Specification of variant interpretation guidelines for inherited retinal dystrophy in Japan.

Accurate interpretation of sequence variants in inherited retinal dystrophy (IRD) is vital given the significant genetic heterogeneity observed in this disorder. To achieve consistent and accurate diagnoses, establishment of standardized guidelines for variant interpretation is essential. The American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for variant interpretation serve as the global "cross-disease" standard for classifying variants in Mendelian hereditary disorders. These guidelines propose a systematic approach for categorizing variants into 5 classes based on various types of evidence, such as population data, computational data, functional data, and segregation data. However, for clinical genetic diagnosis and to ensure standardized diagnosis and treatment criteria, additional specifications based on features associated with each disorder are necessary. In this context, we present a comprehensive framework outlining the newly specified ACMG/AMP rules tailored explicitly to IRD in the Japanese population on behalf of the Research Group on Rare and Intractable Diseases (Ministry of Health, Labour and Welfare of Japan). These guidelines consider disease frequencies, allele frequencies, and both the phenotypic and the genotypic characteristics unique to IRD in the Japanese population. Adjustments and modifications have been incorporated to reflect the specific requirements of the population. By incorporating these IRD-specific factors and refining the existing ACMG/AMP guidelines, we aim to enhance the accuracy and consistency of variant interpretation in IRD cases, particularly in the Japanese population. These guidelines serve as a valuable resource for ophthalmologists and clinical geneticists involved in the diagnosis and treatment of IRD, providing them with a standardized framework to assess and classify genetic variants.

Evaluation of the pathogenic potential of germline DDX41 variants in hematopoietic neoplasms using the ACMG/AMP guidelines.

Clinical use of gene panel testing for hematopoietic neoplasms in areas, such as diagnosis, prognosis prediction, and exploration of treatment options, has increased in recent years. The keys to interpreting gene variants detected in gene panel testing are to distinguish between germline and somatic variants and accurately determine whether the detected variants are pathogenic. If a variant is suspected to be a pathogenic germline variant, it is essential to confirm its consistency with the disease phenotype and gather a thorough family history. Donor eligibility must also be considered, especially if the patient's variant is also detected in the expected donor for hematopoietic stem cell transplantation. However, determining the pathogenicity of gene variants is often complicated, given the current limited availability of databases covering germline variants of hematopoietic neoplasms. This means that hematologists will frequently need to interpret gene variants themselves. Here, we outline how to assess the pathogenicity of germline variants according to criteria from the American College of Medical Genetics and Genomics/Association for Molecular Pathology standards and guidelines for the interpretation of variants using DDX41, a gene recently shown to be closely associated with myeloid neoplasms with a germline predisposition, as an example.

Quantitative thresholds for variant enrichment in 13,845 cases: improving pathogenicity classification in genetic hearing loss.

BACKGROUND: The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines recommend using variant enrichment among cases as "strong" evidence for pathogenicity per the PS4 criterion. However, quantitative support for PS4 thresholds from real-world Mendelian case-control cohorts is lacking. METHODS: To address this gap, we evaluated and established PS4 thresholds using data from the Chinese Deafness Genetics Consortium. A total of 9,050 variants from 13,845 patients with hearing loss (HL) and 6,570 ancestry-matched controls were analyzed. Positive likelihood ratio and local positive likelihood ratio values were calculated to determine the thresholds corresponding to each strength of evidence across three variant subsets. RESULTS: In subset 1, consisting of variants present in both cases and controls with an allele frequency (AF) in cases ≥ 0.0005, an odds ratio (OR) ≥ 6 achieved strong evidence, while OR ≥ 3 represented moderate evidence. For subset 2, which encompassed variants present in both cases and controls with a case AF < 0.0005, and subset 3, comprising variants found only in cases and absent from controls, we defined the PS4_Supporting threshold (OR > 2.27 or allele count ≥ 3) and the PS4_Moderate threshold (allele count ≥ 6), respectively. Reanalysis applying the adjusted PS4 criteria changed the classification of 15 variants and enabled diagnosis of an additional four patients. CONCLUSIONS: Our study quantified evidence strength thresholds for variant enrichment in genetic HL cases, highlighting the importance of defining disease/gene-specific thresholds to improve the precision and accuracy of clinical genetic testing.

