Patterns of mosaicism for sequence and copy-number variants discovered through clinical deep sequencing of disease-related genes in one million individuals.

DNA variants that arise after conception can show mosaicism, varying in presence and extent among tissues. Mosaic variants have been reported in Mendelian diseases, but further investigation is necessary to broadly understand their incidence, transmission, and clinical impact. A mosaic pathogenic variant in a disease-related gene may cause an atypical phenotype in terms of severity, clinical features, or timing of disease onset. Using high-depth sequencing, we studied results from one million unrelated individuals referred for genetic testing for almost 1,900 disease-related genes. We observed 5,939 mosaic sequence or intragenic copy number variants distributed across 509 genes in nearly 5,700 individuals, constituting approximately 2% of molecular diagnoses in the cohort. Cancer-related genes had the most mosaic variants and showed age-specific enrichment, in part reflecting clonal hematopoiesis in older individuals. We also observed many mosaic variants in genes related to early-onset conditions. Additional mosaic variants were observed in genes analyzed for reproductive carrier screening or associated with dominant disorders with low penetrance, posing challenges for interpreting their clinical significance. When we controlled for the potential involvement of clonal hematopoiesis, most mosaic variants were enriched in younger individuals and were present at higher levels than in older individuals. Furthermore, individuals with mosaicism showed later disease onset or milder phenotypes than individuals with non-mosaic variants in the same genes. Collectively, the large compendium of variants, disease correlations, and age-specific results identified in this study expand our understanding of the implications of mosaic DNA variation for diagnosis and genetic counseling.

Scalable detection of technically challenging variants through modified next-generation sequencing.

BACKGROUND: Some clinically important genetic variants are not easily evaluated with next-generation sequencing (NGS) methods due to technical challenges arising from high- similarity copies (e.g., PMS2, SMN1/SMN2, GBA1, HBA1/HBA2, CYP21A2), repetitive short sequences (e.g., ARX polyalanine repeats, FMR1 AGG interruptions in CGG repeats, CFTR poly-T/TG repeats), and other complexities (e.g., MSH2 Boland inversions). METHODS: We customized our NGS processes to detect the technically challenging variants mentioned above with adaptations including target enrichment and bioinformatic masking of similar sequences. Adaptations were validated with samples of known genotypes. RESULTS: Our adaptations provided high-sensitivity and high-specificity detection for most of the variants and provided a high-sensitivity primary assay to be followed with orthogonal disambiguation for the others. The sensitivity of the NGS adaptations was 100% for all of the technically challenging variants. Specificity was 100% for those in PMS2, GBA1, SMN1/SMN2, and HBA1/HBA2, and for the MSH2 Boland inversion; 97.8%-100% for CYP21A2 variants; and 85.7% for ARX polyalanine repeats. CONCLUSIONS: NGS assays can detect technically challenging variants when chemistries and bioinformatics are jointly refined. The adaptations described support a scalable, cost-effective path to identifying all clinically relevant variants within a single sample.

Systematic use of phenotype evidence in clinical genetic testing reduces the frequency of variants of uncertain significance.

Guidelines for variant interpretation include criteria for incorporating phenotype evidence, but this evidence is inconsistently applied. Systematic approaches to using phenotype evidence are needed. We developed a method for curating disease phenotypes as highly or moderately predictive of variant pathogenicity based on the frequency of their association with disease-causing variants. To evaluate this method's accuracy, we retrospectively reviewed variants with clinical classifications that had evolved from uncertain to definitive in genes associated with curated predictive phenotypes. To demonstrate the clinical validity and utility of this approach, we compared variant classifications determined with and without predictive phenotype evidence. The curation method was accurate for 93%-98% of eligible variants. Among variants interpreted using highly predictive phenotype evidence, the percentage classified as pathogenic or likely pathogenic was 80%, compared with 46%-54% had the evidence not been used. Positive results among individuals harboring variants with highly predictive phenotype-guided interpretations would have been missed in 25%-37% of diagnostic tests and 39%-50% of carrier screens had other approaches to phenotype evidence been used. In summary, predictive phenotype evidence associated with specific curated genes can be systematically incorporated into variant interpretation to reduce uncertainty and increase the clinical utility of genetic testing.

