Browse the corpus

BACKGROUND: In population-based research exome sequencing, the path from variant discovery to return of results is not well established. Variants discovered by research exome sequencing have the potential to improve population health. METHODS: Population-based exome sequencing and agnostic ExWAS were performed 5521 Amish individuals. Additional phenotyping and in vitro studies enabled reclassification of a KCNQ1 variant from variant of unknown significance to pathogenic. Results were returned to participants in a community setting. RESULTS: A missense variant was identified in KCNQ1 (c.671C>T, p.T224M), a gene associated with long QT syndrome type 1, which can cause syncope and sudden cardiac death. The p.T224M variant, present in 1/45 Amish individuals is rare in the general population (1/248 566 in gnomAD) and was highly associated with QTc on electro-cardiogram (P=5.53E-24, β=20.2 ms/allele). Because of the potential importance of this variant to the health of the population, additional phenotyping was performed in 88 p.T224M carriers and 54 noncarriers. There was stronger clinical evidence of long QT syndrome in carriers (38.6% versus 5.5%, P=0.0006), greater history of syncope (32% versus 17%, P=0.020), and higher rate of sudden cardiac death in first degree relatives<age 30 (4.5% versus 0%, P=0.026). Expression of p.T224M KCNQ1 in Chinese hamster ovary cells showed near complete loss of protein function. Our clinical and functional data enabled reclassification of p.T224M from a variant of unknown significance to pathogenic. Of the 88 carriers, 93% met criteria for beta-blocker treatment and 5/88 (5.7%) were on medications that may further prolong QTc. Carriers were provided a Clinical Laboratory Improvement Amendments confirmed report, genetic counseling, and treatment recommendations. Follow-up care was coordinated with local physicians. CONCLUSIONS: This work provides a framework by which research exome sequencing can be rapidly translated in a culturally appropriate manner to directly benefit research participants and enable population precision health.

BACKGROUND: Brugada syndrome (BrS) is characterized by the type 1 Brugada ECG pattern. Pathogenic rare variants in SCN5A (mutations) are identified in 20% of BrS families in whom incomplete penetrance and genotype-negative phenotype-positive individuals are observed. E1784K-SCN5A is the most common SCN5A mutation identified. We determined the association of a BrS genetic risk score (BrS-GRS) and SCN5A mutation type on BrS phenotype in BrS families with SCN5A mutations. METHODS: Subjects with a spontaneous type 1 pattern or positive/negative drug challenge from cohorts harboring SCN5A mutations were recruited from 16 centers (n=312). Single nucleotide polymorphisms previously associated with BrS at genome-wide significance were studied in both cohorts: rs11708996, rs10428132, and rs9388451. An additive linear genetic model for the BrS-GRS was assumed (6 single nucleotide polymorphism risk alleles). RESULTS: In the total population (n=312), BrS-GRS ≥4 risk alleles yielded an odds ratio of 4.15 for BrS phenotype ([95% CI, 1.45-11.85]; P=0.0078). Among SCN5A-positive individuals (n=258), BrS-GRS ≥4 risk alleles yielded an odds ratio of 2.35 ([95% CI, 0.89-6.22]; P=0.0846). In SCN5A-negative relatives (n=54), BrS-GRS ≥4 alleles yielded an odds ratio of 22.29 ([95% CI, 1.84-269.30]; P=0.0146). Among E1784K-SCN5A positive family members (n=79), hosting ≥4 risk alleles gave an odds ratio=5.12 ([95% CI, 1.93-13.62]; P=0.0011). CONCLUSIONS: Common genetic variation is associated with variable expressivity of BrS phenotype in SCN5A families, explaining in part incomplete penetrance and genotype-negative phenotype-positive individuals. SCN5A mutation genotype and a BrS-GRS associate with BrS phenotype, but the strength of association varies according to presence of a SCN5A mutation and severity of loss of function.

