involved in all molecular analyses (fellowship project), drafted the work, produced the table and figures, and reviewed the final manuscript.I. S. Conceptualized the work and reviewed the final manuscript.M. H. Pathology review at KHCC and reviewed the final manuscript.O. S. Helped conceptualizing the work as well as planning and coordinating the workflow at KHCC and reviewed the final manuscript.C. S. Helped in validation of germline variants, planned and coordinated the work at Institute of Human Genetics at Heidelberg and reviewed the final manuscript.S. H. Helped in validation of germline variants and reviewed the final manuscript.R. A. and C. P. Helped in bioinformatics analysis and reviewed the final manuscript.A. F. Helped in planning the molecular analysis and reviewed the final manuscript.L. A. Helped in planning the manuscript, editing pedigrees and reviewed the final manuscript.N. A. Helped in identification of pediatric HGG patients, contributed to clinical data and reviewed the final manuscript.M. A. Sh. Genetic counselling for recruited patients and reviewed the final manuscript.R. A. Helped in identification of adult patients, contributed to clinical data and reviewed the final manuscript.S. A., L. F., A. N., A. Q. and H. A. Provided first draft of literature review and reviewed the final manuscript.D. E. R., N. R. F., A. D. and U. T. Provided revision of the work and reviewed the final manuscript.S. M. P. Supervised the whole project and reviewed the final manuscript.C. P. S. Supervised the whole project and reviewed the final manuscript. Data Availability DNA sequencing data were deposited into the German Human Genome-Phenome Archive GHGA repository under accession number GHGAS19356866116009 and are available at the following URL:[https://data.ghga.de/study/GHGAS19356866116009](https:/data.ghga.de/study/GHGAS19356866116009)Access is subject to controlled access procedures in accordance with GHGA data governance policies. References Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Milestones of Lynch syndrome: 1895–2015. Nat Rev Cancer. Mar 2015;15(3):181–94. 10.1038/nrc3878 . Wimmer K et al. Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD), J Med Genet , vol. 51, no. 6, pp. 355 – 65, Jun 2014, 10.1136/jmedgenet-2014-102284 Amayiri N, et al. High frequency of mismatch repair deficiency among pediatric high grade gliomas in Jordan. Int J Cancer. 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Diagnostic criteria for constitutional mismatch repair deficiency (CMMRD): recommendations from the international consensus working group. J Med Genet. Apr 2022;59(4):318–27. 10.1136/jmedgenet-2020-107627 . Talevich E, Shain AH, Botton T, Bastian BC. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing. PLoS Comput Biol. Apr 2016;12(4):e1004873. 10.1371/journal.pcbi.1004873 . Benhamida JK, et al. Reliable Clinical MLH1 Promoter Hypermethylation Assessment Using a High-Throughput Genome-Wide Methylation Array Platform, (in eng). J Mol Diagn. Mar 2020;22(3):368–75. 10.1016/j.jmoldx.2019.11.005 . Niu B et al. MSIsensor: microsatellite instability detection using paired tumor-normal sequence data, Bioinformatics , vol. 30, no. 7, pp. 1015-6, Apr 1 2014, 10.1093/bioinformatics/btt755 Middha S, et al. Reliable Pan-Cancer Microsatellite Instability Assessment by Using Targeted Next-Generation Sequencing Data. JCO Precis Oncol. 2017;2017. 10.1200/PO.17.00084 . Zhu M et al. Large genomic aberrations in MSH2 and MLH1 genes are frequent in Chinese colorectal cancer, (in eng), Cancer Genet Cytogenet , vol. 160, no. 1, pp. 61 – 7, Jul 1 2005, 10.1016/j.cancergencyto.2004.12.008 Li L, et al. Distinct patterns of germ-line deletions in MLH1 and MSH2: the implication of Alu repetitive element in the genetic etiology of Lynch syndrome (HNPCC), (in eng). Hum Mutat. Apr 2006;27(4):388. 10.1002/humu.9417 . Perez-Cabornero L et al. A new strategy to screen MMR genes in Lynch Syndrome: HA-CAE, MLPA and RT-PCR, (in eng), Eur J Cancer , vol. 45, no. 8, pp. 1485-93, May 2009, 10.1016/j.ejca.2009.01.030 Alexandrov LB et al. Signatures of mutational processes in human cancer, Nature , vol. 500, no. 7463, pp. 415 – 21, Aug 22 2013, 10.1038/nature12477 Kayser K, et al. Copy number variation analysis and targeted NGS in 77 families with suspected Lynch syndrome reveals novel potential causative genes. Int J Cancer. Dec 1 2018;143(11):2800–13. 10.1002/ijc.31725 . Shuen AY et al. Functional Repair Assay for the Diagnosis of Constitutional Mismatch Repair Deficiency From Non-Neoplastic Tissue. J Clin Oncol, 37, 6, pp. 461–70, Feb 20 2019, 10.1200/JCO.18.00474 Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. Jun 15 2002;30(12):e57. 10.1093/nar/gnf056 . Campbell BB et al. Comprehensive Analysis of Hypermutation in Human Cancer, Cell , vol. 171, no. 5, pp. 1042–1056 e10, Nov 16 2017. 10.1016/j.cell.2017.09.048 Guo Q, Lakatos E, Bakir IA, Curtius K, Graham TA, Mustonen V. The mutational signatures of formalin fixation on the human genome. Nat Commun. Sep 6 2022;13(1):4487. 10.1038/s41467-022-32041-5 . Trabucco SE, et al. A Novel Next-Generation Sequencing Approach to Detecting Microsatellite Instability and Pan-Tumor Characterization of 1000 Microsatellite Instability-High Cases in 67,000 Patient Samples. J Mol Diagn. Nov 2019;21(6):1053–66. 10.1016/j.jmoldx.2019.06.011 . Chung J, et al. Genomic Microsatellite Signatures Identify Germline Mismatch Repair Deficiency and Risk of Cancer Onset. J Clin Oncol. Feb 1 2023;41(4):766–77. 10.1200/JCO.21.02873 . Additional Declarations No competing interests reported. Supplementary Files BAFplots.pdf CPSpanelgenes.docx MLPAMSH2del7.pdf Table1Clinicalandmolecularcharacterizationofenrolledpatientsinthestudy.