American College Of Medical Genetics And Genomics/Association For Molecular Pathology Research Articles

Abstract 7325: Integration of functional data to classify BRCA1/2 missense variants: An ENIGMA project

Abstract The success of precision cancer prevention and treatment hinges on accurate discrimination between benign and pathogenic germline alleles in cancer susceptibility genes. Variants of uncertain clinical significance (VUS) present a challenge for cancer risk assessment and functional data will be essential to resolve many VUS. To address this, we developed a cloud-based environment to collate, curate, integrate and analyze all published functional data related to BRCA1 and BRCA2 missense VUS. Then, functional evidence criteria for pathogenicity were determined for each assay using the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) classification guidelines and ClinGen criteria. Our approach involved retrieving published articles reporting one or more functional assays of BRCA1/2 missense variants reporting variant impact on various biochemical and cell biological functions. Functional results were recorded using original authors' thresholds and categories, then converted to ordinal variables to harmonize: [0 = no functional impact], [1 = intermediate impact], and [2 = functional impact]. For BRCA1, we updated our previous published integration (Genet Med. 2021;23:306-315) with 14 additional publications, collating results from 53 instances of functional assays reporting functional data on 3,219 unique missense variants. For BRCA2, 28 publications were identified reporting data from 141 instances of functional assays on 4,759 unique missense variants. Utilizing a panel of 542 and 396 known pathogenic and benign reference variants for BRCA1 and BRCA2, respectively, we determined the sensitivity, specificity, and ACMG/AMP odds of pathogenicity for each instance of the assay. The remaining variants were assigned a relevant functional assay code weight for each assay instance. Our study successfully derived ACMG/AMP evidence strength from functional data for 3,040 BRCA1 (94% of 3,219) and 2,704 BRCA2 (57% of 4,759) missense variants. This comprehensive work underscores the utility of functional data in informing classification of BRCA1 and BRCA2 missense VUS. Citation Format: Paulo Lyra, Lucas Dalcolmo, Michael Parsons, Thales Nepomuceno, Samuel Brito, Nam Phuong N. Nguyen, Geise de Oliveira, Joao Paulo da Siva, Laura Caleca, Tanisha Taneja, Chunling Hu, Marcy Richardson, Maria Rossing, Aditi Hazra, Alexandra Martins, Sandrine Caputo, Gael A. Millot, Joanne NgeowYuen Yie, Marcelo A. Carvalho, Melissa Cline, Paolo Radice, Rachael Carlsen, Romy Mesman, Valentina Zampiga, Rehan Villani, Vijay Josef, Shyam Sharan, Kyriaki Michailidou, Amanda B. Spurdle, Fergus Couch, Maaike P.G. Vreeswijk, Alvaro N. Monteiro. Integration of functional data to classify BRCA1/2 missense variants: An ENIGMA project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7325.

Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer-rich regions and 38 spliceogenic variants.

Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss-of-function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6-10 that produced the expected minigene full-length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE-rich intervals by splicing assays in MCF-7 cells. We identified three minimal (10-, 11-, 15-nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild-type minigene and tested functionally. Thirty-eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20-50% of the minigene full-length transcript). Thirty-three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Splicing analysis of 24 potential spliceogenic variants in MMR genes and clinical interpretation based on refined ACMG/AMP criteria.

Lynch syndrome (LS) is a common hereditary cancer syndrome caused by heterozygous germline pathogenic variants in DNA mismatch repair (MMR) genes. Splicing defect constitutes one of the major mechanisms for MMR gene inactivation. Using RT-PCR based RNA analysis, we investigated 24 potential spliceogenic variants in MMR genes and determined their pathogenicity based on refined splicing-related American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria. Aberrant transcripts were confirmed in 19 variants and 17 of which were classified as pathogenic including 11 located outside of canonical splice sites. Most of these variants were previously reported in LS patients without mRNA splicing assessment. Thus, our study provides crucial evidence for pathogenicity determination, allowing for appropriate clinical follow-up. We also found that computational predictions were globally well correlated with RNA analysis results and the use of both SPiP and SpliceAI software appeared more efficient for splicing defect prediction.

