Mutational and splicing landscape in a cohort of 43,000 patients tested for hereditary cancer.

DNA germline genetic testing can identify individuals with cancer susceptibility. However, DNA sequencing alone is limited in its detection and classification of mRNA splicing variants, particularly those located far from coding sequences. Here we address the limitations of splicing variant identification and interpretation by pairing DNA and RNA sequencing and describe the mutational and splicing landscape in a clinical cohort of 43,524 individuals undergoing genetic testing for hereditary cancer predisposition.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

Classification of the canonical splice alteration MUTYH c.934-2A > G is likely benign based on RNA and clinical data.

MUTYH-associated polyposis (MAP) is an autosomal recessive disorder characterized by the development of multiple adenomatous colonic polyps and an increased lifetime risk of colorectal cancer. Germline biallelic pathogenic variants in MUTYH are responsible for MAP. The MUTYH c.934-2A > G (NM_001128425.1) variant, which is also known as c.850-2A > G for NM_001048174.2, has been identified in our laboratory in more than 800 patients, including homozygous and compound heterozygote carriers. The variant was initially classified as a variant of uncertain significance (VUS) because of lack of a MAP phenotype in biallelic carriers. In two unrelated female patients who were heterozygous carriers of this variant, further testing by RNA sequencing identified an aberrant transcript with a deletion of 9 nt at the start of exon 11 (MUTYH r.934_942del9). This event is predicted to lead to an in-frame loss of three amino acids in a noncritical domain of the protein. This was the only splice defect identified in these patients that was not present in the controls, and the aberrant transcript is derived exclusively from the variant allele, strongly supporting the cause of this splice defect as being the intronic variant, MUTYH c.934-2A > G. The splicing analysis demonstrating a small in-frame skipping of three amino acids in a noncritical domain, along with the absence of a MAP phenotype in our internal cohort of biallelic carriers, provides evidence that the variant is likely benign and not of clinical significance.

NUBPL mitochondrial disease: new patients and review of the genetic and clinical spectrum.

BACKGROUND: The nucleotide binding protein-like (NUBPL) gene was first reported as a cause of mitochondrial complex I deficiency (MIM 613621, 618242) in 2010. To date, only eight patients have been reported with this mitochondrial disorder. Five other patients were recently reported to have NUBPL disease but their clinical picture was different from the first eight patients. Here, we report clinical and genetic findings in five additional patients (four families). METHODS: Whole exome sequencing was used to identify patients with compound heterozygous NUBPL variants. Functional studies included RNA-Seq transcript analyses, missense variant biochemical analyses in a yeast model (Yarrowia lipolytica) and mitochondrial respiration experiments on patient fibroblasts. RESULTS: The previously reported c.815-27T>C branch-site mutation was found in all four families. In prior patients, c.166G>A [p.G56R] was always found in cis with c.815-27T>C, but only two of four families had both variants. The second variant found in trans with c.815-27T>C in each family was: c.311T>C [p.L104P] in three patients, c.693+1G>A in one patient and c.545T>C [p.V182A] in one patient. Complex I function in the yeast model was impacted by p.L104P but not p.V182A. Clinical features include onset of neurological symptoms at 3-18 months, global developmental delay, cerebellar dysfunction (including ataxia, dysarthria, nystagmus and tremor) and spasticity. Brain MRI showed cerebellar atrophy. Mitochondrial function studies on patient fibroblasts showed significantly reduced spare respiratory capacity. CONCLUSION: We report on five new patients with NUBPL disease, adding to the number and phenotypic variability of patients diagnosed worldwide, and review prior reported patients with pathogenic NUBPL variants.

Reticular dysgenesis caused by an intronic pathogenic variant in AK2.

