Validation of New Gene Variant Classification Methods:a Field-Test in Diagnostic Cardiogenetics

Background: In the molecular genetic diagnostics of Mendelian disorders, solutions are needed for the major challenge of dealing with the large number of variants of uncertain significance (VUSs) identified using next-generation sequencing (NGS). Recently, promising approaches using constraint metrics to calculate case excess scores (CE), etiological fractions (EF), and gnomAD-derived constraint scores have been reported that estimate the likelihood of rare variants in specific genes or regions that are pathogenic. Our objective is to study the usability of these constraint data into variant interpretation in a diagnostic setting, using our cardiomyopathy cohort. Methods and Results: Patients (N = 2002) referred for clinical genetic diagnostics underwent NGS testing of 55–61 genes associated with cardiomyopathies. Previously classified likely pathogenic (LP) and pathogenic (P) variants were used to validate the use of data from CE, EF, and gnomAD constraint analyses for (re)classification of associated variant types in specific cardiomyopathy subtype-related genes. The classifications corroborated in 94% (354/378) of cases. Next, we reclassified 23 unique VUSs to LP, increasing the diagnostic yield by 1.2%. In addition, 106 unique VUSs (5.3% of patients) were prioritized for co-segregation or functional analyses. Conclusions: Our analysis confirms that the use of constraint metrics data can improve variant interpretation, and we, therefore, recommend using constraint scores on other cohorts and disorders and its inclusion in variant interpretation protocols

Mutational heterogeneity between different regional tumour grades of clear cell renal cell carcinoma

Only a limited number of studies have explored the possible associations between tumour grade and mutated genes in clear cell renal cell carcinoma (ccRCC), and we set out to investigate this further using a multiple sampling and next generation sequencing (NGS) approach in a series of ccRCCs. Multiple regions were sampled from formalin-fixated paraffin-embedded ccRCC tumour blocks from seven patients. In 27 samples from six patients, we performed targeted NGS using a custom 42-gene panel based on the most frequently mutated genes in ccRCC reported in public databases. In four samples from the seventh patient, we performed whole exome sequencing (WES) and array comparative genomic hybridisation for detection of copy number variants (CNVs). Mutated genes and the tumour grades of the samples in which they had been identified were compared both within and between all individual tumours. CNVs were compared across all samples from patient 7. We identified clear genetic heterogeneity within and across tumours, but VHL mutations were seen in all patients. Looking across all samples, we identified eleven genes that were only mutated in samples with one particular tumour grade. However, these genes were never mutated in all samples with that tumour grade. Increasing chromosomal instability corresponded with increasing tumour grade, but we observed minimal association between tumour grade and total mutational load in the WES data. Our study confirms the genetic heterogeneity and tumour grade heterogeneity of ccRCC. Although a relatively small number of samples was analysed, genes were identified that could potentially be specific, though insensitive, markers of higher ccRCC tumour grades

CoNVaDING:Single Exon Variation Detection in Targeted NGS Data

We have developed a tool for detecting single exon copy number variations (CNVs) in targeted next-generation sequencing data: CoNVaDING (Copy Number Variation Detection In Next-generation sequencing Gene panels). CoNVaDING includes a stringent quality control metric, that excludes or flags low quality exons. Since this quality control shows exactly which exons can be reliably analysed and which exons are in need of an alternative analysis method, CoNVaDING is not only useful for CNV detection in a research setting, but also in clinical diagnostics. During the validation phase, CoNVaDING detected all known CNVs in high quality targets in 320 samples analysed, giving 100% sensitivity and 99.998% specificity for 308,574 exons. CoNVaDING outperforms existing tools by exhibiting a higher sensitivity and specificity and by precisely identifying low-quality samples and regions. This article is protected by copyright. All rights reserved.</p

Evaluation of a seven gene mutational profile as a prognostic factor in a population-based study of clear cell renal cell carcinoma

In this study, we investigate the influence of the seven genes (VHL, PBRM1, SETD2, BAP1, KDM5C, MTOR and TP53) most frequently mutated in clear cell renal cell cancer (ccRCC) on cancer-specific survival (CSS) in the prospective Netherlands Cohort Study on diet and cancer. DNA isolated from routinely archived formalin-fixed paraffin-embedded tumour blocks from 252 incident ccRCC cases was available for targeted next generation sequencing. Based on the sequencing quality and the completeness of information on clinical characteristics and follow-up, we could use 110 cases for survival analysis. The association with CSS for each mutated gene in these cases was tested using multivariable Cox proportional hazards models to estimate hazards ratios (HR) and confidence intervals (CIs), and we observed mutations in one or more of the seven genes in 64 out of 110 cases (58%). In the multivariable-adjusted analyses, mutations in VHL and PBRM1 were associated with better CSS (HRs (95% CI) 0.34 (0.13‒0.89) and 0.17 (0.04–0.66), respectively), although these results were not statistically significant after multiple testing correction. No association was observed for the other five genes, which may be attributable to limited power