Clinical implications of VUS reclassification in a single-centre series from application of ACMG/AMP classification rules specified for BRCA1/2

BackgroundBRCA1/2 testing is crucial to guide clinical decisions in patients with hereditary breast/ovarian cancer, but detection of variants of uncertain significance (VUSs) prevents proper management of carriers. The ENIGMA (Evidence-based...

Recommendations for the classification of germline variants in the exonuclease domain of POLE and POLD1

BackgroundGermline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases.MethodsA training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered.ResultsGene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign.ConclusionsOur recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications.

Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants.

Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes. A total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells. Of 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5'UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition. Incorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene.

Specifications and validation of the ACMG/AMP criteria for clinical interpretation of sequence variants in collagen genes associated with joint hypermobility

Deleterious variants in collagen genes are the most common cause of hereditary connective tissue disorders (HCTD). Adaptations of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria are still lacking. A multidisciplinary team was set up for developing specifications of the ACMG/AMP criteria for COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL11A1, COL11A2 and COL12A1, associated with various forms of HCTD featuring joint hypermobility, which is becoming one of the most common reasons of referral for molecular testing in this field. Such specifications were validated against 209 variants, and resulted effective for classifying as pathogenic and likely pathogenic null alleles without downgrading of the PVS1 level of strength and recurrent Glycine substitutions. Adaptations of selected criteria reduced uncertainties on private Glycine substitutions, intronic variants predicted to affect the splicing, and null alleles with a downgraded PVS1 level of strength. Segregation and multigene panel sequencing data mitigated uncertainties on non-Glycine substitutions by the attribution of one or more benignity criteria. These specifications may improve the clinical utility of molecular testing in HCTD by reducing the number of variants with neutral/conflicting interpretations. Close interactions between laboratory and clinicians are crucial to estimate the a priori utility of molecular test and to improve medical reports.

New Insight into the Genotype-Phenotype Correlation of PRPH2-Related Diseases Based on a Large Chinese Cohort and Literature Review.

Variants in PRPH2 are a common cause of inherited retinal dystrophies with high genetic and phenotypic heterogeneity. In this study, variants in PRPH2 were selected from in-house exome sequencing data, and all reported PRPH2 variants were evaluated with the assistance of online prediction tools and the comparative validation of large datasets. All variants were classified based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. Individuals with pathogenic or likely pathogenic variants of PRPH2 were confirmed by Sanger sequencing. Clinical characteristics were summarized. Ten pathogenic or likely pathogenic variants of PRPH2 were identified in 14 families. In our cohort, the most frequent variant was p.G305Afs*19, accounting for 33.3% (5/15) of alleles, in contrast to the literature, where p.R172G (11.6%, 119/1028) was the most common variant. Nine in-house families (63.8%) were diagnosed with retinitis pigmentosa (RP), distinct from the phenotypic spectrum in the literature, which shows that RP accounts for 27.9% (283/1013) and macular degeneration is more common (45.2%, 458/1013). Patients carrying missense variants predicted as damaging by all seven prediction tools and absent in the gnomAD database were more likely to develop RP compared to those carrying missense variants predicted as damaging with fewer tools or with more than one allele number in the gnomAD database (p = 0.001). The population-specific genetic and phenotypic spectra of PRPH2 were explored, and novel insight into the genotype-phenotype correlation of PRPH2 was proposed. These findings demonstrated the importance of assessing PRPH2 variants in distinct populations and the value of providing practical suggestions for the genetic interpretation of PRPH2 variants.

Ataxia with oculomotor apraxia type 2 caused by a novel homozygous mutation in SETX gene, and literature review.

