Rare Loss-of-function Variants Research Articles

Spatial enrichment and genomic analyses reveal the link of NOMO1 with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a severe motor neuron disease with uncertain genetic predisposition in most sporadic cases. Spatial architecture of cell types and gene expression is the basis of cell-cell interactions, biological function and disease pathology, but is not well investigated in human motor cortex, a key ALS relevant brain region. Recent studies indicated single nucleus transcriptomic features of motor neuron vulnerability in ALS motor cortex. However, it remains largely unclear what is the brain regional vulnerability of ALS-associated genes, and what is the genetic link between region-specific genes and ALS risk. Here, we developed an entropy-weighted differential gene expression matrix-based tool (SpatialE) to identify the spatial enrichment of gene sets in spatial transcriptomics (ST). We benchmarked SpatialE against another enrichment tool (Multimodal Intersection Analysis, MIA) using ST data from both human and mouse brain tissues. To investigate regional vulnerability, we analyzed three human motor cortex and two dorsolateral prefrontal cortex tissues for spatial enrichment of ALS-associated genes. We also used Cell2location to estimate the abundance of cell types in ALS-related cortex layers. To dissect the link of regionally expressed genes and ALS risk, we performed burden analyses of rare loss-of-function (LOF) variants detected by whole-genome sequencing in ALS patients and controls, and then analyzed differential gene expression in the TargetALS RNA-seq dataset. SpatialE showed more accurate and specific spatial enrichment of regional cell type markers than MIA in both mouse brain and human dorsolateral prefrontal cortex. Spatial transcriptomic analyses of human motor cortex showed heterogenous cell types and spatial gene expression profiles. We found that 260 manually curated ALS-associated genes are significantly enriched in layer 5 (L5) motor cortex, with abundant expression of upper motor neurons and L5 excitatory neurons. Burden analyses of rare LOF variants in L5-associated genes nominated NOMO1 as a novel ALS-associated gene in a combined sample set of 6,814 ALS patients and 3,324 controls (P = 0.029). Gene expression analyses in central nervous system tissues revealed down-regulation of NOMO1 in ALS, which is consistent with a LOF disease mechanism. In conclusion, our integrated ST and genomic analyses identified regional brain vulnerability in ALS and the association of a L5 gene (NOMO1) with ALS risk.

Cholesterol Contributes to Risk, Severity, and Machine Learning-Driven Diagnosis of Lyme Disease.

Lyme disease is the most prevalent vector-borne disease in the US, yet its host factors are poorly understood and diagnostic tests are limited. We evaluated patients in a large health system to uncover cholesterol's role in the susceptibility, severity, and machine learning-based diagnosis of Lyme disease. A longitudinal health system cohort comprised 1 019 175 individuals with electronic health record data and 50 329 with linked genetic data. Associations of blood cholesterol level, cholesterol genetic scores comprising common genetic variants, and burden of rare loss-of-function (LoF) variants in cholesterol metabolism genes with Lyme disease were investigated. A portable machine learning model was constructed and tested to predict Lyme disease using routine lipid and clinical measurements. There were 3832 cases of Lyme disease. Increasing cholesterol was associated with greater risk of Lyme disease and hypercholesterolemia was more prevalent in Lyme disease cases than in controls. Cholesterol genetic scores and rare LoF variants in CD36 and LDLR were associated with Lyme disease risk. Serological profiling of cases revealed parallel trajectories of rising cholesterol and immunoglobulin levels over the disease course, including marked increases in individuals with LoF variants and high cholesterol genetic scores. The machine learning model predicted Lyme disease solely using routine lipid panel, blood count, and metabolic measurements. These results demonstrate the value of large-scale genetic and clinical data to reveal host factors underlying infectious disease biology, risk, and prognosis and the potential for their clinical translation to machine learning diagnostics that do not need specialized assays.

