Use Of Whole Exome Sequencing Research Articles

P-550 Monoallelic CCNB2 variants may cause oocyte maturation defects and embryo developmental arrest

Abstract Study question Have all the genes that may cause embryo development arrest and oocyte maturation defects been identified in female patients? Summary answer We identified a variant in CCNB2 in our patient. To our knowledge, this is first time that meiotic arrest can be caused by mutated-CCNB2. What is known already Successful reproduction involves the maturation of gametes, fertilization, and early development of the embryo. Oocyte maturation involves a sequence of both morphological and molecular transformations, progressing from germinal vesicle oocytes to metaphase I oocytes and ultimately to metaphase II oocytes. Any disruption in the process of oocyte maturation can result in infertility. With the widespread use of whole exome sequencing (WES), several genes that influence oocyte maturation at different stages have been recently identified. Study design, size, duration We here report the case of repeated ART failure due to oocyte maturation defect and embryo developmental arrest. Participants/materials, setting, methods A 30-year-old female and 38-year-old male applied to our clinic due to unexplained infertility for 4 years. The first ART attempt resulted in 2 of 13 oocytes with late MII. During ART performed in our center, germinal vesicle and meiotic arrest were observed in all oocytes. No mature oocytes were obtained. Her grandfather’s sister was also infertile. WES and Sanger analysis were performed to detect any mutation in known/candidate genes and show familial segregation Main results and the role of chance WES analysis revealed no mutation in genes that were previously reported as a cause of oocyte maturation defect or early embryo development in human. We do a further analysis of genes highly or specifically expressed in oocytes or expressed at low levels yet previous studies have suggested its role in embryo development. We identified a c.787C>T, p.Arg263* heterozygous nonsense variant in the CCNB2 gene that was extremely rare in both multiple databases and our in-house database. Multiple lines of computational evidence support the deleterious effect of the variant on the gene product. This variant was transmitted from her healthy father. CCNNB2 is not expressed in any cells other than oocytes in human. The activity of the M phase-promoting factor (MPF) is crucial for advancing through both mitosis and meiosis. CCNB2 plays a vital role by contributing to the activity of pre-MPF during prophase, necessary for generating enough MPF to proceed through maturation. Furthermore, CCNB2 is significantly involved in mouse oocyte meiosis, playing a key role in both the G2/M and MI/anaphase I transitions. Limitations, reasons for caution New disease-causing variants in this gene should be demonstrated in larger cohorts with female infertility. In addition, further functional studies should be carried out both in CCNB2-mutated human and animals. Wider implications of the findings This study may provide a better understanding background of female infertility and the failure of the ART process. CCNB2 may have a crucial role in the oocyte maturation process and can be utilized as a female infertility genetic marker. Trial registration number NA

The impact of the European Society of Cardiology guidelines and whole exome sequencing on genetic testing in hereditary cardiac diseases.

In the last decade, an incredible improvement has been made in elucidating the genetic bases of cardiomyopathies. Here we report the impact of either the European Society of Cardiology (ESC) guidelines or the use of whole exome sequencing (WES) in terms of a number of variants of uncertain significance (VUS) and missed diagnoses in a series of 260 patients affected by inherited cardiac disorders. Samples were analyzed using a targeted gene panel of 128 cardiac-related genes and/or WES in a subset of patients, with a three-tier approach. Analyzing (i) only a subset of genes related to the clinical presentation, strictly following the ESC guidelines, 20.77% positive test were assessed. The incremental diagnostic rate for (ii) the whole gene panel, and (iii) the WES was 4.71% and 11.67%, respectively. The diverse analytical approaches increased the number of VUSs and incidental findings. Indeed, the use of WES highlights that there is a small percentage of syndromic conditions that standard analysis would not have detected. Moreover, the use of targeted sequencing coupled with "narrow" analytical approach prevents the detection of variants in actionable genes that could allow for preventive treatment. Our data suggest that genetic testing might aid clinicians in the diagnosis of inheritable cardiac disorders.

Case report: Early use of whole exome sequencing unveils HNRNPU-related neurodevelopmental disorder and answers additional clinical questions through reanalysis

This case report chronicles the diagnostic odyssey and resolution of a 27-year-old female with a complex neurodevelopmental disorder (NDD) using Whole Exome Sequencing (WES). The patient presented to a precision medicine clinic with multiple diagnoses including intellectual disability, autism spectrum disorder (ASD), obsessive-compulsive disorder (OCD), tics, seizures, and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Although this patient previously had chromosomal microarray and several single-gene tests, the underlying cause of this patient’s symptoms remained elusive. WES revealed a pathogenic missense mutation in the HNRNPU gene, associated with HNRNPU-related neurodevelopmental disorder (HNRNPU-NDD) and developmental and epileptic encephalopathy-54 (DEE54, OMIM: # 617391). Following this diagnoses, other treating clinicians identified additional indications for genetic testing, however, as the WES data was readily available, the clinical team was able to re-analyze the WES data to address their inquiries without requiring additional tests. This emphasizes the pivotal role of WES in expediting diagnoses, reducing costs, and providing ongoing clinical utility throughout a patient’s life. Accessible WES data in primary care settings can enhance patient care by informing future genetic inquiries, enhancing coordination of care, and facilitating precision medicine interventions, thereby mitigating the burden on families and the healthcare system.