Reassessment of the NF1 variants of unknown significance found during the 20-year activity of a genetics diagnostic laboratory.

The finding of variants of uncertain significance (VUS) in the activity of a diagnostic genetic laboratory is a common issue, which is however provisional and needs to be periodically re-evaluated, due to the continuous advancements in our knowledge of the genetic diseases. Neurofibromatosis type 1, caused by the occurrence of heterozygous pathogenic NF1 variants, is a good model for studying the evolution of VUS, due to the widespread use of genetic testing for the disease, the constant enrichment of the international databases with NF1 variants and the full adult penetrance of the disease, which makes genotyping the parents a crucial step in the diagnostic workflow. The present study retrospectively reviewed and reinterpreted the genetic test results of NF1 in a diagnostic genetic laboratory in the period from January 1, 2000 to December 31, 2020. All the VUS were reinterpreted using the 2015 consensus standards and guidelines for the interpretation. Out of 589 NF1 genetic tests which were performed in the period, a total of 85 VUS were found and reinterpreted in 72 cases (84.7%): 21 (29.2%) were reclassified as benign/likely benign, whereas 51 (70.8%) were recoded as pathogenic/likely pathogenic with a significant trend distribution (Chi square test for trend p = 0.005). Synonymous VUS have mainly been reclassified as class 1 and 2 (7/8, 87.5%), whereas missense variants have been attributed to class 4 and 5 in 38 out of the 58 cases (65.5%). These findings underline an improvement in the classification of variants over time, suggesting that a reinterpretation of the genetic tests should be routinely performed to support the physicians in the clinical diagnosis of genetic diseases.

Molecular characterization and re-interpretation of HNF1A variants identified in Indian MODY subjects towards precision medicine.

Background: HNF1A is an essential component of the transcription factor network that controls pancreatic ß-cell differentiation, maintenance, and glucose stimulated insulin secretion (GSIS). A continuum of protein malfunction is caused by variations in the HNF1A gene, from severe loss-of-function (LOF) variants that cause the highly penetrant Maturity Onset Diabetes of the Young (MODY) to milder LOF variants that are far less penetrant but impart a population-wide risk of type 2 diabetes that is up to five times higher. Before classifying and reporting the discovered variations as relevant in clinical diagnosis, a critical review is required. Functional investigations offer substantial support for classifying a variant as pathogenic, or otherwise as advised by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) ACMG/AMP criteria for variant interpretation. Objective: To determine the molecular basis for the variations in the HNF1A gene found in patients with monogenic diabetes in India. Methods: We performed functional protein analyses such as transactivation, protein expression, DNA binding, nuclear localization, and glucose stimulated insulin secretion (GSIS) assay, along with structural prediction analysis for 14 HNF1A variants found in 20 patients with monogenic diabetes. Results: Of the 14 variants, 4 (28.6%) were interpreted as pathogenic, 6 (42.8%) as likely pathogenic, 3 (21.4%) as variants of uncertain significance, and 1 (7.14%) as benign. Patients harboring the pathogenic/likely pathogenic variants were able to successfully switch from insulin to sulfonylureas (SU) making these variants clinically actionable. Conclusion: Our findings are the first to show the need of using additive scores during molecular characterization for accurate pathogenicity evaluations of HNF1A variants in precision medicine.

Refinement of the assignment to the ACMG/AMP BS3 and PS3 criteria of eight BRCA1 variants of uncertain significance by integrating available functional data with protein interaction assays.