Autosomal recessive inherited ataxia with oculomotor apraxia type 2 (AOA2), caused by SETX gene mutations, is characterized by early-onset, progressive cerebellar ataxia, peripheral neuropathy, oculomotor apraxia and elevated serum α-fetoprotein (AFP). This study aimed to expand and summarize the clinical and genetic characteristics of SETX variants related to AOA2. The biochemical parameters, electromyogram and radiological findings of the patient were evaluated. Whole-exome sequencing (WES) was performed on the patient using next-generation sequencing (NGS), the variants were confirmed by Sanger sequencing and the pathogenicity of the variants was classified according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. We reviewed 57 studies of AOA2 patients with SETX mutations and collected clinical and genetic information. The patient was a 40-year-old Chinese woman who primarily presented with numbness and weakness of the lower limbs in her teenage years. She had elevated AFP, increased serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and decreased anti-Müllerian hormone (AMH) levels. We identified a novel homozygous missense mutation of the SETX gene, c.7118 C>T (p. Thr2373Ile), in the patient via Whole-exome and Sanger sequencing. The variant was located in the DNA/RNA helicase domain and is highly conserved. The protein prediction analysis verified the SETX variant as a damaging alteration and ACMG/AMP guidelines classified it as likely pathogenic. Through a literature review, we identified 229 AOA2 cases with SETX variants, and among the variants, 156 SETX variants were exonic. We found that 107 (46.7%) patients were European, 50 (21.8%) were African and 48 (21.0%) were Asian. Among the Asian patients, five from two families were Mainland Chinese. The main clinical features were cerebellar ataxia (100%), peripheral neuropathy (94.6%), cerebellar atrophy (95.3%) and elevated AFP concentration (92.0%). Most reported SETX mutations in AOA2 patients were missense, frameshift and nonsense mutations. We discovered a novel homozygous variant of the SETX gene as a cause of AOA2 in the current patient and expanded the genotypic spectrum of AOA2. Moreover, the clinical features of AOA2 and genetic findings in SETX were assessed in reported cohorts and are summarized in the present study.

Splicing Analysis of 16 PALB2 ClinVar Variants by Minigene Assays: Identification of Six Likely Pathogenic Variants.

Simple SummaryPALB2 pathogenic variants confer high risk of breast cancer. Here, we have analyzed the impact of PALB2 variants on splicing, a gene expression step that removes introns to form the mature messenger RNA. This process is performed by the splicing machinery through the recognition of specific sequences, namely the 3′ and 5′ splice sites, which determine the exon ends. Variants at these sequences may trigger anomalous splicing and aberrant transcripts that may be associated with a disease. To test the impact of variants on splicing, we used a biotechnological tool called minigene, which replicates, at small-scale, the human gene of interest. Thus, we checked 16 PALB2 variants at the intron/exon boundaries using the minigene mgPALB2_ex1-3. We found that twelve variants disrupted splicing, six of which could be classified as likely pathogenic. These results facilitate the clinical management of carrier patients and families since they may benefit from tailored prevention protocols and therapies.PALB2 loss-of-function variants are associated with significant increased risk of breast cancer as well as other types of tumors. Likewise, splicing disruptions are a common mechanism of disease susceptibility. Indeed, we previously showed, by minigene assays, that 35 out of 42 PALB2 variants impaired splicing. Taking advantage of one of these constructs (mgPALB2_ex1-3), we proceeded to analyze other variants at exons 1 to 3 reported at the ClinVar database. Thirty-one variants were bioinformatically analyzed with MaxEntScan and SpliceAI. Then, 16 variants were selected for subsequent RNA assays. We identified a total of 12 spliceogenic variants, 11 of which did not produce any trace of the expected minigene full-length transcript. Interestingly, variant c.49-1G > A mimicked previous outcomes in patient RNA (transcript ∆(E2p6)), supporting the reproducibility of the minigene approach. A total of eight variant-induced transcripts were characterized, three of which (∆(E1q17), ∆(E3p11), and ∆(E3)) were predicted to introduce a premature termination codon and to undergo nonsense-mediated decay, and five (▼(E1q9), ∆(E2p6), ∆(E2), ▼(E3q48)-a, and ▼(E3q48)-b) maintained the reading frame. According to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, which integrates mgPALB2 data, six PALB2 variants were classified as pathogenic/likely pathogenic, five as VUS, and five as likely benign. Furthermore, five ±1,2 variants were catalogued as VUS because they produced significant proportions of in-frame transcripts of unknown impact on protein function.