Value of genetic testing for pediatric epilepsy: Driving earlier diagnosis of ceroid lipofuscinosis type 2 Batten disease.

This study assessed the effectiveness of genetic testing in shortening the time to diagnosis of late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease. Individuals who received epilepsy gene panel testing through Behind the Seizure® , a sponsored genetic testing program (Cohort A), were compared to children outside of the sponsored testing program during the same period (Cohort B). Two cohorts were analyzed: children aged ≥24 to ≤60 months with unprovoked seizure onset at ≥24 months between December 2016 and January 2020 (Cohort 1) and children aged 0 to ≤60 months at time of testing with unprovoked seizure onset at any age between February 2019 and January 2020 (Cohort 2). The diagnostic yield in Cohort 1A (n = 1814) was 8.4% (n = 153). The TPP1 diagnostic yield within Cohort 1A was 2.9-fold higher compared to Cohort 1B (1.0%, n = 18/1814 vs. .35%, n = 8/2303; p = .0157). The average time from first symptom to CLN2 disease diagnosis was significantly shorter than previously reported (9.8 vs. 22.7 months, p < .001). These findings indicate that facilitated access to early epilepsy gene panel testing helps to increase diagnostic yield for CLN2 disease and shortens the time to diagnosis, enabling earlier intervention.

The global prevalence and ethnic heterogeneity of primary ciliary dyskinesia gene variants: a genetic database analysis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a motile ciliopathy characterised by otosinopulmonary infections. Inheritance is commonly autosomal recessive, with extensive locus and allelic heterogeneity. The prevalence is uncertain. Most genetic studies have been done in North America or Europe. The aim of the study was to estimate the worldwide prevalence and ethnic heterogeneity of PCD. METHODS: We calculated the allele frequency of disease-causing variants in 29 PCD genes associated with autosomal recessive inheritance in 182 681 unique individuals to estimate the global prevalence of PCD in seven ethnicities (African or African American, Latino, Ashkenazi Jewish, Finnish, non-Finnish European, east Asian, and south Asian). We began by aggregating variants that had been interpreted by Invitae, San Francisco, CA, USA, a genetics laboratory with PCD expertise. We then determined the allele frequency of each variant (pathogenic, likely pathogenic, or variant of uncertain significance [VUS]) in the Genome Aggregation Database (gnomAD), a publicly available next-generation sequencing database that aggregates exome and genome sequencing information from a wide variety of large-scale projects and stratifies allele counts by ethnicity. Using the Hardy-Weinberg equilibrium equation, we were able to calculate a lower-end prevalence of PCD for each ethnicity by including only pathogenic and likely pathogenic variants; and upper-end prevalence by also including VUS. This approach was similar to previous work on Li-Fraumeni (TP53 variants) prevalence. We were not diagnosing PCD, but rather estimating prevalence based on known variants. FINDINGS: The overall minimum global prevalence of PCD is calculated to be at least one in 7554 individuals, although this is likely to be an underestimate because some variants currently classified as VUS might be disease-causing and some pathogenic variants might not be detected by our methods. In the overall cohort, Invitae data could be included for variants without gnomAD data for a primary ethnicity. When using only gnomAD allele frequencies to calculate prevalence in individual ethnicities, the estimated prevalence of PCD was lower in each ethnicity compared with the overall cohort. This is because the overall cohort includes additional data from the Invitae database such as copy number variants and other variants not present in gnomAD. With gnomAD we found the expected PCD frequency to be higher in individuals of African ancestry than in most other populations (excluding VUS: 1 in 9906 in African or African American vs 1 in 10 388 in non-Finnish European vs 1 in 14 606 in east Asian vs 1 in 16 309 in Latino; including VUS: 1 in 106 in African or African American vs 1 in 178 in non-Finnish European vs 1 in 196 in Latino vs 1 in 188 in east Asian). In addition, we found that the top 5 genes most commonly implicated in PCD differed across ethnic ancestries and contrasted commonly published findings. INTERPRETATION: PCD appears to be more common than has been recognised, particularly in individuals of African ancestry. We identified gene distributions that differ from those in previous European and North American studies. These results could have an international impact on case identification. Our analytic approach can be expanded as more PCD loci are identified, and could be adapted to study the prevalence of other inherited diseases. FUNDING: None.