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 16 Apr, 2026 Reviews received at journal 12 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers invited by journal 26 Mar, 2026 Editor assigned by journal 13 Mar, 2026 Submission checks completed at journal 11 Mar, 2026 First submitted to journal 11 Mar, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9017459","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":614014143,"identity":"cc927988-f4c6-4cb6-a126-79b41c632bb4","order_by":0,"name":"Olfat Ahmad","email":"","orcid":"","institution":"Hopp Children's Cancer Center (KiTZ)","correspondingAuthor":false,"prefix":"","firstName":"Olfat","middleName":"","lastName":"Ahmad","suffix":""},{"id":614014145,"identity":"2ddc17b9-60c4-43c7-aa3c-9c22f90d64fa","order_by":1,"name":"Iyad Sultan","email":"","orcid":"","institution":"King Hussein Cancer Center 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07:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9017459/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9017459/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105874381,"identity":"8ab02918-495a-47c1-b57d-96a1d07a5feb","added_by":"auto","created_at":"2026-04-01 05:26:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85678,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMLH1 promoter methylation analysis for patients #1, #3, and #10 showing absence of methylation at four relevant CpG sites (cg23658326, cg11600697, cg21490561, and cg00893636), indicated by the red box at the left side of the graph.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9017459/v1/a002b53fa71c87cdef69f2a1.png"},{"id":105874362,"identity":"f3817650-70c9-4e8c-9d7f-8b8d5d045a5a","added_by":"auto","created_at":"2026-04-01 05:26:05","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":994602,"visible":true,"origin":"","legend":"\u003cp\u003eA Apparent overlapping deletions of MSH2 gene detected in three unrelated Jordanian patients as visualized on UCSC genomic browser, likely representing artifacts.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB \u003c/em\u003eApparent overlapping deletions of \u003cem\u003eMLH1\u003c/em\u003e gene detected in three unrelated Jordanian patients as visualized on UCSC genomic browser, likely representing artifacts\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9017459/v1/98c190dedac217899f6c65f4.jpeg"},{"id":105904655,"identity":"9c4710b7-4cac-469d-8d1e-47896b02b4a5","added_by":"auto","created_at":"2026-04-01 10:10:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1791571,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9017459/v1/aa5a580b-a709-4904-a33a-a2452488003d.pdf"},{"id":105874363,"identity":"cfc2eea6-7c94-4de6-8ec5-e8979c1f699b","added_by":"auto","created_at":"2026-04-01 05:26:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1602593,"visible":true,"origin":"","legend":"","description":"","filename":"BAFplots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9017459/v1/a506520213aef5699a558e8c.pdf"},{"id":105874314,"identity":"74705c77-c98c-4f21-828d-c35f8f3013b9","added_by":"auto","created_at":"2026-04-01 05:25:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17371,"visible":true,"origin":"","legend":"","description":"","filename":"CPSpanelgenes.docx","url":"https://assets-eu.researchsquare.com/files/rs-9017459/v1/0eee55e183a5c697eed7912f.docx"},{"id":105874336,"identity":"89bd682f-55ed-4862-9e38-020ff4126f0c","added_by":"auto","created_at":"2026-04-01 05:26:01","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2103538,"visible":true,"origin":"","legend":"","description":"","filename":"MLPAMSH2del7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9017459/v1/1e258613ceed01662f54677d.pdf"},{"id":105874337,"identity":"a7b312e1-d58a-418b-946b-14bad08a47d0","added_by":"auto","created_at":"2026-04-01 05:26:01","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10431450,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Clinicalandmolecularcharacterizationofenrolledpatientsinthestudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-9017459/v1/46885e6f0b5de24072a77953.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Characterization of Mismatch Repair Deficient Tumors In Young Jordanian Patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLynch syndrome (LS) is one of the most common adult-onset hereditary cancer predisposition syndromes (CPSs), caused by heterozygous pathogenic (P) or likely pathogenic (LP) variants in the DNA mismatch repair (MMR) genes \u003cem\u003eMLH1\u003c/em\u003e, \u003cem\u003eMSH2\u003c/em\u003e, \u003cem\u003eMSH6\u003c/em\u003e, and \u003cem\u003ePMS2\u003c/em\u003e, together with monoallelic deletions of the \u003cem\u003eEPCAM\u003c/em\u003e gene vicinal to \u003cem\u003eMSH2\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. On the other hand, constitutional mismatch repair deficiency (CMMRD) syndrome, a more severe manifestation of MMR deficiency (MMRD) due to biallelic P/LP variants in the MMR genes, typically leads to pediatric onset of cancers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Similar to global figures, the rate of suspected LS in young adult Jordanians with colorectal carcinoma (CRC) has been estimated to be 19% by immunohistochemistry (IHC) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, CMMRD is more commonly reported in the Near and Middle East compared to other regions, primarily due to higher rates of consanguinity, which increases the likelihood of inheriting biallelic MMR mutations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For example, while the consanguinity rate in Jordan is estimated to be around 30%, and up to 70% in other Middle and Near Eastern countries, it is less than 4% in most other regions worldwide [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Not surprisingly therefore, previous reports from Jordan showed high frequencies of pediatric MMRD CRC (44%) and high-grade glioma (HGG) (39%) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn both LS and CMMRD, the defective MMR system results in microsatellite instability (MSI) and increased predisposition to tumors with high tumor mutational burden (TMB). While there are some differences in the tumor spectrum caused by the two syndromes, both predispose mainly to CRC and brain tumors, among other malignancies. Despite the significant contribution of MMRD to CRC and HGG in Jordanian pediatric and young adult populations, molecular characterization of MMRD-associated tumors from Jordan and the broader Middle East remains limited [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this case series, we aim to provide insights into molecular characterization of young Jordanian patients (\u0026lt;\u0026thinsp;45 years) with MMRD-associated HGG and/or CRC. For cases in which no pathogenic MMR variant was identified, an extended hereditary CPS gene panel was additionally analyzed.