The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel.

PURPOSE: Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS: The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS: The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION: Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

Scaling resolution of variant classification differences in ClinVar between 41 clinical laboratories through an outlier approach.

ClinVar provides open access to variant classifications shared from many clinical laboratories. Although most classifications are consistent across laboratories, classification differences exist. To facilitate resolution of classification differences on a large scale, clinical laboratories were encouraged to reassess outlier classifications of variants with medically significant differences (MSDs). Outliers were identified by first comparing ClinVar submissions from 41 clinical laboratories to detect variants with MSDs between the laboratories (650 variants). Next, MSDs were filtered for variants with ≥3 classifications (244 variants), of which 87.6% (213 variants) had a majority consensus in ClinVar, thus allowing for identification of outlier classifications in need of reassessment. Laboratories with outlier classifications were sent a custom report and encouraged to reassess variants. Results were returned for 204 (96%) variants, of which 62.3% (127) were resolved. Of those 127, 64.6% (82) were resolved due to reassessment prompted by this study and 35.4% (45) resolved by a previously completed reassessment. This study demonstrates a scalable approach to classification resolution and capitalizes on the value of data sharing within ClinVar. These activities will help the community move toward more consistent variant classifications, which will improve the care of patients with, or at risk for, genetic disorders.

Evaluation of a 27-gene inherited cancer panel across 630 consecutive patients referred for testing in a clinical diagnostic laboratory.

BACKGROUND: Extensive clinical and genetic heterogeneity of inherited cancers has allowed multi-gene panel testing to become an efficient means for identification of patients with an inherited predisposition to a broad spectrum of syndromic and nonsyndromic forms of cancer. This study reports our experience with a 27-gene inherited cancer panel on a cohort of 630 consecutive individuals referred for testing at our laboratory with the following objectives: 1. Determine the rates for positive cases and those with variants of uncertain clinical significance (VUS) relative to data published in the recent literature, 2. Examine heterogeneity among the constituent genes on the panel, and 3. Review test uptake in the cohort relative to other reports describing outcomes for expanded panel testing. METHODS: Clinical and genomic data were reviewed on 630 individuals tested on a panel of 27 genes selected on the basis of high (≥ 40%) or moderate to low (≤ 40%) lifetime risk of hereditary cancer. These patients were not enriched for adherence to the National Comprehensive Cancer Network (NCCN) criteria for Hereditary Breast and Ovarian Cancer (HBOC) or Lynch Syndrome (LS) and constitute a referral laboratory cohort. RESULTS: Sixty-five individuals with variants classified as pathogenic or likely pathogenic across 14 genes were identified for an overall positive rate of 10.3%. Although a family history of cancer constituted a major reason for referral, accounting for 84% of our cohort, excluding patients with a known familial variant did not have a significant impact on the observed positive rate (9% vs 10.3%). More than half (58%) of the pathogenic or likely pathogenic variants were observed in high or moderate to low risk genes on the panel, while only 42% occurred in classic HBOC or LS-associated genes. CONCLUSION: These results provide the actual percentage of family or personal history of cancer that can be attributed to pathogenic or likely pathogenic variants in one or more of the genes on our panel and corroborate the utility of multi-gene panels over sequential testing to identify individuals with an inherited predisposition to cancer.

BRCA Share: A Collection of Clinical BRCA Gene Variants.

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

Exploring the landscape of pathogenic genetic variation in the ExAC population database: insights of relevance to variant classification.

PURPOSE: We evaluated the Exome Aggregation Consortium (ExAC) database as a control cohort to classify variants across a diverse set of genes spanning dominant and recessively inherited disorders. METHODS: The frequency of pathogenic variants in ExAC was compared with the estimated maximal pathogenic allele frequency (MPAF), based on the disease prevalence, penetrance, inheritance, allelic and locus heterogeneity of each gene. Additionally, the observed carrier frequency and the ethnicity-specific variant distribution were compared between ExAC and the published literature. RESULTS: The carrier frequency and ethnic distribution of pathogenic variants in ExAC were concordant with reported estimates. Of 871 pathogenic/likely pathogenic variants across 19 genes, only 3 exceeded the estimated MPAF. Eighty-four percent of variants with ExAC frequencies above the estimated MPAF were classified as "benign." Additionally, 20% of the cardiac and 19% of the Lynch syndrome gene variants originally classified as "VUS" occurred with ExAC frequencies above the estimated MPAF, making these suitable for reassessment. CONCLUSIONS: The ExAC database is a useful source for variant classification and is not overrepresented for pathogenic variants in the genes evaluated. However, the mutational spectrum, pseudogenes, genetic heterogeneity, and paucity of literature should be considered in deriving meaningful classifications using ExAC.Genet Med 18 8, 850-854.

Clinical characterization of the mutational landscape of 24,639 real-world samples from patients with myeloid malignancies.