The clinical screening of cancer predisposition genes has led to the identification of a large number of variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is limited by the amount of necessary data, which are difficult to obtain for rare variants. The guidelines for variant interpretation of the American College of Medical Genetics and Genomics along with the Association for Molecular Pathology (ACMG/AMP) state that "well-established" functional studies provide strong support of a pathogenic or benign impact (criteria PS3 and BS3, respectively) and can be used as evidence type to reach a final classification. Moreover, the Clinical Genome Resource Sequence Variant Interpretation Working Group developed rule specifications to refine the PS3/BS3 criteria. Recently, Lira PC et al. developed the "Hi Set" approach that generated PS3/BS3 codes for over two-thousands BRCA1 VUS. While highly successful, this approach did not discriminate a group of variants with conflicting evidences. Here, we aimed to implement the outcomes of the "Hi-set" approach applying Green Fluorescent Protein (GFP)-reassembly assays, assessing the effect of variants in the RING and BRCT domains of BRCA1 on the binding of these domains with the UbcH5a or ABRAXAS proteins, respectively. The analyses of 26 clinically classified variants, including 13 tested in our previous study, showed 100% sensitivity and specificity in identifying pathogenic and benign variants for both the RING/UbcH5a and the BRCTs/ABRAXAS interactions. We derived the strength of evidences generated by the GFP-reassembly assays corresponding to moderate for both PS3 and BS3 criteria assessment. The GFP-reassembly assays were applied to the functional characterization of 8 discordant variants from the study by Lyra et al. The outcomes of these analyses, combined with those reported in the "Hi Set" study, allowed the assignment of ACMG/AMP criteria in favor or against pathogenicity for all 8 examined variants. The above findings were validated with a semi-quantitative Mammalian Two-Hybrid approach, and totally concordant results were observed. Our data contributes in shedding light on the functional significance of BRCA1 VUS and on their clinical interpretation within the ACMG/AMP framework.

MARGINAL: An Automatic Classification of Variants in BRCA1 and BRCA2 Genes Using a Machine Learning Model.

Implementation of next-generation sequencing (NGS) for the genetic analysis of hereditary diseases has resulted in a vast number of genetic variants identified daily, leading to inadequate variant interpretation and, consequently, a lack of useful clinical information for treatment decisions. Herein, we present MARGINAL 1.0.0, a machine learning (ML)-based software for the interpretation of rare BRCA1 and BRCA2 germline variants. MARGINAL software classifies variants into three categories, namely, (likely) pathogenic, of uncertain significance and (likely) benign, implementing the criteria established by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP). We first annotated BRCA1 and BRCA2 variants using various sources. Then, we automatically implemented the ACMG-AMP criteria, and we finally constructed the ML model for variant classification. To maximize accuracy, we compared the performance of eight different ML algorithms in a classification scheme based on a serial combination of two classifiers. The model showed high predictive abilities with maximum accuracy of 92% and 98%, recall of 92% and 98% and specificity of 90% and 98% for the first and second classifiers, respectively. Our results indicate that using a gene and disease-specific ML automated software for clinical variant evaluation can minimize conflicting interpretations.

Need for revision of the ACMG/AMP guidelines for interpretation of X-linked variants.

The guidelines provided by American College of Medical Genetics and Genomics (ACMG) and the Association of Molecular Pathology (AMP) (ACMG/AMP guidelines) suggest a framework for the classification of clinical variants. However, the interpretations can be inconsistent, with each definition sometimes proving to be ambiguous. In particular, there can be difficulty with interpretation of variants related to the X-linked recessive trait. To confirm whether there are biases in the interpretation of inherited traits, we reanalyzed variants reported prior to the release of the ACMG/AMP guidelines. As expected, the interpretation ratio as pathogenic or likely pathogenic was significantly lower for variants related to the X-linked recessive trait. Evaluation of variants related to the X-linked recessive trait, hence, need to consider whether the variant is identified only in males in accordance with the X-linked recessive trait. The ACMG/AMP guidelines should be revised to eliminate the bias revealed in this study.