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients Cohort\u003c/h2\u003e \u003cp\u003eThis is a retrospective analysis of Jordanian patients conducted as a collaboration between King Hussein Cancer Center (KHCC), the German Cancer Research Center (DKFZ) and the Institute of Human Genetics Heidelberg, Germany. Patients younger than 45 years, clinically diagnosed with a biopsy-proven CRC or brain tumor at KHCC from January 1991 until October 2021 were recruited and shortlisted according to their MMR status as suggested by IHC of the corresponding four MMR proteins (MLH1, MSH2, MSH6 and PMS2), or if they scored\u0026thinsp;\u0026ge;\u0026thinsp;3 on CMMRD criteria regardless of staining pattern [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Institutional Review Board (IRB) approval from KHCC was obtained for contacting living patients, collecting their pedigrees, and obtaining blood samples for germline analysis at DKFZ. In addition, DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) sections and shipped to DKFZ for molecular analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePathology Review\u003c/h3\u003e\n\u003cp\u003eAt KHCC, pathology reports and tumor blocks were reviewed for MMRD by IHC of the corresponding four MMR proteins (PMS2, MSH6, MLH1, and MSH2). Ventana Benchmark \u0026copy; automated immunostainer \u0026copy; was used to run the IHC stains according to the manufacturer\u0026rsquo;s instructions. Positive and negative controls were applied on each slide. A tumor was considered MMR proficient if nuclear staining of all of the four MMR proteins was detected in the studied tumor cells, regardless of the intensity of staining or the percentage of positive cell nuclei. MMR-deficient tumors exhibited an absence of any detectable nuclear signal in either one or two MMR proteins in the tumor cells by definition; the absence of nuclear MMR staining of both tumor and normal cells is a hallmark of CMMRD, while intact nuclear staining of normal cells suggested LS.\u003c/p\u003e\n\u003ch3\u003eMolecular Analysis\u003c/h3\u003e\n\u003cp\u003eNext generation sequencing was performed on leukocyte DNA using a NovaSeq 6000 PE 150 S4 platform employing Iow coverage whole genome sequencing (lcWGS) and whole exome sequencing (WES) (Illumina, San Diego, CA, USA). Data were processed and aligned to the GRCh37/hg19 reference human genome assembly using the Biomedical Genomics Workbench (Qiagen, Hilden, Germany), and assessed using Ingenuity Variant Analysis (Qiagen) and DKFZ in-house NGS data analysis workflow. To predict the pathogenicity/impairment of genetic variants, we utilized several computational tools, including SIFT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sift.jcvi.org/\u003c/span\u003e\u003cspan address=\"http://sift.jcvi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and PolyPhen-2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genetics.bwh.harvard.edu/pph2/\u003c/span\u003e\u003cspan address=\"http://genetics.bwh.harvard.edu/pph2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Additionally, we referred to Varsome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://landing.varsome.com/varsome\u003c/span\u003e\u003cspan address=\"https://landing.varsome.com/varsome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ClinVar (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/clinvar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and gnomAD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"https://gnomad.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases to gather further insights into the clinical significance and pathogenicity of the identified variants.\u003c/p\u003e \u003cp\u003eTo assess copy number variants (CNVs) we initially implemented CNVkit v2.1.0 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], to identify the variants and generate B allele frequency (BAF) plots. For the six patients who showed a CNV in \u003cem\u003eMSH2\u003c/em\u003e or \u003cem\u003eMLH1\u003c/em\u003e by rough computational mapping of potentially deleted regions identified by CNVkit, Multiplex Ligation-dependent Probe Amplification (MLPA) analysis was performed for confirmation. For QC of complete hybridization and ligation in the MLPA process all samples were evaluated for correct ratio and peak height of the three-quality control (QC) peaks. Using the probe set P003-D1* (MRC-Holland) for CNV detection within \u003cem\u003eMLH1/MSH2\u003c/em\u003e (including \u003cem\u003eEPCAM\u003c/em\u003e exon 9). A 50% intensity- reduced signal for the probe matching \u003cem\u003eMSH2\u003c/em\u003e exon 7 was obtained. In addition, an alternative MLPA kit (P248-B1) containing a specific probe matching a region in intron 7 (51 nt downstream of exon 7) likewise showed a 50% intensity-reduced signal that confirmed this deletion. This also allowed a crude mapping of the extent of the \u003cem\u003eMSH2\u003c/em\u003e deletion, with limitations due to this methodology. No other sample showed deletions/duplications detectable by MLPA analysis.\u003c/p\u003e \u003cp\u003eFor the cases in which no single nucleotide variant (SNV), indel, or CNV was identified in the MMR genes (n\u0026thinsp;=\u0026thinsp;3) or cases that has a VUS variant of MMR gene (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eMLH1\u003c/em\u003e promoter methylation status was evaluated using the Illumina EPIC methylation array. To visualize methylation patterns, a heatmap of \u003cem\u003eMLH1\u003c/em\u003e-associated CpG sites was generated in RStudio (v.4.2.0), enabling assessment of methylation levels across the most relevant CpG sites cg23658326, cg11600697, cg21490561, and cg00893636 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, a CPS panel comprising 213 cancer predisposition genes (listed in Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTMB was calculated based on WES data, as the number of high-quality (quality score\u0026thinsp;\u0026ge;\u0026thinsp;8) somatic functional SNVs and Indels in exonic regions divided by the total size of the exonic regions in the genome (total targeted regions overlapping annotated exons), multiplied by 1\u0026nbsp;million (TMB\u0026thinsp;=\u0026thinsp;mutations/ genome \u0026times; 1,000,000). Target regions were derived from the Agilent 7 (without Untranslated Region (UTRs)) exome enrichment kit, and annotations were based on Gencode version 19. Mutational signatures were