Application of whole exome sequencing in the diagnosis of muscular disorders: a study of Taiwanese pediatric patients.

Muscular dystrophies and congenital myopathies encompass various inherited muscular disorders that present diagnostic challenges due to clinical complexity and genetic heterogeneity. This study aimed to investigate the use of whole exome sequencing (WES) in diagnosing muscular disorders in pediatric patients in Taiwan. Out of 161 pediatric patients suspected to have genetic/inherited myopathies, 115 received a molecular diagnosis through conventional tests, single gene testing, and gene panels. The remaining 46 patients were divided into three groups: Group 1 (multiplex ligation-dependent probe amplification-negative Duchenne muscular dystrophy) with three patients (6.5%), Group 2 (various forms of muscular dystrophies) with 21 patients (45.7%), and Group 3 (congenital myopathies) with 22 patients (47.8%). WES analysis of these groups found pathogenic variants in 100.0% (3/3), 57.1% (12/21), and 68.2% (15/22) of patients in Groups 1 to 3, respectively. WES had a diagnostic yield of 65.2% (30 patients out of 46), detecting 30 pathogenic or potentially pathogenic variants across 28 genes. WES enables the diagnosis of rare diseases with symptoms and characteristics similar to congenital myopathies and muscular dystrophies, such as muscle weakness. Consequently, this approach facilitates targeted therapy implementation and appropriate genetic counseling.

A bird’s eye view on the use of whole exome sequencing in rare congenital ophthalmic diseases

Phenotypic and genotypic heterogeneity in congenital ocular diseases, especially in anterior segment dysgenesis (ASD), have created challenges for proper diagnosis and classification of diseases. Over the last decade, genomic research has indeed boosted our understanding in the molecular basis of ASD and genes associated with both autosomal dominant and recessive patterns of inheritance have been described with a wide range of expressivity. Here we describe the molecular characterization of a cohort of 162 patients displaying isolated or syndromic congenital ocular dysgenesis. Samples were analyzed with diverse techniques, such as direct sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing (WES), over 20 years. Our data reiterate the notion that PAX6 alterations are primarily associated with ASD, mostly aniridia, since the majority of the cohort (66.7%) has a pathogenic or likely pathogenic variant in the PAX6 locus. Unexpectedly, a high fraction of positive samples (20.3%) displayed deletions involving the 11p13 locus, either partially/totally involving PAX6 coding region or abolishing its critical regulatory region, underlying its significance. Most importantly, the use of WES has allowed us to both assess variants in known ASD genes (i.e., CYP1B1, ITPR1, MAB21L1, PXDN, and PITX2) and to identify rarer phenotypes (i.e., MIDAS, oculogastrointestinal-neurodevelopmental syndrome and Jacobsen syndrome). Our data clearly suggest that WES allows expanding the analytical portfolio of ocular dysgenesis, both isolated and syndromic, and that is pivotal for the differential diagnosis of those conditions in which there may be phenotypic overlaps and in general in ASD.

Whole Exome Sequencing as a First-Line Molecular Genetic Test in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathies (DEE) are severe neurodevelopmental disorders characterized by recurrent, usually early-onset, epileptic seizures accompanied by developmental impairment often related to both underlying genetic etiology and abnormal epileptiform activity. Today, next-generation sequencing technologies (NGS) allow us to sequence large portions of DNA quickly and with low costs. The aim of this study is to evaluate the use of whole-exome sequencing (WES) as a first-line molecular genetic test in a sample of subjects with DEEs characterized by early-onset drug-resistant epilepsies, associated with global developmental delay and/or intellectual disability (ID). We performed 82 WESs, identifying 35 pathogenic variants with a detection rate of 43%. The identified variants were highlighted on 29 different genes including, 3 new candidate genes (KCNC2, STXBP6, DHRS9) for DEEs never identified before. In total, 23 out of 35 (66%) de novo variants were identified. The most frequently identified type of inheritance was autosomal dominant de novo (60%) followed by autosomal recessive in homozygosity (17%) and heterozygosity (11%), autosomal dominant inherited from parental mosaicism (6%) and X-linked dominant de novo (6%). The most frequent mutations identified were missense (75%) followed by frameshift deletions (16%), frameshift duplications (5%), and splicing mutations (3%). Considering the results obtained in the present study we support the use of WES as a form of first-line molecular genetic testing in DEEs.