Reticular dysgenesis is a form of severe combined immunodeficiency (SCID) caused by biallelic pathogenic variants in AK2 Here we present the case of a boy diagnosed with SCID following a positive newborn screen (NBS). Genetic testing revealed a homozygous variant: AK2 c.330 + 5G > A. In silico analyses predicted weakened native donor splice site. However, this variant was initially classified as a variant of uncertain significance (VUS) given lack of direct evidence. To determine the impact on splicing, we analyzed RNA from the proband and his parents, using massively parallel RNA-seq of cloned RT-PCR products. Analysis showed that c.330 + 5G > A results in exon 3 skipping, which encodes a critical region of the AK2 protein. With these results, the variant was upgraded to pathogenic, and the patient was given a diagnosis of reticular dysgenesis. Interpretation of VUS at noncanonical splice site nucleotides presents a challenge. RNA sequencing provides an ideal platform to perform qualitative and quantitative assessment of intronic VUS, which can lead to reclassification if a significant impact on mRNA is observed. Genetic disorders of hematopoiesis and immunity represent fruitful areas to apply RNA-based analysis for variant interpretation given the high expression of RNA in blood.

Splicing profile by capture RNA-seq identifies pathogenic germline variants in tumor suppressor genes.

Germline variants in tumor suppressor genes (TSGs) can result in RNA mis-splicing and predisposition to cancer. However, identification of variants that impact splicing remains a challenge, contributing to a substantial proportion of patients with suspected hereditary cancer syndromes remaining without a molecular diagnosis. To address this, we used capture RNA-sequencing (RNA-seq) to generate a splicing profile of 18 TSGs (APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, and TP53) in 345 whole-blood samples from healthy donors. We subsequently demonstrated that this approach can detect mis-splicing by comparing splicing profiles from the control dataset to profiles generated from whole blood of individuals previously identified with pathogenic germline splicing variants in these genes. To assess the utility of our TSG splicing profile to prospectively identify pathogenic splicing variants, we performed concurrent capture DNA and RNA-seq in a cohort of 1000 patients with suspected hereditary cancer syndromes. This approach improved the diagnostic yield in this cohort, resulting in a 9.1% relative increase in the detection of pathogenic variants, demonstrating the utility of performing simultaneous DNA and RNA genetic testing in a clinical context.

Correction: DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

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DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

PURPOSE: Gross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications. METHODS: The DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. RESULTS: DBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic. CONCLUSION: DBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.

Comparison of pelvic muscle architecture between humans and commonly used laboratory species.

INTRODUCTION AND HYPOTHESIS: Pelvic floor muscles (PFM) are deleteriously affected by vaginal birth, which contributes to the development of pelvic floor disorders. To mechanistically link these events, experiments using animal models are required, as access to human PFM tissue is challenging. In choosing an animal model, a comparative study of PFM design is necessary, since gross anatomy alone is insufficient to guide the selection. METHODS: Human PFM architecture was measured using micromechanical dissection and then compared with mouse (n = 10), rat (n = 10), and rabbit (n = 10) using the Architectural Difference Index (ADI) (parameterizing a combined measure of sarcomere length-to-optimal-sarcomere ratio, fiber-to-muscle-length ratio, and fraction of total PFM mass and physiological cross-sectional area (PCSA) contributed by each muscle). Coccygeus (C), iliocaudalis (IC), and pubocaudalis (PC) were harvested and subjected to architectural measurements. Parameters within species were compared using repeated measures analysis of variance (ANOVA) with post hoc Tukey's tests. The scaling relationships of PFM across species were quantified using least-squares regression of log-10-transformed variables. RESULTS: Based on the ADI, rat was found to be the most similar to humans (ADI = 2.5), followed by mouse (ADI = 3.3). When animals' body mass was regressed against muscle mass, muscle length, fiber length, and PCSA scaling coefficients showed a negative allometric relationship or smaller increase than predicted by geometric scaling. CONCLUSION: In terms of muscle design among commonly used laboratory animals, rat best approximates the human PFM, followed by mouse. Negative allometric scaling of PFM architectural parameters is likely due to the multifaceted function of these muscles.