Integrating TREC/KREC assay and some cytokines in the evaluation of the immune status of patients with DiGeorge Syndrome

Integrating TREC/KREC assay and some cytokines in the evaluation of the immune status of patients with DiGeorge Syndrome | 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 Research Article Integrating TREC/KREC assay and some cytokines in the evaluation of the immune status of patients with DiGeorge Syndrome Assem Metwally Abo-Shanab, Haiam Abdel Raouf, Alaaeldin G. Fayez, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4231044/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 Aim The study aimed to offer better genetic evaluation and consultation for DiGeorge syndrome (DGS) patients by combining screening of 22q11.2 and immunologic studies. A basic immune profile including the basic CD panel and immunoglobulins estimation was performed. TRECS and KRECS expression were studied in addition to measuring serum IL33, Obestatin, HLA-G, and Procalcitonin serum levels. Methods All investigations were performed for DGS patients (n = 33) and the matched control group (n = 45). Polymorphic 22q11.2 markers mapping was performed by PCR-STR technique. Lymphocyte subsets immunophenotyping was done using flow cytometry, while measurement of serum immunoglobulins was estimated using nephelometry. Real-time PCR was the method used for TRECs and KRECs measurement. Serum IL33, Obestatin, HLA-G, and Procalcitonin levels were determined using an Enzyme-linked immunosorbent assay (ELISA). Data was coded, tabulated, and statistically analyzed using SPSS version 19.0 software. Results In our case–control study, KREC expression was significantly elevated in DGS compared to healthy controls (P = 0.0008). There was also a significant increase in immunoglobulin levels in DGS. CD8% as well as CD8 absolute count in the patients with DGS were significantly lower than in the healthy control (P = 0.01273 and 0.05358 respectively). There were no significant differences in IL33, Obestatin, HLA-G, and Procalcitonin levels between DGS patients compared to the control group. Our results concerning the distinct segment of 22q11.2 as a DGS susceptibility region revealed an informative novel atypical interstitial homozygous deletion. This deletion included D22S944 and COMT absence, and D22S941 and D22S264 presence. Out of 33 DGS patients, three patients showed deletion in the D22S944 marker only in the presence of D22S941, and D22S264 markers. Therefore, we could assume that D22S944 is a common deleted marker in non-isolated DGS patients. Conclusion Combining 22q11.2 region screening, immune profile studies, and TRECS and KRECS expression offers a new comprehensive approach for DGS patients. This approach provides a better strategy for genetic consultation for DGS patients. Moreover, this study may be the first to show a small interstitial 22q11.2 deletion stereotype in a DGS patient and also showed that the smallest deletion at the 22q11.2 region is enough to confer the DGS phenotype. DiGeorge Syndrome TREC KREC HLA-G Procalcitonin Obestatin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Chromosome 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion syndrome in humans. It occurs in almost 1:4000 live births and was traditionally identified as DiGeorge syndrome which is classified as a primary immunodeficiency. It is described by T-cell lymphopenia and thymic dysplasia [ 1 ]. The condition can be grouped into two categories, complete and partial DiGeorge syndrome. Individuals with a complete absence of the thymus are considered to have complete DiGeorge syndrome [ 2 ]. In DiGeorge syndrome, there is dysregulation of T–B-cell interactions. Complete DiGeorge syndrome was found to suffer from life-threatening severe T-cell lymphopenia. Whereas partial DiGeorge syndrome is characterized by decreased thymic output show and T-cell lymphopenia. This is exemplified in a low number of T-cell receptor excision circles (TRECs) [ 3 ]. This resulted from recent thymic emigrant T cells and a low number of naïve T cells [ 4 ]. The humoral immune compartment in DiGeorge is also affected. This is reflected in hypogammaglobulinemia, diminished response to vaccination, (Patel et al, 2012), and dysfunctional maturation of B-cells [ 5 ]. This leads to increased vulnerability to infections, atopy, and autoimmune disease. There is a wide variability of phenotypic features. Hallmark features include cardiac anomalies and hypoparathyroidism, in addition to thymic aplasia or hypoplasia. Other phenotypic features include feeding and swallowing anomalies, palatal defects, renal abnormalities, and others [ 2 ]. Patients don't necessarily manifest the typical features of the syndrome and severity differs considerably between patients. Even if the mutation is familiarly inherited or presented in identical twins, the deletion may be expressed with completely different phenotypes [ 6 ]. Immune deficiency in such patients cannot be evaluated depending on clinical phenotype. Some degree of T cell lymphopenia is encountered in around 70% of patients with 22q11.2del [ 7 ]. Therefore, immunologic assessment of affected subjects with 22q11.2del and any other developmental thymus deformity (DTD) is crucial to define immune status and assess infection susceptibility. T cell markers (CD3, CD4, CD8), B cell markers (CD19 or CD20), and natural killer cell markers (CD16 or CD56) should be included in the primary immunologic evaluation required for diagnosis. This is to be requested along with IgG, IgM, and IgA estimation. Moreover, T cell receptor excision circles (TREC) assay is performed if available; abnormal results increase the probability of significant T cell lymphopenia (TCL) [ 8 ]. Actual thymus-derived naïve T cells (RTEs) can be evaluated by T cell receptor excision circles (TREC) assay. It serves as an excellent marker for this task. During V(D)J recombination of TCR in the thymus, circulating remnants of the DNA excision by-products are formed. These remnants are thus measured. Therefore, whether the presence or degree of thymic T cell production, both are directly reflected by (TREC) assay. Neonates who may have abnormal TREC assay reflect remarkable TCL [ 9 ]. Nevertheless, an assay can also detect congenital athymia as well as other severe forms of thymic hypoplasia [ 10 ]. Nevertheless, and in the same context, kappa-deleting element recombination circles (KREC), can be used to detect newly developed B cells. A conjoint TREC/KREC assay has been used to detect patients with severe B cell disorders [ 11 ]. In another study, TREC was only identified in severe cases with deep lymphopenia, whereas children with incomplete DiGeorge syndrome had normal range TREC/KREC levels both at birth and postnatally. However, a decline of both TREC and KREC levels with age was noticed significantly in DGS patients than in controls. Therefore, their assay is complementary to routine investigations and follow-up of patients. Based on the del22q11 background, their levels may reflect age-related immune changes along the course of the disease [ 4 ]. The humoral immune system is sometimes found intact in 22q11.2 del subjects. Yet, immunoglobulin (Ig) A deficiencies improper response to vaccines, and transient hypogammaglobulinemia were sometimes encountered. Recently, humoral abnormalities as well as Immunoglobulins have been studied in 22q11.2 del [ 12 ]. IgG deficiency may develop even in individuals with normal immune function early in life. It may develop a picture very similar to CVID both clinically and biologically. Moreover, the US Immunodeficiency Network (USIDNET) performed a conjoint study with the European Society for Immunodeficiencies (ESID), where nearly half the subjects with confirmed 22q11.2del, had IgG, IgA, and IgM abnormalities [ 8 ]. Obestatin, a ghrelin gene product, was primarily defined as an appetite suppressant; its role in metabolism remains an ongoing debate. It is thought to be involved in the regulation of metabolic homeostasis and cardiovascular function. Moreover, some previous studies noted that obestatin increased beta cell mass and improved lipid metabolism; thus, inversely correlated with fasting blood glucose [ 13 ]. (Cowan et al, 2016) Moreover, in obese individuals, Obestatin levels were found reduced and negative with body mass index (BMI) [ 14 , 15 ]. In this same context, regarding adults with 22q11.2DS, there is evidence of obesity, global medical multimorbidity, and some other features, such as early onset Parkinson’s disease in these subjects [ 16 , 17 ]. Procalcitonin (PCT) is one of the biomarkers released in response to bacterial infections. It has an important role in the diagnosis and management of sepsis and lower respiratory tract infection.18– 23. In the same context, it is worth noting that Infections were observed in DGS and the most common infection was noted as pneumonia [ 18 ]. IL-33 is a member of the IL-1 family of cytokines. It is found in multiple organs and is mainly localized to barrier epithelial cells and endothelial cells. Fibroblasts, myofibroblasts, and airway smooth muscle cells were also found to express Il-33 [ 19 , 20 ]. (Byers et al, 2013 Calderon et al, 2023). It has a crucial role in innate and adaptive immune responses. It drives type 2 T-helper responses and has the ability to activate basophils, mast cells, and eosinophils [ 21 ]. It is released into the extracellular space by endothelial cells following cellular insult, necrosis, secondary to cigarette smoke, pollutants, allergens, and viral or bacterial exposure, hence acting as an innate immune indicator of danger (alarmin). Accordingly, IL-33 is considered to perform some immunologically important functions in the cortex and medulla of the thymus during mammalian development [ 22 ]. (Mamoor 2020) Therefore, IL-33 in addition to acting as a nuclear transcription factor, has pro-inflammatory effects as well [ 20 , 23 ]. Infection-induced IL-33 release was found to induce thymic involution and thus T cell aging which consequently impairs host control of severe infection. Moreover, T cell immune deficiency or increased liability to infections has been related to impaired thymus function in many conditions in children such as DiGeorge syndrome, preterm newborn, and severe combined immunodeficiency [ 24 , 25 ]. (Zdrojewicz et al, 2016 and Xu et al, 2022). HLA-G is an HLA antigen having immunomodulatory properties. CD4 and CD8 T lymphocytes express it as well as NK cells, monocytes, and dendritic cells. T cells expressing the immunomodulatory m HLA-G molecule are identified as distinct subpopulations of Treg lymphocytes. These were found present in the peripheral blood of 0.1–8.3% of healthy subjects, and primarily originate from the thymus. Studies confirmed their importance in health and disease [ 26 ]. Moreover, quantitative, and qualitative derangements of HLA-G were detected in several immune conditions which highly suggests that HLA-G immune cells may be involved in the pathogenesis of such disorders [ 27 ]. Patients and methods Patients This study is a case-control study that was conducted in the period from November 2019 to May 2022. It was performed in line with the principles of the Declaration of Helsinki. The study included thirty-three patients with DiGeorge syndrome (DGS) (22q11.2DS). In addition, forty-five healthy subjects, matched for age and gender, were included as a control group. All patients were recruited among patients referred to Genetics Clinics at the Medical Research Centre of Excellency, National Research Centre (NRC), Dokki, Giza, Egypt. This study's Approval was granted by the Ethics Committee of the National Research Centre (NRC), Cairo, Egypt. (Ethics No. 19267-1). Written informed consent was obtained from the parents of the participants for including their children in the study. Blood samples Five milliliters of fresh peripheral blood samples from all patients and controls were drawn. 2.5 ml of blood taken were divided into two sterile EDTA-containing tubes. 