Clinical use of whole exome sequencing in children with developmental delay/intellectual disability

BackgroundIdentifying the underlying etiology of developmental delay/intellectual disability (DD/ID) is challenging but important. The genetic diagnosis of unexplained DD/ID helps in the treatment and prognosis of the disability in patients. In this study, we reported our experience of using whole exome sequencing (WES) of children with unexplained DD/ID. MethodsWe conducted a retrospective analysis of WES results of children under 19 years of age with unexplained DD/ID between January 2020 and December 2021. The demographic data of all patients and variants identified through WES were evaluated. Furthermore, we evaluated the clinical characteristics that influenced the identification of genetic causes. ResultsForty-one patients with DD/ID were included, of whom 21 (51.2 %) were male. The average age at symptom onset was 1.6 ± 1.3 years, and the duration from symptom onset to diagnosis was 3.1 ± 3.7 years. Hypotonia was the most common symptom (17 patients, 41.5 %), and epilepsy was confirmed in 10 patients (24.4 %). Twenty-two pathogenic/likely pathogenic variants were identified in 20 patients, and three variants of uncertain significance were identified in three patients. Family-based trio Sanger sequencing for candidate variants of 12 families was conducted; 10 variants were de novo, one variant paternally inherited, and two variants compound heterozygous. The diagnostic yield of WES for DD/ID was 48.8 % and was significantly high in patients with an early onset of DD/ID and facial dysmorphism. In contrast, patients with autism spectrum disorder (ASD) were more likely to have negative WES results compared with others without ASD. ConclusionThe diagnostic yield of WES was 48.8 %. We conclude that patients’ characteristics, such as dysmorphic features and the age of symptom onset, can predict the likelihood that WES will identify a causal variant of a phenotype.

Unique coexistence of SHOX and PTHLH gene mutations in a 12-year-old boy with syndromic short stature. Case report with literature review

Despite the availability of advanced genetic testing that enables accurate and reliable genotype assessment, clinicians continue to face diagnostic problems, especially in patients with potentially pathogenic mutations in different genes. SHOX gene mutations are the most common cause of proportional monogenic short stature, whereas the PTHLH gene encodes a parathormone-related protein that plays a crucial role in the regulation of cell growth, calcium ion transport, and bone development. The authors present the case of a 12-year-old boy with a short stature and a mosaic of developmental malformations; nephrocalcinosis; and calcium and magnesium metabolism disorders; with a unique coexistence of mutations in the SHOX and PTHLH genes. The presented case extends the clinical spectrum associated with these rare mutations and shows the usefulness of whole exome sequencing in diagnosing patients with atypical phenotypes.

OP80 Diagnostic Molecular Sequencing Of DNA (Exomes And Genomes) Is Not Perfect: Implications For HTA

IntroductionThe recent release of powerful next-generation sequencing platforms, which can provide whole exome sequencing (WES) or whole genome sequencing (WGS) in quicker timelines and at reduced costs, has resulted in proposals for these diagnostic testing methods to be routinely integrated into clinical practice in multiple settings. However, the complexities of these diagnostic approaches, and the minimal comparative evidence available on them, creates difficulties in the evaluation of their diagnostic performance. Novel approaches need to be developed to improve the health technology assessment (HTA) of WES and WGS.MethodsSeveral HTAs on genetic testing and the use of WES or WGS in fetal medicine were reviewed. Information on factors associated with this diagnostic modality that affect typical test accuracy assessment (e.g., sensitivity and specificity) was extracted. The multiple steps required for completing a WES or WGS test, and the potential for the introduction of errors (type I or type II) at each of these steps, were mapped and examples provided. The clinical and economic implications associated with imperfect and uncertain test accuracy were described.ResultsLimited data on analytical and clinical validity were identified. WES and WGS are multistep processes and errors were found in sampling, molecular sequencing, bioinformatic filtering, and variant interpretation; therefore, the assumption that WES or WGS is 100 percent sensitive or specific is not reasonable. Although alternative evidence-based estimates are unlikely to be available, the inevitability of such errors, and their implications in terms of comparative effectiveness, safety, and cost effectiveness, should be described in HTAs.ConclusionsWhile unknown diagnostic accuracy remains an issue with WES and WGS testing, formal sensitivity analysis of test performance characteristics should be conducted as part of HTAs. A checklist has been developed to assist those involved in HTA and policy to understand the potential for inaccurate test results in clinical practice, and the risk-benefit implications of these diagnostic errors for patients.