Myeloid neoplasms represent a broad spectrum of hematological disorders for which somatic mutation status in key driver genes is important for diagnosis, prognosis and treatment. Here we summarize the findings of a targeted, next generation sequencing laboratory developed test in 24,639 clinical myeloid samples. Data were analyzed comprehensively and as part of individual cohorts specific to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Overall, 48,015 variants were detected, and variants were found in all 50 genes in the panel. The mean number of mutations per patient was 1.95. Mutation number increased with age (Spearman's rank correlation coefficient, ρ = 0.29, P < 0.0001) and was higher in patients with AML than MDS or MPN (Student's t-test, P < 0.0001). TET2 was the most common mutation detected (19.1% of samples; 4,695/24,639) including 7.7% (1,908/24,639) with multi-hit TET2 mutations. Mutation frequency was correlated between patients with cytopenias and MDS (Spearman's, ρ = 0.97, P < 2.2×10-16) with the MDS diagnostic gene SF3B1 being the only notable outlier. This large retrospective study shows the utility of NGS testing to inform clinical decisions during routine clinical care and highlights the mutational landscape of a broad population of myeloid patients.

Technical standards and guidelines for spinal muscular atrophy testing.

Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron (SMN1) gene, affecting approximately 1 in 10,000 live births. The disease is characterized by progressive symmetrical muscle weakness resulting from the degeneration and loss of anterior horn cells in the spinal cord and brainstem nuclei. The disease is classified on the basis of age of onset and clinical course. Two almost identical SMN genes are present on 5q13: the SMN1 gene, which is the spinal muscular atrophy-determining gene, and the SMN2 gene. The homozygous absence of the SMN1 exon 7 has been observed in the majority of patients and is being used as a reliable and sensitive spinal muscular atrophy diagnostic test. Although SMN2 produces less full-length transcript than SMN1, the number of SMN2 copies has been shown to modulate the clinical phenotype. Carrier detection relies on the accurate determination of the SMN1 gene copies. This document follows the outline format of the general Standards and Guidelines for Clinical Laboratories. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and methods of analysis.

Cystic fibrosis carrier testing in an ethnically diverse US population.

BACKGROUND: The incidence of cystic fibrosis (CF) and the frequency of specific disease-causing mutations vary among populations. Affected individuals experience a range of serious clinical consequences, notably lung and pancreatic disease, which are only partially dependent on genotype. METHODS: An allele-specific primer-extension reaction, liquid-phase hybridization to a bead array, and subsequent fluorescence detection were used in testing for carriers of 98 CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations among 364 890 referred individuals with no family history of CF. RESULTS: One in 38 individuals carried one of the 98 CFTR mutations included in this panel. Of the 87 different mutations detected, 18 were limited to a single ethnic group. African American, Hispanic, and Asian individuals accounted for 33% of the individuals tested. The mutation frequency distribution of Caucasians was significantly different from that of each of these ethnic groups (P < 1 × 10⁻¹°). CONCLUSIONS: Carrier testing using a broad mutation panel detects differences in the distribution of mutations among ethnic groups in the US.

REEP1 mutation spectrum and genotype/phenotype correlation in hereditary spastic paraplegia type 31.

Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for REEP1 mutations and copy number variations. We identified 13 novel and 2 known REEP1 mutations in 16 familial and sporadic patients by direct sequencing analysis. Twelve out of 16 mutations were small insertions, deletions or splice site mutations. These changes would result in shifts of the open-reading-frame followed by premature termination of translation and haploinsufficiency. Interestingly, we identified two disease associated variations in the 3'-UTR of REEP1 that fell into highly conserved micro RNA binding sites. Copy number variation analysis in a subset of 133 HSP index patients revealed a large duplication of REEP1 that involved exons 2-7 in an Irish family. Clinically most SPG31 patients present with a pure spastic paraplegia; rare complicating features were restricted to symptoms or signs of peripheral nerve involvement. Interestingly, the distribution of age at onset suggested a bimodal pattern with the appearance of initial symptoms of disease either before the age of 20 years or after the age of 30 years. The overall mutation rate in our clinically heterogeneous sample was 3.0%; however, in the sub-sample of pure HSP REEP1 mutations accounted for 8.2% of all patients. These results firmly establish REEP1 as a relatively frequent autosomal dominant HSP gene for which genetic testing is warranted. We also establish haploinsufficiency as the main molecular genetic mechanism in SPG31, which should initiate and guide functional studies on REEP1 with a focus on loss-of-function mechanisms. Our results should be valid as a reference for mutation frequency, spectrum of REEP1 mutations, and clinical phenotypes associated with SPG31.

Ovarian failure in ataxia with oculomotor apraxia type 2.

Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive disorder associated with mutations in the Senataxin (SETX) gene. Clinical manifestations (ataxia, peripheral neuropathy, oculomotor apraxia) of this disease have previously been limited to the nervous system. We describe a patient homozygous for a novel mutation of SETX who manifested not only ataxia but also ovarian failure.