Genetics, Functions, and Clinical Impact of Presenilin-1 (PSEN1) Gene.

Presenilin-1 (PSEN1) has been verified as an important causative factor for early onset Alzheimer's disease (EOAD). PSEN1 is a part of γ-secretase, and in addition to amyloid precursor protein (APP) cleavage, it can also affect other processes, such as Notch signaling, ß-cadherin processing, and calcium metabolism. Several motifs and residues have been identified in PSEN1, which may play a significant role in γ-secretase mechanisms, such as the WNF, GxGD, and PALP motifs. More than 300 mutations have been described in PSEN1; however, the clinical phenotypes related to these mutations may be diverse. In addition to classical EOAD, patients with PSEN1 mutations regularly present with atypical phenotypic symptoms, such as spasticity, seizures, and visual impairment. In vivo and in vitro studies were performed to verify the effect of PSEN1 mutations on EOAD. The pathogenic nature of PSEN1 mutations can be categorized according to the ACMG-AMP guidelines; however, some mutations could not be categorized because they were detected only in a single case, and their presence could not be confirmed in family members. Genetic modifiers, therefore, may play a critical role in the age of disease onset and clinical phenotypes of PSEN1 mutations. This review introduces the role of PSEN1 in γ-secretase, the clinical phenotypes related to its mutations, and possible significant residues of the protein.

The pathogenicity classification of PAH gene variants in the Iranian population.

Till now not many studies have been conducted to classify PAH gene variants according to American College of Medical Genetics and Genomics (ACMG-AMP) guidelines. The aim of this study was to collect all PAH gene variants reported among Iranian population and investigate their pathogenicity based on ACMG-AMP guidelines. Systematic collection of PAH gene variants, verification of variants, in silico analysis, and application of ACMG-AMP guidelines were the main steps in performing the present study. A total of 267 unique variants were identified; according to ACMG-AMP guidelines, 90, 40, 71, 14, and 52 variants were classified as pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), and benign (B), respectively. The need to pay more attention to synonymous and missense variants with low or no impact on protein function as well as intronic variants, whether they are deep or are close to intron/exon boundaries, was a highlight of this study. Due to the fact that few functional studies are performed on these variants, it is suggested that they be analyzed first using bioinformatics tools, and if positive results are obtained, then functional studies can be designed.

SOD-1 Variants in Amyotrophic Lateral Sclerosis: Systematic Re-Evaluation According to ACMG-AMP Guidelines.

Amyotrophic lateral sclerosis (ALS) is the most common type of motor neuron disease whose causes are unclear. The first ALS gene associated with the autosomal dominant form of the disease was SOD1. This gene has a high rate of rare variants, and an appropriate classification is essential for a correct ALS diagnosis. In this study, we re-evaluated the classification of all previously reported SOD1 variants (n = 202) from ALSoD, project MinE, and in-house databases by applying the ACMG-AMP criteria to ALS. New bioinformatics analysis, frequency rating, and a thorough search for functional studies were performed. We also proposed adjusting criteria strength describing how to apply them to SOD1 variants. Most of the previously reported variants have been reclassified as likely pathogenic and pathogenic based on the modified weight of the PS3 criterion, highlighting how in vivo or in vitro functional studies are determining their interpretation and classification. Furthermore, this study reveals the concordance and discordance of annotations between open databases, indicating the need for expert review to adapt the study of variants to a specific disease. Indeed, in complex diseases, such as ALS, the oligogenic inheritance, the presence of genes that act as risk factors and the reduced penetration must be considered. Overall, the diagnosis of ALS remains clinical, and improving variant classification could support genetic data as diagnostic criteria.

Evaluating the impact of in silico predictors on clinical variant classification.