Multigene Panel Testing in a Large Cohort of Adults With Epilepsy: Diagnostic Yield and Clinically Actionable Genetic Findings.

BACKGROUND AND OBJECTIVES: Although genetic testing among children with epilepsy has demonstrated clinical utility and become a part of routine testing, studies in adults are limited. This study reports the diagnostic yield of genetic testing in adults with epilepsy. METHODS: Unrelated individuals aged 18 years and older who underwent diagnostic genetic testing for epilepsy using a comprehensive, next-generation sequencing-based, targeted gene panel (range 89-189 genes) were included in this cross-sectional study. Clinical information, provided at the discretion of the ordering clinician, was reviewed and analyzed. Diagnostic yield was calculated for all individuals including by age at seizure onset and comorbidities based on clinician-reported information. The proportion of individuals with clinically actionable genetic findings, including instances when a specific treatment would be indicated or contraindicated due to a diagnostic finding, was calculated. RESULTS: Among 2,008 individuals, a diagnostic finding was returned for 218 adults (10.9%), with clinically actionable findings in 55.5% of diagnoses. The highest diagnostic yield was in adults with seizure onset during infancy (29.6%, 0-1 year), followed by in early childhood (13.6%, 2-4 years), late childhood (7.0%, 5-10 years), adolescence (2.4%, 11-17 years), and adulthood (3.7%, ≥18 years). Comorbid intellectual disability (ID) or developmental delay resulted in a high diagnostic yield (16.0%), most notably for females (19.6% in females vs 12.3% in males). Among individuals with pharmacoresistant epilepsy, 13.5% had a diagnostic finding, and of these, 57.4% were clinically actionable genetic findings. DISCUSSION: These data reinforce the utility of genetic testing for adults with epilepsy, particularly for those with childhood-onset seizures, ID, and pharmacoresistance. This is an important consideration due to longer survival and the complexity of the transition from pediatric to adult care. In addition, more than half of diagnostic findings in this study were considered clinically actionable, suggesting that genetic testing could have a direct impact on clinical management and outcomes.

Re-evaluating cancer risks associated with the CHEK2 p.Ser428Phe Ashkenazi Jewish founder pathogenic variant.

A missense variant (p.Ser428Phe [S428F]) in the CHEK2 gene is reportedly associated with a 2-3 fold increase in breast cancer risk in Ashkenazi Jews. This study aimed to re-evaluate cancer risks conferred by the CHEK2 S428F variant in Ashkenazi Jews. De-identified data from CHEK2 S428F variant carriers sequenced with multigene panels were analyzed. Overall, 486/341,531 (0.14%) cases of all ethnicities diagnosed with any cancer type were CHEK2 S428F carriers, of whom 243/9980 self-identified as Ashkenazi Jews and carried this risk variant only. Compared with ethnically matched non-cancer controls, across all cancer cases, this variant was not more prevalent (p = 0.271). Specifically, variant prevalence was not different in breast cancer cases compared with controls. Though the variant was shown to be enriched in pancreatic cancer cases (p = 0.008), sample size was small. The CHEK2 S428F variant was not overrepresented in Ashkenazi Jews with breast cancer and most other cancer types analyzed, except for pancreatic cancer, compared with ethnically matched non- cancer controls. These findings should prompt reevaluating ethnic-specific CHEK2 S428F cancer attributable risk.