Risk Variants in the Exomes of Children With Critical Illness

Diagnostic genetic testing can lead to changes in management in the pediatric intensive care unit. Genetic risk in children with critical illness but nondiagnostic exome sequencing (ES) has not been explored. To assess the association between loss-of-function (LOF) variants and pediatric critical illness. This genetic association study examined ES first screened for causative variants among 267 children at the Morgan Stanley Children's Hospital of NewYork-Presbyterian, of whom 22 were otherwise healthy with viral respiratory failure; 18 deceased children with bronchiolitis from the Office of the Chief Medical Examiner of New York City, of whom 14 were previously healthy; and 9990 controls from the Institute for Genomic Medicine at Columbia University Irving Medical Center. The ES data were generated between January 1, 2015, and December 31, 2020, and analyzed between January 1, 2017, and September 2, 2022. Critical illness. Odds ratios and P values for genes and gene-sets enriched for rare LOF variants and the loss-of-function observed/expected upper bound fraction (LOEUF) score at which cases have a significant enrichment. This study included 285 children with critical illness (median [range] age, 4.1 [0-18.9] years; 148 [52%] male) and 9990 controls. A total of 228 children (80%) did not receive a genetic diagnosis. After quality control (QC), 231 children harbored excess rare LOF variants in genes with a LOEUF score of 0.680 or less (intolerant genes) (P = 1.0 × 10-5). After QC, 176 children without a diagnosis harbored excess ultrarare LOF variants in intolerant genes but only in those without a known disease association (odds ratio, 1.8; 95% CI, 1.3-2.5). After QC, 25 children with viral respiratory failure harbored excess ultrarare LOF variants in intolerant genes but only in those without a known disease association (odds ratio, 2.8; 95% CI, 1.1-6.6). A total of 114 undiagnosed children were enriched for de novo LOF variants in genes without a known disease association (observed, 14; expected, 6.8; enrichment, 2.05). In this genetic association study, excess LOF variants were observed among critically ill children despite nondiagnostic ES. Variants lay in genes without a known disease association, suggesting future investigation may connect phenotypes to causative genes.

Rare loss of function variants in the hepatokine gene INHBE protect from abdominal obesity

Identifying genetic variants associated with lower waist-to-hip ratio can reveal new therapeutic targets for abdominal obesity. We use exome sequences from 362,679 individuals to identify genes associated with waist-to-hip ratio adjusted for BMI (WHRadjBMI), a surrogate for abdominal fat that is causally linked to type 2 diabetes and coronary heart disease. Predicted loss of function (pLOF) variants in INHBE associate with lower WHRadjBMI and this association replicates in data from AMP-T2D-GENES. INHBE encodes a secreted protein, the hepatokine activin E. In vitro characterization of the most common INHBE pLOF variant in our study, indicates an in-frame deletion resulting in a 90% reduction in secreted protein levels. We detect associations with lower WHRadjBMI for variants in ACVR1C, encoding an activin receptor, further highlighting the involvement of activins in regulating fat distribution. These findings highlight activin E as a potential therapeutic target for abdominal obesity, a phenotype linked to cardiometabolic disease.

Penetrance estimation of SORL1 loss‐of‐function variants adjusted on APOE genotypes suggest a non‐monogenic inheritance

AbstractBackground SORL1 rare loss‐of‐function (LoF) variants are a strong risk factor for Alzheimer disease (AD) with odds ratios above 30 for protein‐truncating variants (PTVs). Accordingly, some pedigrees suggest a high penetrance with individuals developing AD before 65 years. However, the age‐dependent penetrance of these variants is unknown, precluding an accurate use for genetic counselling or inclusion in preventive trials. Importantly, both rarity of these variants and co‐occurrence with other risk factors, including the common APOE4 allele, make penetrance estimations difficult based on classical strategies.MethodWe selected LoF SORL1 rare variants (PTVs and missense variants with a LoF effect following in vitro analyses) from exome sequencing data of >1,300 EOAD cases from France (Rouen‐CNRMAJ). To evaluate the age‐related penetrance of AD associated with SORL1 LoF variants, we first built a model based on family information. To ensure an accurate adjustment for APOE genotypes, our model combines a literature‐based baseline for SORL1 non carriers to an additive effect of SORL1 LoF variants computed from our pedigrees. Since pedigrees include relatives with missing genotypes, we applied an Expectation‐Maximization (EM) algorithm to take full advantage of the available information. Probands’ phenotype information was excluded to avoid ascertainment biases. We applied this first method to 34 pedigrees (69 affected relatives, 270 unaffected relatives, 45 relatives with available genotype information). Additionally, we used sequencing data from 7306 cases and 5852 controls to compute a population‐based estimate of the age‐dependent lifetime risk associated with both APOE4 allele and SORL1 rare PTVs or predicted damaging missense variants.ResultIn both models, 100% penetrance was reached before 75 years for carriers of SORL1 LoF variants who were also homozygous for APOE4. Penetrance remained incomplete for heterozygous E4 carriers, even at age 75, and was lower for non‐E4 carriers. We are providing penetrance estimation curves and 95%IC for the first time for SORL1 rare variants and at the digenic level.ConclusionOur results suggest that SORL1 rare LoF variants should be considered in a non‐monogenic model, different from APP and PSEN1/2 mutations. Such curves appear critical before these variants are used for genetic counseling and preventive clinical trials.