PURPOSE: According to the American College of Medical Genetics and Genomics/Association of Medical Pathology (ACMG/AMP) guidelines, in silico evidence is applied at the supporting strength level for pathogenic (PP3) and benign (BP4) evidence. Although PP3 is commonly used, less is known about the effect of these criteria on variant classification outcomes. METHODS: A total of 727 missense variants curated by Clinical Genome Resource expert groups were analyzed to determine how often PP3 and BP4 were applied and their impact on variant classification. The ACMG/AMP categorical system of variant classification was compared with a quantitative point-based system. The pathogenicity likelihood ratios of REVEL, VEST, FATHMM, and MPC were calibrated using a gold standard set of 237 pathogenic and benign variants (classified independent of the PP3/BP4 criteria). RESULTS: The PP3 and BP4 criteria were applied by Variant Curation Expert Panels to 55% of missense variants. Application of those criteria changed the classification of 15% of missense variants for which either criterion was applied. The point-based system resolved borderline classifications. REVEL and VEST performed best at a strength level consistent with moderate evidence. CONCLUSION: We show that in silico criteria are commonly applied and often affect the final variant classifications. When appropriate thresholds for in silico predictors are established, our results show that PP3 and BP4 can be used at a moderate strength.

Identification of a novel pathogenic variant in PALB2 and BARD1 genes by a multigene sequencing panel in triple negative breast cancer in Morocco.

Pathogenic variants (PVs) in BRCA genes have been mainly associated with an increasing risk of triple negative breast cancer (TNBC). The contribution of PVs in non-BRCA genes to TNBC seems likely since the processing of homologous recombination repair of double-strand DNA breaks involves several genes. Here, we investigate the susceptibility of genetic variation of the BRCA and non-BRCA genes in 30 early-onset Moroccan women with TNBC. Methods: Targeted capture-based next generation sequencing (NGS) method was performed with a multigene panel testing (MGPT) for variant screening. Panel sequencing was performed with genes involved in hereditary predisposition to cancer and candidate genes whose involvement remains unclear using Illumina MiSeq platform. Interpretation was conducted by following the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) criteria. Results: PVs were identified in 20% (6/30) of patients with TNBC. Of these, 16.7% (5/30) carried a BRCA PV [10% (3/30) in BRCA1, 6.7% (2/30) in BRCA2] and 6.6% (2/30) carried a non-BRCA PV. The identified PVs in BRCA genes (BRCA1 c.798_799delTT, BRCA1 c.3279delC, BRCA2 c.1310_1313del, and BRCA2 c.1658T>G) have been reported before and were classified as pathogenic. The identified founder PVs BRCA1 c.798_799del and BRCA2 c.1310_1313delAAGA represented 10% (3/30). Our MGPT allowed identification of several sequence variations in most investigated genes, among which we found novel truncating variations in PALB2 and BARD1 genes. The PALB2 c.3290dup and BARD1 c.1333G>T variants are classified as pathogenic. We also identified 42 variants of unknown/uncertain significance (VUS) in 70% (21/30) of patients with TNBC, including 50% (21/42) missense variants. The highest VUS rate was observed in ATM (13%, 4/30). Additionally, 35.7% (15/42) variants initially well-known as benign, likely benign or conflicting interpretations of pathogenicity have been reclassified as VUS according to ACMG-AMP. Conclusions: PALB2 and BARD1 along with BRCA genetic screening could be helpful for a larger proportion of early-onset TNBC in Morocco.

APP, PSEN1, and PSEN2 Variants in Alzheimer's Disease: Systematic Re-evaluation According to ACMG Guidelines.