Novel PHEX gene locus-specific database: Comprehensive characterization of vast number of variants associated with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemia, is caused by disrupting variants in the PHEX gene, located on the X chromosome. XLH is inherited in an X-linked pattern with complete penetrance observed for both males and females. Patients experience lifelong symptoms resulting from chronic hypophosphatemia, including impaired bone mineralization, skeletal deformities, growth retardation, and diminished quality of life. This chronic condition requires life-long management with disease-specific therapies, which can improve patient outcomes especially when initiated early in life. To centralize and disseminate PHEX variant information, we have established a new PHEX gene locus-specific database, PHEX LSDB. As of April 30, 2021, 870 unique PHEX variants, compiled from an older database of PHEX variants, a comprehensive literature search, a sponsored genetic testing program, and XLH clinical trials, are represented in the PHEX LSDB. This resource is publicly available on an interactive, searchable website (https://www.rarediseasegenes.com/), which includes a table of variants and associated data, graphical/tabular outputs of genotype-phenotype analyses, and an online submission form for reporting new PHEX variants. The database will be updated regularly with new variants submitted on the website, identified in the published literature, or shared from genetic testing programs.

Comprehensive Genetic Testing for Pediatric Hypertrophic Cardiomyopathy Reveals Clinical Management Opportunities and Syndromic Conditions.

Hypertrophic cardiomyopathy (HCM) has historically been diagnosed phenotypically. Through genetic testing, identification of a molecular diagnosis (MolDx) is increasingly common but the impact on pediatric patients is unknown. This was a retrospective study of next-generation sequencing data for 602 pediatric patients with a clinician-reported history of HCM. Diagnostic yield was stratified by gene and self-reported race/ethnicity. A MolDx of HCM was identified in 242 (40%) individuals. Sarcomeric genes were the highest yielding, but pathogenic and/or likely pathogenic (P/LP) variants in syndromic genes were found in 36% of individuals with a MolDx, often in patients without documented clinical suspicion for a genetic syndrome. Among all MolDx, 73% were in genes with established clinical management recommendations and 2.9% were in genes that conferred eligibility for clinical trial enrollment. Black patients were the least likely to receive a MolDx. In the current era, genetic testing can impact management of HCM, beyond diagnostics or prognostics, through disease-specific guidelines or clinical trial eligibility. Genetic testing frequently can help identify syndromes in patients for whom syndromes may not be suspected. These findings highlight the importance of pursuing broad genetic testing, independent of suspicion based on phenotype. Lower rates of MolDx in Black patients may contribute to health inequities. Further research is needed evaluating the genetics of HCM in underrepresented/underserved populations. Additionally, research related to the impact of genetic testing on clinical management of other diseases is warranted.

Molecular Diagnoses of X-Linked and Other Genetic Hypophosphatemias: Results From a Sponsored Genetic Testing Program.