1 ml for CBC and the remaining amount was used for flow cytometric analysis which was done within 24 hours of collection as well as DNA extraction which was done simultaneously or on refrigerated samples stored at 2–8°C within one week of collection. Another 2.5 ml of blood was centrifuged at a rate of 3000 rounds per minute for 10 min to separate the serum which was collected and immediately frozen in 0.2 ml aliquots at -80°C until assayed for immunoglobulin levels by nephelometry and for ELISA tests. Determination of Immunoglobulins (IgA, IgM, and IgG) Measurement of serum immunoglobulin was performed using the method of nephelometry [ 28 ]. Flow cytometric analysis Anticoagulated blood was stained using the whole-blood lysis method. Whole blood was stained with monoclonal antibodies directed against the surface antigens: CD3 FITC labeled MoAbs for T lymphocytes, CD16 labeled with PE for NK cells, FITC labeled CD4 for helper T lymphocytes, PE-labeled CD8 for cytotoxic T lymphocyte and FITC labeled CD19 for B lymphocytes (BD Biosciences, USA.). Lymphocyte subsets immunophenotyping was done using flow cytometry (BD Accuri™ C6 Cytometer, USA) [ 29 ]. Real-time PCR for TRECs and KRECs measurement Genomic DNA was isolated from peripheral blood leukocytes by QIAamp DNAMini Kit (50 preps), catalog number 51304, Germany ( https://www.qiagen.com/eg/ ). The assay was run on 7500 Fast Real-Time PCR (Applied Biosystems) using the specific primers and probes for TRECs, KRECs, and β-actin. RT-PCR was carried out using TaqMan Universal PCR Master Mix II. 10 µl PCR reaction mix was added to the 5 µl DNA samples, 1 µl forward primer (Applied Biosystems, USA.), 1 µl reverse primer (Applied Biosystems, USA.), 1 µl Taqman TAMRA probe and 2 µl H2O in a sterile 48-well PCR plate, using real-time cycler conditions of initial activation stage at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 seconds, and a combined primer/probe annealing and elongation at 60°C for 1 min. TRECs, KRECs, and β-actin copy numbers have been obtained by extrapolating the respective sample quantities from the standard curve obtained by serial dilutions of human genomic DNA (Promega, USA.). The initial copy numbers of TRECs, KRECs, and β-actin were calculated from the following equation: "Copies of the gene of interest = mass of gDNA / mass of haploid genome". The number of TRECs and KRECs in each sample was calculated per µl of extracted DNA. β-actin copy number was used to judge the successful amplification of each sample. Enzyme-linked immunosorbent assay (ELISA) Serum IL33, Obestatin, HLA-G, and Procalcitonin levels of all study subjects were determined using Human Procalcitonin ELISA kit (EIAab, Co., Ltd, East Lake Hi-Tech Development Zone, Wuhan, China) and Human IL33, Obestatin and HLA-G ELISA kits (NOVA, Beijing, China) by following the manufacturer’s protocol. Polymorphic 22q11.2 markers mapping The PCR-STR technique was performed using specific primers for microsatellite genotyping that were directly retrieved from Electronic PCR ( www.ncbi.nlm.nih.gov/sutils/e-pcr/ ; NCBI, USA). Three consecutive polymorphic loci were used and located in the usually 3-Mb deleted region: D22S941, D22S944, and D22S264, plus the COMT marker. The PCR products were visualized on 4% agarose gel. Statistical methods The collected data were coded, tabulated, and statistically analyzed using SPSS version 19.0 software (SPSS Inc., Chicago, Illinois, USA). T-test was used for comparing the parametric results among groups. The Mann-Whitney U Test was used for comparing the non-parametric results among groups. Data were presented as median and range. All analyses were two-tailed, the level of significance was taken at P value < 0.05 is significant, otherwise is non-significant. Results This case–control study included two groups: group I (33 children clinically diagnosed as DGS and Group II (45 healthy controls). The demographic and clinical characteristics of DGS patients and volunteer control subjects are presented in Table 1 . All the DGS patients in the study had congenital heart diseases (CHDs). Table 1 Demographic and clinical characteristics Characteristic Normal healthy controls DiGeorge P value No. of cases 45 33 Gender, no. Male % 22 (48.9%) 16 ((48.5%) 0.462 Female % 23 (51.1%) 17 (51.5%) Age (years) mean ± SD 3.9 ± 2.86 2.7 ± 1.9 0.297 P value calculated using T-test KRECs expression was significantly elevated in DGS patients as compared with the healthy controls (P = 0.0008) (Fig. 1 ). There was also a significant increase in immunoglobulins level between DGS patients and the healthy controls (IgA P = 0.014, IgG P = 0.0019& IgM P = 0.0032) (Fig. 2 ). * P < 0.05 Significant versus controls (by Mann Whitney U Test) Table 2 summarizes laboratory findings such as TLC, lymph, CD3, CD16, CD4, and CD8 in addition to CD19 in the DGS group and control group. CD8% as well as CD8 absolute count in the patients with DGS were significantly lower than in the healthy control (P = 0.01273 and 0.05358 respectively). The results in Table 3 showed that there were no significant differences in IL33, Obestatin, HLA-G, and Procalcitonin levels between DGS patients compared with the control group. Table 2 Comparison of the laboratory data measured between cases and controls. Variables DGS Control P value Median (Range) Median (Range) TLC 8250 (3600–14200) 6050 (4140–10890) 0.1765 lymph % 39.7 (32–61) 52.6 (23-87.5) 0.05949 Absolute lymph 3688.5 (2752–6958) 3592.9 (1288–9100) 0.6472 CD3% 60.4 (20-70.8) 59.1 (31.7–73.1) 0.5588 Absolute CD3 1734.2 (883.4-4181.8) 1861.2 (807.6-3778.8) 0.6999 CD16% 14.7 (5-38.9) 15.2 (7-37.4) 0.6129 Absolute CD16 422.3 (159.6-1793.3) 421 (182.6–1950) 0.7445 CD4% 29.9 (12.6–43.5) 27.8 (22-40.7) 0.8776 Absolute CD4 1055.5 (590-3012.8) 1046 (288.5–2265) 1.0236 CD8% 18.5 (5-43.1) 26 (7.3–36.8) 0.01273* Absolute CD8 548.7 (212.3-1071.5) 922 (273–1738) 0.05358* CD19% 17.6 (10.3–37.4) 16 (4.4–41) 0.4412 Absolute CD19 571.2 (284.8-2177.9) 538 (90.7–3731) 0.6999 Results were expressed as Median (Range). * Significant versus controls (by Mann Whitney U Test) Table 3 Biomarkers comparing apparently healthy control with DiGeorge patients. Variables DGS Control P value Median (Range) Median (Range) IL 33 pg/ml 178 (43–271) 134.9 (5.4-231.8) 0.05911 Obestatin pg/ml 951.7 (335–4901) 835 (135–3635) 0.4735 HLA-G ng/m 42.3 (20.5-125.1) 47.3 (17.7-141.8) 0.8212 Procalcitonin pg/ml 197.5(50–400) 202.5 (105–281) 0.37 Results were expressed as Median (Range). * Significant versus controls (by Mann Whitney U Test) A Novel atypical interstitial homozygous 22q11.2 microdeletion suggested three DGS-susceptibility genes. Using three polymorphic DNA markers, an informative novel atypical interstitial homozygous deletion was found. This deletion included D22S944 and COMT absence, and D22S941 and D22S264 presence. The COMT gene, the nearest gene to the distal region of D22S264, was used to determine the minimal breakpoint loci. The distance of the identified novel deleted region was approximately 0.35 Mb leading to the deletion of definitely 3 genes: GP1BB, TBX1, and COMT (Figs. 3 and 4 ). Out of 33 DGS patients, three patients showed deletion in the D22S944 marker only in the presence of D22S941, and D22S264 markers as shown in Table 4 . Therefore, we could assume that D22S944 is a common deleted marker in non-isolated DGS patients. None of the healthy controls had deletions. Table 4 STS-PCR results for three genetic polymorphic STS markers in the 22q11.2 region. Case no. D22S941 (224–260 bps) D22S944 (158-178bps) COMT D22S264 Notes 1 + Del + + 2 + + + + 3 + + + + 4 + + + + 5 + + + + 6 + + + + 7 + + + + 8 + + + + 9 + + + + 10 + + + + 11 + Del + + 12 + + + + 13 + + + + 14 + Del + + 15 + + + + 16 + Del + + 17 + + + + 18 + + + + 19 + + + + 20 + + + + 21 + + + + 22 + + + + 23 + Del Del + Novel atypical interstitial nested microdeletion about 0.35Mb 24 + + + + 25 + + + + 26 + + + + 27 + + + + 28 + + + + 29 + + + + 30 + + + + 31 + + + + 32 + + + + 33 + + + + Discussion DiGeorge Syndrome (DGS) (22q11.2DS) is considered the most common microdeletion syndrome in humans. It occurs in almost 1:4000 live births. Phenotypic features vary considerably; nearly 75–80% of DGS patients exhibit disorders of the immune system, which may cause them to suffer from immune instabilities such as autoimmune disease, susceptibility to infections, and atopy [ 1 ]. Our results showed that KREC expression was significantly elevated in DGS patients as compared with the healthy controls (P = 0.0008). On the contrary, Dar et al., 2015 and Lingman Framme et al, 2014 stated that in DGS patients, KREC levels don’t vary much from those of healthy controls, which suggests normal bone marrow output of B cells [ 30 , 31 ]. Nevertheless, DGS patients with low TREC counts have long-term impairment of thymic output. These patients were considerably more liable to viral infections, which is similar to the setting of more severe T-cell lymphopenia [ 31 , 32 ]. Moreover, the link between lower TREC levels and recurrent infections continues regardless of age [ 30 ]. Our research found a significant increase in immunoglobulin levels among DGS patients compared to healthy controls (IgA P = 0.014, IgG P = 0.0019& IgM P = 0.0032) unlike Patel K et al., 2012 who stated that after 3 years of age, almost 6% of DGS patients have low IgG levels [ 12 ]. In the same context, Al-Herz et al., 2004 and Kung SJ et al., 2007 reported selective IgM deficiency associated with DGS although less common [ 33 , 34 ]. However, in one cohort, the most common humoral defect reported was low IgM [ 35 ]. In DGS, low levels of IgG, IgA, IgM, and defective antibody responses to vaccines were described. Severe infections observed in DGS were attributed to humoral immune deficiency [ 12 , 36 , 37 ]. In 17% of cases, humoral immunity instabilities were witnessed [ 38 ]. Some autoimmune problems including juvenile rheumatoid arthritis (JRA) reported IgA deficiency in nearly 13% of patients with DGS [ 39 ]. Despite the finding that hypogammaglobulinemia was found to present in the first year of life, it usually resolves. On the other hand, hypergammaglobulinemia was reported in some cases after the age of five. Surprisingly, the majority of affected individuals show functional antibody defects despite the normal antibody function and antibody avidity [ 36 , 39 ]. Impaired T-cell production resulting from thymic hypoplasia leads to immunodeficiency. It's quite apparent that lower cells of thymic lineage are present in newborns with DGS. Sixty-seven percent of individuals had reduced T-cell production and more than 18% showed compromised T-cell function in previous studies [ 39 ]. In another study including 1,421 DGS patients, abnormal T-cell populations were found in half the included subjects [ 38 ]. Over time, the production of T-cells improves and in the first year of life, children much improve despite the considerable T-cell defects they were suffering from. Whereas individuals with minor T-cell decrease showed better performance against pathogens [ 39 ]. Regarding our study, CD8% as well as CD8 absolute count in the patients with DGS were significantly lower than healthy controls (P = 0.01273 and 0.05358 respectively). Another study conducted with partial DGS patients had the same opinion. It stated that 20 out of 25 patients had low CD8 + T cells and recurrent bacterial and viral infections [ 36 ]. On the other hand, researchers demonstrated normalization of CD4 + T cell numbers in most patients up to 3 years [ 40 ]. In another observational study, 81% of DGS patients reported normal levels of CD8 + T at the age of 2 years when compared to healthy controls [ 18 ]. Our results showed that IL33 showed no significant difference in DGS patients compared with the control group. In an experimental model of DGS, Handel E et al., 2022, explored the changes in the non-epithelial stromal compartment of the thymus across different developmental stages [ 41 ]. High levels of complexity within the thymic mesenchyme were revealed using Single-cell sequencing. A Uniform Manifold Approximation and Projection (UMAP) analysis of non-TEC (thymic epithelial cell) stroma recognized cluster incorporated two distinct medullary fibroblast subtypes, the medullary fibroblast subtypes 1 (MedFb1) and 2 (MedFb2). Both subtypes also comprised transcripts for IL-33 and Cxcl16, which are important for dendritic cell activation and NKT cell migration, respectively [ 42 , 43 ]. In about 40% of DGS patients, obesity has been reported with onset often during childhood or adolescence [ 16 , 44 , 45 ]. In obesity, Nakahara T et al., 2008, reported a negative correlation of plasma obestatin concentrations with body mass index, insulin resistance index, and plasma leptin concentrations [ 46 ]. Regarding our research, there was no statistically significant difference in obestatin levels between DGS patients and controls. Eberle et al., 2008 stated that serious infections and lymphoproliferative complications were more frequent in high-risk patients with DGS [ 47 ]. Procalcitonin levels begin rising within 3– 4 h of bacterial infection and usually peak between 12 and 36 h [ 48 ]. Nargis W et al., 2014 observed 75% of diagnostic accuracy, 72% of specificity, and sensitivity of 76% for procalcitonin. They concluded as well that procalcitonin is better than CRP regarding accuracy in the identification and assessment of sepsis severity. As for our results, there were no significant differences in procalcitonin levels between DGS patients and apparently healthy controls [ 49 ]. Persistent low numbers of CD4 + CD45RA + and CD8 + T cells in DGS patients rendered them more prone to lethal infections and lymphoproliferative disorders during the follow-up period [ 47]. Feger U et al., 2006 defined novel subsets of T cells expressing the immunomodulatory molecule HLA-G as CD4 HLA-GC distinct Treg cells [ 26 ]. Through blocking cell cycle progression, sHLA-G5 has the ability to inhibit CD4 and CD8 T cell alloproliferation [ 50 ]. However, our results showed no significant differences in HLA-G levels between patients and controls. We analyzed the 22q11.2 region in extracted DNA from the peripheral blood of 33 DGS patients and 20 controls who were referred from our clinical genetics department at the National Research Centre (NRC). Using a set of polymorphic STR markers with known locations, we found one patient with novel atypical nested homozygous microdeletion flanked by D22S941 and D22S264 markers. None of the healthy controls had deletions. Our results suggest a distinct segment of 22q11.2 as a DGS susceptibility region which is located between markers D22S941 and D22S264. Using the published DNA sequence of human chromosome 22, we estimated this region to contain three main genes: GP1BB, TBX1, and COMT. It is still not known which of those genes are directly responsible for DGS pathogenesis. Therefore, Further