The ClinGen Brain Malformation Variant Curation Expert Panel: Rules for somatic variants in AKT3, MTOR, PIK3CA, and PIK3R2

PurposePostzygotic (somatic) variants in the mTOR pathway genes cause a spectrum of distinct developmental abnormalities. Accurate classification of somatic variants in this group of disorders is crucial for affected individuals and their families. MethodsThe ClinGen Brain Malformation Variant Curation Expert Panel was formed to curate somatic variants associated with developmental brain malformations. We selected the genes AKT3, MTOR, PIK3CA, and PIK3R2 as the first set of genes to provide additional specifications to the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) sequence variant interpretation guidelines, which currently focus solely on germline variants. ResultsA total of 24 of the original 28 ACMG/AMP criteria required modification. Several modifications used could be applied to other genes and disorders in which somatic variants play a role: 1) using variant allele fraction differences as evidence that somatic mutagenesis occurred as a proxy for de novo variation, 2) incorporating both somatic and germline evidence, and 3) delineating phenotype on the basis of variable tissue expression. ConclusionWe have established a framework for rigorous interpretation of somatic mosaic variants, addressing issues unique to somatic variants that will be applicable to many genes and conditions.

Minigene-based splicing analysis and ACMG/AMP-based tentative classification of 56 ATM variants.

The ataxia telangiectasia‐mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss‐of‐function variants are associated with 2‐fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large‐scale sequencing project BRIDGES. A total of 381 variants at the intron–exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4–9, 11–17, 25–29, and 49–52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF‐7/HeLa cells. Forty‐eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full‐length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi‐exon skipping. Twenty‐seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)‐based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL‐transcript due to the use of a noncanonical GG‐5’‐splice‐site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage‐sensitive expression model in which variants producing ≥30% of FL‐transcripts would be predicted benign, while variants producing ≤13% of FL‐transcripts might be pathogenic. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Mutation update of SERPING1 related to hereditary angioedema in the Chinese population

BackgroundHereditary angioedema (HAE) is a rare disease characterized by recurrent attacks of severe swellings of the skin and submucosa. More than 900 variants of the SERPING1 gene associated with HAE have been identified. However, only approximately 50 variants have been identified in the Chinese population. This study aimed to update the mutational spectrum in Chinese HAE patients and provide evidence for the accurate diagnosis of HAE.MethodsA total of 97 unrelated HAE patients were enrolled in the study. Sanger sequencing and multiple ligation-dependent probe amplification analysis were used to identify the variants in the SERPING1 gene. The variants were reviewed in a number of databases, including the Human Gene Mutation Database (HGMD) (http://www.hgmd.cf.ac.uk/) and the Leiden Open Variation Database (LOVD, https://databases.lovd.nl/shared/variants/SERPING1). The American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) criteria was used to determine the pathogenicity of the variants.ResultsOf the 97 patients, 76 different variants were identified in 90 of them and no disease-causing variants were identified in the remaining 7 patients. Among the 76 variants, 35 variants were novel and submitted to ClinVar. Missense and in-frame variants were the most common variants (36.8%), followed by frameshift (28.9%), nonsense (14.5%), splice site (13.2%) variants, and gross deletions/duplications (6.6%).ConclusionsOur findings broaden the mutational spectrum of SERPING1 and provide evidence for accurate diagnosis and predictive genetic counseling.

Classification of BRCA2 Variants of Uncertain Significance (VUS) Using an ACMG/AMP Model Incorporating a Homology-Directed Repair (HDR) Functional Assay.

The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer-testing laboratory. The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2. Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS. Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C.

Simple SummaryLoss-of-function variants of the RAD51C gene are known to confer a risk of breast and ovarian cancers. In this study, we analyzed the impact of RAD51C variants on splicing, a highly regulated gene expression step by which introns are removed and exons are sequentially joined. Exon recognition is guided by specific sequences, the 3′ and 5′ splice sites, which define the exon boundaries. Variants of these sequences of susceptibility genes may lead to aberrant splicing and abnormal transcripts that may trigger a disease. Splicing can be tested using a biotechnological tool called minigenes, which mimic the human gene of interest. Thus, we checked 20 RAD51C splice-site variants using the minigene mgR51C_ex2-8. We found that they all disrupted the splicing mechanism, and 16 variants could be classified as likely pathogenic. Our findings are clinically actionable, and variant carriers may benefit from tailored prevention protocols and therapies.RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1–4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants.