X-linked hypophosphatemia (XLH), a dominant disorder caused by pathogenic variants in the PHEX gene, affects both sexes of all ages and results in elevated serum fibroblast growth factor 23 (FGF23) and below-normal serum phosphate. In XLH, rickets, osteomalacia, short stature, and lower limb deformity may be present with muscle pain and/or weakness/fatigue, bone pain, joint pain/stiffness, hearing difficulty, enthesopathy, osteoarthritis, and dental abscesses. Invitae and Ultragenyx collaborated to provide a no-charge sponsored testing program using a 13-gene next-generation sequencing panel to confirm clinical XLH or aid diagnosis of suspected XLH/other genetic hypophosphatemia. Individuals aged ≥6 months with clinical XLH or suspected genetic hypophosphatemia were eligible. Of 831 unrelated individuals tested between February 2019 and June 2020 in this cross-sectional study, 519 (62.5%) individuals had a pathogenic or likely pathogenic variant in PHEX (PHEX-positive). Among the 312 PHEX-negative individuals, 38 received molecular diagnoses in other genes, including ALPL, CYP27B1, ENPP1, and FGF23; the remaining 274 did not have a molecular diagnosis. Among 319 patients with a provider-reported clinical diagnosis of XLH, 88.7% (n = 283) had a reportable PHEX variant; 81.5% (n = 260) were PHEX-positive. The most common variant among PHEX-positive individuals was an allele with both the gain of exons 13-15 and c.*231A>G (3'UTR variant) (n = 66/519). Importantly, over 80% of copy number variants would have been missed by traditional microarray analysis. A positive molecular diagnosis in 41 probands (4.9%; 29 PHEX positive, 12 non-PHEX positive) resulted in at least one family member receiving family testing. Additional clinical or family member information resulted in variant(s) of uncertain significance (VUS) reclassification to pathogenic/likely pathogenic (P/LP) in 48 individuals, highlighting the importance of segregation and clinical data. In one of the largest XLH genetic studies to date, 65 novel PHEX variants were identified and a high XLH diagnostic yield demonstrated broad insight into the genetic basis of XLH. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

ATP1A3-Encoded Sodium-Potassium ATPase Subunit Alpha 3 D801N Variant Is Associated With Shortened QT Interval and Predisposition to Ventricular Fibrillation Preceded by Bradycardia.

Background Pathogenic variation in the ATP1A3-encoded sodium-potassium ATPase, ATP1A3, is responsible for alternating hemiplegia of childhood (AHC). Although these patients experience a high rate of sudden unexpected death in epilepsy, the pathophysiologic basis for this risk remains unknown. The objective was to determine the role of ATP1A3 genetic variants on cardiac outcomes as determined by QT and corrected QT (QTc) measurements. Methods and Results We analyzed 12-lead ECG recordings from 62 patients (male subjects=31, female subjects=31) referred for AHC evaluation. Patients were grouped according to AHC presentation (typical versus atypical), ATP1A3 variant status (positive versus negative), and ATP1A3 variant (D801N versus other variants). Manual remeasurements of QT intervals and QTc calculations were performed by 2 pediatric electrophysiologists. QTc measurements were significantly shorter in patients with positive ATP1A3 variant status (P<0.001) than in patients with genotype-negative status, and significantly shorter in patients with the ATP1A3-D801N variant than patients with other variants (P<0.001). The mean QTc for ATP1A3-D801N was 344.9 milliseconds, which varied little with age, and remained <370 milliseconds throughout adulthood. ATP1A3 genotype status was significantly associated with shortened QTc by multivariant regression analysis. Two patients with the ATP1A3-D801N variant experienced ventricular fibrillation, resulting in death in 1 patient. Rare variants in ATP1A3 were identified in a large cohort of genotype-negative patients referred for arrhythmia and sudden unexplained death. Conclusions Patients with AHC who carry the ATP1A3-D801N variant have significantly shorter QTc intervals and an increased likelihood of experiencing bradycardia associated with life-threatening arrhythmias. ATP1A3 variants may represent an independent cause of sudden unexplained death. Patients with AHC should be evaluated to identify risk of sudden death.

Elucidating clinical phenotypic variability associated with the polyT tract and TG repeats in CFTR.