Modeling TCIRG1 Neutropenia By Utilizing Patient Derived Induced Pluripotent Stem Cells

Background: Severe congenital neutropenia (SCN) is a hereditary hematopoietic disorder characterized by recurrent infections and leukemic transformation. We previously identified a novel heterozygous missense mutation (R736S) in TCIRG1 causing SCN in a large multigenerational family (PMID: 24753205). We also identified a statistically significant correlation in individuals with lower ANC with rare missense variants in TCIRG1 in a large cohort of population-based genome screening (PMID: 27229898). Recently, we have discovered another TCIRG1 heterozygousvariant: R736C in two unrelated families with congenital neutropenia. Independently, Shinwari et al identified a novel TCIRG1 mutation, V52L, which was correlated with the neutropenic phenotype (PMID: 35573728). Homozygous mutations of TCIRG1 are causing autosomal recessive osteopetrosis (ARO), which is characterized by osteopetrosis and secondary hematological and neurological abnormalities. The Genome Aggregation Database (gnomAD) has no data about TCIRG1 R736S and R736C variants accruing in the healthy population. This is in contrast of numerous healthy individuals with a various heterozygous variants throughout the TCIRG1 gene. Aims: We aim to generate in-vitro model of TCIRG1 neutropenia by utilizing patient derived induced pluripotent stem cells (iPSCs) and characterize the hematological abnormalities caused by mutant TCIRG1 expression. We also aim to characterize the incidence of TCIRG1-associated neutropenia and the prevalence of TCIRG1 variants by sharing this new information within the scientific community through identifying more cases/families with this disease. Methods: Medical history, differential WBC counts and whole exome sequencing (WES) was used to identify the genetic basis of neutropenia in the family with R736S mutation. The R736C mutation in the new families also were identified by WES of the index cases. Medical history, blood counts and Sanger sequencing identified their affected relatives. iPSC lines of 4 affected individuals from 2 families harboring TCIRG1 R736S and R736C mutations and healthy volunteer were generated using standard methodology. S732F benign heterozygous TCIRG1 variant was generated by utilizing CRISPR/Cas9 knock-In gene editing using a healthy volunteer iPSC line. iPSCs hematopoietic differentiation was achieved by using STEMdiff Hematopoietic Kit. Resultant CD34+ cells were differentiated towards neutrophils using a published protocol (PMID: 35795780). Cell proliferation, survival and differentiation characteristics were measured by automated cell counter and flow cytometry, cell viability staining and surface markers analysis for granulocytic differentiation. Results: The discovery of these families came through the reputation and work of the Severe Congenital Neutropenia International Registry (SCNIR) and the expanding use of WES to identify the causes of hereditary disorders. With the identification of the index cases, we have identified several affected family members in each family. In all three families the severity of neutropenia is variable and correlates with the incidence of recurrent bacterial infections. Myeloid differentiation of CD34+ cells derived from iPSCs of 4 patients from two families with R736S and R736C mutations revealed maturation, survival and proliferation abnormalities compared to volunteer cells and cell line harboring benign S732F heterozygous TCIRG1 mutation. All patient cells had from 2 to 3-fold drop in cell proliferation and cell viability drop by over 70% for the patients with R736S and up to 90% with R736C mutation compared to the controls. The myeloid differentiation in the patients' cells, measured by flow cytometry and assessed by CD66b+/CD14+ and CD11b+/CD15+ markers was impaired by 38% and 23% respectively for the patients with R736S and 43% and 24% for the patients harboring R736C mutation. Conclusions: The pathogenesis of TCIRG1-associated neutropenia is currently unknown. Carriers of pathogenic variants from these families appear to have neutropenia with variable severity. Our preliminary studies show that patient iPSC derived hematopoietic cells closely recapitulate the neutropenic phenotype. Population based genomic studies; careful family histories and WES will identify new patients with this disorder.