BACKGROUND: LMNA is a known causative gene of dilated cardiomyopathy and familial conduction disturbance. Nonsense-mediated mRNA decay, normally caused by nonsense mutations, is a safeguard process to protect cells from deleterious effects of inappropriate proteins from mutated genes. Nonsense-mediated mRNA decay induced by nonstop codon mutations is rare. We investigated the effect of an LMNA missense mutation identified in 2 families affected by cardiac laminopathy. METHODS: Genomic DNA and total RNA were isolated from patients' peripheral blood lymphocytes or cardiac tissue. LMNA-coding exons were screened by direct sequencing. Complementary DNAs were generated by a reverse transcription-polymerase chain reaction from total RNA. Quantitative polymerase chain reaction was performed to quantify the LMNA complementary DNA amount by using specific primers for lamins A and C. A minigene splicing reporter experiment was performed to assess the effect of detected variants on RNA splicing. The protein expressions of both isoforms were analyzed by Western blotting. RESULTS: We detected a missense variant c.936 G>C (p. Q312H) at the end of exon 5 of LMNA by genomic DNA sequencing in 2 unrelated families affected by dilated cardiomyopathy and cardiac conduction disturbance. This variant was previously reported in a French family suffering from muscular dystrophy and cardiac conduction disturbance. Sequencing of complementary DNA demonstrated that the mutated allele was absent. By quantitative polymerase chain reaction assay, we confirmed a 90% reduction in LMNA complementary DNA. The minigene splicing reporter assay demonstrated a splicing error by the variant. Western blot analysis revealed that lamin A and C expressions were reduced far >50%. CONCLUSIONS: We report an LMNA missense mutation found in 2 families, which disrupted a normal splicing site, led to nonsense-mediated mRNA decay, and resulted in severe cardiac laminopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a genetically heterogeneous cardiac disease characterized by progressive ventricular enlargement and reduced systolic function. Here, we report genetic and functional analyses implicating the rat sarcoma signaling protein, SOS1 (Son of sevenless homolog 1), in DCM pathogenesis. METHODS: Exome sequencing was performed on 412 probands and family members from our DCM cohort, identifying several SOS1 variants with potential disease involvement. As several lines of evidence have implicated dysregulated rat sarcoma signaling in the pathogenesis of DCM, we assessed functional impact of each variant on the activation of ERK (extracellular signal-regulated kinase), AKT (protein kinase B), and JNK (c-Jun N-terminal kinase) pathways. Relative expression levels were determined by Western blot in HEK293T cells transfected with variant or wild-type human SOS1 expression constructs. RESULTS: A rare SOS1 variant [c.571G>A, p.(Glu191Lys)] was found to segregate alongside an A-band TTN truncating variant in a pedigree with aggressive, early-onset DCM. Reduced disease severity in the absence of the SOS1 variant suggested its potential involvement as a genetic risk factor for DCM in this family. Exome sequencing identified 5 additional SOS1 variants with potential disease involvement in 4 other families [c.1820T>C, p.(Ile607Thr); c.2156G>C, p.(Gly719Ala); c.2230A>G, p.(Arg744Gly); c.2728G>C, p.(Asp910His); c.3601C>T, p.(Arg1201Trp)]. Impacted amino acids occupied a number of functional domains relevant to SOS1 activity, including the N-terminal histone fold, as well as the C-terminal REM (rat sarcoma exchange motif), CDC25 (cell division cycle 25), and PR (proline-rich) tail domains. Increased phosphorylated ERK expression relative to wild-type levels was seen for all 6 SOS1 variants, paralleling known disease-relevant SOS1 signaling profiles. CONCLUSIONS: These data support gain-of-function variation in SOS1 as a contributing factor to isolated DCM.

BACKGROUND: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. METHODS: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS-mediated cardiac hypertrophy. RESULTS: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca2+ handling, including increased frequency of irregular Ca2+ transients and changes in Ca2+ handling kinetics. CONCLUSIONS: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.