generated utilizing paired normal/tumor WES data via COSMIC signature assignment tool \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cancer.sanger.ac.uk/signatures/assignment/\u003c/span\u003e\u003cspan address=\"https://cancer.sanger.ac.uk/signatures/assignment/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. MSI was assessed on the basis of NGS data of WES utilizing MSIsensor \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ding-lab/msisensor\u003c/span\u003e\u003cspan address=\"https://github.com/ding-lab/msisensor\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We used a cut-off MSIsensor score\u0026thinsp;\u0026ge;\u0026thinsp;10 to define MSI high (MSI-H) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFourteen Jordanian patients, all aged younger than 45 years, from 12 families, were enrolled in the study. They presented with CRCs (n\u0026thinsp;=\u0026thinsp;11) and/or brain tumors (n\u0026thinsp;=\u0026thinsp;4) associated with suspected MMRD by IHC staining or clinical features, with one patient diagnosed with both CRC and a HGG (patient #12, Table 1). The cohort included five children: two siblings with CRCs (ages 13, and 15) and three with brain tumors (ages 9, 13, and 17 years). Nine patients exhibited IHC staining patterns compatible with LS. Three patients had IHC staining pattern consistent with CMMRD, and two patients with normal IHC staining but suggestive overall CMMRD score, according to the recommendations from the international consensus working group [8].\u003c/p\u003e\n\u003cdiv\u003e\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable abbreviations\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eNumber (No), Identifier (ID), Microsatellite Instability (MSI), and Tumor Mutational Burden (TMB), Immunohistochemistry (IHC), Colorectal Cancer (CRC), Lynch Syndrome (LS), Male (M), High (H), Chromosome (Chr), Large (lg), Deletion (Del), Genome Reference Consortium Human Build 37 (GRCh37), Not available (NA), Kilo base (kb), Codon (c.), Protein (p.), MLPA (Multiplex Ligation-dependent Probe Amplification), Duplication (Dup), Frameshift (Fs), Insertion (ins), Low (L), Likely Pathogenic (LP), Missense Mutation (ms), Untranslated region (UTR), Pathogenic (P), Constitutional Mismatch Repair Deficiency (CMMRD).\u003c/p\u003e\n\u003ch3\u003ePedigrees\u0026rsquo; Key:\u003c/h3\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eIdentified Germline Variants of MMR Genes\u003c/h2\u003e\n \u003cp\u003eFive distinct frameshift (fs) indels of \u003cem\u003eMMR\u003c/em\u003e genes were identified in five unrelated patients, including 3 \u003cem\u003eMLH1\u003c/em\u003e variants (patients # 5, 8 and 9), one \u003cem\u003eMSH2\u003c/em\u003e variant (patient #4) and another \u003cem\u003ePMS2\u003c/em\u003e variant (patient #14).\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #4 (Table\u0026nbsp;1) is a 33-year-old male with CRC whose brother has also developed CRC. He carries \u003cem\u003eMSH2\u003c/em\u003e variant NM_000251.3:c.1321dup (p.Thr441fs), which is fs variant classified as P variant in ClinVar.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient # 5 (Table 1) is a 28-year-old male with CRC and a family history of various tumors including CRC, gastric, esophageal, endometrial, brain, and breast cancers. He has a fs variant in \u003cem\u003eMLH1\u003c/em\u003e gene ENST00000231790.2:c.1164_1165insGAAT(p.Ser388fs), which is classified as P variant in Clinvar.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #8 (Table 1) is a 37-year-old male with a family history notable for multiple relatives affected predominantly by CRC and two cases of breast cancer. He carries the \u003cem\u003eMLH1\u003c/em\u003e variant NM_000249.4:c.901del (p.(Gln301ArgfsTer66)), a fs alteration confirmed to be P variant in Clinvar.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #9 (Table 1) is a 43-year-old female with a family history of various gastrointestinal malignancies, together with breast, lung and parathyroid tumors. She has \u003cem\u003eMLH1\u003c/em\u003e variant NM_000249.4:c.1836_1839del (p.(Val612del)), which is a fs deletion, confirmed by expert panel to be LP in Clinvar.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #14 (Table 1) is a 9-year-old with a HGG and a family history of CRC, uterine cancer in the mother and thyroid tumors. She has a \u003cem\u003ePMS2\u003c/em\u003e variant ENST00000441476.2:c.1269dup (p.(Gln424AlafsTer12)). As endometrial cancer is an integrate tumor of LS, it is likely that the mother is a carrier of this variant.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eThree missense (ms) variants were identified across three families, including two families with affected sibling pairs (five patients in total). These included a P \u003cem\u003eMSH6\u003c/em\u003e variant, a LP \u003cem\u003ePMS2\u003c/em\u003e variant, and a VUS in \u003cem\u003eMSH6\u003c/em\u003e:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #11 (Table 1) is a 17-year-old girl who presented with a HGG, carries a pathogenic \u003cem\u003eMSH6\u003c/em\u003e variant ENST00000234420.5:c.2314C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.(Arg772Trp)), which is confirmed as P variant in Clinvar. Her parents have no history of cancer; however, the family is consanguineous. Notably, the maternal grandmother was diagnosed with uterine cancer at age 60, and the paternal grandfather developed colorectal cancer at age 40. This background highlights the need for genetic screening of both currently healthy parents.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatients #6 and #7 (Table 1) are sisters aging 13 and 15 years when presented with CRC, who are identified to carry an ms variant of \u003cem\u003ePMS2\u003c/em\u003e gene NM_000535.7:c.903G\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Lys301Asn), classified as LP in Clinvar. The 15-year-old girl had a pilomatrixoma as well, and they had a family history of leukemia, uterine, pancreatic and skin malignancy. Similar to patient #11, the parents must have been yet undetected carriers of one of the \u003cem\u003ePMS2\u003c/em\u003e variants. Although the children\u0026rsquo;s parents were not tested yet it is conceivable that both of them could be carrier of this \u003cem\u003ePMS2\u003c/em\u003e missense variant.