The strategies of classifying APP, PSEN1, and PSEN2 variants varied substantially in the previous studies. We aimed to re-evaluate these variants systematically according to the American college of medical genetics and genomics and the association for molecular pathology (ACMG-AMP) guidelines. In our study, APP, PSEN1, and PSEN2 variants were collected by searching Alzforum and PubMed database with keywords "PSEN1," "PSEN2," and "APP." These variants were re-evaluated based on the ACMG-AMP guidelines. We compared the number of pathogenic/likely pathogenic variants of APP, PSEN1, and PSEN2. In total, 66 APP variants, 323 PSEN1 variants, and 63 PSEN2 variants were re-evaluated in our study. 94.91% of previously reported pathogenic variants were re-classified as pathogenic/likely pathogenic variants, while 5.09% of them were variants of uncertain significance (VUS). PSEN1 carried the most prevalent pathogenic/likely pathogenic variants, followed by APP and PSEN2. Significant statistically difference was identified among these three genes when comparing the number of pathogenic/likely pathogenic variants (P < 2.2 × 10-16). Most of the previously reported pathogenic variants were re-classified as pathogenic/likely pathogenic variants while the others were re-evaluated as VUS, highlighting the importance of interpreting APP, PSEN1, and PSEN2 variants with caution according to ACMG-AMP guidelines.

Cancer SIGVAR: A semiautomated interpretation tool for germline variants of hereditary cancer-related genes.

Cancer is one of the most important health issues globally and the accuracy of interpretation of cancer-related variants is critical for the clinical management of hereditary cancer. ClinGen Sequence Variant Interpretation Working Groups have developed many adaptations of American College of Medical Genetics and Genomics and the Association of Molecular Pathologists guidelines to improve the consistency of interpretation. We combined the most recent adaptations to expand the number of the criteria from 28 to 48 and developed a tool called Cancer SIGVAR to help genetic counselors interpret the clinical significance of cancer germline variants. Our tool can accept VCF files as input and realize fully automated interpretation based on 21 criteria and semiautomated interpretation based on 48 criteria. We validated the performance of our tool with the ClinVar and CLINVITAE benchmark databases, achieving an average consistency for pathogenic and benign assessment up to 93.71% and 79.38%, respectively. We compared Cancer SIGVAR with two similar tools, InterVar and PathoMAN, and analyzed the main differences in criteria and implementation. Furthermore, we selected 911 variants from another two in-house benchmark databases, and semiautomated interpretation reached an average classification consistency of 98.35%. Our findings highlight the need to optimize automated interpretation tools based on constantly updated guidelines. Cancer SIGVAR is publicly available at http://cancersigvar.bgi.com/.

Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls.

PURPOSE: Stringent variant interpretation guidelines can lead to high rates of variants of uncertain significance (VUS) for genetically heterogeneous disease like long QT syndrome (LQTS) and Brugada syndrome (BrS). Quantitative and disease-specific customization of American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines can address this false negative rate. METHODS: We compared rare variant frequencies from 1847 LQTS (KCNQ1/KCNH2/SCN5A) and 3335 BrS (SCN5A) cases from the International LQTS/BrS Genetics Consortia to population-specific gnomAD data and developed disease-specific criteria for ACMG/AMP evidence classes-rarity (PM2/BS1 rules) and case enrichment of individual (PS4) and domain-specific (PM1) variants. RESULTS: Rare SCN5A variant prevalence differed between European (20.8%) and Japanese (8.9%) BrS patients (p = 5.7 × 10-18) and diagnosis with spontaneous (28.7%) versus induced (15.8%) Brugada type 1 electrocardiogram (ECG) (p = 1.3 × 10-13). Ion channel transmembrane regions and specific N-terminus (KCNH2) and C-terminus (KCNQ1/KCNH2) domains were characterized by high enrichment of case variants and >95% probability of pathogenicity. Applying the customized rules, 17.4% of European BrS and 74.8% of European LQTS cases had (likely) pathogenic variants, compared with estimated diagnostic yields (case excess over gnomAD) of 19.2%/82.1%, reducing VUS prevalence to close to background rare variant frequency. CONCLUSION: Large case-control data sets enable quantitative implementation of ACMG/AMP guidelines and increased sensitivity for inherited arrhythmia genetic testing.

The role of clinical response to treatment in determining pathogenicity of genomic variants.

