The frequency of a variant in the general population is a key criterion used in the clinical interpretation of sequence variants. With certain exceptions, such as founder mutations, the rarity of a ...variant is a prerequisite for pathogenicity. However, defining the threshold at which a variant should be considered "too common" is challenging and therefore diagnostic laboratories have typically set conservative allele frequency thresholds.
Recent publications of large population sequencing data, such as the Exome Aggregation Consortium (ExAC) database, provide an opportunity to characterize with accuracy and precision the frequency distributions of very rare disease-causing alleles. Allele frequencies of pathogenic variants in ClinVar, as well as variants expected to be pathogenic through the nonsense-mediated decay (NMD) pathway, were analyzed to study the burden of pathogenic variants in 79 genes of clinical importance.
Of 1364 BRCA1 and BRCA2 variants that are well characterized as pathogenic or that are expected to lead to NMD, 1350 variants had an allele frequency of less than 0.0025%. The remaining 14 variants were previously published founder mutations. Importantly, we observed no difference in the distributions of pathogenic variants expected to be lead to NMD compared to those that are not. Therefore, we expanded the analysis to examine the distributions of NMD expected variants in 77 additional genes. These 77 genes were selected to represent a broad set of clinical areas, modes of inheritance, and penetrance. Among these variants, most (97.3%) had an allele frequency of less than 0.01%. Furthermore, pathogenic variants with allele frequencies greater than 0.01% were well characterized in publications and included many founder mutations.
The observations made in this study suggest that, with certain caveats, a very low allele frequency threshold can be adopted to more accurately interpret sequence variants.
Multiple-gene sequencing is entering practice, but its clinical value is unknown. We evaluated the performance of a customized germline-DNA sequencing panel for cancer-risk assessment in a ...representative clinical sample.
Patients referred for clinical BRCA1/2 testing from 2002 to 2012 were invited to donate a research blood sample. Samples were frozen at -80° C, and DNA was extracted from them after 1 to 10 years. The entire coding region, exon-intron boundaries, and all known pathogenic variants in other regions were sequenced for 42 genes that had cancer risk associations. Potentially actionable results were disclosed to participants.
In total, 198 women participated in the study: 174 had breast cancer and 57 carried germline BRCA1/2 mutations. BRCA1/2 analysis was fully concordant with prior testing. Sixteen pathogenic variants were identified in ATM, BLM, CDH1, CDKN2A, MUTYH, MLH1, NBN, PRSS1, and SLX4 among 141 women without BRCA1/2 mutations. Fourteen participants carried 15 pathogenic variants, warranting a possible change in care; they were invited for targeted screening recommendations, enabling early detection and removal of a tubular adenoma by colonoscopy. Participants carried an average of 2.1 variants of uncertain significance among 42 genes.
Among women testing negative for BRCA1/2 mutations, multiple-gene sequencing identified 16 potentially pathogenic mutations in other genes (11.4%; 95% CI, 7.0% to 17.7%), of which 15 (10.6%; 95% CI, 6.5% to 16.9%) prompted consideration of a change in care, enabling early detection of a precancerous colon polyp. Additional studies are required to quantify the penetrance of identified mutations and determine clinical utility. However, these results suggest that multiple-gene sequencing may benefit appropriately selected patients.
We investigated the frequencies and characteristics of intragenic copy-number variants (CNVs) in a deep sampling of disease genes associated with monogenic disorders.
Subsets of 1507 genes were ...tested using next-generation sequencing to simultaneously detect sequence variants and CNVs in >143,000 individuals referred for genetic testing. We analyzed CNVs in gene panels for hereditary cancer syndromes and cardiovascular, neurological, or pediatric disorders.
Our analysis identified 2844 intragenic CNVs in 384 clinically tested genes. CNVs were observed in 1.9% of the entire cohort but in a disproportionately high fraction (9.8%) of individuals with a clinically significant result. CNVs accounted for 4.7–35% of pathogenic variants, depending on clinical specialty. Distinct patterns existed among CNVs in terms of copy number, location, exons affected, clinical classification, and genes affected. Separately, analysis of de-identified data for 599 genes unrelated to the clinical phenotype yielded 4054 CNVs. Most of these CNVs were novel rare events, present as duplications, and enriched in genes associated with recessive disorders or lacking loss-of-function mutational mechanisms.