Gene dosage studies are required. The majority 22q11.2 deleted region (3 Mb) contains approximately 50 genes, and several miRNAs as shown in (Fig. 5 ). TBX1 and COMT genes are considered the most relevant genes to DGS [ 51 ]. TBX1 gene encodes a T-box transcription factor, which is known to have an essential role in early vertebrate development. Using FISH analysis with KB1764E3 probe which encompassed TBX1, CDCrel-1, and GP1BB genes, no deletions were observed in 13 patients with DGS [ 52 ]. McQuade et al., 1999 detected small deletions including TBX1 and COMT genes in a patient with the DGS phenotype [ 53 ]. However, our results agree with MacQuade et al., 1999 study, but we detect novel homozygous 22q11.2 deletion encompassing TBX1, COMT, and in addition to GP1BB using Polymorphic STS markers mapping as illustrated in (Fig. 5 ). GP1BB [Glycoprotein 1b platelet subunit beta] was previously detected in DGS patients with atypical features [ 54 – 56 ]. Using SNP‑array analysis, Huang et al., 2015 found four 22q11.2DS patients shared the same deletion breakpoints which included TBX1, COMT, DGCR2, GP1BB, RTN4R, PRODH, SNAP29, and SERP genes [ 57 ]. It is worth noting that DGS has phenotypic heterogeneity and severity variability. So, some patients are mildly affected, whereas others have severe features that could be due in part to the presence of genetic modifiers and dosage of the deleted gene's haploinsufficiency. It was reported that Phenotypic variability in DGS is associated with (i) behavioral traits including autistic spectrum disorders (ASD) are apparently more frequent in LCRA-B deleted individuals [ 58 , 59 ], (ii) schizophrenia (SCZ) and attention deficit hyperactivity disorder (ADHD) phenotypes are apparently in LCRA-D deleted region including COMT, PRODH, GNB1L, TBX1, SEPT5/GP1BB, ZDHHC8, PI4KA, and ARVCF genes [ 60 – 62 ]. Conclusions Our results may be given merit to adopt a new comprehensive investigation strategy for DGS patients that combines screening of 22q11.2 region, immunoglobulins level patterns, and TRECS and KRECS expression. This investigation strategy can provide better genetic consultations for DGS patients. However, the current study may be the first to show a small interstitial 22q11.2 deletion stereotype in a DGS patient, but it has shown that the smallest deletion at the 22q11.2 region is enough to confer the DGS phenotype. Further investigating of other DGS-relevant pathogenesis factors and their correlation with the patient’s clinical manifestations can help to provide a better explanatory model for the clinical variability of DGS disease. Declarations Ethical Approval This study was conducted in accordance with the principles outlined in the Declaration of Helsinki. The research protocol and procedures were approved by the Ethics Committee of the National Research Centre (NRC), Cairo, Egypt, under Ethics No. 19267-1. Informed consent was obtained from the parents of all participants for their inclusion in the study. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Availability of Data and Materials The datasets generated and analyzed during the current study. However, a deidentified version of the dataset may be available from the corresponding author upon reasonable request. Author Contributions Statement N.K. & H.A. conceptualized the study and designed the research methodology. E.A. & N.E. collected and analyzed the data. A.F. performed statistical analysis and interpretation of results. A.A. drafted the manuscript and prepared the figures. R.M. & I.H. critically reviewed and revised the manuscript for intellectual content. All authors approved the final version of the manuscript for submission. References Davies EG. Immunodeficiency in DiGeorge syndrome and options for treating cases with complete athymia. Frontiers in immunology. 2013 Oct 31;4:322. Biggs SE, Gilchrist B, May KR. Chromosome 22q11. 2 Deletion (DiGeorge Syndrome): Immunologic Features, Diagnosis, and Management. Current Allergy and Asthma Reports. 2023 Apr;23(4):213-22. Klocperk A, Paračková Z, Bloomfield M, Rataj M, Pokorný J, Unger S, Warnatz K, Šedivá A. Follicular helper T cells in DiGeorge syndrome. Frontiers in Immunology. 2018 Jul 23;9:1730. Froňková E, Klocperk A, Svatoň M, Nováková M, Kotrová M, Kayserová J, Kalina T, Keslová P, Votava F, Vinohradská H, Freiberger T. The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome. Plos one. 2014 Dec 8;9(12):e114514. Klocperk A, Mejstříková E, Kayserová J, Kalina T, Šedivá A. Low marginal zone-like B lymphocytes and natural antibodies characterize skewed B-lymphocyte subpopulations in del22q11 DiGeorge patients. Clinical Immunology. 2015 Dec 1;161(2):144-9. Digilio MC, Angioni A, De Santis M, Lombardo A, Giannotti A, Dallapiccola B, Marino B. Spectrum of clinical variability in familial deletion 22q11. 2: from full manifestation to extremely mild clinical anomalies. Clinical genetics. 2003 Apr;63(4):308-13. Markert ML. Defects in thymic development. InStiehm's Immune Deficiencies 2020 Jan 1 (pp. 357-379). Academic Press. Mustillo PJ, Sullivan KE, Chinn IK, Notarangelo LD, Haddad E, Davies EG, de la Morena MT, Hartog N, Yu JE, Hernandez-Trujillo VP, Ip W. Clinical practice guidelines for the immunological management of chromosome 22q11. 2 deletion syndrome and other defects in thymic development. Journal of Clinical Immunology. 2023 Feb;43(2):247-70. Lewis DB, Haines C, Ross D. Protein tyrosine kinase 7: a novel surface marker for human recent thymic emigrants with potential clinical utility. Journal of Perinatology. 2011 Apr;31(1):S72-81. Pierdominici M, Mazzetta F, Caprini E, Marziali M, Digilio MC, Marino B, Aiuti A, Amati F, Russo G, Novelli G, Pandolfi F. Biased T-cell receptor repertoires in patients with chromosome 22q11. 2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome). Clinical & Experimental Immunology. 2003 May;132(2):323-31. Borte S, Wang N, Óskarsdóttir S, von Döbeln U, Hammarström L. Newborn screening for primary immunodeficiencies: beyond SCID and XLA. Annals of the New York Academy of Sciences. 2011 Dec;1246(1):118-30. Patel K, Akhter J, Kobrynski L, Gathman B, Davis O, Sullivan KE, International DiGeorge Syndrome Immunodeficiency Consortium. Immunoglobulin deficiencies: the B-lymphocyte side of DiGeorge Syndrome. The Journal of pediatrics. 2012 Nov 1;161(5):950-3. Cowan E, Burch KJ, Green BD, Grieve DJ. Obestatin as a key regulator of metabolism and cardiovascular function with emerging therapeutic potential for diabetes. British Journal of Pharmacology. 2016 Jul;173(14):2165-81. Zou CC, Liang L, Wang CL, Fu JF, Zhao ZY. The change in ghrelin and obestatin levels in obese children after weight reduction. Acta Paediatrica. 2009 Jan;98(1):159-65. Zhang N, Yuan C, Li Z, Li J, Li X, Li C, Li R, Wang SR. Meta-analysis of the relationship between obestatin and ghrelin levels and the ghrelin/obestatin ratio with respect to obesity. The American journal of the medical sciences. 2011 Jan 1;341(1):48-55. Voll SL, Boot E, Butcher NJ, Cooper S, Heung T, Chow EW, Silversides CK, Bassett AS. Obesity in adults with 22q11. 2 deletion syndrome. Genetics in Medicine. 2017 Feb;19(2):204-8. Malecki SL, Van Mil S, Graffi J, Breetvelt E, Corral M, Boot E, Chow EW, Sanches M, Verma AA, Bassett AS. A genetic model for multimorbidity in young adults. Genetics in Medicine. 2020 Jan 1;22(1):132-41. Suksawat Y, Sathienkijkanchai A, Veskitkul J, Jirapongsananuruk O, Visitsunthorn N, Vichyanond P, Pacharn P. Resolution of primary immune defect in 22q11. 2 deletion syndrome. Journal of Clinical Immunology. 2017 May;37:375-82. Byers DE, Alexander-Brett J, Patel AC, Agapov E, Dang-Vu G, Jin X, Wu K, You Y, Alevy Y, Girard JP, Stappenbeck TS. Long-term IL-33–producing epithelial progenitor cells in chronic obstructive lung disease. The Journal of clinical investigation. 2013 Sep 3;123(9):3967-82. Calderon AA, Dimond C, Choy DF, Pappu R, Grimbaldeston MA, Mohan D, Chung KF. Targeting interleukin-33 and thymic stromal lymphopoietin pathways for novel pulmonary therapeutics in asthma and COPD. European Respiratory Review. 2023 Mar 31;32(167). Ramesh N, Gawli K, Kiran Pasupulleti V, Unniappan S. Metabolic and cardiovascular actions of nesfatin-1: implications in health and disease. Current Pharmaceutical Design. 2017 Mar 1;23(10):1453-64. Mamoor S. Interleukin 33 is differentially expressed in the thymus by developmental stage and cell type. Liew FY, Girard JP, Turnquist HR. Interleukin-33 in health and disease. Nature Reviews Immunology. 2016 Nov;16(11):676-89. Zdrojewicz Z, Pachura E, Pachura P. The thymus: a forgotten, but very important organ. Advances in clinical and experimental medicine: official organ Wroclaw Medical University. 2016 Mar 1;25(2):369-75. Xu L, Wei C, Chen Y, Wu Y, Shou X, Chen W, Lu D, Sun H, Li W, Yu B, Wang X. IL-33 induces thymic involution-associated naive T cell aging and impairs host control of severe infection. Nature Communications. 2022 Nov 12;13(1):6881. Feger U, Tolosa E, Huang YH, Waschbisch A, Biedermann T, Melms A, Wiendl H. HLA-G expression defines a novel regulatory T-cell subset present in human peripheral blood and sites of inflammation. Blood, The Journal of the American Society of Hematology. 2007 Jul 15;110(2):568-77. Contini P, Murdaca G, Puppo F, Negrini S. HLA-G expressing immune cells in immune mediated diseases. Frontiers in Immunology. 2020 Aug 28; 11:1613. Aksu G, Genel F, Koturoglu G, Kurugol Z, Kutukculer N. Serum immunoglobulin (IgG, IgM, IgA) and IgG subclass concentrations in healthy children: a study using nephelometric technique. Turkish Journal of Pediatrics. 2006 Jan 1;48(1):19. Rahman M. Introduction to flowcytometry. J Clin Invest. 2006; 45:345–361. Dar N, Gothelf D, Korn D, Frisch A, Weizman A, Michaelovsky E, Carmel M, Yeshayahu Y, Dubnov-Raz G, Pessach IM, Simon AJ. Thymic and bone marrow output in individuals with 22q11. 2 deletion syndrome. Pediatric Research. 2015 Apr;77(4):579-85. Lingman Framme J, Borte S, von Döbeln U, Hammarström L, Óskarsdóttir S. Retrospective analysis of TREC based newborn screening results and clinical phenotypes in infants with the 22q11 deletion syndrome. Journal of clinical immunology. 2014 May;34:514-9. Framme JL, Lundqvist C, Lundell AC, van Schouwenburg PA, Lemarquis AL, Thörn K, Lindgren S, Gudmundsdottir J, Lundberg V, Degerman S, Zetterström RH. Long-term follow-up of newborns with 22q11 deletion syndrome and low TRECs. Journal of Clinical Immunology. 2022 Apr;42(3):618-33. Al‐Herz W, McGeady SJ, Gripp KW. 22q11. 2 deletion syndrome and selective IgM deficiency: an association of a common chromosomal abnormality with a rare immunodeficiency. American Journal of Medical Genetics Part A. 2004 May 15;127(1):99-100. Kung SJ, Gripp KW, Stephan MJ, Fairchok MP, McGeady SJ. Selective IgM deficiency and 22q11. 2 deletion syndrome. Annals of Allergy, Asthma & Immunology. 2007 Jul 1;99(1):87-92. Giardino G, Radwan N, Koletsi P, Morrogh DM, Adams S, Ip W, Worth A, Jones A, Meyer-Parsonson I, Gaspar HB, Gilmour K. Clinical and immunological features in a cohort of patients with partial DiGeorge syndrome followed at a single center. Blood, The Journal of the American Society of Hematology. 2019 Jun 13;133(24):2586-96. Gennery AR, Barge D, O'sullivan JJ, Flood TJ, Abinun M, Cant AJ. Antibody deficiency and autoimmunity in 22q11. 2 deletion syndrome. Archives of disease in childhood. 2002 Jun 1;86(6):422-5. Finocchi A, Di Cesare S, Romiti ML, Capponi C, Rossi P, Carsetti R, Cancrini C. Humoral immune responses and CD27+ B cells in children with DiGeorge syndrome (22q11. 2 deletion syndrome). Pediatric Allergy and Immunology. 2006 Aug;17(5):382-8. Campbell IM, Sheppard SE, Crowley TB, McGinn DE, Bailey A, McGinn MJ, Unolt M, Homans JF, Chen EY, Salmons HI, Gaynor JW. What is new with 22q? An update from the 22q and You Center at the Children's Hospital of Philadelphia. American Journal of Medical Genetics Part A. 2018 Oct;176(10):2058-69. Sullivan KE. Chromosome 22q11. 2 deletion syndrome and DiGeorge syndrome. Immunological Reviews. 2019 Jan;287(1):186-201. Sedivá1ADEFG A, Bartůňková1AG J, Zachová1B R, Poloučková1B A, Hrušák1BC O, Janda1BCEF A, Kočárek2BCD E, Novotná2BC D, Novotná2BC K, Klein3B T. Early development of immunity in diGeorge syndrome. Med Sci Monit. 2005;11(4):187. Handel AE, Cheuk S, Dhalla F, Maio S, Hübscher T, Rota I, Deadman ME, Ekwall O, Lütolf M, Weinberg K, Holländer G. Developmental dynamics of the neural crest–mesenchymal axis in creating the thymic microenvironment. Science Advances. 2022 May 13;8(19): eabm9844. Germanov E, Veinotte L, Cullen R, Chamberlain E, Butcher EC, Johnston B. Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells. The Journal of Immunology. 2008 Jul 1;181(1):81-91. Kurokawa M, Matsukura S, Kawaguchi M, Ieki K, Suzuki S, Watanabe S, Homma T, Yamaguchi M, Takeuchi H, Adachi M. Interleukin-33-activated dendritic cells induce the production of thymus and activation-regulated chemokine and macrophage-derived chemokine. International archives of allergy and immunology. 2013 May 1;161(Suppl. 2):52-7. Bassett AS, Chow EW, Husted J, Weksberg R, Caluseriu O, Webb GD, Gatzoulis MA. Clinical features of 78 adults with 22q11 deletion syndrome. American Journal of Medical Genetics Part A. 2005 Nov 1;138(4):307-13. Guzman ML, Delgado I, Lay‐Son G, Willans E, Puga A, Repetto GM. Growth in Chilean infants with chromosome 22q11 microdeletion syndrome. American Journal of Medical Genetics Part A. 2012 Nov;158(11):2682-6. Nakahara T, Harada T, Yasuhara D, Shimada N, Amitani H, Sakoguchi T, Kamiji MM, Asakawa A, Inui A. Plasma obestatin concentrations are negatively correlated with body mass index, insulin resistance index, and plasma leptin concentrations in obesity and anorexia nervosa. Biological psychiatry. 