Biallelic pathogenic variants in CFTR manifest as cystic fibrosis (CF) or other CFTR-related disorders (CFTR-RDs). The 5T allele, causing alternative splicing and reduced protein activity, is modulated by the adjacent TG repeat element, though previous data have been limited to small, selective cohorts. Here, the risk and spectrum of phenotypes associated with the CFTR TG-T5 haplotype variants (TG11T5, TG12T5, and TG13T5) in the absence of the p.Arg117His variant are evaluated. Individuals who received physician-ordered next-generation sequencing of CFTR were included. TG[11-13]T5 variant frequencies (biallelic or with another CF-causing variant [CFvar]) were calculated. Clinical information reported by the ordering provider or the individual was examined. Among 548,300 individuals, the T5 minor allele frequency (MAF) was 4.2% (TG repeat distribution: TG11 = 68.1%, TG12 = 29.5%, TG13 = 2.4%). When present with a CFvar, each TG[11-13]T5 variant was significantly enriched in individuals with a high suspicion of CF or CFTR-RD (personal/family history of CF/CFTR-RD) compared to those with a low suspicion for CF or CFTR-RD (hereditary cancer screening, CFTR not requisitioned). Compared to CFvar/CFvar individuals, those with TG[11-13]T5/CFvar generally had single-organ involvement, milder symptoms, variable expressivity, and reduced penetrance. These data improve our understanding of disease risks associated with TG[11-13]T5 variants and have important implications for reproductive genetic counseling.

PIGG variant pathogenicity assessment reveals characteristic features within 19 families.

PURPOSE: Phosphatidylinositol Glycan Anchor Biosynthesis, class G (PIGG) is an ethanolamine phosphate transferase catalyzing the modification of glycosylphosphatidylinositol (GPI). GPI serves as an anchor on the cell membrane for surface proteins called GPI-anchored proteins (GPI-APs). Pathogenic variants in genes involved in the biosynthesis of GPI cause inherited GPI deficiency (IGD), which still needs to be further characterized. METHODS: We describe 22 individuals from 19 unrelated families with biallelic variants in PIGG. We analyzed GPI-AP surface levels on granulocytes and fibroblasts for three and two individuals, respectively. We demonstrated enzymatic activity defects for PIGG variants in vitro in a PIGG/PIGO double knockout system. RESULTS: Phenotypic analysis of reported individuals reveals shared PIGG deficiency-associated features. All tested GPI-APs were unchanged on granulocytes whereas CD73 level in fibroblasts was decreased. In addition to classic IGD symptoms such as hypotonia, intellectual disability/developmental delay (ID/DD), and seizures, individuals with PIGG variants of null or severely decreased activity showed cerebellar atrophy, various neurological manifestations, and mitochondrial dysfunction, a feature increasingly recognized in IGDs. Individuals with mildly decreased activity showed autism spectrum disorder. CONCLUSION: This in vitro system is a useful method to validate the pathogenicity of variants in PIGG and to study PIGG physiological functions.

Limited-Variant Screening vs Comprehensive Genetic Testing for Familial Hypercholesterolemia Diagnosis.

Importance: Familial hypercholesterolemia (FH) is the most common inherited cardiovascular disease and carries significant morbidity and mortality risks. Genetic testing can identify affected individuals, but some array-based assays screen only a small subset of known pathogenic variants. Objective: To identify the number of clinically significant variants associated with FH that would be missed by an array-based, limited-variant screen when compared with next-generation sequencing (NGS)-based comprehensive testing. Design, Setting, and Participants: This cross-sectional study compared comprehensive genetic test results for clinically significant variants associated with FH with results for a subset of 24 variants screened by a limited-variant array. Data were deidentified next-generation sequencing results from indication-based or proactive gene panels. Individuals receiving next-generation sequencing-based genetic testing, either for an FH indication between November 2015 and June 2020 or as proactive health screening between February 2016 and June 2020 were included. Ancestry was reported by clinicians who could select from preset options or enter free text on the test requisition form. Main Outcomes and Measures: Number of pathogenic or likely pathogenic (P/LP) variants identified. Results: This study included 4563 individuals who were referred for FH diagnostic testing and 6482 individuals who received next-generation sequencing of FH-associated genes as part of a proactive genetic test. Among individuals in the indication cohort, the median (interquartile range) age at testing was 49 (32-61) years, 55.4% (2528 of 4563) were female, and 63.6% (2902 of 4563) were self-reported White/Caucasian. In the indication cohort, the positive detection rate would have been 8.4% (382 of 4563) for a limited-variant screen compared with the 27.0% (1230 of 4563) observed with the next-generation sequencing-based comprehensive test. As a result, 68.9% (848 of 1230) of individuals with a P/LP finding in an FH-associated gene would have been missed by the limited screen. The potential for missed findings in the indication cohort varied by ancestry; among individuals with a P/LP finding, 93.7% (59 of 63) of self-reported Black/African American individuals and 84.7% (122 of 144) of Hispanic individuals would have been missed by the limited-variant screen, compared with 33.3% (4 of 12) of Ashkenazi Jewish individuals. In the proactive cohort, the prevalence of clinically significant FH variants was approximately 1:191 per the comprehensive test, and 61.8% (21 of 34) of individuals with an FH-associated P/LP finding would have been missed by a limited-variant screen. Conclusions and Relevance: Limited-variant screens may falsely reassure the majority of individuals at risk for FH that they do not carry a disease-causing variant, especially individuals of self-reported Black/African American and Hispanic ancestry.