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatients #12 and #13 (Table 1) are siblings: a 22-year-old brother diagnosed with CRC and HGG, and his 13-year-old sister diagnosed with HGG. Their mother died from a brain tumor, and two maternal uncles were also diagnosed with brain tumors in their twenties. Based on this strong family history, the siblings were enrolled in the study despite negative IHC results for MMRD. Germline analysis came back positive only for a shared VUS in \u003cem\u003eMSH6\u003c/em\u003e gene, ENST00000234420.10: c.1217G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Cys406Tyr), which makes it worth reporting.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #2 (Table 1) is a 30-year-old female, who is diagnosed with CRC. er family history is highly suggestive of a hereditary CPS with several family members affected with gastrointestinal, uterine, sarcomas and other malignancies. No SNVs or indels were identified in her sample by sequencing. A large deletion of approximately 74 kb in \u003cem\u003eMSH2\u003c/em\u003e was suggested by lcWGS (see sequencing artifacts section below), and MLPA analysis subsequently confirmed a heterozygous deletion involving exon 7 only (Supplementary Information (SI)). This deletion represents a known pathogenic copy number variant predicted to cause a frameshift and is associated with LS [13\u0026ndash;15].\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n\u003c/div\u003e\n\u003ch3\u003eAdditional Testing for Patients with No Validated MMR Variants\u003c/h3\u003e\n\u003cp\u003eFor the two siblings (Patients #12 and #13) who carried only a VUS in \u003cem\u003eMSH6\u003c/em\u003e (ENST00000234420.10:c.1217G\u0026thinsp;\u0026gt;\u0026thinsp;A; p.Cys406Tyr), as well as for Patients #1, #3, and #10 (Table\u0026nbsp;1), in whom no SNVs, indels, or CNVs were identified, a CPS gene panel (specified in the methods section) was performed and did not reveal any P/LP variants. For the adult patients with CRC in this group (Patients #1, #3, and #10), \u003cem\u003eMLH1\u003c/em\u003e promoter hypermethylation analysis was performed (Fig.\u0026nbsp;1) and yielded negative results in all cases.\u003c/p\u003e\n\u003cp\u003eTaken together, although not proven by functional tests, these findings suggest a possible contribution of the \u003cem\u003eMSH6\u003c/em\u003e VUS in the two siblings (Patients #12 and #13). In contrast, in Patients #1, #3, and #10\u0026mdash;who were enrolled based solely on young age at diagnosis (39, 44, and 38 years, respectively) and loss of MMR protein expression by IHC, but without strong clinical suspicion\u0026mdash;the observed IHC pattern is more likely attributable to a somatic event.\u003c/p\u003e\n\u003cp\u003eFigure 1. \u003cem\u003eMLH1 promoter methylation analysis for patients #1, #3, and #10 showing absence of methylation at four relevant CpG sites (cg23658326, cg11600697, cg21490561, and cg00893636), indicated by the red box at the left side of the graph.\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003eApparent large deletions likely representing sequencing artifacts\u003c/h3\u003e\n\u003cp\u003eIn addition to the identified sequence variants, the next generation sequencing (NGS) data suggested large deletions in six patients: three involving \u003cem\u003eMSH2\u003c/em\u003e and three involving \u003cem\u003eMLH1\u003c/em\u003e. However, MLPA analysis confirmed only part of one deletion in a single patient (patient #2, Table\u0026nbsp;1), suggesting that most of these findings may represent sequencing artifacts rather than true constitutional deletions. Furthermore, the three cases with presumed \u003cem\u003eMSH2\u003c/em\u003e deletion have overlapping segment of 74.6 kb (chr2:47,635,736\u0026ndash;47,710,309, GRCh37) (Fig.\u0026nbsp;2-A), and the three patients with presumed \u003cem\u003eMLH1\u003c/em\u003e deletion have an overlapping area spanning approximately 53 kb (chr3:37,037,285\u0026ndash;37,090,106, GRCh37) (Fig.\u0026nbsp;2-B), originally calculated from NGS based BAF plots. The recurrence of highly overlapping genomic regions across unrelated patients further supports the interpretation of these events as technical artifacts. This conclusion is also consistent with the overall clinical and molecular characterization of the tumors, as discussed below on a case-by-case basis.\u003c/p\u003e\n\u003cp\u003eFigure 2-B Apparent overlapping deletions of \u003cem\u003eMLH1\u003c/em\u003e gene detected in three unrelated Jordanian patients as visualized on UCSC genomic browser, likely representing artifacts.\u003c/p\u003e\n\u003cp\u003eThe following cases showed putative artifacts in \u003cem\u003eMSH2. BAF plots for all patients are available in SI\u003c/em\u003e:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #1 (Table 1) exhibited a deletion spanning approximately 390 kb (chr2:47,641,377\u0026ndash;48,033,551, GRCh37), resulting in loss of \u003cem\u003eMSH2\u003c/em\u003e from intron 4 onward as well as deletion of the first eight exons of \u003cem\u003eMSH6\u003c/em\u003e. As explained in previous paragraph, no additional molecular events were identified: specifically, no SNVs or indels in the MMR genes, no pathogenic variants in other CPS genes, and no \u003cem\u003eMLH1\u003c/em\u003e promoter hypermethylation. The absence of strong clinical suspicion and patient\u0026rsquo;s enrolment based solely on young age (39 years), the IHC staining pattern could be attributable to a somatic event.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #2 (Table 1) showed a 74 kb deletion (chr2:47,635,736\u0026ndash;47,710,309, GRCh37), which encompasses most of the gene from intron 2 to parts of the 3\u0026rsquo;UTR of \u003cem\u003eMSH2\u003c/em\u003e and overlaps with the first deletion. MLPA analysis carried out with two different probe kits only confirmed a large deletion of exon 7 and of 51nt of DNA downstream of exon 7 exclusively, as explained above.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #10 (Table 1) presented with a 112 kb deletion (chr2:47,635,736\u0026ndash;47,748,646, GRCh37) encompassing intron 2 of \u003cem\u003eMSH2\u003c/em\u003e and extending into the \u003cem\u003eKCNK12\u003c/em\u003e gene, overlapping part of the deleted region observed in Patients #1 and #2. As explained in previous paragraph, and similar to patient #1, no additional molecular germline events were identified. Apart from his age at diagnosis (38 years), this patient did not exhibit strong clinical suspicion for a hereditary CPS. His tumor mutational burden was low (TMB 2.1), and MSI status was also low (0.13%). The loss of MMR protein expression on IHC may therefore reflect a somatic rather than germline event.