Rare, Damaging DNA Variants in CORIN and Risk of Coronary Artery Disease: Insights From Functional Genomics and Large-Scale Sequencing Analyses

Corin is a protease expressed in cardiomyocytes that plays a key role in salt handling and intravascular volume homeostasis via activation of natriuretic peptides. It is unknown if Corin loss-of-function (LOF) is causally associated with risk of coronary artery disease (CAD). We analyzed all coding CORIN variants in an Italian case-control study of CAD. We functionally tested all 64 rare missense mutations in Western Blot and Mass Spectroscopy assays for proatrial natriuretic peptide cleavage. An expanded rare variant association analysis for Corin LOF mutations was conducted in whole exome sequencing data from 37 799 CAD cases and 212 184 controls. We observed LOF variants in CORIN in 8 of 1803 (0.4%) CAD cases versus 0 of 1725 controls (P, 0.007). Of 64 rare missense variants profiled, 21 (33%) demonstrated <30% of wild-type activity and were deemed damaging in the 2 functional assays for Corin activity. In a rare variant association study that aggregated rare LOF and functionally validated damaging missense variants from the Italian study, we observed no association with CAD-21 of 1803 CAD cases versus 12 of 1725 controls with adjusted odds ratio of 1.61 ([95% CI, 0.79-3.29]; P=0.17). In the expanded sequencing dataset, there was no relationship between rare LOF variants with CAD was also observed (odds ratio, 1.15 [95% CI, 0.89-1.49]; P=0.30). Consistent with the genetic analysis, we observed no relationship between circulating Corin concentrations with incident CAD events among 4744 participants of a prospective cohort study-sex-stratified hazard ratio per SD increment of 0.96 ([95% CI, 0.87-1.07], P=0.48). Functional testing of missense mutations improved the accuracy of rare variant association analysis. Despite compelling pathophysiology and a preliminary observation suggesting association, we observed no relationship between rare damaging variants in CORIN or circulating Corin concentrations with risk of CAD.

Hypercholesterolemia risk associated Abca6 does not regulate lipoprotein metabolism in mice or hamster

Hypercholesterolemia has strong heritability and about 40–60% of hypercholesterolemia is caused by genetic risk factors. A number of monogenic genes have been identified so far for familial hypercholesterolemia (FH). However, in the general population, more than 90% of individuals with LDL cholesterol over 190 mg/dL do not carry known FH mutations. Large scale whole-exome sequencing has identified thousands of variants that are predicted to be loss-of-function (LoF) and each individual has a median of about twenty rare LoF variants and several hundreds more common LoF variants. However, majority of those variants have not been characterized and their functional consequence remains largely unknown. Rs77542162 is a common missense variant in ABCA6 and is strongly associated with hypercholesterolemia in different populations. ABCA6 is a cholesterol responsive gene and has been suggested to play a role in lipid metabolism. However, whether and how rs77542162 and ABCA6 regulate lipoprotein metabolism remain unknown. In current study, we systemically characterized the function of rs77542162 and ABCA6 in cultured cells and in vivo of rodents. We found that Abca6 is specifically expressed on the basolateral surface of hepatocytes in mouse liver. The rs77542162 variant disrupts ABCA6 protein stability and results in loss of functional protein. However, we found no evidence that Abca6 plays a role in lipoprotein metabolism in either normal mice or hypercholesterolemia mice or hamsters. Thus, our results suggest that Abca6 does not regulate lipoprotein metabolism in rodents and highlight the challenge and importance of functional characterization of disease-associated variants in animal models.

Enrichment of loss-of-function and copy number variants in ventricular cardiomyopathy genes in 'lone' atrial fibrillation.