THU160 A Prospective Genetic Analysis Of Children With Idiopathic Short Stature (ISS) Using Whole-exome Sequencing (WES): First Results

Abstract Disclosure: L.D. Cellin: None. N.L. Menezes De Andrade: None. R.C. Rezende: None. V. Souza: None. N.C. Dantas: None. E. Quedas: None. M.F. Funari: None. G. Vasques: None. R.D. Scalco: None. A.C. Malaquias: None. A. Jorge: Consulting Fee; Self; Novo Nordisk. Grant Recipient; Self; BioMarin. Introduction: Children classified as idiopathic short stature (ISS) may have a monogenic cause that can explain their growth disorder. In this context, genetic tests emerge as a new diagnostic tool. Objectives: To determine the usefulness of WES for the genetic investigation of children with ISS. Patients and Methods: We sequentially enrolled 95 children with ISS without previous genetic testing, except for karyotyping for girls. In WES analysis, we prioritized rare variants (MAF<0.001), classified as loss-of-function (LoF) or missense predicted to be deleterious on in silico predictions. All variants were categorized according to ACMG/AMP criteria. We analyzed genes previously associated with growth disorders. CNV analysis was performed based on WES data; additionally, MLPA for SHOX was performed to assess deletion in regulatory regions. In positive cases, we carry out segregation in the family. Results: Our cohort was characterized by male preponderance (67%), markedly short stature (height SDS -2.6±0.7, 20% with height SDS<-3), and in 48% of cases, at least one of the parents have short stature. We identified 12 pathogenic or likely pathogenic (P/LP) variants by WES, including two gene deletions (SHOX and PTHLH) and 10 point mutations in ACAN (2x), IHH, PRKG2, LTBP3, ERF, THRA, GHR, PTPN11, and NF1. Additionally, two deletions involving the regulatory region of the SHOX gene were identified by MLPA. One patient had two P/LP alterations, totaling a diagnostic yield of 13.7% (12.6% if only WES results were considered). It is noteworthy that in addition to genes already associated with ISS (ACAN, SHOX, IHH, GHR, PTPN11, NF1), we observed a greater diversity of genes, including resistance to thyroid hormones (THRA) and milder forms of skeletal dysplasia (LTBP3, PTHLH, PRKG2). All but one of the variants were in the heterozygous state, including two de novo variants and four inherited from a parent with short stature. Fifteen patients have variants of uncertain significance (VUS) in genes already associated with growth disorder: IHH (3x), FGFR3 (2x), NPR2 (2x), LZTR1 (2x), MMP13, COL11A1, COL11A2, EXT2, IGF1R, and WNT5. Furthermore, four patients have variants of interest in new candidate genes (MAU2, MAP3K4, NR2E1, C6ORF136). Conclusion: The diagnostic yield was similar to other studies in children with ISS, but a greater diversity of genetic causes was observed, with those involving growth-plate genes and the RAS-MAPK pathway being more frequent. The large number of VUS highlights the need for further studies and new tools to classify these variants definitively. Presentation: Thursday, June 15, 2023

Comparative yield of molecular diagnostic algorithms for autism spectrum disorder diagnosis in India: evidence supporting whole exome sequencing as first tier test

BackgroundAutism spectrum disorder (ASD) affects 1 in 100 children globally with a rapidly increasing prevalence. To the best of our knowledge, no data exists on the genetic architecture of ASD in India. This study aimed to identify the genetic architecture of ASD in India and to assess the use of whole exome sequencing (WES) as a first-tier test instead of chromosomal microarray (CMA) for genetic diagnosis.MethodsBetween 2020 and 2022, 101 patient-parent trios of Indian origin diagnosed with ASD according to the Diagnostic and Statistical Manual, 5th edition, were recruited. All probands underwent a sequential genetic testing pathway consisting of karyotyping, Fragile-X testing (in male probands only), CMA and WES. Candidate variant validation and parental segregation analysis was performed using orthogonal methods.ResultsOf 101 trios, no probands were identified with a gross chromosomal anomaly or Fragile-X. Three (2.9%) and 30 (29.7%) trios received a confirmed genetic diagnosis from CMA and WES, respectively. Amongst diagnosis from WES, SNVs were detected in 27 cases (90%) and CNVs in 3 cases (10%), including the 3 CNVs detected from CMA. Segregation analysis showed 66.6% (n = 3 for CNVs and n = 17 for SNVs) and 16.6% (n = 5) of the cases had de novo and recessive variants respectively, which is in concordance with the distribution of variant types and mode of inheritance observed in ASD patients of non-Hispanic white/ European ethnicity. MECP2 gene was the most recurrently mutated gene (n = 6; 20%) in the present cohort. Majority of the affected genes identified in the study cohort are involved in synaptic formation, transcription and its regulation, ubiquitination and chromatin remodeling.ConclusionsOur study suggests de novo variants as a major cause of ASD in the Indian population, with Rett syndrome as the most commonly detected disorder. Furthermore, we provide evidence of a significant difference in the diagnostic yield between CMA (3%) and WES (30%) which supports the implementation of WES as a first-tier test for genetic diagnosis of ASD in India.

Extending and outlining the genotypic and phenotypic spectrum of novel mutations of NALCN gene in IHPRF1 syndrome: identifying recurrent urinary tract infection.