Spinal Muscular Atrophy: Overview of Molecular Diagnostic Approaches.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease and the most common genetic cause of infant mortality, affecting ∼1 in 10,000 live births. The disease is characterized by progressive symmetrical muscle weakness resulting from the degeneration and loss of anterior horn cells in the spinal cord and brain stem nuclei. The disease is classified on the basis of age of onset and clinical course. SMA is caused by mutations in the telomeric copy of the survival motor neuron 1 (SMN1) gene, but all patients retain a centromeric copy of the gene, SMN2. The homozygous absence of the SMN1 exon 7 has been observed in the majority of patients and is being utilized as a reliable and sensitive SMA diagnostic test. In the majority of cases, the disease severity correlates inversely with an increased SMN2 gene copy number. Carrier detection, in the deletion cases, relies on the accurate determination of the SMN1 gene copies. Since SMA is one of the most common lethal genetic disorders, with a carrier frequency of 1 in 40 to 1 in 60, direct carrier dosage testing has been beneficial to many families. This unit attempts to highlight the molecular genetics of SMA with a focus on the advantages and limitations of the current molecular technologies.

Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens.

Spinal muscular atrophy (SMA) is a leading inherited cause of infant death with a reported incidence of ~1 in 10,000 live births and is second to cystic fibrosis as a common, life-shortening autosomal recessive disorder. The American College of Medical Genetics has recommended population carrier screening for SMA, regardless of race or ethnicity, to facilitate informed reproductive options, although other organizations have cited the need for additional large-scale studies before widespread implementation. We report our data from carrier testing (n = 72,453) and prenatal diagnosis (n = 121) for this condition. Our analysis of large-scale population carrier screening data (n = 68,471) demonstrates the technical feasibility of high throughput testing and provides mutation carrier and allele frequencies at a level of accuracy afforded by large data sets. In our United States pan-ethnic population, the calculated a priori carrier frequency of SMA is 1/54 with a detection rate of 91.2%, and the pan-ethnic disease incidence is calculated to be 1/11,000. Carrier frequency and detection rates provided for six major ethnic groups in the United States range from 1/47 and 94.8% in the Caucasian population to 1/72 and 70.5% in the African American population, respectively. This collective experience can be utilized to facilitate accurate pre- and post-test counseling in the settings of carrier screening and prenatal diagnosis for SMA.

Opportunities and challenges associated with clinical diagnostic genome sequencing: a report of the Association for Molecular Pathology.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.

Laboratory guidelines for detection, interpretation, and reporting of maternal cell contamination in prenatal analyses a report of the association for molecular pathology.

This document summarizes laboratory guidelines for the detection, interpretation, and reporting of maternal cell contamination in prenatal analyses.

Population carrier screening for spinal muscular atrophy a position statement of the association for molecular pathology.

Spinal muscular atrophy is a common and often fatal autosomal recessive disorder for which carrier screening is available. The Association for Molecular Pathology has evaluated recent opinions regarding population carrier screening, reviewed the current literature, and developed a position statement that includes specific recommendations addressing both diagnostic and practical issues that affect implementation.

Fragile X premutation in a woman with cognitive impairment, tremor, and history of premature ovarian failure.

Few women with Fragile X tremor ataxia syndrome (FXTAS) have been reported. They have milder manifestations at a later age than men. This gender difference may be related to the X inactivation pattern in women. We describe a woman who presented to her geriatrician with poor memory and was found to have ataxia and tremor. Additional queries yielded history of premature ovarian failure. Genetic testing showed heterozygous fragile X mental retardation gene premutation with 103 CGG repeats in the abnormal allele and 31 CGG repeats in the normal allele. Also, the X inactivation pattern was skewed with the active X chromosome predominantly having the premutation allele. We believe that FXTAS is more common in women than is generally thought and that many such patients masquerade as dementia of old age. Action tremor and ataxia associated with a history suggestive of premature ovarian failure should raise suspicions for FXTAS.

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids.

The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273-279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis. These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells.

The frequency of short-chain acyl-CoA dehydrogenase gene variants in the US population and correlation with the C(4)-acylcarnitine concentration in newborn blood spots.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a clinically heterogeneous disorder. The clinical phenotype varies from fatal metabolic decompensation in early life to subtle adult onset, some patients remain asymptomatic. Two mutations (511C>T; 625G>A) have been described in exons 5 and 6 of the SCAD gene. Although they alter the structural and catalytic properties of the SCAD protein, these variants are not true disease-causing mutations but confer disease susceptibility. Previous studies found these gene variants to be common in Europeans. We aimed to establish the frequency of these variants in the US population and to determine whether the presence of these variants correlates with elevated butyrylcarnitine (C(4)-acylcarnitine) concentrations in newborn blood spots. Based on the analysis of 694 samples, we found that the allele frequency of the 625G>A variant was significantly higher (22%) than that of the 511C>T variant (3%). These gene variants were detected in either homozygous or compound heterozygous form in 7% of the study population. Additionally, the frequency of the 625G>A allele in the Hispanic population (30%) was significantly higher than that of the African-American (9%) and Asian (13%) subpopulations. A previously unreported variant, IVS 5 (-10) C>T, was identified in three African-American newborns (0.3%). The C(4)-acylcarnitine concentration in blood spots was significantly higher in subjects homozygous for the 625A variant when compared to those homozygous for the wild type (p<0.0001). However, none of the observed genotypes was associated with a concentration of C(4)-acylcarnitine that would be consistent with a biochemical diagnosis of SCAD deficiency.