Atrial fibrillation (AF) is a complex heritable disease whose genetic underpinnings remain largely unexplained, though recent work has suggested that the arrhythmia may develop secondary to an underlying atrial cardiomyopathy. We sought to evaluate for enrichment of loss-of-function (LOF) and copy number variants (CNVs) in genes implicated in ventricular cardiomyopathy in 'lone' AF. Whole-exome sequencing was performed in 255 early onset 'lone' AF cases, defined as arrhythmia onset prior to 60 years of age in the absence of known clinical risk factors. Subsequent evaluations were restricted to 195 cases of European genetic ancestry, as defined by principal component analysis, and focused on a pre-defined set of 43 genes previously implicated in ventricular cardiomyopathy. Bioinformatic analysis identified 6 LOF variants (3.1%), including 3 within the TTN gene, among cases in comparison with 4 of 503 (0.80%) controls [odds ratio: 3.96; 95% confidence interval (CI): 1.11-14.2; P = 0.033]. Further, two AF cases possessed a novel heterozygous 8521 base pair TTN deletion, confirmed with Sanger sequencing and breakpoint validation, which was absent from 4958 controls (P = 0.0014). Subsequent cascade screening in two families revealed evidence of co-segregation of a LOF variant with 'lone' AF. 'Lone' AF cases are enriched in rare LOF variants from cardiomyopathy genes, findings primarily driven by TTN, and a novel TTN deletion, providing additional evidence to implicate atrial cardiomyopathy as an AF genetic sub-phenotype. Our results also highlight that AF may develop in the context of these variants in the absence of a discernable ventricular cardiomyopathy.

Heterozygous ABCG5 Gene Deficiency and Risk of Coronary Artery Disease.

Familial sitosterolemia is a rare Mendelian disorder characterized by hyperabsorption and decreased biliary excretion of dietary sterols. Affected individuals typically have complete genetic deficiency-homozygous loss-of-function (LoF) variants-in the ABCG5 or ABCG8 genes and have substantially elevated plasma sitosterol and LDL (low-density lipoprotein) cholesterol (LDL-C) levels. The impact of partial genetic deficiency of ABCG5 or ABCG8-as occurs in heterozygous carriers of LoF variants-on LDL-C and risk of coronary artery disease (CAD) has remained uncertain. We first recruited 9 sitosterolemia families, identified causative LoF variants in ABCG5 or ABCG8, and evaluated the associations of these ABCG5 or ABCG8 LoF variants with plasma phytosterols and lipid levels. We next assessed for LoF variants in ABCG5 or ABCG8 in CAD cases (n=29 321) versus controls (n=357 326). We tested the association of rare LoF variants in ABCG5 or ABCG8 with blood lipids and risk for CAD. Rare LoF variants were defined as protein-truncating variants with minor allele frequency <0.1% in ABCG5 or ABCG8. In sitosterolemia families, 7 pedigrees harbored causative LoF variants in ABCG5 and 2 pedigrees in ABCG8. Homozygous LoF variants in either ABCG5 or ABCG8 led to marked elevations in sitosterol and LDL-C. Of those sitosterolemia families, heterozygous carriers of ABCG5 LoF variants exhibited increased sitosterol and LDL-C levels compared with noncarriers. Within large-scale CAD case-control cohorts, prevalence of rare LoF variants in ABCG5 and in ABCG8 was ≈0.1% each. ABCG5 heterozygous LoF variant carriers had significantly elevated LDL-C levels (25 mg/dL [95% CI, 14-35]; P=1.1×10-6) and were at 2-fold increased risk of CAD (odds ratio, 2.06 [95% CI, 1.27-3.35]; P=0.004). By contrast, ABCG8 heterozygous LoF carrier status was not associated with increased LDL-C or risk of CAD. Although familial sitosterolemia is traditionally considered as a recessive disorder, we observed that heterozygous carriers of an LoF variant in ABCG5 had significantly increased sitosterol and LDL-C levels and a 2-fold increase in risk of CAD.

Missense variants in the spectrin repeat domain of DSP are associated with arrhythmogenic cardiomyopathy: A family report and systematic review.

Rare loss of function variants in DSP, which codes for the desmosomal protein desmoplakin, have been implicated in dilated and arrhythmogenic right ventricular cardiomyopathies. We present a family with arrhythmogenic cardiomyopathy associated with a novel missense variant in DSP (NM_004415.4): c.877G>A, p.(Glu293Lys). The phenotype is characterized by predominant involvement of the left ventricle with systolic dysfunction, fibrosis, and life-threatening arrhythmias. We performed a systematic review of literature collecting all cardiomyopathy cases with rare missense variants in DSP. We demonstrate that the distribution of missense variants across the protein domains in cardiomyopathy cases differs from that in gnomAD (p = .04), with a case enrichment of rare missense variants in the spectrin repeat domain (36/78 [46%] in cases vs. 449/1495 [30%] in gnomAD; p = .004). Our findings highlight the predominance of cardiac arrhythmia and left ventricular involvement in desmoplakin cardiomyopathy and pinpoint to a potential mutation hotspot in DSP thereby facilitating missense variant interpretation in the diagnostic setting.