Infantile hypotonia with psychomotor retardation and characteristic facies 1 (IHPRF1) is caused by biallelic mutations in the NALCN gene, the major ion channel responsible for the background Na + conduction in neurons. Through whole-exome sequencing (WES), we report three novel homozygous variants in three families, including c.1434 + 1G > A, c.3269G > A, and c.2648G > T, which are confirmed and segregated by Sanger sequencing. Consequently, intron 12's highly conserved splice donor location is disrupted by the pathogenic c.1434 + 1G > A variation, most likely causing the protein to degrade through nonsense-mediated decay (NMD). Subsequently, a premature stop codon is thus generated at amino acid 1090 of the protein as a result of the pathogenic c.3269G > A; p.W1090* variation, resulting in NMD or truncated protein production. Lastly, the missense mutation c.2648G > T; p.G883V can play a critical role in the interplay of functional domains. This study introduces recurrent urinary tract infections for the first time, broadening the phenotypic range of IHPRF1 syndrome in addition to the genotypic spectrum. This trait may result from insufficient bladder emptying, which may be related to the NALCN channelosome's function in background Na + conduction. This work advances knowledge about the molecular genetic underpinnings of IHPRF1 and introduces a novel phenotype through the widespread use of whole exome sequencing.

Use of whole exome sequencing for identification of genetic variants related to Growth Hormone Deficiency and Short Stature: A Family-Based Study.

Genetic polymorphisms in genes involved in growth process and Vitamin-D metabolism form a significant etiology behind growth hormone deficiency and short stature. The aim of this study was to explore for known and unknown genes and variants related to growth hormone and short stature in a family based study using whole exome sequencing (WES). This family-based study included a family with members diagnosed with growth hormone deficiency, short stature and Vitamin-D deficiency (four boys affected and four boys non-affected). The participants were recruited from King Abdulaziz University Hospital (Jeddah, Saudi Arabia) and referred to King Fahad Centre for Medical Research (Jeddah, Saudi Arabia from April 2022 to June 2022. The consanguineous parents and one of the affected boys (aged 16 years old) underwent WES. Several variants in RNPC3, ACAN, GC, VDR and LRP2 were identified in index cases but not in controls. Novel frameshift and splice region variants in RNPC3 (c.358dupA, p.Arg120fs) were detected. Other missense variants were also observed including variants in ACAN (c.2591C>T, c.2789G>T, c.2815T>A, c.4207A>G, c.4523A>C and c.7119C>G), GC (rs4588 and rs7041) and LRP2 (rs2075252 and rs1991517). A start loss variant in VDR (rs2228570) with high impact was also observed. Our findings suggest a potential association of these variants with growth hormone deficiency and short stature. In this study, novel pathogenic variants in RNPC3 were revealed as well as other variants in ACAN and in genes related to Vitamin-D metabolism (GC, VDR and LRP2) that some or all might be associated with growth hormone deficiency. Further large-scale studies are required to address the association of these variants with growth hormone deficiency and its subsequent short stature.