2008 Aug 1;64(3):252-5. Eberle P, Berger C, Junge S, Dougoud S, Büchel EV, Riegel M, Schinzel A, Seger R, Güngör T. Persistent low thymic activity and non-cardiac mortality in children with chromosome 22q11· 2 microdeletion and partial DiGeorge syndrome. Clinical & Experimental Immunology. 2009 Feb;155(2):189-98. Covington EW, Roberts MZ, Dong J. Procalcitonin monitoring as a guide for antimicrobial therapy: a review of current literature. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2018 May;38(5):569-81. Nargis W, Ibrahim MD, Ahamed BU. Procalcitonin versus C-reactive protein: Usefulness as biomarker of sepsis in ICU patient. International journal of critical illness and injury science. 2014 Jul;4(3):195. Bahri R, Hirsch F, Josse A, Rouas-Freiss N, Bidere N, Vasquez A, Carosella ED, Charpentier B, Durrbach A. Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes. The Journal of Immunology. 2006 Feb 1;176(3):1331-9. Verhoeven L, Reitsma P, Siegel LS. Cognitive and linguistic factors in reading acquisition. Reading and writing. 2011 Apr;24:387-94. Yagi H, Furutani Y, Hamada H, Sasaki T, Asakawa S, Minoshima S, Ichida F, Joo K, Kimura M, Imamura SI, Kamatani N. Role of TBX1 in human del22q11. 2 syndrome. The Lancet. 2003 Oct 25;362(9393):1366-73. McQuade L, Christodoulou J, Budarf M, Sachdev R, Wilson M, Emanuel B, Colley A. Patient with a 22q11. 2 deletion with no overlap of the minimal DiGeorge syndrome critical region (MDGCR). American journal of medical genetics. 1999 Sep 3;86(1):27-33. Hayashi T, Suzuki K. Molecular pathogenesis of Bernard-Soulier syndrome. InSeminars in thrombosis and hemostasis 2000 (Vol. 26, No. 01, pp. 053-060). Copyright© 2000 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.:+ 1 (212) 584-4663). Kato T, Kosaka K, Kimura M, Imamura SI, Yamada O, Iwai K, Ando M, Joh-o K, Kuroe K, Ohtake A, Takao A. Thrombocytopenia in patients with 22q11. 2 deletion syndrome and its association with glycoprotein Ib-β. Genetics in Medicine. 2003 Mar 1;5(2):113-9. Kunishima S, Imai T, Kobayashi R, Kato M, Ogawa S, Saito H. B ernard–S oulier syndrome caused by a hemizygous GPIb β mutation and 22q11. 2 deletion. Pediatrics International. 2013 Aug;55(4):434-7. Huang L, Xie Y, Zhou Y, Luo Y, Huang X, Xu Z, Cai D, Fang Q. Clinical and molecular cytogenetic studies of an unrecognised 22q11. 2 deletion in three families. Experimental and Therapeutic Medicine. 2015 Mar 1;9(3):823-8. Burnside RD. 22q11. 21 deletion syndromes: a review of proximal, central, and distal deletions and their associated features. Cytogenetic and Genome Research. 2015 Aug 8;146(2):89-99. Clements CC, Wenger TL, Zoltowski AR, Bertollo JR, Miller JS, de Marchena AB, Mitteer LM, Carey JC, Yerys BE, Zackai EH, Emanuel BS. Critical region within 22q11. 2 linked to higher rate of autism spectrum disorder. Molecular Autism. 2017 Dec;8:1-7. Jungerius BJ, Hoogendoorn ML, Bakker SC, Van't Slot R, Bardoel AF, Ophoff RA, Wijmenga C, Kahn RS, Sinke RJ. An association screen of myelin-related genes implicates the chromosome 22q11 PIK4CA gene in schizophrenia. Molecular psychiatry. 2008 Nov;13(11):1060-8. Hiroi N, Takahashi T, Hishimoto A, Izumi T, Boku S, Hiramoto T. Copy number variation at 22q11. 2: from rare variants to common mechanisms of developmental neuropsychiatric disorders. Molecular psychiatry. 2013 Nov;18(11):1153-65. McDonald-McGinn DM, Sullivan KE, Marino B, Philip N, Swillen A, Vorstman JA, Zackai EH, Emanuel BS, Vermeesch JR, Morrow BE, Scambler PJ. 22q11. 2 deletion syndrome. Nature reviews Disease primers. 2015 Nov 19;1(1):1-9. Morrow BE, McDonald‐McGinn DM, Emanuel BS, Vermeesch JR, Scambler PJ. Molecular genetics of 22q11. 2 deletion syndrome. American journal of medical genetics Part A. 2018 Oct;176(10):2070-81. Additional Declarations No competing interests reported. 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. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4231044","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292074765,"identity":"dd8f8819-e6b2-46b6-bd07-275c4629db93","order_by":0,"name":"Assem Metwally Abo-Shanab","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYDADfgYGNhK1SDaQrMXgALFa+PsPH93AuOOevPGN5GcPPlQwyPOLHcCvReJGWtoNxjPFhttupJkbzjjDYDhzdgIBa27wmN1gbEtIMLuRYCbN28aQYHCbgBb58+e/gbUYz0j/RpwWgwM5bGAtBhI5RNpieCMN7DCgN96USc44I0HYL3LnDz8DaZHnb0/fJvGhwkaeX5qAFhBg/gMiBcAqJQgrRwD+A6SoHgWjYBSMgpEEAAfMQaUzKqESAAAAAElFTkSuQmCC","orcid":"","institution":"National Research Centre","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Assem","middleName":"Metwally","lastName":"Abo-Shanab","suffix":""},{"id":292074766,"identity":"d0b71589-a3b7-4eb9-98c5-ebb33065f981","order_by":1,"name":"Haiam Abdel Raouf","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haiam","middleName":"Abdel","lastName":"Raouf","suffix":""},{"id":292074767,"identity":"02bcd39c-7a70-4527-a77f-99c1e6ea9501","order_by":2,"name":"Alaaeldin G. Fayez","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alaaeldin","middleName":"G.","lastName":"Fayez","suffix":""},{"id":292074768,"identity":"5b693650-76b4-4b15-934a-aefd40b4e7fc","order_by":3,"name":"Iman Helwa","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Iman","middleName":"","lastName":"Helwa","suffix":""},{"id":292074769,"identity":"6e1904c8-38e9-4128-971d-a77ee07c5d87","order_by":4,"name":"Engy A. Ashaat","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Engy","middleName":"A.","lastName":"Ashaat","suffix":""},{"id":292074770,"identity":"2095954a-1b76-4e31-acec-4ae6b7bd12f0","order_by":5,"name":"Naglaa Kholoussi","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naglaa","middleName":"","lastName":"Kholoussi","suffix":""},{"id":292074771,"identity":"2c231cef-b8af-4ad9-ac34-6d4971de4824","order_by":6,"name":"Nora N. Esmaiel","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nora","middleName":"N.","lastName":"Esmaiel","suffix":""},{"id":292074773,"identity":"55f4ea25-ab2b-4392-bed5-ccc71490e9ce","order_by":7,"name":"Rania Fawzy Mahmoud Abdelkawy","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rania","middleName":"Fawzy Mahmoud","lastName":"Abdelkawy","suffix":""}],"badges":[],"createdAt":"2024-04-07 11:14:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4231044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4231044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55251812,"identity":"d2d5b6a1-6478-44a9-8ead-118bd98dd4cf","added_by":"auto","created_at":"2024-04-24 17:43:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30923,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of TRECs and KRECs in DGS patients and control subjects.\u003c/p\u003e\n\u003cp\u003e* P\u0026lt; 0.05 Significant versus controls (by Mann Whitney U Test)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4231044/v1/05abf152e7635be5b2fe9117.png"},{"id":55251859,"identity":"a0cbc98e-6ef5-406f-8f47-fd173ddef939","added_by":"auto","created_at":"2024-04-24 17:43:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22761,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoglobulins median levels in subjects with DGS patients compared with control subjects.\u003c/p\u003e\n\u003cp\u003e* P\u0026lt; 0.05 Significant versus controls (by Mann Whitney U Test)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4231044/v1/e81e14a13564464a590047fb.png"},{"id":55252557,"identity":"d5d28bf7-5621-454a-963d-df14ed2cd22f","added_by":"auto","created_at":"2024-04-24 17:51:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48023,"visible":true,"origin":"","legend":"\u003cp\u003eGel images showing the amplified markers products in duplicate PCR reactions of D22S944 and COMT markers [I and II] for the affected case, and PCR reaction of COMT marker for control case [III] showed deletion of D22S944 and COMT markers in the affected case.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4231044/v1/4021ea8b6c31ffb2d8488484.png"},{"id":55251815,"identity":"e8dc2b82-1cbf-453b-a8e1-c6a3f07af733","added_by":"auto","created_at":"2024-04-24 17:43:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11493,"visible":true,"origin":"","legend":"\u003cp\u003eOrder of STRs and genes located on the detected nested 22q11.2 microdeletion. (-) indicates the deleted loci, while (+) indicates the undeleted loci. The triangle marked white is the detected deletion breakpoint.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4231044/v1/87dc6be2036ca583f1623aac.png"},{"id":55251814,"identity":"48ce686e-80d4-4e30-8f8e-ffb8285e9066","added_by":"auto","created_at":"2024-04-24 17:43:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201397,"visible":true,"origin":"","legend":"\u003cp\u003eThis image was adapted from an image published previously\u003cstrong\u003e \u003c/strong\u003e[62, 63].\u003cstrong\u003e \u003c/strong\u003eThe 3 Mb 22q11.2 region (hg19 assembly, coordinates) is revealed as a line that spans the 22q11.2 region. Four low copy repeats (LCR22), termed LCR22A, LCR22B, LCR22C and LCR22D are indicated. Below the line representing the 22q11.2 region, most of the known coding and noncoding genes are presented. In a clear blue font, TBX1 is indicated. A clear red font indicates genes linked to recessive genetic conditions. A star indicates noncoding genes. The size and position of reported 22q11.2 deletions are indicated by a grey box, and novel, in the current study, 22q11.2 deletion is indicated by a black box. The frequencies of deletions were obtained from McDonald-McGinn and colleagues in the special AJMG issue.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4231044/v1/5b88cdfa66396578650204c6.png"},{"id":55252811,"identity":"71a5ef07-411a-46eb-b490-c50146514f55","added_by":"auto","created_at":"2024-04-24 17:59:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":763166,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4231044/v1/c5ac9d25-64f2-4214-86b8-9b3450aecaff.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrating TREC/KREC assay and some cytokines in the evaluation of the immune status of patients with DiGeorge Syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChromosome 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion syndrome in humans. It occurs in almost 1:4000 live births and was traditionally identified as DiGeorge syndrome which is classified as a primary immunodeficiency. It is described by T-cell lymphopenia and thymic dysplasia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe condition can be grouped into two categories, complete and partial DiGeorge syndrome. Individuals with a complete absence of the thymus are considered to have complete DiGeorge syndrome [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn DiGeorge syndrome, there is dysregulation of T\u0026ndash;B-cell interactions. Complete DiGeorge syndrome was found to suffer from life-threatening severe T-cell lymphopenia. Whereas partial DiGeorge syndrome is characterized by decreased thymic output show and T-cell lymphopenia. This is exemplified in a low number of T-cell receptor excision circles (TRECs) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis resulted from recent thymic emigrant T cells and a low number of na\u0026iuml;ve T cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The humoral immune compartment in DiGeorge is also affected. This is reflected in hypogammaglobulinemia, diminished response to vaccination, (Patel et al, 2012), and dysfunctional maturation of B-cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This leads to increased vulnerability to infections, atopy, and autoimmune disease. There is a wide variability of phenotypic features. Hallmark features include cardiac anomalies and hypoparathyroidism, in addition to thymic aplasia or hypoplasia. Other phenotypic features include feeding and swallowing anomalies, palatal defects, renal abnormalities, and others [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePatients don't necessarily manifest the typical features of the syndrome and severity differs considerably between patients. Even if the mutation is familiarly inherited or presented in identical twins, the deletion may be expressed with completely different phenotypes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImmune deficiency in such patients cannot be evaluated depending on clinical phenotype. Some degree of T cell lymphopenia is encountered in around 70% of patients with 22q11.2del [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, immunologic assessment of affected subjects with 22q11.2del and any other developmental thymus deformity (DTD) is crucial to define immune status and assess infection susceptibility. T cell markers (CD3, CD4, CD8), B cell markers (CD19 or CD20), and natural killer cell markers (CD16 or CD56) should be included in the primary immunologic evaluation required for diagnosis. This is to be requested along with IgG, IgM, and IgA estimation. Moreover, T cell receptor excision circles (TREC) assay is performed if available; abnormal results increase the probability of significant T cell lymphopenia (TCL) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eActual thymus-derived na\u0026iuml;ve T cells (RTEs) can be evaluated by T cell receptor excision