Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

Clinical Genetics of Inherited Arrhythmogenic Disease in the Pediatric Population

Sudden death is a rare event in the pediatric population but with a social shock due to its presentation as the first symptom in previously healthy children. Comprehensive autopsy in pediatric cases identify an inconclusive cause in 40–50% of cases. In such cases, a diagnosis of sudden arrhythmic death syndrome is suggested as the main potential cause of death. Molecular autopsy identifies nearly 30% of cases under 16 years of age carrying a pathogenic/potentially pathogenic alteration in genes associated with any inherited arrhythmogenic disease. In the last few years, despite the increasing rate of post-mortem genetic diagnosis, many families still remain without a conclusive genetic cause of the unexpected death. Current challenges in genetic diagnosis are the establishment of a correct genotype–phenotype association between genes and inherited arrhythmogenic disease, as well as the classification of variants of uncertain significance. In this review, we provide an update on the state of the art in the genetic diagnosis of inherited arrhythmogenic disease in the pediatric population. We focus on emerging publications on gene curation for genotype–phenotype associations, cases of genetic overlap and advances in the classification of variants of uncertain significance. Our goal is to facilitate the translation of genetic diagnosis to the clinical area, helping risk stratification, treatment and the genetic counselling of families.

Arrhythmia Variant Associations and Reclassifications in the eMERGE-III Sequencing Study

Background: Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results (RoR) is unclear. In addition, the majority of discovered variants are currently classified as Variants of Uncertain Significance (VUS), limiting clinical actionability. Methods: The eMERGE-III study is a multi-center prospective cohort which included 21,846 participants without prior indication for cardiac genetic testing. Participants were sequenced for 109 Mendelian disease genes, including 10 linked to arrhythmia syndromes. Variant carriers were assessed with Electronic Health Record (EHR)-derived phenotypes and follow-up clinical examination. Selected VUS (n=50) were characterized in vitro with automated electrophysiology experiments in HEK293 cells. Results: As previously reported, 3.0% of participants had pathogenic or likely pathogenic (P/LP) variants in the 109 genes. Herein, we report 120 participants (0.6%) with P/LP arrhythmia variants. Compared to non-carriers, arrhythmia P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their EHRs. Fifty four participants had variant results returned. Nineteen of these 54 participants had inherited arrhythmia syndrome diagnoses (primarily long QT syndrome), and 12/19 of these diagnoses were made only after variant results were returned (0.05%). After in vitro functional evaluation of 50 variants of uncertain significance (VUS), we reclassified 11 variants: 3 to likely benign and 8 to P/LP. Conclusions: Genome sequencing in a large population without indication for arrhythmia genetic testing identified phenotype-positive carriers of variants in congenital arrhythmia syndrome disease genes. As large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, EHR phenotypes, and in vitro functional studies.

Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

Most myopathic lamin variants aggregate: a functional genomics approach for assessing variants of uncertain significance

Hundreds of LMNA variants have been associated with several distinct disease phenotypes. However, genotype–phenotype relationships remain largely undefined and the impact for most variants remains unknown. We performed a functional analysis for 178 variants across five structural domains using two different overexpression models. We found that lamin A aggregation is a major determinant for skeletal and cardiac laminopathies. An in vitro solubility assay shows that aggregation-prone variants in the immunoglobulin-like domain correlate with domain destabilization. Finally, we demonstrate that myopathic-associated LMNA variants show aggregation patterns in induced pluripotent stem cell derived-cardiomyocytes (iPSC-CMs) in contrast to non-myopathic LMNA variants. Our data-driven approach (1) reveals that striated muscle laminopathies are predominantly protein misfolding diseases, (2) demonstrates an iPSC-CM experimental platform for characterizing laminopathic variants in human cardiomyocytes, and (3) supports a functional assay to aid in assessing pathogenicity for myopathic variants of uncertain significance.