#5124 GENETIC GLOMERULAR DISEASES IN THE ADULT PATIENT: A WORK IN PROGRESS – SINGLE CENTRE COHORT

Abstract Background and Aims Recent advances in genetic molecular techniques have allowed the expansion of knowledge in glomerular diseases of genetic cause, including the identification of genes involved in focal and segmental glomerular sclerosis (FSGS). The prevalence of adult-onset genetic FSGS varies widely depending on the population studied. In central Europe, NPHS2 and COL4A3, COL4A4 e COL4A5 genes are postulated to be the most frequent genes implicated in genetic causes of adult FSGS. This study aims to characterize patients submitted to genetic study of glomerular diseases in the Nephrogenetics Clinic of our centre. Method We conducted a single-centre retrospective analysis of the patients submitted to genetic study of glomerular diseases in the Nephrogenetics Clinic of our centre from 2016 to 2022. Adult patients with a glomerular disease phenotype suggestive of a genetic cause were studied, using a predetermined phenotype-targeted panel of genes, through massive parallel sequencing. The number of genes included in this panel has increased (from 21 genes in 2016 to 49 genes since 2018); given this change, patients with a negative result on the first approach repeated screening with the broader panel. Additionally, patients presenting with advanced chronic kidney disease (CKD) without an identifiable phenotype, studied with a larger panel for CKD causing genes, are reported. In patients with a negative result but highly suggestive presentation, whole exome sequencing (WES) was considered, after multidisciplinary discussion with a Geneticist's evaluation. Patients with variants of unknown significance (VUS) were referred to a Genetics’ consultation for genetic counselling and segregation studies. Results Of the 80 patients studied (comprising 74 families), 52.5% were female (n=42) and 79% (n=63) of Caucasian ascent. Mean age was 45.4 ± 16.0 years. Nine patients were studied with the larger gene panel for CKD. 85% (n=68) had CKD (all stages) at presentation, with proteinuria (ranging from sub-nephrotic to nephrotic range) and 15% had asymptomatic urinary changes. 50% (n=40) of patients had positive family history, and 31% (n=25) had a syndromic presentation and/or extra renal manifestations. 27.5% (n=22) had a renal biopsy, and the most prevalent result was FSGS (11%; n=9). Genetic variants were identified in 70% (n=56) of the patients; but pathogenicity is not fully established in all: genotype-phenotype correlations, including inheritance pattern and histological correlation, and segregation studies (when indicated and possible) are ongoing. In 5 patients results are pending. Pathogenic / likely pathogenic variants were identified in at least 47.5% (n=38) patients. The most frequently implicated genes were: COL4A3, COL4A4 e COL4A5 (n=21), high risk APOL1 (n=8); NPSH2 (n=5), IFN2 (n=6, 3 pathogenic, 1 variant of unknown significance (VUS), 2 under segregation studies) and MYH9 (n=3). COL4A3, COL4A4 e COL4A5 mutations occur with Alport syndrome, but also with histologically documented FSGS, sub-nephrotic and nephrotic proteinuria, nephrotic syndrome and asymptomatic urinary changes. In our cohort, NPHS2 p.R229Q was identified in 5 patients (6%), but only with pathogenic significance in 3 patients, and APOL1 high risk haplotype was co-identified with COL4 variants in 3 patients. Conclusion The genetic study of a cohort of patients suspected of genetic FSGS allowed the identification of genetic variants in the genes of interest in a high percentage of cases. The most frequently implicated genes were COL4A3, COL4A4 e COL4A5, NPHS2, INF2 and APOL1 high risk haplotype. The high percentage of gene variants identified in our cohort is probably due to the selection of a population with a high likelihood of genetic FSGS. Further studies will clarify the causal and/or modifier effect of some genes, namely COL4A3, COL4A4 e COL4A5. The future use of WES in cases of high likelihood of genetic cause will probably allow the identification on novel candidate genes and increase the clinical benefits of a genetic diagnosis of FSGS.

Var∣Decrypt: a novel and user-friendly tool to explore and prioritize variants in whole-exome sequencing data

BackgroundHigh-throughput sequencing (HTS) offers unprecedented opportunities for the discovery of causative gene variants in multiple human disorders including cancers, and has revolutionized clinical diagnostics. However, despite more than a decade of use of HTS-based assays, extracting relevant functional information from whole-exome sequencing (WES) data remains challenging, especially for non-specialists lacking in-depth bioinformatic skills.ResultsTo address this limitation, we developed Var∣Decrypt, a web-based tool designed to greatly facilitate WES data browsing and analysis. Var∣Decrypt offers a wide range of gene and variant filtering possibilities, clustering and enrichment tools, providing an efficient way to derive patient-specific functional information and to prioritize gene variants for functional analyses. We applied Var∣Decrypt on WES datasets of 10 acute erythroid leukemia patients, a rare and aggressive form of leukemia, and recovered known disease oncogenes in addition to novel putative drivers. We additionally validated the performance of Var∣Decrypt using an independent dataset of ~ 90 multiple myeloma WES, recapitulating the identified deregulated genes and pathways, showing the general applicability and versatility of Var∣Decrypt for WES analysis.ConclusionDespite years of use of WES in human health for diagnosis and discovery of disease drivers, WES data analysis still remains a complex task requiring advanced bioinformatic skills. In that context, there is a need for user-friendly all-in-one dedicated tools for data analysis, to allow biologists and clinicians to extract relevant biological information from patient datasets. Here, we provide Var∣Decrypt (trial version accessible here: https://vardecrypt.com/app/vardecrypt), a simple and intuitive Rshiny application created to fill this gap. Source code and detailed user tutorial are available at https://gitlab.com/mohammadsalma/vardecrypt.

Graph-ETMB: A graph neural network-based model for tumour mutation burden estimation

As a critical indicator of how easily the human immune system recognizes tumour cells, tumour mutational burden (TMB) is widely used to identify the potential effectiveness of immune checkpoint inhibitor therapy. However, the difficulties associated with the whole exome sequencing (WES) process, such as high tissue sampling requirements, high costs, and long turnaround times, have hindered the widespread clinical use of WES. Furthermore, the mutation landscape varies across cancer types, and the distribution of TMBs varies across cancer subtypes. Therefore, there is an urgent clinical need to develop a small cancer-specific panel to estimate TMB accurately, predict immunotherapy response cost-effectively and assist physicians in precise decision-making. This paper uses a graph neural network framework (Graph-ETMB) to address the cancer specificity problem in TMB. The correlation and tractability between mutated genes are described through message-passing and aggregation algorithms between graph networks. Then the graph neural network is trained in the lung adenocarcinoma data through a semi-supervised approach, resulting in a mutation panel containing 20 genes with a length of only 0.16 Mb. The number of genes to be detected is smaller than most commercial panels currently in clinical use. In addition, the efficacy of the designed panel in predicting immunotherapy response was further determined in an independent validation dataset, exploring the association between TMB and immunotherapy efficacy.