SMA Identified: Clinical and Molecular Findings From a Sponsored Testing Program for Spinal Muscular Atrophy in More Than 2,000 Individuals.

Background: Spinal muscular atrophy (SMA) linked to chromosome 5q is an inherited progressive neuromuscular disorder with a narrow therapeutic window for optimal treatment. Although genetic testing provides a definitive molecular diagnosis that can facilitate access to effective treatments, limited awareness and other barriers may prohibit widespread testing. In this study, the clinical and molecular findings of SMA Identified-a no-charge sponsored next-generation sequencing (NGS)-based genetic testing program for SMA diagnosis-are reported. Methods: Between March 2018 and March 2020, unrelated individuals who had a confirmed or suspected SMA diagnosis or had a family history of SMA were eligible. All individuals underwent diagnostic genetic testing for SMA at clinician discretion. In total, 2,459 individuals were tested and included in this analysis. An NGS-based approach interrogated sequence and copy number of SMN1 and SMN2. Variants were confirmed by multiplex ligation-dependent probe amplification sequencing. Individuals were categorized according to genetic test results: diagnostic (two pathogenic SMN1 variants), nearly diagnostic (SMN1 exon-7 deletion with a variant of uncertain significance [VUS] in SMN1 or SMN2), indeterminate VUS (one VUS in SMN1 or SMN2), carrier (heterozygous SMN1 deletion only), or negative (no pathogenic variants or VUS in SMN1 or SMN2). Diagnostic yield was calculated. Genetic test results were analyzed based on clinician-reported clinical features and genetic modifiers (SMN2 copy number and SMN2 c.859G>C). Results: In total, 2,459 unrelated individuals (mean age 24.3 ± 23.0 years) underwent diagnostic testing. The diagnostic yield for diagnostic plus nearly diagnostic results was 31.3% (n = 771/2,459). Age of onset and clinical presentation varied considerably for individuals and was dependent on SMN2 copy number. Homozygous deletions represented the most common genetic etiology (96.2%), with sequence variants also observed in probands with clinical diagnoses of SMA. Conclusions: Using a high-yield panel test in a no-charge sponsored program early in the diagnostic odyssey may open the door for medical interventions in a substantial number of individuals with SMA. These findings have potential implications for clinical management of probands and their families.

One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation.

PURPOSE: To evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions. METHODS: An interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)-based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients. RESULTS: In the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests. CONCLUSION: The analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.

Spectrum of splicing variants in disease genes and the ability of RNA analysis to reduce uncertainty in clinical interpretation.

