Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next-generation sequencing (NGS). This requires pathogenicity classification of... Show moreEach year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next-generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5-tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as "consensus" when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled "conflicting", while other nonconsensus observations were labeled "no consensus". We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis. Show less
Variations in the DMD gene that affect dystrophin production underlie both the severe Duchenne and the milder Becker muscular dystrophies (DMD and BMD, respectively). Depending on their location,... Show moreVariations in the DMD gene that affect dystrophin production underlie both the severe Duchenne and the milder Becker muscular dystrophies (DMD and BMD, respectively). Depending on their location, deletions and duplications involving one or more exons of a gene can have a range of consequences. This overview, summarizing the important points to consider, was drafted in response to frequent questions we receive about deletions/duplications involving the dystrophin encoding DMD gene. Although directed at DMD, the observations made can be applied to many other genes. The overview is meant primarily for healthcare professionals involved with interpreting the results of genetic analyses in clinical practice. Show less
The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently... Show moreThe multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. Show less
Wu, H.Y.; Vonk, K.K.D.; Maarel, S.M. van der; Santen, G.W.E.; Daxinger, L. 2019
Increasing use of next-generation sequencing technologies in clinical diagnostics allows large-scale discovery of genetic variants, but also results in frequent identification of variants of... Show moreIncreasing use of next-generation sequencing technologies in clinical diagnostics allows large-scale discovery of genetic variants, but also results in frequent identification of variants of unknown significance (VUSs). Their classification into disease-causing and neutral variants is often hampered by the absence of robust functional tests. Here, we demonstrate that a luciferase reporter assay, in combination with ChIP-qPCR, reliably separates pathogenic ZBTB24 missense variants in the context of immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome from natural variants in healthy individuals and patients of other diseases. Application of our assay to two published ZBTB24 missense VUSs indicates that they are likely not to cause ICF2 syndrome. Furthermore, we show that rare gnomAD ZBTB24 missense variants in key residues of the C2H2-ZF domain lead to a loss of function phenotype that resembles ICF2, suggesting that these individuals are carriers of ICF syndrome. In summary, we have developed a robust functional test to validate missense variants in ZBTB24. Show less
Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric... Show moreConstitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome. Show less
Oliveira, J.; Gruber, A.; Cardoso, M.; Taipa, R.; Fineza, I.; Goncalves, A.; ... ; Sousa, M. 2018