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe following cases showed putative artifacts in \u003cem\u003eMLH1. BAF plots for all patients are available in SI\u003c/em\u003e:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #3 (Table 1) exhibited a deletion spanning approximately 53 kb (chr3:37,037,285\u0026ndash;37,090,106, GRCh37), affecting exons 2\u0026ndash;17 (parts of intron 1 to parts of intron 17). As explained in previous paragraph, and similar to patients #1 and 10, no additional molecular germline events were identified. Given the absence of strong clinical suspicion and his enrolment based solely on young age (44 years), the IHC staining pattern could be attributable to a somatic event.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #13 (Table 1) showed an approximately 132 kb (chr3:37,038,204\u0026ndash;37,170,640, GRCh37), encompassing most parts of intron 2 far into the 3\u0026apos; untranslated region (UTR) and the noncoding UBE2F pseudogene 1 (UBE2FP1). However, the patient\u0026rsquo;s sibling (patient #12, Table 1) did not show the same deletion. Both siblings had normal IHC despite the suggestive clinical picture of CMMRD. Indeed, both siblings showed a VUS in \u003cem\u003eMSH6\u003c/em\u003e gene ENST00000234420.10: c.1217G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.(Cys406Tyr)), which might be causative of their clinical picture.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePatient #11 (Table 1) showed an approximately 54 kb in size (chr3: 37,038,204\u0026ndash;37,093,204, GRCh37), affecting most parts of intron 2 to the 3\u0026apos; UTR. This variant cannot explain the IHC loss of \u003cem\u003eMSH2\u003c/em\u003e, \u003cem\u003eMSH6\u003c/em\u003e. In addition, this patient had a P variant of \u003cem\u003eMSH6\u003c/em\u003e gene ENST00000234420.10: c.2314C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.(Arg772Trp)), which clearly explains the patient\u0026rsquo;s clinical picture including her diagnosis with HGG at the age of 17 years among other features.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eAdditional Molecular Findings\u003c/h2\u003e\n \u003cp\u003eTMB varied significantly among patients, ranging from 2.1 to 352 variant/Mb, with a median of 23 variant/Mb and a mode of 19 variant/Mb. Notably, four tumors exhibited a TMB of less than 10 variant/Mb, while one had a TMB exceeding 100 variant/Mb.\u003c/p\u003e\n \u003cp\u003eMSI on the basis of NGS data of WES was low in eight of 14 patients, including six of the proved 11 MMRD tumors, with scores ranging from 0.13% to 26% (mean: 10.6%). This limitation might be related to FFPE material as explained in the discussion below.\u003c/p\u003e\n \u003cp\u003eMutation signatures in all the tumor cases showed an overrepresentation of C\u0026thinsp;\u0026gt;\u0026thinsp;T transitions (Table 1), similar to signature 6 which has been associated with mismatch repair-deficient tumor tissue [16].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNine of the enrolled fourteen patients have a P/LP variant in an MMR gene. In addition, a pair of enrolled siblings have a VUS of \u003cem\u003eMSH6\u003c/em\u003e gene. Three patients (patient #1, #3 and #10, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) have not shown any SNV, indel, or CNV of MMR genes and were suggested to be somatic cases of MMR deficiency. Indeed, a substantial proportion of patients who meet the clinical criteria of LS or CMMRD, do not show germline mutations in any of the respective genes. Two studies of 28 and 38 cases of suspected CMMRD tumors reported 25% and 26% of the cases to have no identified P or LP variants [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, it is estimated that only 53\u0026ndash;70% of suspected LS cases show germline variants, with the higher end of the range relating to cases with IHC loss of \u003cem\u003eMSH2\u003c/em\u003e/\u003cem\u003eMSH6\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Other cancer predisposition genes explained some cases [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In our cases series, all the three patients for whom no variants were identified, have weak clinical suspicion and were enrolled because of their age, and suggestive IHC results. Nevertheless, we examined them for other CPS and negative results were obtained. In addition, none of those cases showed evidence of \u003cem\u003eMLH1\u003c/em\u003e promoter hypermethylation.\u003c/p\u003e \u003cp\u003eThe two siblings (patients #12 and #13) exhibit strong clinical suspicion for CMMRD despite negative IHC stains of MMR genes, with no SNV, indel or CNV of MMR genes identified. The fact that both of them carry the same VUS variant of \u003cem\u003eMSH6\u003c/em\u003e gene ENST00000234420.10:c.1217G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.(Cys406Tyr)) makes it worth reporting. Functional genomic testing for this variant can be helpful to confirm the diagnosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApparent large deletions likely representing sequencing artifacts were identified in 6 patients, 3 showing overlapping deletions of \u003cem\u003eMLH1\u003c/em\u003e and 3 showing overlapping deletions in \u003cem\u003eMSH2\u003c/em\u003e. At first, the deletions looked similar to each other which raised our suspicion for artifacts (BAF plots, SI). Moreover, two patients already carry other germline MMR events that could explain their situation (patients #11 and #13). Accordingly, MLPA[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], which is a sensitive technique for detecting large CNVs through quantitative analysis of specific regions of the MMR genes was conducted. Only one patient of the six proved to have part of the identified sequencing deletion of \u003cem\u003eMSH2\u003c/em\u003e confirmed by MLPA, leaving three cases (patient #1, #3 and #10, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) suspected to be somatic MMRD given the absence of other germline events and the weak clinical suspicion.