Universal intragenic CNV analysis adds substantial clinical sensitivity to genetic testing. Clinically relevant CNVs have distinct properties that distinguish them from CNVs contributing to normal variation in human disease genes.
Waldenström's macroglobulinemia is a lymphoplasmacytic lymphoma. Genetic analysis has revealed a common mutation (L265P) in
MYD88
in more than 90% of patients with this disease. The mutation appears ...to activate NF-κB.
Waldenström's macroglobulinemia is an IgM-secreting lymphoplasmacytic lymphoma (LPL).
1
,
2
Clinical manifestations of Waldenström's macroglobulinemia include cytopenia resulting from bone marrow infiltration by lymphoplasmacytic cells, paraprotein-related cryoglobulinemia, the cold agglutinin syndrome, demyelinating neuropathy, and symptomatic hyperviscosity.
3
The oncogenic basis of Waldenström's macroglobulinemia has not been defined. Familial clustering of Waldenström's macroglobulinemia and other B-cell disorders suggests that genetic factors play a role in the pathogenesis of Waldenström's macroglobulinemia in certain patients.
4
–
6
IgM monoclonal gammopathy of unknown significance (MGUS) is characterized by the presence of a monoclonal IgM protein and the absence of bone marrow disease involvement on histologic examination.
1
IgM . . .
PurposeClinVar is increasingly used as a resource for both genetic variant interpretation and clinical practice. However, controversies exist regarding the consistency of classifications in ClinVar, ...and questions remain about how best to use these data. Our study systematically examined ClinVar to identify common sources of discordance and thus inform ongoing practices.MethodsWe analyzed variants that had multiple classifications in ClinVar, excluding benign polymorphisms. Classifications were categorized by potential actionability and pathogenicity. Consensus interpretations were calculated for each variant, and the properties of the discordant outlier classifications were summarized.ResultsOur study included 74,065 classifications of 27,224 unique variants in 1,713 genes. We found that (i) concordance rates differed among clinical areas and variant types; (ii) clinical testing methods had much higher concordance than basic literature curation and research efforts; (iii) older classifications had greater discordance than newer ones; and (iv) low-penetrance variants had particularly high discordance.ConclusionRecent variant classifications from clinical testing laboratories have high overall concordance in many (but not all) clinical areas. ClinVar can be a reliable resource supporting variant interpretation, quality assessment, and clinical practice when factors uncovered in this study are taken into account. Ongoing improvements to ClinVar may make it easier to use, particularly for nonexpert users.
Bioinformatics pipelines are an integral component of next-generation sequencing (NGS). Processing raw sequence data to detect genomic alterations has significant impact on disease management and ...patient care. Because of the lack of published guidance, there is currently a high degree of variability in how members of the global molecular genetics and pathology community establish and validate bioinformatics pipelines. Improperly developed, validated, and/or monitored pipelines may generate inaccurate results that may have negative consequences for patient care. To address this unmet need, the Association of Molecular Pathology, with organizational representation from the College of American Pathologists and the American Medical Informatics Association, has developed a set of 17 best practice consensus recommendations for the validation of clinical NGS bioinformatics pipelines. Recommendations include practical guidance for laboratories regarding NGS bioinformatics pipeline design, development, and operation, with additional emphasis on the role of a properly trained and qualified molecular professional to achieve optimal NGS testing quality.
Gene panels for hereditary breast and ovarian cancer risk assessment are gaining acceptance, even though the clinical utility of these panels is not yet fully defined. Technical questions remain, ...however, about the performance and clinical interpretation of gene panels in comparison with traditional tests. We tested 1105 individuals using a 29-gene next-generation sequencing panel and observed 100% analytical concordance with traditional and reference data on >750 comparable variants. These 750 variants included technically challenging classes of sequence and copy number variation that together represent a significant fraction (13.4%) of the pathogenic variants observed. For BRCA1 and BRCA2 , we also compared variant interpretations in traditional reports to those produced using only non-proprietary resources and following criteria based on recent (2015) guidelines. We observed 99.8% net report concordance, albeit with a slightly higher variant of uncertain significance rate. In 4.5% of BRCA-negative cases, we uncovered pathogenic variants in other genes, which appear clinically relevant. Previously unseen variants requiring interpretation accumulated rapidly, even after 1000 individuals had been tested. We conclude that next-generation sequencing panel testing can provide results highly comparable to traditional testing and can uncover potentially actionable findings that may be otherwise missed. Challenges remain for the broad adoption of panel tests, some of which will be addressed by the accumulation of large public databases of annotated clinical variants.