circles (TREC) assay. It serves as an excellent marker for this task. During V(D)J recombination of TCR in the thymus, circulating remnants of the DNA excision by-products are formed. These remnants are thus measured. Therefore, whether the presence or degree of thymic T cell production, both are directly reflected by (TREC) assay. Neonates who may have abnormal TREC assay reflect remarkable TCL [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, an assay can also detect congenital athymia as well as other severe forms of thymic hypoplasia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, and in the same context, kappa-deleting element recombination circles (KREC), can be used to detect newly developed B cells. A conjoint TREC/KREC assay has been used to detect patients with severe B cell disorders [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn another study, TREC was only identified in severe cases with deep lymphopenia, whereas children with incomplete DiGeorge syndrome had normal range TREC/KREC levels both at birth and postnatally. However, a decline of both TREC and KREC levels with age was noticed significantly in DGS patients than in controls. Therefore, their assay is complementary to routine investigations and follow-up of patients. Based on the del22q11 background, their levels may reflect age-related immune changes along the course of the disease [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe humoral immune system is sometimes found intact in 22q11.2 del subjects. Yet, immunoglobulin (Ig) A deficiencies improper response to vaccines, and transient hypogammaglobulinemia were sometimes encountered. Recently, humoral abnormalities as well as Immunoglobulins have been studied in 22q11.2 del [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIgG deficiency may develop even in individuals with normal immune function early in life. It may develop a picture very similar to CVID both clinically and biologically. Moreover, the US Immunodeficiency Network (USIDNET) performed a conjoint study with the European Society for Immunodeficiencies (ESID), where nearly half the subjects with confirmed 22q11.2del, had IgG, IgA, and IgM abnormalities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eObestatin, a ghrelin gene product, was primarily defined as an appetite suppressant; its role in metabolism remains an ongoing debate. It is thought to be involved in the regulation of metabolic homeostasis and cardiovascular function. Moreover, some previous studies noted that obestatin increased beta cell mass and improved lipid metabolism; thus, inversely correlated with fasting blood glucose [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. (Cowan et al, 2016)\u003c/p\u003e \u003cp\u003eMoreover, in obese individuals, Obestatin levels were found reduced and negative with body mass index (BMI) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this same context, regarding adults with 22q11.2DS, there is evidence of obesity, global medical multimorbidity, and some other features, such as early onset Parkinson\u0026rsquo;s disease in these subjects [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProcalcitonin (PCT) is one of the biomarkers released in response to bacterial infections. It has an important role in the diagnosis and management of sepsis and lower respiratory tract infection.18\u0026ndash; 23. In the same context, it is worth noting that Infections were observed in DGS and the most common infection was noted as pneumonia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-33 is a member of the IL-1 family of cytokines. It is found in multiple organs and is mainly localized to barrier epithelial cells and endothelial cells. Fibroblasts, myofibroblasts, and airway smooth muscle cells were also found to express Il-33 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. (Byers et al, 2013 Calderon et al, 2023).\u003c/p\u003e \u003cp\u003eIt has a crucial role in innate and adaptive immune responses. It drives type 2 T-helper responses and has the ability to activate basophils, mast cells, and eosinophils [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is released into the extracellular space by endothelial cells following cellular insult, necrosis, secondary to cigarette smoke, pollutants, allergens, and viral or bacterial exposure, hence acting as an innate immune indicator of danger (alarmin). Accordingly, IL-33 is considered to perform some immunologically important functions in the cortex and medulla of the thymus during mammalian development [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. (Mamoor 2020)\u003c/p\u003e \u003cp\u003eTherefore, IL-33 in addition to acting as a nuclear transcription factor, has pro-inflammatory effects as well [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInfection-induced IL-33 release was found to induce thymic involution and thus T cell aging which consequently impairs host control of severe infection. Moreover, T cell immune deficiency or increased liability to infections has been related to impaired thymus function in many conditions in children such as DiGeorge syndrome, preterm newborn, and severe combined immunodeficiency [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. (Zdrojewicz et al, 2016 and Xu et al, 2022).\u003c/p\u003e \u003cp\u003eHLA-G is an HLA antigen having immunomodulatory properties. CD4 and CD8 T lymphocytes express it as well as NK cells, monocytes, and dendritic cells. T cells expressing the immunomodulatory m HLA-G molecule are identified as distinct subpopulations of Treg lymphocytes. These were found present in the peripheral blood of 0.1\u0026ndash;8.3% of healthy subjects, and primarily originate from the thymus. Studies confirmed their importance in health and disease [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, quantitative, and qualitative derangements of HLA-G were detected in several immune conditions which highly suggests that HLA-G immune cells may be involved in the pathogenesis of such disorders [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e"},{"header":"Patients and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePatients\u003c/h2\u003e\n \u003cp\u003eThis study is a case-control study that was conducted in the period from November 2019 to May 2022. It was performed in line with the principles of the Declaration of Helsinki. The study included thirty-three patients with DiGeorge syndrome (DGS) (22q11.2DS). In addition, forty-five healthy subjects, matched for age and gender, were included as a control group. All patients were recruited among patients referred to Genetics Clinics at the Medical Research Centre of Excellency, National Research Centre (NRC), Dokki, Giza, Egypt. This study\u0026apos;s Approval was granted by the Ethics Committee of the National Research Centre (NRC), Cairo, Egypt. (Ethics No. 19267-1). Written informed consent was obtained from the parents of the participants for including their children in the study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eBlood samples\u003c/h2\u003e\n \u003cp\u003eFive milliliters of fresh peripheral blood samples from all patients and controls were drawn. 2.5 ml of blood taken were divided into two sterile EDTA-containing tubes. 1 ml for CBC and the remaining amount was used for flow cytometric analysis which was done within 24 hours of collection as well as DNA extraction which was done simultaneously or on refrigerated samples stored at 2\u0026ndash;8\u0026deg;C within one week of collection. Another 2.5 ml of blood was centrifuged at a rate of 3000 rounds per minute for 10 min to separate the serum which was collected and immediately frozen in 0.2 ml aliquots at -80\u0026deg;C until assayed for immunoglobulin levels by nephelometry and for ELISA tests.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eDetermination of Immunoglobulins (IgA, IgM, and IgG)\u003c/h2\u003e\n \u003cp\u003eMeasurement of serum immunoglobulin was performed using the method of nephelometry [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eFlow cytometric analysis\u003c/h2\u003e\n \u003cp\u003eAnticoagulated blood was stained using the whole-blood lysis method. Whole blood was stained with monoclonal antibodies directed against the surface antigens: CD3 FITC labeled MoAbs for T lymphocytes, CD16 labeled with PE for NK cells, FITC labeled CD4 for helper T lymphocytes, PE-labeled CD8 for cytotoxic T lymphocyte and FITC labeled CD19 for B lymphocytes (BD Biosciences, USA.). Lymphocyte subsets immunophenotyping was done using flow cytometry (BD Accuri\u0026trade; C6 Cytometer, USA) [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eReal-time PCR for TRECs and KRECs measurement\u003c/h2\u003e\n \u003cp\u003eGenomic DNA was isolated from peripheral blood leukocytes by QIAamp DNAMini Kit (50 preps), catalog number 51304, Germany (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.qiagen.com/eg/\u003c/span\u003e\u003c/span\u003e). The assay was run on 7500 Fast Real-Time PCR (Applied Biosystems) using the specific primers and probes for TRECs, KRECs, and \u0026beta;-actin. RT-PCR was carried out using TaqMan Universal PCR Master Mix II. 10 \u0026micro;l PCR reaction mix was added to the 5 \u0026micro;l DNA samples, 1 \u0026micro;l forward primer (Applied Biosystems, USA.), 1 \u0026micro;l reverse primer (Applied Biosystems, USA.), 1 \u0026micro;l Taqman TAMRA probe and 2 \u0026micro;l H2O in a sterile 48-well PCR plate, using real-time cycler conditions of initial activation stage at 95\u0026deg;C for 10 min, followed by 45 cycles of denaturation at 95\u0026deg;C for 15 seconds, and a combined primer/probe annealing and elongation at 60\u0026deg;C for 1 min. TRECs, KRECs, and \u0026beta;-actin copy numbers have been obtained by extrapolating the respective sample quantities from the standard curve obtained by serial dilutions of human genomic DNA (Promega, USA.). The initial copy numbers of TRECs, KRECs, and \u0026beta;-actin were calculated from the following equation:\u003c/p\u003e\n \u003cp\u003e\u0026quot;Copies of the gene of interest\u0026thinsp;=\u0026thinsp;mass of gDNA / mass of haploid genome\u0026quot;.\u003c/p\u003e\n \u003cp\u003eThe number of TRECs and KRECs in each sample was calculated per \u0026micro;l of extracted DNA. \u0026beta;-actin copy number was used to judge the successful amplification of each sample.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\n \u003cp\u003eSerum IL33, Obestatin, HLA-G, and Procalcitonin levels of all study subjects were determined using Human Procalcitonin ELISA kit (EIAab, Co., Ltd, East Lake Hi-Tech Development Zone, Wuhan, China) and Human IL33, Obestatin and HLA-G ELISA kits (NOVA, Beijing, China) by following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003ePolymorphic 22q11.2 markers mapping\u003c/h2\u003e\n \u003cp\u003eThe PCR-STR technique was performed using specific primers for microsatellite genotyping that were directly retrieved from Electronic PCR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.ncbi.nlm.nih.gov/sutils/e-pcr/\u003c/span\u003e\u003c/span\u003e; NCBI, USA). Three consecutive polymorphic loci were used and located in the usually 3-Mb deleted region: D22S941, D22S944, and D22S264, plus the COMT marker. The PCR products were visualized on 4% agarose gel.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical methods\u003c/h2\u003e\n \u003cp\u003eThe collected data were coded, tabulated, and statistically analyzed using SPSS version 19.0 software (SPSS Inc., Chicago, Illinois, USA). T-test was used for comparing the parametric results among groups. The Mann-Whitney U Test was used for comparing the non-parametric results among groups. Data were presented as median and range. All analyses were two-tailed, the level of significance was taken at P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is significant, otherwise is non-significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThis case\u0026ndash;control study included two groups: group I (33 children clinically diagnosed as DGS and Group II (45 healthy controls). The demographic and clinical characteristics of DGS patients and volunteer control subjects are presented in Table \u003cspan\u003e1\u003c/span\u003e. All the DGS patients in the study had congenital heart diseases (CHDs).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eDemographic and clinical characteristics\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal healthy controls\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDiGeorge\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of cases\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender, no.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22 (48.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16 ((48.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.462\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23 (51.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17 (51.