\u003c/p\u003e \u003cp\u003eTMB in our cohort exhibited a wide range, with four tumors having normal TMB unexpectedly, with only one of which suspected to be a somatic case of CRC (patient #10). The majority displayed hypermutation (\u0026gt;\u0026thinsp;10 variants/Mb) and one was classified as ultra-hypermutated (\u0026gt;\u0026thinsp;100 variants/Mb). Hypermutation and ultra-hypermutation have been strongly linked to MMRD, which are characteristic of LS and CMMRD, with the latter being more likely to demonstrate extreme mutational loads [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Unexpectedly, six of the proved MMRD tumors of 11 patients exhibited low MSI. This might be related to the quality of DNA extracted from old FFPE blocks dating back to 1991 for the oldest block (patient #5, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to Qingli Guo et al. \u0026ldquo;Application of MSIsensor to an unrepaired CRC sample could lead to miscalling of MSI status for the tumour\u0026rdquo; [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, tumor purity (percentage of tumor cells per slide) is a prerequisite for successful MSI analysis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Utilizing the more sensitive Low-Pass Genomic Instability Characterisation Assay (LOGIC) would be helpful in such scenario [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, this study highlights the importance of surveillance in affected Jordanian families. Notably, the presence of a germline MMR variant in affected children may serve as an indicator of cancer risk in their often asymptomatic parents. This is illustrated by patients #6, #7 and #11, who developed tumors while parents were still clinically unaffected. In addition, the study underscores the limitations of using FFPE material for molecular characterization of MMRD tumors and highlights the need to confirm sequencing-based CNVs by MLPA analysis. Another limitation is the unavailability of parental DNA samples, which precluded discrimination between biallelic and monoallelic inheritance.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLynch syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPSs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCancer predisposition syndromes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLikely pathogenic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMismatch repair\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCMMRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConstitutional mismatch repair deficiency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMismatch repair deficiency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColorectal carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh grade glioma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicrosatellite instability\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor mutational burden\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKing Hussein Cancer Center\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDKFZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGerman Cancer Research Center\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIRB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstitutional Review Board\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFFPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFormalin-Fixed Paraffin-Embedded\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003elcWGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLow coverage whole genome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWES\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhole exome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eUBE2FP1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eUBE2F\u003c/em\u003e pseudogene 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCNV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCopy number variant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eB allele frequency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMultiplex ligation-dependent probe amplification\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuality control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSNV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle nucleotide variant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUTR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUntranslated region\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSI-H\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicrosatellite instability- high\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003efs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFrameshift\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ems\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSupplementary Information\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNext generation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003ePatients were recruited from King Hussein Cancer Center (KHCC). The study was approved by the Institutional Review Board of KHCC (ethics approval number: 140F-2021) and was conducted in accordance with the ethical standards of the institutional research committee and the principles of the Declaration of Helsinki. Freely given, informed consent to participate in the study and to publish the results was obtained from all participants prior to inclusion. For participants under the age of 16, informed consent was obtained from a parent or legal guardian.\u003c/p\u003e \u003cp\u003e Written informed consent for publication of pseudonymized clinical and molecular data was obtained from all participants or their legal guardians, where applicable. Participants were informed about the purpose of the study and the intended publication of their data in a scientific journal.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to Publish\u003c/h2\u003e \u003cp\u003e As indicated in the ethics statement, informed consent was obtained from participants and/or their parents including consent to participate and to publish their results.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Statement\u003c/h2\u003e \u003cp\u003e This research was partially supported by King Hussein Cancer Center (KHCC), Amman, Jordan, where patients were recruited, pathology blocks were cut and reviewed and DNA was extracted. Molecular analysis was supported by the German Cancer Research Center (DKFZ), Heidelberg, Germany, as part of the fellowship project of O.A.