EP364: Lessons from pathogenicity classifications for 251 RYR1 variants associated with malignant hyperthermia susceptibility using variant curation expert panel revised criteria

The American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) have suggested criteria for variant pathogenicity classification. The ClinGen malignant hyperthermia susceptibility (MHS) variant curation expert panel (VCEP) has specified the ACMG/AMP criteria for variants in RYR1 associated with MHS. The specified criteria, and a pilot analysis of 84 variants, have been published. Here we have updated the specified criteria and expanded the set of RYR1 variants classified for pathogenicity using the RYR1-specific ACMG/AMP criteria.

EP471: The role of clinical exome sequencing in genetic diagnosing of skeletal dysplasia in fetus detected by prenatal ultrasound in Vietnam

Skeletal dysplasia is typically diagnosed using a combination of radiographic imaging, clinical examinations, and molecular testing. CES (Clinical Exome Sequencing) plays an important role in the genetic diagnosing of skeletal dysplasia. However, detailed molecular-based epidemiologic studies on this disorder through prenatal sonography in Vietnam have not yet appeared. This study aimed to comprehensively characterize the molecular genetic landscape in unselected 98 Vietnamese cases suspected of skeletal dysplasia through prenatal screening, particularly novel variants as well as fetal phenotypes. Our CES includes 168 clinically relevant genes. Ninety-eight participants detected skeletal dysplasia through prenatal ultrasound were recruited from June August 2020 to August 2021. Amniotic fluid samples were screened by CNV-seq to detect abnormal copy number variants (CNVs) and CES to detect Single Nucleotide Variants (SNVs). Variants were interpreted according to American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. The predominant sign on ultrasound is short and/or bowing long bones. The other findings are bipedal varus, aplasia/dysplasia of nasal bones, absence of limb bones, deformities in metacarpal bones and part of the distal phalanx, fixed joints of wrist and knee, digit malformation, bilateral rocker-bottom feet, osteogenesis imperfecta, skull bone anomalies, and spina bifida. Out of the total 98 participants with skeletal dysplasia by ultrasound screening, 95 (97%) fetuses had normal copy number variants and the remaining three (roughly 3%) individuals had CNV anomalies. Among 95 cases with normal CNV, further NGS analysis revealed 47/95 (49%) fetuses harbored a variety of single nucleotide variants (SNV). These SNVs were categorized as “pathogenic and likely pathogenic” in 27/47 (57.4%), “variants of uncertain significance” in 20/47 (42.6%). A total of 50 variants in 23 different genes associated with skeletal dysplasia were identified. In particular, 21 novel variants including 8 pathogenic/likely pathogenic variants and 13 variants of uncertain significance were identified and reported. Remarkably, most variants were frequently located in genes FGFR3, FLNB, COG4, COL10A1, COL1A2, and COL2A1 of which the common phenotype is short and/or bowing long bones. Among three cases with aberrant CNVs, trisomy 21, the mosaic of trisomy 16, and submicroscopic microdeletions in chromosomes 11 were identified. To our knowledge, this is the first study in Vietnam that utilizes the targeted next-generation gene sequencing combined with CNV analysis in diagnosing prenatal cases of suspected skeletal dysplasia with an overall diagnostic yield of 51%. Molecular findings are crucial to guide medical treatment and management and to inform assessment of risk for familial recurrence. Multiple novel variants reported in this study will substantially expand the genetic archive of genes associated with skeletal dysplasia. Furthermore, we suggest more research to inform the pathogenicity of our VUS and novel variants.