Use of whole-exome sequencing to identify novel monogenic gene mutations and genotype-phenotype correlations in Chinese Han children with urolithiasis.

The incidence of urolithiasis (UL) in children has been increasing. Although the pathogenesis of pediatric UL is controversial and remains unclear, multiple monogenic causes of UL have been identified. We aim to investigate the prevalence of inherited UL causes and explore the genotype-phenotype correlation in a Chinese pediatric group. In this study, we analyzed the DNA of 82 pediatric UL patients using exome sequencing (ES). The data of metabolic evaluation and genomic sequencing were subsequently analyzed together. We detected 54 genetic mutations in 12 of 30 UL-related genes. A total of 15 detected variants were described as pathogenic mutations, and 12 mutations were considered likely pathogenic. Molecular diagnoses were made in 21 patients with pathogenic or likely pathogenic variants. Six novel mutations that were not previously reported were identified in this cohort. Calcium oxalate stones were detected in 88.9% cases (8/9) with hyperoxaluria-related mutations, while 80% of individuals (4/5) with cystinuria-causing defects were diagnosed with cystine stones. Our study highlights the significant genetic abnormalities in pediatric UL and demonstrates the diagnostic power of ES for screening patients with UL.

An absolute approach to using whole exome DNA and RNA workflow for cancer biomarker testing.

The concept of personalized medicine in cancer has emerged rapidly with the advancement of genome sequencing and the identification of clinically relevant variants that contribute to disease prognosis and facilitates targeted therapy options. In this study, we propose to validate a whole exome-based tumor molecular profiling for DNA and RNA from formalin-fixed paraffin-embedded (FFPE) tumor tissue. The study included 166 patients across 17 different cancer types. The scope of this study includes the identification of single-nucleotide variants (SNVs), insertions/deletions (INDELS), copy number alterations (CNAs), gene fusions, tumor mutational burden (TMB), and microsatellite instability (MSI). The assay yielded a mean read depth of 200×, with >80% of on-target reads and a mean uniformity of >90%. Clinical maturation of whole exome sequencing (WES) (DNA and RNA)- based assay was achieved by analytical and clinical validations for all the types of genomic alterations in multiple cancers. We here demonstrate a limit of detection (LOD) of 5% for SNVs and 10% for INDELS with 97.5% specificity, 100% sensitivity, and 100% reproducibility. The results were >98% concordant with other orthogonal techniques and appeared to be more robust and comprehensive in detecting all the clinically relevant alterations. Our study demonstrates the clinical utility of the exome-based approach of comprehensive genomic profiling (CGP) for cancer patients at diagnosis and disease progression. The assay provides a consolidated picture of tumor heterogeneity and prognostic and predictive biomarkers, thus helping in precision oncology practice. The primary intended use of WES (DNA+RNA) assay would be for patients with rare cancers as well as for patients with unknown primary tumors, and this category constitutes nearly 20-30% of all cancers. The WES approach may also help us understand the clonal evolution during disease progression to precisely plan the treatment in advanced stage disease.

Detection of ATXN2 Expansions in an Exome Dataset: An Underdiagnosed Cause of Parkinsonism.

CAG-repeat expansions in Ataxin 2 (ATXN2) are known to cause spinocerebellar ataxia type 2 (SCA2), but CAA interrupted expansions may also result in autosomal dominant Parkinson's disease (AD PD). However, because of technical limitations, such expansions are not explored in whole exome sequencing (WES) data. To identify ATXN2 expansions using WES data from PD cases. We explored WES data from a cohort of 477 index cases with PD using ExpansionHunter (Illumina DRAGEN Bio-IT Platform, San Diego, CA). Putative expansions were confirmed by combining polymerase chain reaction and fragment length analysis followed by sub-cloning and sequencing methods. Using ExpansionHunter, we identified three patients from two families with AD PD carrying either ATXN2 22/39 or 22/37 repeats, both interrupted by four CAA repeats. These findings demonstrate the usefulness of WES to detect pathogenic CAG repeat expansions, which were found in 1.7% of AD PD in the ATXN2 gene in our exome dataset.