The complexities of gene expression pose challenges for the clinical interpretation of splicing variants. To better understand splicing variants and their contribution to hereditary disease, we evaluated their prevalence, clinical classifications, and associations with diseases, inheritance, and functional characteristics in a 689,321-person clinical cohort and two large public datasets. In the clinical cohort, splicing variants represented 13% of all variants classified as pathogenic (P), likely pathogenic (LP), or variants of uncertain significance (VUSs). Most splicing variants were outside essential splice sites and were classified as VUSs. Among all individuals tested, 5.4% had a splicing VUS. If RNA analysis were to contribute supporting evidence to variant interpretation, we estimated that splicing VUSs would be reclassified in 1.7% of individuals in our cohort. This would result in a clinically significant result (i.e., P/LP) in 0.1% of individuals overall because most reclassifications would change VUSs to likely benign. In ClinVar, splicing VUSs were 4.8% of reported variants and could benefit from RNA analysis. In the Genome Aggregation Database (gnomAD), splicing variants comprised 9.4% of variants in protein-coding genes; most were rare, precluding unambiguous classification as benign. Splicing variants were depleted in genes associated with dominant inheritance and haploinsufficiency, although some genes had rare variants at essential splice sites or had common splicing variants that were most likely compatible with normal gene function. Overall, we describe the contribution of splicing variants to hereditary disease, the potential utility of RNA analysis for reclassifying splicing VUSs, and how natural variation may confound clinical interpretation of splicing variants.

Prioritizing genes for systematic variant effect mapping.

MOTIVATION: When rare missense variants are clinically interpreted as to their pathogenicity, most are classified as variants of uncertain significance (VUS). Although functional assays can provide strong evidence for variant classification, such results are generally unavailable. Multiplexed assays of variant effect can generate experimental 'variant effect maps' that score nearly all possible missense variants in selected protein targets for their impact on protein function. However, these efforts have not always prioritized proteins for which variant effect maps would have the greatest impact on clinical variant interpretation. RESULTS: Here, we mined databases of clinically interpreted variants and applied three strategies, each building on the previous, to prioritize genes for systematic functional testing of missense variation. The strategies ranked genes (i) by the number of unique missense VUS that had been reported to ClinVar; (ii) by movability- and reappearance-weighted impact scores, to give extra weight to reappearing, movable VUS and (iii) by difficulty-adjusted impact scores, to account for the more resource-intensive nature of generating variant effect maps for longer genes. Our results could be used to guide systematic functional testing of missense variation toward greater impact on clinical variant interpretation. AVAILABILITY AND IMPLEMENTATION: Source code available at: https://github.com/rothlab/mave-gene-prioritization. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Incorporating Spinal Muscular Atrophy Analysis by Next-Generation Sequencing into a Comprehensive Multigene Panel for Neuromuscular Disorders.

Background: Spinal muscular atrophy (SMA) is traditionally molecularly diagnosed by multiplex ligation-dependent probe amplification or quantitative polymerase chain reaction (qPCR). SMA analyses are not routinely incorporated into gene panel analyses for individuals with suspected SMA or broader neuromuscular indications. Aim: We sought to determine whether a next-generation sequencing (NGS) approach that integrates SMA analyses into a multigene neuromuscular disorders panel could detect undiagnosed SMA. Materials and Methods: Sequence and copy number variants of the SMN1/SMN2 genes were simultaneously analyzed in samples from 5304 unselected individuals referred for testing using an NGS-based 122-gene neuromuscular panel. This diagnostic approach was validated using DNA from 68 individuals who had been previously diagnosed with SMA via quantitative PCR for SMN1/SMN2. Results: Homozygous loss of SMN1 was detected in 47 unselected individuals. Heterozygous loss of SMN1 was detected in 118 individuals; 8 had an indeterminate variant in "SMN1 or SMN2" that supported an SMA diagnosis but required additional disambiguation. Of the remaining SMA carriers, 44 had pathogenic variants in other genes. Concordance rates between NGS and qPCR were 100% and 93% for SMN1 and SMN2 copy numbers, respectively. Where there was disagreement, phenotypes were more consistent with the SMN2 results from NGS. Conclusion: Integrating NGS-based SMA testing into a multigene neuromuscular panel allows a single assay to diagnose SMA while comprehensively assessing the spectrum of variants that can occur in individuals with broad differential diagnoses or nonspecific/overlapping neuromuscular features.