A novel missense variant in ESRRB gene causing autosomal recessive non-syndromic hearing loss: in silico analysis of a case

BackgroundHereditary hearing loss (HHL) is a common heterogeneous disorder affecting all ages, ethnicities, and genders. The most common form of HHL is autosomal recessive non-syndromic hearing loss (ARNSHL), in which there is no genotype–phenotype correlation in the majority of cases. This study aimed to identify the genetic causes of hearing loss (HL) in a family with Iranian Azeri Turkish ethnicity negative for gap junction beta-2 (GJB2), gap junction beta-6 (GJB6), and mitochondrially encoded 12S rRNA (MT-RNR1) deleterious mutations.MethodsTargeted genome sequencing method was applied to detect genetic causes of HL in the family. Sanger sequencing was employed to verify the segregation of the variant. Finally, we used bioinformatics tools and American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines to determine whether the detected variant might affect the corresponding protein or not.ResultsA novel homozygous missense mutation, c.499G>A (p.G167R), was identified in exon 5 of the ESRRB (estrogen-related receptor beta) gene. Healthy and affected family members confirmed the co-segregation of the variant with ARNSHL. Eventually, the variant's pathogenicity was confirmed by the in silico analysis and the ACMG/AMP guidelines.ConclusionThe study suggests that the detected variant, c.499G>A, plays a crucial role in the development of ARNSHL, emphasizing the clinical significance of the ESRRB gene in ARNSHL patients. Additionally, it would be helpful for genetic counseling and clinical management of ARNSHL patients and providing preventive opportunities.

Identification and Computational Analysis of Rare Variants of Known Hearing Loss Genes Present in Five Deaf Members of a Pakistani Kindred.

Hearing loss (HL) is the most common neurosensory defect in humans that affects the normal communication. Disease is clinically and genetically heterogeneous, rendering challenges for the molecular diagnosis of affected subjects. This study highlights the phenotypic and genetic complexity of inherited HL in a large consanguineous Pakistan kindred. Audiological evaluation of all affected individuals revealed varying degree of mild to profound sensorineural HL. Whole exome (WES) of four family members followed by Sanger sequencing revealed candidate disease-associated variants in five known deafness genes: GJB2 (c.231G>A; p.(Trp77 *)), SLC26A4 (c.1337A>G; p.(Gln446Arg)), CDH23 (c.2789C>T; p.(Pro930Leu)), KCNQ4 (c.1672G>A; p.(Val558Met)) and MPDZ (c.4124T>C; p.(Val1375Ala)). All identified variants replaced evolutionary conserved residues, were either absent or had low frequencies in the control databases. Our in silico and 3-Dimensional (3D) protein topology analyses support the damaging impact of identified variants on the encoded proteins. However, except for the previously established “pathogenic” and “likely pathogenic” categories for the c.231G>A (p.(Trp77 *)) allele of GJB2 and c.1377A>G (p.(Gln446Arg)) of SLC26A4, respectively, all the remaining identified variants were classified as “uncertain significance” based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant pathogenicity guidelines. Our study highlights the complexity of genetic traits in consanguineous families, and the need of combining the functional studies even with the comprehensive profiling of multiple family members to improve the genetic diagnosis in complex inbred families.

Clinical impact of re-evaluating genes and variants implicated in dilated cardiomyopathy

PurposeAccurate interpretation of variants detected in dilated cardiomyopathy (DCM) is crucial for patient care but has proven challenging. We applied a set of proposed refined American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria for DCM, reclassified all detected variants in robust genes, and associated these results to patients' phenotype. MethodsThe study included 902 DCM probands from the Maastricht Cardiomyopathy Registry who underwent genetic testing. Two gene panel sizes (extended n = 48; and robust panel n = 14) and two standards of variant classification (standard versus the proposed refined ACMG/AMP criteria) were applied to compare genetic yield. ResultsA pathogenic or likely pathogenic (P/LP) variant was found in 17.8% of patients, and a variant of uncertain significance (VUS) was found in 32.8% of patients when using method 1 (extended panel (n = 48) + standard ACMG/AMP), compared to respectively 16.9% and 12.9% when using method 2 (robust panel (n = 14) + standard ACMG/AMP), and respectively 14% and 14.5% using method 3 (robust panel (n = 14) + refined ACMG/AMP). Patients with P/LP variants had significantly lower event-free survival compared to genotype-negative DCM patients. ConclusionStringent gene selection for DCM genetic testing reduced the number of VUS while retaining ability to detect similar P/LP variants. The number of genes on diagnostic panels should be limited to genes that have the highest signal to noise ratio.