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years) mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.297\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eP value calculated using T-test\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eKRECs expression was significantly elevated in DGS patients as compared with the healthy controls (P\u0026thinsp;=\u0026thinsp;0.0008) (Fig. \u003cspan\u003e1\u003c/span\u003e). There was also a significant increase in immunoglobulins level between DGS patients and the healthy controls (IgA P\u0026thinsp;=\u0026thinsp;0.014, IgG P\u0026thinsp;=\u0026thinsp;0.0019\u0026amp; IgM P\u0026thinsp;=\u0026thinsp;0.0032) (Fig. \u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 Significant versus controls (by Mann Whitney U Test)\u003c/p\u003e\n\u003cp\u003eTable \u003cspan\u003e2\u003c/span\u003e summarizes laboratory findings such as TLC, lymph, CD3, CD16, CD4, and CD8 in addition to CD19 in the DGS group and control group. CD8% as well as CD8 absolute count in the patients with DGS were significantly lower than in the healthy control (P\u0026thinsp;=\u0026thinsp;0.01273 and 0.05358 respectively). The results in Table \u003cspan\u003e3\u003c/span\u003e showed that there were no significant differences in IL33, Obestatin, HLA-G, and Procalcitonin levels between DGS patients compared with the control group.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eComparison of the laboratory data measured between cases and controls.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDGS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (Range)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (Range)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTLC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8250 (3600\u0026ndash;14200)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6050 (4140\u0026ndash;10890)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1765\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003elymph %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.7 (32\u0026ndash;61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.6 (23-87.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.05949\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute lymph\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3688.5 (2752\u0026ndash;6958)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3592.9 (1288\u0026ndash;9100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6472\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD3%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.4 (20-70.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.1 (31.7\u0026ndash;73.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5588\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute CD3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1734.2 (883.4-4181.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1861.2 (807.6-3778.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD16%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.7 (5-38.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.2 (7-37.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6129\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute CD16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e422.3 (159.6-1793.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e421 (182.6\u0026ndash;1950)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7445\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD4%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.9 (12.6\u0026ndash;43.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.8 (22-40.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8776\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute CD4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1055.5 (590-3012.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1046 (288.5\u0026ndash;2265)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0236\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD8%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.5 (5-43.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26 (7.3\u0026ndash;36.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.01273*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute CD8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e548.7 (212.3-1071.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e922 (273\u0026ndash;1738)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.05358*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD19%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.6 (10.3\u0026ndash;37.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16 (4.4\u0026ndash;41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4412\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute CD19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e571.2 (284.8-2177.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e538 (90.7\u0026ndash;3731)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eResults were expressed as Median (Range).\u003c/p\u003e\n\u003cp\u003e* Significant versus controls (by Mann Whitney U Test)\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eBiomarkers comparing apparently healthy control with DiGeorge patients.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDGS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (Range)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian (Range)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL 33 pg/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e178 (43\u0026ndash;271)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134.9 (5.4-231.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.05911\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eObestatin pg/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e951.7 (335\u0026ndash;4901)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e835 (135\u0026ndash;3635)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4735\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHLA-G ng/m\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.3 (20.5-125.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.3 (17.7-141.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8212\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eProcalcitonin pg/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e197.5(50\u0026ndash;400)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e202.5 (105\u0026ndash;281)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eResults were expressed as Median (Range).\u003c/p\u003e\n\u003cp\u003e* Significant versus controls (by Mann Whitney U Test)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA Novel atypical interstitial homozygous 22q11.2 microdeletion suggested three DGS-susceptibility genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing three polymorphic DNA markers, an informative novel atypical interstitial homozygous deletion was found. This deletion included D22S944 and COMT absence, and D22S941 and D22S264 presence. The COMT gene, the nearest gene to the distal region of D22S264, was used to determine the minimal breakpoint loci. The distance of the identified novel deleted region was approximately 0.35 Mb leading to the deletion of definitely 3 genes: GP1BB, TBX1, and COMT (Figs. \u003cspan\u003e3\u003c/span\u003e and \u003cspan\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eOut of 33 DGS patients, three patients showed deletion in the D22S944 marker only in the presence of D22S941, and D22S264 markers as shown in Table \u003cspan\u003e4\u003c/span\u003e. Therefore, we could assume that D22S944 is a common deleted marker in non-isolated DGS patients. None of the healthy controls had deletions.\u003c/p\u003e\n\u003cdiv\u003e\u003c/div\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eSTS-PCR results for three genetic polymorphic STS markers in the 22q11.2 region.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCase no.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD22S941\u003c/p\u003e\n \u003cp\u003e(224\u0026ndash;260 bps)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD22S944\u003c/p\u003e\n \u003cp\u003e(158-178bps)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCOMT\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD22S264\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNotes\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNovel atypical interstitial nested microdeletion about 0.35Mb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDiGeorge Syndrome (DGS) (22q11.2DS) is considered the most common microdeletion syndrome in humans. It occurs in almost 1:4000 live births. Phenotypic features vary considerably; nearly 75\u0026ndash;80% of DGS patients exhibit disorders of the immune system, which may cause them to suffer from immune instabilities such as autoimmune disease, susceptibility to infections, and atopy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results showed that KREC expression was significantly elevated in DGS patients as compared with the healthy controls (P\u0026thinsp;=\u0026thinsp;0.0008). On the contrary, Dar et al., 2015 and Lingman Framme et al, 2014 stated that in DGS patients, KREC levels don\u0026rsquo;t vary much from those of healthy controls, which suggests normal bone marrow output of B cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Nevertheless, DGS patients with low TREC counts have long-term impairment of thymic output. These patients were considerably more liable to viral infections, which is similar to the setting of more severe T-cell lymphopenia [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, the link between lower TREC levels and recurrent infections continues regardless of age [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur research found a significant increase in immunoglobulin levels among DGS patients compared to healthy controls (IgA P\u0026thinsp;=\u0026thinsp;0.014, IgG P\u0026thinsp;=\u0026thinsp;0.0019\u0026amp; IgM P\u0026thinsp;=\u0026thinsp;0.0032) unlike Patel K et al., 2012 who stated that after 3 years of age, almost 6% of DGS patients have low IgG levels [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the same context, Al-Herz et al., 2004 and Kung SJ et al., 2007 reported selective IgM deficiency associated with DGS although less common [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, in one cohort, the most common humoral defect reported was low IgM [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn DGS, low levels of IgG, IgA, IgM, and defective antibody responses to vaccines were described. Severe infections observed in DGS were attributed to humoral immune deficiency [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 17% of cases, humoral immunity instabilities were witnessed [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Some autoimmune problems including juvenile rheumatoid arthritis (JRA) reported IgA deficiency in nearly 13% of patients with DGS [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Despite the finding that hypogammaglobulinemia was found to present in the first year of life, it usually resolves. On the other hand, hypergammaglobulinemia was reported in some cases after the age of five. Surprisingly, the majority of affected individuals show functional antibody defects despite the normal antibody function and antibody avidity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImpaired T-cell production resulting from thymic hypoplasia leads to immunodeficiency. It's quite apparent that lower cells of thymic lineage are present in newborns with DGS. Sixty-seven percent of individuals had reduced T-cell production and more than 18% showed compromised T-cell function in previous studies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In another study including 1,421 DGS patients, abnormal T-cell populations were found in half the included subjects [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Over time, the production of T-cells improves and in the first year of life, children much improve despite the considerable T-cell defects they were suffering from. Whereas individuals with minor T-cell decrease showed better performance against pathogens [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding our study, CD8% as well as CD8 absolute count in the patients with DGS were significantly lower than healthy controls (P\u0026thinsp;=\u0026thinsp;0.01273 and 0.05358 respectively). Another study conducted with partial DGS patients had the same opinion. It stated that 20 out of 25 patients had low CD8\u0026thinsp;+\u0026thinsp;T cells and recurrent bacterial and viral infections [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, researchers demonstrated normalization of CD4\u0026thinsp;+\u0026thinsp;T cell numbers in most patients up to 3 years [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In another observational study, 81% of DGS patients reported normal levels of CD8\u0026thinsp;+\u0026thinsp;T at the age of 2 years when compared to healthy controls [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results showed that IL33 showed no significant difference in DGS patients compared with the control group. In an experimental model of DGS, Handel E et al., 2022, explored the changes in the non-epithelial stromal compartment of the thymus across different developmental stages [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. High levels of complexity within the thymic mesenchyme were revealed using Single-cell sequencing. A Uniform Manifold Approximation and Projection (UMAP) analysis of non-TEC (thymic epithelial cell) stroma recognized cluster incorporated two distinct medullary fibroblast subtypes, the medullary fibroblast subtypes 1 (MedFb1) and 2 (MedFb2). Both subtypes also comprised transcripts for IL-33 and Cxcl16, which are important for dendritic cell activation and NKT cell migration, respectively [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn about 40% of DGS patients, obesity has been reported with onset often during childhood or adolescence [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In obesity, Nakahara T et al., 2008, reported a negative correlation of plasma obestatin concentrations with body mass index, insulin resistance index, and plasma leptin concentrations [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Regarding our research, there was no statistically significant difference in obestatin levels between DGS patients and controls.