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eO. A. Conceptualized the work, was involved in all molecular analyses (fellowship project), drafted the work, produced the table and figures, and reviewed the final manuscript.I. S. Conceptualized the work and reviewed the final manuscript.M. H. Pathology review at KHCC and reviewed the final manuscript.O. S. Helped conceptualizing the work as well as planning and coordinating the workflow at KHCC and reviewed the final manuscript.C. S. Helped in validation of germline variants, planned and coordinated the work at Institute of Human Genetics at Heidelberg and reviewed the final manuscript.S. H. Helped in validation of germline variants and reviewed the final manuscript.R. A. and C. P. Helped in bioinformatics analysis and reviewed the final manuscript.A. F. Helped in planning the molecular analysis and reviewed the final manuscript.L. A. Helped in planning the manuscript, editing pedigrees and reviewed the final manuscript.N. A. Helped in identification of pediatric HGG patients, contributed to clinical data and reviewed the final manuscript.M. A. Sh. Genetic counselling for recruited patients and reviewed the final manuscript.R. A. Helped in identification of adult patients, contributed to clinical data and reviewed the final manuscript.S. A., L. F., A. N., A. Q. and H. A. Provided first draft of literature review and reviewed the final manuscript.D. E. R., N. R. F., A. D. and U. T. Provided revision of the work and reviewed the final manuscript.S. M. P. Supervised the whole project and reviewed the final manuscript.C. P. S. Supervised the whole project and reviewed the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eDNA sequencing data were deposited into the German Human Genome-Phenome Archive GHGA repository under accession number GHGAS19356866116009 and are available at the following URL:[https://data.ghga.de/study/GHGAS19356866116009](https:/data.ghga.de/study/GHGAS19356866116009)Access is subject to controlled access procedures in accordance with GHGA data governance policies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Milestones of Lynch syndrome: 1895\u0026ndash;2015. Nat Rev Cancer. 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Genomic Microsatellite Signatures Identify Germline Mismatch Repair Deficiency and Risk of Cancer Onset. J Clin Oncol. Feb 1 2023;41(4):766\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.21.02873\u003c/span\u003e\u003cspan address=\"10.1200/JCO.21.02873\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mismatch repair deficiency (MMRD), Constitutional mismatch repair deficiency (CMMRD), Lynch syndrome, Colorectal cancer, High-grade glioma, Jordan / Middle East","lastPublishedDoi":"10.21203/rs.3.rs-9017459/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9017459/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn Jordan, approximately 19% of colorectal carcinomas (CRCs) in patients younger than 45 years show mismatch repair deficiency (MMRD) by immunohistochemistry (IHC). MMRD in pediatric high-grade gliomas (HGG) and CRC in Jordan appears substantially more frequent than global estimates (39% and 44%, respectively), likely reflecting high consanguinity rates and an increased contribution of constitutional MMRD (CMMRD). However, molecular characterization of MMRD-associated tumors in the Middle East remains limited.\u003c/p\u003e \u003cp\u003eWe analyzed 14 Jordanian patients (\u0026lt;\u0026thinsp;45 years) from 12 families with clinical or IHC evidence of MMRD, including four brain tumors and 11 CRCs; five patients were children. Pathogenic or likely pathogenic MMR variants were identified in nine families, including one affected sibling pair, comprising five frameshift variants, three single nucleotide variants, and one large deletion. A variant of uncertain significance in \u003cem\u003eMSH6\u003c/em\u003e was detected in another sibling pair. Apparent large deletions suggestive of sequencing artifacts were observed in six patients, underscoring the need for confirmatory copy number analysis by multiplex ligation-dependent probe amplification (MLPA). Three adult cases lacked germline MMR variants, \u003cem\u003eMLH1\u003c/em\u003e hypermethylation, or alterations in other cancer predisposition genes and were considered likely somatic.\u003c/p\u003e \u003cp\u003eTumor mutational burden (TMB) ranged from 2 to 352 variants/Mb (median: 23). Microsatellite instability (MSI) was low in seven tumors, likely due to degraded DNA from older Formalin-Fixed Paraffin-Embedded (FFPE) samples. Among tumors with low TMB and MSI, only one lacked an identifiable germline MMR variant. All tumors showed overrepresentation of C\u0026thinsp;\u0026gt;\u0026thinsp;T transitions, consistent with MMRD-associated mutational signature 6.\u003c/p\u003e \u003cp\u003eThis study represents the first comprehensive molecular characterization of MMRD-associated tumors in Jordan.\u003c/p\u003e","manuscriptTitle":"Molecular Characterization of Mismatch Repair Deficient Tumors In Young Jordanian Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 05:24:51","doi":"10.21203/rs.3.rs-9017459/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-16T14:49:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T16:01:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90747791155824213598254786278902354796","date":"2026-03-31T06:25:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191674175519509658507566939036770747997","date":"2026-03-30T14:53:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-26T10:21:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-13T10:23:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T23:24:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2026-03-11T15:32:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c222c6aa-64c9-4370-9e77-fe95b089a638","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T05:24:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 05:24:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9017459","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9017459","identity":"rs-9017459","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}