BACKGROUND: Tachycardia-induced cardiomyopathy (TIC) is a reversible cardiomyopathy induced by tachyarrhythmia, and the genetic background of the TIC is not well understood. The hyperpolarization-activated cyclic nucleotide-gated channel gene HCN4 is highly expressed in the conduction system where it is involved in heart rate control. We speculated that the HCN4 gene is associated with TIC. METHODS: We enrolled 930 Japanese patients with atrial fibrillation (AF) for screening, 350 Japanese patients with AF for replication, and 1635 non-AF controls. In the screening AF set, we compared HCN4 single-nucleotide polymorphism genotypes between AF subjects with TIC (TIC, n=73) and without TIC (non-TIC, n=857). Of 17 HCN4 gene-tag single-nucleotide polymorphisms, rs7172796, rs2680344, rs7164883, rs11631816, and rs12905211 were significantly associated with TIC. Among them, only rs7164883 was independently associated with TIC after conditional analysis (TIC versus non-TIC: minor allele frequency, 26.0% versus 9.7%; P=1.62×10-9; odds ratio=3.2). RESULTS: We confirmed this association of HCN4 single-nucleotide polymorphism rs7164883 with TIC in the replication set (TIC=41 and non-TIC=309; minor allele frequency, 28% versus 9.9%; P=1.94×10-6; odds ratio=3.6). The minor allele frequency of rs7164883 was similar in patients with AF and non-AF controls (11% versus 10.9%; P=0.908). CONCLUSIONS: The HCN4 gene single-nucleotide polymorphism rs7164883 may be a new genetic marker for TIC in patients with AF.

BACKGROUND: APOL1 renal risk variants are strongly associated with chronic kidney disease in Black adults, but reported associations with cardiovascular disease (CVD) have been conflicting. METHODS: We examined associations of APOL1 with incident coronary heart disease (n=323), ischemic stroke (n=331), and the composite CVD outcome (n=500) in 10 605 Black participants of the REGARDS study (Reasons for Geographic and Racial Differences in Stroke). Primary analyses compared individuals with APOL1 high-risk genotypes to APOL1 low-risk genotypes in Cox proportional hazards models adjusted for CVD risk factors and African ancestry. RESULTS: APOL1 high-risk participants were younger and more likely to have albuminuria at baseline than APOL1 low-risk participants. The risk of incident stroke, coronary heart disease, or composite CVD end point did not significantly differ by APOL1 genotype status in multivariable models. The association of APOL1 genotype with incident composite CVD differed by diabetes mellitus status (Pinteraction=0.004). In those without diabetes mellitus, APOL1 high-risk genotypes associated with greater risk of incident composite CVD (hazard ratio, 1.67; 95% confidence interval, 1.12-2.47) compared with those with APOL1 low-risk genotypes in multivariable adjusted models. This latter association was driven by ischemic strokes (hazard ratio, 2.32; 95% confidence interval, 1.33-4.07), in particular, those related to small vessel disease (hazard ratio, 5.10; 95% confidence interval, 1.55-16.56). There was no statistically significant association of APOL1 genotypes with incident CVD in subjects with diabetes mellitus. The APOL1 high-risk genotype was associated with higher stroke risk in individuals without but not those with chronic kidney disease in fully adjusted models. CONCLUSIONS: APOL1 high-risk status is associated with CVD events in community-dwelling Black adults without diabetes mellitus.

BACKGROUND: Accurately predicting the impact of rare nonsynonymous variants on disease risk is an important goal in precision medicine. Variants in the cardiac sodium channel SCN5A (protein NaV1.5; voltage-dependent cardiac Na+ channel) are associated with multiple arrhythmia disorders, including Brugada syndrome and long QT syndrome. Rare SCN5A variants also occur in ≈1% of unaffected individuals. We hypothesized that in vitro electrophysiological functional parameters explain a statistically significant portion of the variability in disease penetrance. METHODS: From a comprehensive literature review, we quantified the number of carriers presenting with and without disease for 1712 reported SCN5A variants. For 356 variants, data were also available for 5 NaV1.5 electrophysiological parameters: peak current, late/persistent current, steady-state V1/2 of activation and inactivation, and recovery from inactivation. RESULTS: We found that peak and late current significantly associate with Brugada syndrome (P<0.001; ρ=-0.44; Spearman rank test) and long QT syndrome disease penetrance (P<0.001; ρ=0.37). Steady-state V1/2 activation and recovery from inactivation associate significantly with Brugada syndrome and long QT syndrome penetrance, respectively. Continuous estimates of disease penetrance align with the current American College of Medical Genetics classification paradigm. CONCLUSIONS: NaV1.5 in vitro electrophysiological parameters are correlated with Brugada syndrome and long QT syndrome disease risk. Our data emphasize the value of in vitro electrophysiological characterization and incorporating counts of affected and unaffected carriers to aid variant classification. This quantitative analysis of the electrophysiological literature should aid the interpretation of NaV1.5 variant electrophysiological abnormalities and help improve NaV1.5 variant classification.