\u003c/p\u003e \u003cp\u003eEberle et al., 2008 stated that serious infections and lymphoproliferative complications were more frequent in high-risk patients with DGS [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Procalcitonin levels begin rising within 3\u0026ndash; 4 h of bacterial infection and usually peak between 12 and 36 h [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Nargis W et al., 2014 observed 75% of diagnostic accuracy, 72% of specificity, and sensitivity of 76% for procalcitonin. They concluded as well that procalcitonin is better than CRP regarding accuracy in the identification and assessment of sepsis severity. As for our results, there were no significant differences in procalcitonin levels between DGS patients and apparently healthy controls [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePersistent low numbers of CD4\u0026thinsp;+\u0026thinsp;CD45RA\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells in DGS patients rendered them more prone to lethal infections and lymphoproliferative disorders during the follow-up period [ 47]. Feger U et al., 2006 defined novel subsets of T cells expressing the immunomodulatory molecule HLA-G as CD4 HLA-GC distinct Treg cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Through blocking cell cycle progression, sHLA-G5 has the ability to inhibit CD4 and CD8 T cell alloproliferation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, our results showed no significant differences in HLA-G levels between patients and controls.\u003c/p\u003e \u003cp\u003eWe analyzed the 22q11.2 region in extracted DNA from the peripheral blood of 33 DGS patients and 20 controls who were referred from our clinical genetics department at the National Research Centre (NRC). Using a set of polymorphic STR markers with known locations, we found one patient with novel atypical nested homozygous microdeletion flanked by D22S941 and D22S264 markers. None of the healthy controls had deletions.\u003c/p\u003e \u003cp\u003eOur results suggest a distinct segment of 22q11.2 as a DGS susceptibility region which is located between markers D22S941 and D22S264. Using the published DNA sequence of human chromosome 22, we estimated this region to contain three main genes: GP1BB, TBX1, and COMT. It is still not known which of those genes are directly responsible for DGS pathogenesis. Therefore, Further Gene dosage studies are required.\u003c/p\u003e \u003cp\u003eThe majority 22q11.2 deleted region (3 Mb) contains approximately 50 genes, and several miRNAs as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). TBX1 and COMT genes are considered the most relevant genes to DGS [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. TBX1 gene encodes a T-box transcription factor, which is known to have an essential role in early vertebrate development. Using FISH analysis with KB1764E3 probe which encompassed TBX1, CDCrel-1, and GP1BB genes, no deletions were observed in 13 patients with DGS [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. McQuade et al., 1999 detected small deletions including TBX1 and COMT genes in a patient with the DGS phenotype [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, our results agree with MacQuade et al., 1999 study, but we detect novel homozygous 22q11.2 deletion encompassing TBX1, COMT, and in addition to GP1BB using Polymorphic STS markers mapping as illustrated in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGP1BB [Glycoprotein 1b platelet subunit beta] was previously detected in DGS patients with atypical features [\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Using SNP‑array analysis, Huang et al., 2015 found four 22q11.2DS patients shared the same deletion breakpoints which included TBX1, COMT, DGCR2, GP1BB, RTN4R, PRODH, SNAP29, and SERP genes [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is worth noting that DGS has phenotypic heterogeneity and severity variability. So, some patients are mildly affected, whereas others have severe features that could be due in part to the presence of genetic modifiers and dosage of the deleted gene's haploinsufficiency. It was reported that Phenotypic variability in DGS is associated with (i) behavioral traits including autistic spectrum disorders (ASD) are apparently more frequent in LCRA-B deleted individuals [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], (ii) schizophrenia (SCZ) and attention deficit hyperactivity disorder (ADHD) phenotypes are apparently in LCRA-D deleted region including COMT, PRODH, GNB1L, TBX1, SEPT5/GP1BB, ZDHHC8, PI4KA, and ARVCF genes [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results may be given merit to adopt a new comprehensive investigation strategy for DGS patients that combines screening of 22q11.2 region, immunoglobulins level patterns, and TRECS and KRECS expression. This investigation strategy can provide better genetic consultations for DGS patients. However, the current study may be the first to show a small interstitial 22q11.2 deletion stereotype in a DGS patient, but it has shown that the smallest deletion at the 22q11.2 region is enough to confer the DGS phenotype. Further investigating of other DGS-relevant pathogenesis factors and their correlation with the patient\u0026rsquo;s clinical manifestations can help to provide a better explanatory model for the clinical variability of DGS disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the principles outlined in the Declaration of Helsinki. The research protocol and procedures were approved by the Ethics Committee of the National Research Centre (NRC), Cairo, Egypt, under Ethics No. 19267-1. Informed consent was obtained from the parents of all participants for their inclusion in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study. However, a deidentified version of the dataset may be available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.K. \u0026amp; H.A. conceptualized the study and designed the research methodology.\u003c/p\u003e\n\u003cp\u003eE.A. \u0026amp; N.E. collected and analyzed the data.\u003c/p\u003e\n\u003cp\u003eA.F. performed statistical analysis and interpretation of results.\u003c/p\u003e\n\u003cp\u003eA.A. drafted the manuscript and prepared the figures.\u003c/p\u003e\n\u003cp\u003eR.M. \u0026amp; I.H. critically reviewed and revised the manuscript for intellectual content.\u003c/p\u003e\n\u003cp\u003eAll authors approved the final version of the manuscript for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDavies EG. Immunodeficiency in DiGeorge syndrome and options for treating cases with complete athymia. Frontiers in immunology. 2013 Oct 31;4:322.\u003c/li\u003e\n\u003cli\u003eBiggs SE, Gilchrist B, May KR. Chromosome 22q11. 2 Deletion (DiGeorge Syndrome): Immunologic Features, Diagnosis, and Management. Current Allergy and Asthma Reports. 2023 Apr;23(4):213-22.\u003c/li\u003e\n\u003cli\u003eKlocperk A, Paračkov\u0026aacute; Z, Bloomfield M, Rataj M, Pokorn\u0026yacute; J, Unger S, Warnatz K, \u0026Scaron;ediv\u0026aacute; A. Follicular helper T cells in DiGeorge syndrome. Frontiers in Immunology. 2018 Jul 23;9:1730.\u003c/li\u003e\n\u003cli\u003eFroňkov\u0026aacute; E, Klocperk A, Svatoň M, Nov\u0026aacute;kov\u0026aacute; M, Kotrov\u0026aacute; M, Kayserov\u0026aacute; J, Kalina T, Keslov\u0026aacute; P, Votava F, Vinohradsk\u0026aacute; H, Freiberger T. The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome. Plos one. 2014 Dec 8;9(12):e114514.\u003c/li\u003e\n\u003cli\u003eKlocperk A, Mejstř\u0026iacute;kov\u0026aacute; E, Kayserov\u0026aacute; J, Kalina T, \u0026Scaron;ediv\u0026aacute; A. Low marginal zone-like B lymphocytes and natural antibodies characterize skewed B-lymphocyte subpopulations in del22q11 DiGeorge patients. Clinical Immunology. 2015 Dec 1;161(2):144-9.\u003c/li\u003e\n\u003cli\u003eDigilio MC, Angioni A, De Santis M, Lombardo A, Giannotti A, Dallapiccola B, Marino B. Spectrum of clinical variability in familial deletion 22q11. 2: from full manifestation to extremely mild clinical anomalies. Clinical genetics. 2003 Apr;63(4):308-13.\u003c/li\u003e\n\u003cli\u003eMarkert ML. Defects in thymic development. InStiehm\u0026apos;s Immune Deficiencies 2020 Jan 1 (pp. 357-379). Academic Press.\u003c/li\u003e\n\u003cli\u003eMustillo PJ, Sullivan KE, Chinn IK, Notarangelo LD, Haddad E, Davies EG, de la Morena MT, Hartog N, Yu JE, Hernandez-Trujillo VP, Ip W. Clinical practice guidelines for the immunological management of chromosome 22q11. 2 deletion syndrome and other defects in thymic development. Journal of Clinical Immunology. 2023 Feb;43(2):247-70.\u003c/li\u003e\n\u003cli\u003eLewis DB, Haines C, Ross D. Protein tyrosine kinase 7: a novel surface marker for human recent thymic emigrants with potential clinical utility. Journal of Perinatology. 2011 Apr;31(1):S72-81.\u003c/li\u003e\n\u003cli\u003ePierdominici M, Mazzetta F, Caprini E, Marziali M, Digilio MC, Marino B, Aiuti A, Amati F, Russo G, Novelli G, Pandolfi F. Biased T-cell receptor repertoires in patients with chromosome 22q11. 2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome). Clinical \u0026amp; Experimental Immunology. 2003 May;132(2):323-31.\u003c/li\u003e\n\u003cli\u003eBorte S, Wang N, \u0026Oacute;skarsd\u0026oacute;ttir S, von D\u0026ouml;beln U, Hammarstr\u0026ouml;m L. Newborn screening for primary immunodeficiencies: beyond SCID and XLA. Annals of the New York Academy of Sciences. 2011 Dec;1246(1):118-30.\u003c/li\u003e\n\u003cli\u003ePatel K, Akhter J, Kobrynski L, Gathman B, Davis O, Sullivan KE, International DiGeorge Syndrome Immunodeficiency Consortium. Immunoglobulin deficiencies: the B-lymphocyte side of DiGeorge Syndrome. The Journal of pediatrics. 2012 Nov 1;161(5):950-3.\u003c/li\u003e\n\u003cli\u003eCowan E, Burch KJ, Green BD, Grieve DJ. Obestatin as a key regulator of metabolism and cardiovascular function with emerging therapeutic potential for diabetes. British Journal of Pharmacology. 2016 Jul;173(14):2165-81.\u003c/li\u003e\n\u003cli\u003eZou CC, Liang L, Wang CL, Fu JF, Zhao ZY. The change in ghrelin and obestatin levels in obese children after weight reduction. Acta Paediatrica. 2009 Jan;98(1):159-65.\u003c/li\u003e\n\u003cli\u003eZhang N, Yuan C, Li Z, Li J, Li X, Li C, Li R, Wang SR. Meta-analysis of the relationship between obestatin and ghrelin levels and the ghrelin/obestatin ratio with respect to obesity. The American journal of the medical sciences. 2011 Jan 1;341(1):48-55.\u003c/li\u003e\n\u003cli\u003eVoll SL, Boot E, Butcher NJ, Cooper S, Heung T, Chow EW, Silversides CK, Bassett AS. Obesity in adults with 22q11. 2 deletion syndrome. Genetics in Medicine. 2017 Feb;19(2):204-8.\u003c/li\u003e\n\u003cli\u003eMalecki SL, Van Mil S, Graffi J, Breetvelt E, Corral M, Boot E, Chow EW, Sanches M, Verma AA, Bassett AS. A genetic model for multimorbidity in young adults. Genetics in Medicine. 2020 Jan 1;22(1):132-41.\u003c/li\u003e\n\u003cli\u003eSuksawat Y, Sathienkijkanchai A, Veskitkul J, Jirapongsananuruk O, Visitsunthorn N, Vichyanond P, Pacharn P. Resolution of primary immune defect in 22q11. 2 deletion syndrome. Journal of Clinical Immunology. 2017 May;37:375-82.\u003c/li\u003e\n\u003cli\u003eByers DE, Alexander-Brett J, Patel AC, Agapov E, Dang-Vu G, Jin X, Wu K, You Y, Alevy Y, Girard JP, Stappenbeck TS. Long-term IL-33\u0026ndash;producing epithelial progenitor cells in chronic obstructive lung disease. The Journal of clinical investigation. 2013 Sep 3;123(9):3967-82.\u003c/li\u003e\n\u003cli\u003eCalderon AA, Dimond C, Choy DF, Pappu R, Grimbaldeston MA, Mohan D, Chung KF. Targeting interleukin-33 and thymic stromal lymphopoietin pathways for novel pulmonary therapeutics in asthma and COPD. European Respiratory Review. 2023 Mar 31;32(167).\u003c/li\u003e\n\u003cli\u003eRamesh N, Gawli K, Kiran Pasupulleti V, Unniappan S. Metabolic and cardiovascular actions of nesfatin-1: implications in health and disease. Current Pharmaceutical Design. 2017 Mar 1;23(10):1453-64.\u003c/li\u003e\n\u003cli\u003eMamoor S. Interleukin 33 is differentially expressed in the thymus by developmental stage and cell type.\u003c/li\u003e\n\u003cli\u003eLiew FY, Girard JP, Turnquist HR. Interleukin-33 in health and disease. Nature Reviews Immunology. 2016 Nov;16(11):676-89.\u003c/li\u003e\n\u003cli\u003eZdrojewicz Z, Pachura E, Pachura P. The thymus: a forgotten, but very important organ. Advances in clinical and experimental medicine: official organ Wroclaw Medical University. 2016 Mar 1;25(2):369-75.\u003c/li\u003e\n\u003cli\u003eXu L, Wei C, Chen Y, Wu Y, Shou X, Chen W, Lu D, Sun H, Li W, Yu B, Wang X. IL-33 induces thymic involution-associated naive T cell aging and impairs host control of severe infection. Nature Communications. 2022 Nov 12;13(1):6881.\u003c/li\u003e\n\u003cli\u003eFeger U, Tolosa E, Huang YH, Waschbisch A, Biedermann T, Melms A, Wiendl H. HLA-G expression defines a novel regulatory T-cell subset present in human peripheral blood and sites of inflammation. Blood, The Journal of the American Society of Hematology. 