In This Issue

HomeCirculation ResearchVol. 126, No. 2In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published16 Jan 2020https://doi.org/10.1161/RES.0000000000000321Circulation Research. 2020;126:159is related toMonogenic and Polygenic Contributions to Atrial Fibrillation RiskMetabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesisis related toActivated Endothelial TGFβ1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1TGFβ Signaling and Defective Thrombus Resolution (p 162)Aberrant TGFβ1 signaling is a likely driver of chronic thromboembolic pulmonary hypertension, say Bochenek et al.Download figureDownload PowerPointChronic thromboembolic pulmonary hypertension (CTEPH) is a rare, life-threatening form of pulmonary hypertension characterized by persistent blood clots in the lung arteries that ultimately result in the formation of fibrotic lesions containing irregular vasculature. The mechanisms underlying CTEPH are largely unknown, but because the cytokine TGFβ1 regulates fibrosis and angiogenesis, and because mutations affecting TGFβ1 signaling have been implicated in another form of pulmonary hypertension, Bochenek and colleagues hypothesized this factor’s involvement. Sure enough, they found that, in CTEPH patient tissue, components of the TGFβ1 pathway were upregulated, including TGFβ1 itself and endothelin-1—a vasoconstrictor and promoter of fibrosis. The team also showed that mice engineered to lack TGFβ1 in platelet cells had faster resolution of experimental thrombosis than did control animals. Furthermore, inhibition of endothelin-1 activity prevented human and mouse endothelial cells converting to a fibrotic phenotype. The results suggest the TGFβ1-to-endothelin-1 pathway plays a pathological role in CTEPH and lends supporting evidence that the clinical use of endothelin-1 receptor blockers—prescribed to some patients with CTEPH—is a worthwhile therapy.ACC2 Deletion Prevents Aspartate Synthesis(p 182)Boosting fatty acid oxidation prevents aspartate synthesis and cardiac hypertrophy, report Ritterhoff et al.Download figureDownload PowerPointCardiac hypertrophy, in which the heart and its component cells become enlarged, can be a normal physiological process to cope with increased demand—for example, in pregnancy or elite athletes—but can also be pathological—caused by high blood pressure and other cardiovascular conditions. Pathological hypertrophy is associated with a metabolic switch in the heart from primarily fatty acid-based to glucose-based. And, boosting fatty acid oxidation (FAO) can protect against hypertrophy in cultured cardiomyocytes and animals. The mechanism underlying this protection is unknown, however. Now, Ritterhoff and colleagues show that in addition to increased glucose consumption, hypertrophic rat cardiomyocytes ramp up their levels of aspartate—a molecule involved in cell growth via synthesis of nucleic acids, RNAs, and proteins. And, boosting FAO (by genetic manipulation) prevents both the aspartate rise and hypertrophy in the cells as well as in live mice. Adding aspartate to such FAO-boosted cells could reinstate the hypertrophic phenotype. The discovery of this relationship between glucose metabolism, aspartate, and FAO may inform the design of new therapies aimed at manipulating heart cell metabolism to manage hypertrophy, say the authors.Genetic Basis of Atrial Fibrillation (p 200)Choi et al examine poly-and mono-genetic predisposition to atrial fibrillation.Download figureDownload PowerPointAffecting ≈30 million individuals worldwide, atrial fibrillation (AF) is a common cardiac arrhythmia associated with an increased risk of heart failure, stroke, dementia, and death. While the condition can be idiopathic, there is also evidence of inheritance. Indeed, a quarter of patients have a parent, sibling, or child with the condition. Genome-wide association studies have identified numerous common genetic variants linked to AF, and the more of these variants a person carries, the greater the risk—referred to as their polygenic risk score (PRS). Studies of families with AF have also revealed rarer monogenic variants, such as loss-of-function (LOF) mutations in the sarcomeric gene titin (TTN). Now, Choi and colleagues look at these common polygenic and rare monogenic variants together to examine their relative contributions. Studying the genomes of 1546 AF patients and more than 41 000 controls, the team discovered a significant association between AF and rare LOF variants at TTN. Indeed, 14% of individuals with such variants had AF. However, the team also showed that while TTN variants were highly penetrant, PRS scores accounted for considerably more of the variance in AF susceptibility. Thus, the study indicates that, generally speaking, a person’s PRS is a better indicator of overall AF risk. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMonogenic and Polygenic Contributions to Atrial Fibrillation RiskSeung Hoan Choi, et al. Circulation Research. 2020;126:200-209Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate BiosynthesisJulia Ritterhoff, et al. Circulation Research. 2020;126:182-196Activated Endothelial TGFβ1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1Magdalena L. Bochenek, et al. Circulation Research. 2020;126:162-181 January 17, 2020Vol 126, Issue 2 Advertisement Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000321 Originally publishedJanuary 16, 2020 PDF download Advertisement