Functional investigation of two simultaneous or separately segregating DSP variants within a single family support the theory of a dose-dependent disease severity

Desmoplakin (DP) is an important component of desmosomes, essential in cell-cell connecting structures in stress-bearing tissues. Over many hundreds of pathogenic variants in DSP have been associated with different cutaneous and cardiac phenotypes or a combination, known as a cardiocutaneous syndrome. Of less than 5% of the reported DSP variants, the effect on the protein has been investigated. Here, we describe and have performed RNA, protein and tissue analysis in a large family where DSPc.273+5G>A/c.6687delA segregated with palmoplantar keratoderma (PPK), woolly hair and lethal cardiomyopathy, while DSPWT/c.6687delA segregated with PPK and milder cardiomyopathy. hiPSC-derived cardiomyocytes and primary keratinocytes from carriers were obtained for analysis. Unlike the previously reported nonsense variants in the last exon of DSP that bypassed the nonsense-mediated mRNA surveillance system leading to protein truncation, variant c.6687delA was shown to cause loss of protein expression. Patients carrying both variants and having a considerably more severe phenotype were shown to have 70% DP protein reduction, while patients carrying only c.6687delA had 50% protein reduction and a milder phenotype. Analysis of RNA from patient cells did not show any splicing effect of the c.273+5G>A variant. However, a minigene splicing assay clearly showed alternative spliced transcripts originating from this variant. This study shows the importance of RNA and protein analyses to pinpoint the exact effect of DSP variants instead of solely relying on predictions. In addition, the particular pattern of inheritance, with simultaneous or separately segregating DSP variants within the same family, strongly supports the theory of a dose-dependent disease severity

Functional Studies on Primary Tubular Epithelial Cells Indicate a Tumor Suppressor Role of SETD2 in Clear Cell Renal Cell Carcinoma

SET domain-containing 2 (SETD2) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma (ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1, whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no β-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC

Diagnostic yield of targeted next generation sequencing in 2002 Dutch cardiomyopathy patients

BACKGROUND: Next-generation sequencing (NGS) is increasingly used for clinical evaluation of cardiomyopathy patients as it allows for simultaneous screening of multiple cardiomyopathy-associated genes. Adding copy number variant (CNV) analysis of NGS data is not routine yet and may contribute to the diagnostic yield. OBJECTIVES: Determine the diagnostic yield of our targeted NGS gene panel in routine clinical diagnostics of Dutch cardiomyopathy patients and explore the impact of exon CNVs on diagnostic yield. METHODS: Patients (N = 2002) referred for clinical genetic analysis underwent diagnostic testing of 55-61 genes associated with cardiomyopathies. Samples were analyzed and evaluated for single nucleotide variants (SNVs), indels and CNVs. CNVs identified in the NGS data and suspected of being pathogenic based on type, size and location were confirmed by additional molecular tests. RESULTS: A (likely) pathogenic (L)P variant was detected in 22.7% of patients, including 3 with CNVs and 25 where a variant was identified in a gene currently not associated with the patient's cardiomyopathy subtype. Only 15 out of 2002 patients (0.8%) were found to carry two (L)P variants. CONCLUSION: The yield of routine clinical diagnostics of cardiomyopathies was relatively low when compared to literature. This is likely due to the fact that our study reports the outcome of patients in daily routine diagnostics, therefore also including patients not fully fulfilling (subtype specific) cardiomyopathy criteria. This may also explain why (L)P variants were identified in genes not associated with the reported subtype. The added value of CNV analysis was shown to be limited but not negligible

Feasibility of Follow-Up Studies and Reclassification in Spinocerebellar Ataxia Gene Variants of Unknown Significance

Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative disorders with autosomal dominant inheritance. Genetic testing for SCA leads to diagnosis, prognosis and risk assessment for patients and their family members. While advances in sequencing and computing technologies have provided researchers with a rapid expansion in the genetic test content that can be used to unravel the genetic causes that underlie diseases, the large number of variants with unknown significance (VUSes) detected represent challenges. To minimize the proportion of VUSes, follow-up studies are needed to aid in their reclassification as either (likely) pathogenic or (likely) benign variants. In this study, we addressed the challenge of prioritizing VUSes for follow-up using (a combination of) variant segregation studies, 3D protein modeling, in vitro splicing assays and functional assays. Of the 39 VUSes prioritized for further analysis, 13 were eligible for follow up. We were able to reclassify 4 of these VUSes to LP, increasing the molecular diagnostic yield by 1.1%. Reclassification of VUSes remains difficult due to limited possibilities for performing variant segregation studies in the classification process and the limited availability of routine functional tests

Recurrent Coding Sequence Variation Explains only A Small Fraction of the Genetic Architecture of Colorectal Cancer

Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10-7), and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10-7); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10-