PURPOSE: The 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants provide a framework to standardize terminology in the classification of variants uncovered through genetic testing. We aimed to assess the validity of utilizing clinical response to therapies specifically targeted to a suspected disease in clarifying variant pathogenicity. METHODS: Five families with disparate clinical presentations and different genetic diseases evaluated and treated in multiple diagnostic settings are summarized. RESULTS: Extended evaluations indicated possible genetic diagnoses and assigned candidate causal variants, but the cumulative clinical, biochemical, and molecular information in each instance was not completely consistent with the identified disease. Initiation of treatment specific to the suspected diagnoses in the affected individuals led to clinical improvement in all five families. CONCLUSION: We propose that the effect of therapies that are specific and targeted to treatable genetic diseases embodies an in vivo physiological response and could be considered as additional criteria within the 2015 ACMG/AMP guidelines in determining genomic variant pathogenicity.

Integration of functional assay data results provides strong evidence for classification of hundreds of BRCA1 variants of uncertain significance.

PURPOSE: BRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification. METHODS: We have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification. RESULTS: Integration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach. CONCLUSION: High quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.

A Collaborative Effort to Define Classification Criteria for ATM Variants in Hereditary Cancer Patients.

BACKGROUND: Gene panel testing by massive parallel sequencing has increased the diagnostic yield but also the number of variants of uncertain significance. Clinical interpretation of genomic data requires expertise for each gene and disease. Heterozygous ATM pathogenic variants increase the risk of cancer, particularly breast cancer. For this reason, ATM is included in most hereditary cancer panels. It is a large gene, showing a high number of variants, most of them of uncertain significance. Hence, we initiated a collaborative effort to improve and standardize variant classification for the ATM gene. METHODS: Six independent laboratories collected information from 766 ATM variant carriers harboring 283 different variants. Data were submitted in a consensus template form, variant nomenclature and clinical information were curated, and monthly team conferences were established to review and adapt American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria to ATM, which were used to classify 50 representative variants. RESULTS: Amid 283 different variants, 99 appeared more than once, 35 had differences in classification among laboratories. Refinement of ACMG/AMP criteria to ATM involved specification for twenty-one criteria and adjustment of strength for fourteen others. Afterwards, 50 variants carried by 254 index cases were classified with the established framework resulting in a consensus classification for all of them and a reduction in the number of variants of uncertain significance from 58% to 42%. CONCLUSIONS: Our results highlight the relevance of data sharing and data curation by multidisciplinary experts to achieve improved variant classification that will eventually improve clinical management.

A multi-dimensional analysis of genotype-phenotype discordance in malignant hyperthermia susceptibility.

BACKGROUND: Malignant hyperthermia (MH) susceptibility is an inherited condition, diagnosed either by the presence of a pathogenic genetic variant or by in vitro caffeine-halothane contracture testing. Through a multi-dimensional approach, we describe the implications of discordance between genetic and in vitro test results in a patient with a family history of possible MH. METHODS: The patient, whose brother had a possible MH reaction, underwent the caffeine-halothane contracture test (CHCT) according to the North American MH Group protocol. Screening of the complete RYR1 and CACNA1S transcripts was done using Sanger sequencing. Additional functional analyses included skinned myofibre calcium-induced calcium release sensitivity, calcium signalling assays in cultured myotubes, and in silico evaluation of the effect of any genetic variants on their chemical environment. RESULTS: The patient's CHCT result was negative but she carried an RYR1 variant c.1209C>G, p.Ile403Met, that is listed as pathogenic by the European Malignant Hyperthermia Group. Functional tests indicated a gain-of-function effect with a weak impact, and the variant was predicted to affect the folding stability of the 3D structure of the RyR1 protein. Based on American College of Medical Genetics and Genomics/Association of Molecular Pathology guidelines, this variant would be characterised as a variant of uncertain significance. CONCLUSIONS: Available data do not confirm or exclude an increased risk of MH for this patient. Further research is needed to correlate RyR1 functional assays, including the current gold standard testing for MH susceptibility, with clinical phenotypes. The pathogenicity of genetic variants associated with MH susceptibility should be re-evaluated.