The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant ...benchmark sets, but these exclude nearly 400 medically relevant genes due to their repetitiveness or polymorphic complexity. Here, we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single-nucleotide variations, 3,600 insertions and deletions and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes, including CBS, CRYAA and KCNE1. When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.
The practice of genetic testing for hereditary breast and/or ovarian cancer (HBOC) is rapidly evolving owing to the recent introduction of multigene panels. While these tests may identify 40% to 50% ...more individuals with hereditary cancer gene mutations than does testing for BRCA1/2 alone, whether finding such mutations will alter clinical management is unknown.
To define the potential clinical effect of multigene panel testing for HBOC in a clinically representative cohort.
Observational study of patients seen between 2001 and 2014 in 3 large academic medical centers. We prospectively enrolled 1046 individuals who were appropriate candidates for HBOC evaluation and who lacked BRCA1/2 mutations.
We carried out multigene panel testing on all participants, then determined the clinical actionability, if any, of finding non-BRCA1/2 mutations in these and additional comparable individuals.
We evaluated the likelihood of (1) a posttest management change and (2) an indication for additional familial testing, considering gene-specific consensus management guidelines, gene-associated cancer risks, and personal and family history.
Among 1046 study participants, 40 BRCA1/2-negative patients (3.8%; 95% CI, 2.8%-5.2%) harbored deleterious mutations, most commonly in moderate-risk breast and ovarian cancer genes (CHEK2, ATM, and PALB2) and Lynch syndrome genes. Among these and an additional 23 mutation-positive individuals enrolled from our clinics, most of the mutations (92%) were consistent with the spectrum of cancer(s) observed in the patient or family, suggesting that these results are clinically significant. Among all 63 mutation-positive patients, additional disease-specific screening and/or prevention measures beyond those based on personal and family history alone would be considered for most (33 52% of 63; 95% CI, 40.3%-64.2%). Furthermore, additional familial testing would be considered for those with first-degree relatives (42 72% of 58; 95% CI, 59.8%-82.2%) based on potential management changes for mutation-positive relatives. This clinical effect was not restricted to a few of the tested genes because most identified genes could change clinical management for some patients.
In a clinically representative cohort, multigene panel testing for HBOC risk assessment yielded findings likely to change clinical management for substantially more patients than does BRCA1/2 testing alone. Multigene testing in this setting is likely to alter near-term cancer risk assessment and management recommendations for mutation-affected individuals across a broad spectrum of cancer predisposition genes.
Background
An estimated 5–10% of breast and ovarian cancers are due to hereditary causes such as hereditary breast and ovarian cancer (HBOC) syndrome. Medicare, the third-party payer that covers 44 ...million patients in the United States, has implemented a set of clinical criteria to determine coverage for the testing of the
BRCA1
and
BRCA2
genes. These criteria, developed to identify carriers of
BRCA1/2
variants, have not been evaluated in the panel testing era. This study investigated a series of Medicare patients undergoing genetic testing for HBOC to determine the efficacy of genetic testing criteria in identifying patients with hereditary risk.
Methods
This study retrospectively examined de-identified data from a consecutive series of Medicare patients undergoing genetic testing based on personal and family history of breast and gynecologic cancer. Ordering clinicians indicated whether patients did or did not meet established criteria for
BRCA1/2
genetic testing. The genetic test results were compared between the group that met the criteria and the group that did not. Patients in families with known pathogenic (P) or likely pathogenic (LP) variants were excluded from the primary analysis.
Results
Among 4196 unique Medicare patients, the rate of P/LP variants for the patients who met the criteria for genetic testing was 10.5%, and for those who did not, the rate was 9% (
p
= 0.26).
Conclusions
The results of this study indicate that a substantial number of Medicare patients with clinically actionable genetic variants are being missed by current testing criteria and suggest the need for significant expansion and simplification of the testing criteria for HBOC.
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