Monogenic and Polygenic Contributions to Atrial Fibrillation Risk

Genome-wide association studies have identified over 100 genetic loci for atrial fibrillation (AF); recent work described an association between loss-of-function (LOF) variants in TTN and early-onset AF. We sought to determine the contribution of rare and common genetic variation to AF risk in the general population. The UK Biobank is a population-based study of 500 000 individuals including a subset with genome-wide genotyping and exome sequencing. In this case-control study, we included AF cases and controls of genetically determined white-European ancestry; analyses were performed using a logistic mixed-effects model adjusting for age, sex, the first 4 principal components of ancestry, empirical relationships, and case-control imbalance. An exome-wide, gene-based burden analysis was performed to examine the relationship between AF and rare, high-confidence LOF variants in genes with ≥10 LOF carriers. A polygenic risk score for AF was estimated using the LDpred algorithm. We then compared the contribution of AF polygenic risk score and LOF variants to AF risk. The study included 1546 AF cases and 41 593 controls. In an analysis of 9099 genes with sufficient LOF variant carriers, a significant association between AF and rare LOF variants was observed in a single gene, TTN (odds ratio, 2.71, P=2.50×10-8). The association with AF was more significant (odds ratio, 6.15, P=3.26×10-14) when restricting to LOF variants located in exons highly expressed in cardiac tissue (TTNLOF). Overall, 0.44% of individuals carried TTNLOF variants, of whom 14% had AF. Among individuals in the highest 0.44% of the AF polygenic risk score only 9.3% had AF. In contrast, the AF polygenic risk score explained 4.7% of the variance in AF susceptibility, while TTNLOF variants only accounted for 0.2%. Both monogenic and polygenic factors contribute to AF risk in the general population. While rare TTNLOF variants confer a substantial AF penetrance, the additive effect of many common variants explains a larger proportion of genetic susceptibility to AF.

Exome Sequencing in BRCA1- and BRCA2-Negative Greek Families Identifies MDM1 and NBEAL1 as Candidate Risk Genes for Hereditary Breast Cancer.

Approximately 10% of breast cancer (BC) cases are hereditary BC (HBC), with HBC most commonly encountered in the context of hereditary breast and ovarian cancer (HBOC) syndrome. Although thousands of loss-of-function (LoF) alleles in over 20 genes have been associated with HBC susceptibility, the genetic etiology of approximately 50% of cases remains unexplained, even when polygenic risk models are considered. We focused on one of the least-studied European populations and applied whole-exome sequencing (WES) to 52 individuals from 17 Greek HBOC families, in which at least one patient was negative for known HBC risk variants. Initial screening revealed pathogenic variants in known cancer genes, including BARD1:p.Trp91* detected in a cancer-free individual, and MEN1:p.Glu260Lys detected in a BC patient. Gene- and variant-based approaches were applied to exome data to identify candidate risk variants outside of known risk genes. Findings were verified in a collection of Canadian HBOC patients of European ancestry (FBRCAX), in an independent group of Canadian BC patients (CHUM-BC) and controls (CARTaGENE), as well as in individuals from The Cancer Genome Atlas (TCGA) and the UK Biobank (UKB). Rare LoF variants were uncovered in MDM1 and NBEAL1 in Greek and Canadian HBOC patients. We also report prioritized missense variants SETBP1:c.4129G > C and C7orf34:c.248C > T. These variants comprise promising candidates whose role in cancer pathogenicity needs to be explored further.