2007 Jul 15;110(2):568-77.\u003c/li\u003e\n\u003cli\u003eContini P, Murdaca G, Puppo F, Negrini S. HLA-G expressing immune cells in immune mediated diseases. Frontiers in Immunology. 2020 Aug 28; 11:1613.\u003c/li\u003e\n\u003cli\u003eAksu G, Genel F, Koturoglu G, Kurugol Z, Kutukculer N. Serum immunoglobulin (IgG, IgM, IgA) and IgG subclass concentrations in healthy children: a study using nephelometric technique. Turkish Journal of Pediatrics. 2006 Jan 1;48(1):19.\u003c/li\u003e\n\u003cli\u003eRahman M. Introduction to flowcytometry. J Clin Invest. 2006; 45:345\u0026ndash;361.\u003c/li\u003e\n\u003cli\u003eDar N, Gothelf D, Korn D, Frisch A, Weizman A, Michaelovsky E, Carmel M, Yeshayahu Y, Dubnov-Raz G, Pessach IM, Simon AJ. Thymic and bone marrow output in individuals with 22q11. 2 deletion syndrome. Pediatric Research. 2015 Apr;77(4):579-85.\u003c/li\u003e\n\u003cli\u003eLingman Framme J, Borte S, von D\u0026ouml;beln U, Hammarstr\u0026ouml;m L, \u0026Oacute;skarsd\u0026oacute;ttir S. Retrospective analysis of TREC based newborn screening results and clinical phenotypes in infants with the 22q11 deletion syndrome. Journal of clinical immunology. 2014 May;34:514-9.\u003c/li\u003e\n\u003cli\u003eFramme JL, Lundqvist C, Lundell AC, van Schouwenburg PA, Lemarquis AL, Th\u0026ouml;rn K, Lindgren S, Gudmundsdottir J, Lundberg V, Degerman S, Zetterstr\u0026ouml;m RH. Long-term follow-up of newborns with 22q11 deletion syndrome and low TRECs. Journal of Clinical Immunology. 2022 Apr;42(3):618-33.\u003c/li\u003e\n\u003cli\u003eAl‐Herz W, McGeady SJ, Gripp KW. 22q11. 2 deletion syndrome and selective IgM deficiency: an association of a common chromosomal abnormality with a rare immunodeficiency. American Journal of Medical Genetics Part A. 2004 May 15;127(1):99-100.\u003c/li\u003e\n\u003cli\u003eKung SJ, Gripp KW, Stephan MJ, Fairchok MP, McGeady SJ. Selective IgM deficiency and 22q11. 2 deletion syndrome. Annals of Allergy, Asthma \u0026amp; Immunology. 2007 Jul 1;99(1):87-92.\u003c/li\u003e\n\u003cli\u003eGiardino G, Radwan N, Koletsi P, Morrogh DM, Adams S, Ip W, Worth A, Jones A, Meyer-Parsonson I, Gaspar HB, Gilmour K. Clinical and immunological features in a cohort of patients with partial DiGeorge syndrome followed at a single center. Blood, The Journal of the American Society of Hematology. 2019 Jun 13;133(24):2586-96.\u003c/li\u003e\n\u003cli\u003eGennery AR, Barge D, O\u0026apos;sullivan JJ, Flood TJ, Abinun M, Cant AJ. Antibody deficiency and autoimmunity in 22q11. 2 deletion syndrome. Archives of disease in childhood. 2002 Jun 1;86(6):422-5.\u003c/li\u003e\n\u003cli\u003eFinocchi A, Di Cesare S, Romiti ML, Capponi C, Rossi P, Carsetti R, Cancrini C. Humoral immune responses and CD27+ B cells in children with DiGeorge syndrome (22q11. 2 deletion syndrome). Pediatric Allergy and Immunology. 2006 Aug;17(5):382-8.\u003c/li\u003e\n\u003cli\u003eCampbell IM, Sheppard SE, Crowley TB, McGinn DE, Bailey A, McGinn MJ, Unolt M, Homans JF, Chen EY, Salmons HI, Gaynor JW. What is new with 22q? An update from the 22q and You Center at the Children\u0026apos;s Hospital of Philadelphia. American Journal of Medical Genetics Part A. 2018 Oct;176(10):2058-69.\u003c/li\u003e\n\u003cli\u003eSullivan KE. Chromosome 22q11. 2 deletion syndrome and DiGeorge syndrome. Immunological Reviews. 2019 Jan;287(1):186-201.\u003c/li\u003e\n\u003cli\u003eSediv\u0026aacute;1ADEFG A, Bartůňkov\u0026aacute;1AG J, Zachov\u0026aacute;1B R, Poloučkov\u0026aacute;1B A, Hru\u0026scaron;\u0026aacute;k1BC O, Janda1BCEF A, Koč\u0026aacute;rek2BCD E, Novotn\u0026aacute;2BC D, Novotn\u0026aacute;2BC K, Klein3B T. Early development of immunity in diGeorge syndrome. Med Sci Monit. 2005;11(4):187.\u003c/li\u003e\n\u003cli\u003eHandel AE, Cheuk S, Dhalla F, Maio S, H\u0026uuml;bscher T, Rota I, Deadman ME, Ekwall O, L\u0026uuml;tolf M, Weinberg K, Holl\u0026auml;nder G. Developmental dynamics of the neural crest\u0026ndash;mesenchymal axis in creating the thymic microenvironment. Science Advances. 2022 May 13;8(19): eabm9844.\u003c/li\u003e\n\u003cli\u003eGermanov E, Veinotte L, Cullen R, Chamberlain E, Butcher EC, Johnston B. Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells. The Journal of Immunology. 2008 Jul 1;181(1):81-91.\u003c/li\u003e\n\u003cli\u003eKurokawa M, Matsukura S, Kawaguchi M, Ieki K, Suzuki S, Watanabe S, Homma T, Yamaguchi M, Takeuchi H, Adachi M. Interleukin-33-activated dendritic cells induce the production of thymus and activation-regulated chemokine and macrophage-derived chemokine. International archives of allergy and immunology. 2013 May 1;161(Suppl. 2):52-7.\u003c/li\u003e\n\u003cli\u003eBassett AS, Chow EW, Husted J, Weksberg R, Caluseriu O, Webb GD, Gatzoulis MA. Clinical features of 78 adults with 22q11 deletion syndrome. American Journal of Medical Genetics Part A. 2005 Nov 1;138(4):307-13.\u003c/li\u003e\n\u003cli\u003eGuzman ML, Delgado I, Lay‐Son G, Willans E, Puga A, Repetto GM. Growth in Chilean infants with chromosome 22q11 microdeletion syndrome. American Journal of Medical Genetics Part A. 2012 Nov;158(11):2682-6.\u003c/li\u003e\n\u003cli\u003eNakahara T, Harada T, Yasuhara D, Shimada N, Amitani H, Sakoguchi T, Kamiji MM, Asakawa A, Inui A. Plasma obestatin concentrations are negatively correlated with body mass index, insulin resistance index, and plasma leptin concentrations in obesity and anorexia nervosa. Biological psychiatry. 2008 Aug 1;64(3):252-5.\u003c/li\u003e\n\u003cli\u003eEberle P, Berger C, Junge S, Dougoud S, B\u0026uuml;chel EV, Riegel M, Schinzel A, Seger R, G\u0026uuml;ng\u0026ouml;r T. Persistent low thymic activity and non-cardiac mortality in children with chromosome 22q11\u0026middot; 2 microdeletion and partial DiGeorge syndrome. Clinical \u0026amp; Experimental Immunology. 2009 Feb;155(2):189-98.\u003c/li\u003e\n\u003cli\u003eCovington EW, Roberts MZ, Dong J. Procalcitonin monitoring as a guide for antimicrobial therapy: a review of current literature. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2018 May;38(5):569-81.\u003c/li\u003e\n\u003cli\u003eNargis W, Ibrahim MD, Ahamed BU. Procalcitonin versus C-reactive protein: Usefulness as biomarker of sepsis in ICU patient. International journal of critical illness and injury science. 2014 Jul;4(3):195.\u003c/li\u003e\n\u003cli\u003eBahri R, Hirsch F, Josse A, Rouas-Freiss N, Bidere N, Vasquez A, Carosella ED, Charpentier B, Durrbach A. Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes. The Journal of Immunology. 2006 Feb 1;176(3):1331-9.\u003c/li\u003e\n\u003cli\u003eVerhoeven L, Reitsma P, Siegel LS. Cognitive and linguistic factors in reading acquisition. Reading and writing. 2011 Apr;24:387-94.\u003c/li\u003e\n\u003cli\u003eYagi H, Furutani Y, Hamada H, Sasaki T, Asakawa S, Minoshima S, Ichida F, Joo K, Kimura M, Imamura SI, Kamatani N. Role of TBX1 in human del22q11. 2 syndrome. The Lancet. 2003 Oct 25;362(9393):1366-73.\u003c/li\u003e\n\u003cli\u003eMcQuade L, Christodoulou J, Budarf M, Sachdev R, Wilson M, Emanuel B, Colley A. Patient with a 22q11. 2 deletion with no overlap of the minimal DiGeorge syndrome critical region (MDGCR). American journal of medical genetics. 1999 Sep 3;86(1):27-33.\u003c/li\u003e\n\u003cli\u003eHayashi T, Suzuki K. Molecular pathogenesis of Bernard-Soulier syndrome. InSeminars in thrombosis and hemostasis 2000 (Vol. 26, No. 01, pp. 053-060). Copyright\u0026copy; 2000 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.:+ 1 (212) 584-4663).\u003c/li\u003e\n\u003cli\u003eKato T, Kosaka K, Kimura M, Imamura SI, Yamada O, Iwai K, Ando M, Joh-o K, Kuroe K, Ohtake A, Takao A. Thrombocytopenia in patients with 22q11. 2 deletion syndrome and its association with glycoprotein Ib-\u0026beta;. Genetics in Medicine. 2003 Mar 1;5(2):113-9.\u003c/li\u003e\n\u003cli\u003eKunishima S, Imai T, Kobayashi R, Kato M, Ogawa S, Saito H. B ernard\u0026ndash;S oulier syndrome caused by a hemizygous GPIb \u0026beta; mutation and 22q11. 2 deletion. Pediatrics International. 2013 Aug;55(4):434-7.\u003c/li\u003e\n\u003cli\u003eHuang L, Xie Y, Zhou Y, Luo Y, Huang X, Xu Z, Cai D, Fang Q. Clinical and molecular cytogenetic studies of an unrecognised 22q11. 2 deletion in three families. Experimental and Therapeutic Medicine. 2015 Mar 1;9(3):823-8.\u003c/li\u003e\n\u003cli\u003eBurnside RD. 22q11. 21 deletion syndromes: a review of proximal, central, and distal deletions and their associated features. Cytogenetic and Genome Research. 2015 Aug 8;146(2):89-99.\u003c/li\u003e\n\u003cli\u003eClements CC, Wenger TL, Zoltowski AR, Bertollo JR, Miller JS, de Marchena AB, Mitteer LM, Carey JC, Yerys BE, Zackai EH, Emanuel BS. Critical region within 22q11. 2 linked to higher rate of autism spectrum disorder. Molecular Autism. 2017 Dec;8:1-7.\u003c/li\u003e\n\u003cli\u003eJungerius BJ, Hoogendoorn ML, Bakker SC, Van\u0026apos;t Slot R, Bardoel AF, Ophoff RA, Wijmenga C, Kahn RS, Sinke RJ. An association screen of myelin-related genes implicates the chromosome 22q11 PIK4CA gene in schizophrenia. Molecular psychiatry. 2008 Nov;13(11):1060-8.\u003c/li\u003e\n\u003cli\u003eHiroi N, Takahashi T, Hishimoto A, Izumi T, Boku S, Hiramoto T. Copy number variation at 22q11. 2: from rare variants to common mechanisms of developmental neuropsychiatric disorders. Molecular psychiatry. 2013 Nov;18(11):1153-65.\u003c/li\u003e\n\u003cli\u003eMcDonald-McGinn DM, Sullivan KE, Marino B, Philip N, Swillen A, Vorstman JA, Zackai EH, Emanuel BS, Vermeesch JR, Morrow BE, Scambler PJ. 22q11. 2 deletion syndrome. Nature reviews Disease primers. 2015 Nov 19;1(1):1-9.\u003c/li\u003e\n\u003cli\u003eMorrow BE, McDonald‐McGinn DM, Emanuel BS, Vermeesch JR, Scambler PJ. Molecular genetics of 22q11. 2 deletion syndrome. American journal of medical genetics Part A. 2018 Oct;176(10):2070-81.\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":"DiGeorge Syndrome, TREC, KREC, HLA-G, Procalcitonin, Obestatin","lastPublishedDoi":"10.21203/rs.3.rs-4231044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4231044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eThe study aimed to offer better genetic evaluation and consultation for DiGeorge syndrome (DGS) patients by combining screening of 22q11.2 and immunologic studies. A basic immune profile including the basic CD panel and immunoglobulins estimation was performed. TRECS and KRECS expression were studied in addition to measuring serum IL33, Obestatin, HLA-G, and Procalcitonin serum levels.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAll investigations were performed for DGS patients (n\u0026thinsp;=\u0026thinsp;33) and the matched control group (n\u0026thinsp;=\u0026thinsp;45). Polymorphic 22q11.2 markers mapping was performed by PCR-STR technique. Lymphocyte subsets immunophenotyping was done using flow cytometry, while measurement of serum immunoglobulins was estimated using nephelometry. Real-time PCR was the method used for TRECs and KRECs measurement. Serum IL33, Obestatin, HLA-G, and Procalcitonin levels were determined using an Enzyme-linked immunosorbent assay (ELISA). Data was coded, tabulated, and statistically analyzed using SPSS version 19.0 software.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn our case\u0026ndash;control study, KREC expression was significantly elevated in DGS compared to healthy controls (P\u0026thinsp;=\u0026thinsp;0.0008). There was also a significant increase in immunoglobulin levels in DGS. CD8% as well as CD8 absolute count in the patients with DGS were significantly lower than in the healthy control (P\u0026thinsp;=\u0026thinsp;0.01273 and 0.05358 respectively). There were no significant differences in IL33, Obestatin, HLA-G, and Procalcitonin levels between DGS patients compared to the control group. Our results concerning the distinct segment of 22q11.2 as a DGS susceptibility region revealed an informative novel atypical interstitial homozygous deletion. This deletion included D22S944 and COMT absence, and D22S941 and D22S264 presence. Out of 33 DGS patients, three patients showed deletion in the D22S944 marker only in the presence of D22S941, and D22S264 markers. Therefore, we could assume that D22S944 is a common deleted marker in non-isolated DGS patients.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCombining 22q11.2 region screening, immune profile studies, and TRECS and KRECS expression offers a new comprehensive approach for DGS patients. This approach provides a better strategy for genetic consultation for DGS patients. Moreover, this study may be the first to show a small interstitial 22q11.2 deletion stereotype in a DGS patient and also showed that the smallest deletion at the 22q11.2 region is enough to confer the DGS phenotype.\u003c/p\u003e","manuscriptTitle":"Integrating TREC/KREC assay and some cytokines in the evaluation of the immune status of patients with DiGeorge Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-24 17:43:13","doi":"10.21203/rs.3.rs-4231044/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":"b807b0ba-68c9-4419-ac15-d96600e03e72","owner":[],"postedDate":"April 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-27T16:56:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-24 17:43:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4231044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4231044","identity":"rs-4231044","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}