484 Whole Genome Sequencing Analysis of Gastroparesis

INTRODUCTION: Gastroparesis is a disorder characterized by delayed gastric emptying of solid food in the absence of mechanical obstruction of the stomach, resulting in the cardinal symptoms of early satiety, postprandial fullness, nausea, vomiting, belching and bloating. To ascertain the genetic risk factors for gastroparesis we conducted the first to date whole genome sequencing (WGS) study of gastroparesis cohort. METHODS: We investigated the frequency and effect of rare loss-of-function (LOF) variants in patients with idiopathic and diabetic gastroparesis enrolled in a clinical study, VLY686-2301. The dataset consisted of 119 WGS samples. RESULTS: Among rare LOF variants, we report an increased frequency of a frameshift mutation within Motilin Receptor (MLNR) gene, variant rs562138828. Motilin is a 22 amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract. The protein encoded by this gene is a motilin receptor which is a member of the G-protein coupled receptor 1 family. We have shown an increased frequency of a frameshift mutation with MAF 0.01 as compared 0.0009 AF in GNOMAD (P-value = 0.01) with Odds Ratio of 21.9. We detect 4/119 gastroparesis patients carry the variant of interest that results in p.Leu202ArgfsTer105. Noteworthy is the fact that the 4 cases were equally split among idiopathic and diabetic, so this possible Gastroparesis risk factor appears to be agnostic as to condition. The finding may be of direct relevance to treatment as individuals with the identified mutation may respond differently to gastroparesis treatments especially those targeting MLNR. Among other rare LOF, we identified a case of CHD7 discussed in literature in gastro-related context and a case of CFTR duodenal stenosis pathogenic variant. The CFTR variant has been seen in pancreatitis and is likely causative of Cystic Fibrosis Gut which is characterized by increased mucous viscosity and development of intestinal inflammation, dysbiosis, and dysmotility. CONCLUSION: Whole genome sequencing of gastroparesis patient samples showed enrichment for rare variants in the MTLR in cases compared with controls. The identified LOF variants within the region can serve as a risk factor for disease as well as inform treatments, especially given the knowledge of different response to treatment.

RARE LOSS-OF-FUNCTION VARIANT ANALYSIS IN LONE ATRIAL FIBRILLATION

Atrial fibrillation (AF) is a complex heritable disorder that substantially increases the risk for stroke, sudden death and all-cause mortality. Lone AF is a subset defined by early diagnosis (less than 60 years) and with no evidence of cardiopulmonary disease. Genome-wide association studies have associated loci near genes for structural integrity and function of muscles with AF susceptibility. In addition, some studies have identified rare loss-of-function (LoF) variants in dilated cardiomyopathy (DCM) genes in lone AF. The increased burden of these variants in lone AF patients may indicate a shared predisposition between AF and DCM. We performed a retrospective study on 252 adult patients and a control group of 503 healthy European subjects. Exome sequencing and microarray assays were performed at the McGill Genome Center while exome data from the controls was retrieved from 1KG Consortium. Principal component analysis was performed to define a group with common ancestry using SVS software. Variants within candidate DCM genes were filtered by minor allele frequency of ≤0.1%, a Combined Annotation Dependent Depletion (CADD) Phred score of >20 and a LoF variant type (frameshift, splice acceptor, splice donor and stop gain). These factors were retrieved from public annotation databases and applied using VarSeq software for variant analysis. All variants were confirmed with Sanger sequencing. Cascade screening was attempted on all cases positive for variants. The AF cohort, an ethnically homogenous composite of 181 lone AF cases, had 6 heterozygous LoF variants, including 4 novel variants, in 6 individuals, while the controls had 4 in 503 individuals. Half of variants in the cases were in the TTN gene (3/6). The prevalence of rare LoF variants in the lone AF cohort was 3.3% while in the control cohort 0.79%, the odds ratio of rare LoF variants found in lone AF patients compared to controls is 4.28 (95%CI: 1.19-15.33, P-value: 2.5E-02). Cascade screening in one family demonstrated co-segregation of one of the rare TTN variants and AF. Patients with lone AF carry a significant burden of heterozygous LoF variants in DCM genes. Our results are in agreement with a previous study identifying TTN variants in lone AF patients. Further, we identified novel variants in different DCM genes and confirmed the co-segregation of a TTN variant and AF in one family. Our findings may indicate a common genetic predisposition between lone AF and DCM, which may help elucidate novel pathophysiologic mechanisms, and improve diagnosis and treatment.

Whole exome sequencing analysis in severe chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide, is substantially influenced by genetic factors. Alpha-1 antitrypsin deficiency demonstrates that rare coding variants of large effect can influence COPD susceptibility. To identify additional rare coding variants in patients with severe COPD, we conducted whole exome sequencing analysis in 2543 subjects from two family-based studies (Boston Early-Onset COPD Study and International COPD Genetics Network) and one case-control study (COPDGene). Applying a gene-based segregation test in the family-based data, we identified significant segregation of rare loss of function variants in TBC1D10A and RFPL1 (P-value<2x10-6), but were unable to find similar variants in the case-control study. In single-variant, gene-based and pathway association analyses, we were unable to find significant findings that replicated or were significant in meta-analysis. However, we found that the top results in the two datasets were in proximity to each other in the protein-protein interaction network (P-value=0.014), suggesting enrichment of these results for similar biological processes. A network of these association results and their neighbors was significantly enriched in the transforming growth factor beta-receptor binding and cilia-related pathways. Finally, in a more detailed examination of candidate genes, we identified individuals with putative high-risk variants, including patients harboring homozygous mutations in genes associated with cutis laxa and Niemann-Pick Disease Type C. Our results likely reflect heterogeneity of genetic risk for COPD along with limitations of statistical power and functional annotation, and highlight the potential of network analysis to gain insight into genetic association studies.

T52 - The Potential Role of Low Frequency And Rare Variants In Tardive Dyskinesia Genetics

Background Tardive dyskinesia (TD) is a chronic, irreversible side effect of antipsychotic-treated schizophrenia patients TD occurrence is influenced by both clinical and demographic variables, as well as genetic factors. Contribution of common variants to TD susceptibility has been investigated in recent years, including by the genome-wide association study approach, but results are inconsistent. In order to discover the involvement of low frequency and rare variants in this phenotype, we implemented whole exome sequencing (WES) method. Methods We whole exome sequenced 20 Ashkenazi Jewish schizophrenia patients with severe TD, and 18 patients without any manifestation of TD (total AIMS score of zero) despite more than 10 years of exposure to antipsychotics. For prioritization, we concentrated on Loss of Function (LoF) rare variants. Results We were able to identify several interesting rare (1%-5%) and extremely rare ( Discussion WES may be implemented in pharmacogenetics studies of antipsychotics. Our preliminary results point to the role of low frequency and rare LoF variants in TD.

Testing for Modes of Inheritance Involving Compound Heterozygotes

Functional variants change the protein product or the expression of genes. Due to the latest advances in sequencing technology, most known functional variants can now be assayed in a cost-effective manner. However, to fully use the information from functional variants, researchers need to model the joint effect of these variants. In this article, we propose methods that model the action/interaction of loss-of-function (LOF) mutations, i.e., those mutations that eliminate the protein product of a gene. When multiple LOFs occur in the same causal gene/region, their effect on a phenotype might depend on whether these mutations lie on the same DNA strand/haplotype. When compared to LOFs occurring on the same strand, if these mutations lie on different strands, both copies of the gene are impaired and the impact on the relevant phenotypes is likely to be more severe. To use the information from LOF strand colocalization, we propose three methods that utilize the information from the estimated number of affected strands. We compare the performance of the proposed and competing methods by using simulations of common and rare LOF variants. Two of the proposed methods exhibited desirable power profiles, the first for both common and rare LOFs and the second only for common LOFs. One of the existing methods, collapsed double heterozygosity, exhibits good power to detect compound models for rare variants, especially when no haplotype harbors two or more rare alleles. Consequently, we recommend these three methods to be used for the analysis of functional variants coming from sequencing studies.

Detecting Low Frequent Loss-of-Function Alleles in Genome Wide Association Studies with Red Hair Color as Example

Multiple loss-of-function (LOF) alleles at the same gene may influence a phenotype not only in the homozygote state when alleles are considered individually, but also in the compound heterozygote (CH) state. Such LOF alleles typically have low frequencies and moderate to large effects. Detecting such variants is of interest to the genetics community, and relevant statistical methods for detecting and quantifying their effects are sorely needed. We present a collapsed double heterozygosity (CDH) test to detect the presence of multiple LOF alleles at a gene. When causal SNPs are available, which may be the case in next generation genome sequencing studies, this CDH test has overwhelmingly higher power than single SNP analysis. When causal SNPs are not directly available such as in current GWA settings, we show the CDH test has higher power than standard single SNP analysis if tagging SNPs are in linkage disequilibrium with the underlying causal SNPs to at least a moderate degree (r2>0.1). The test is implemented for genome-wide analysis in the publically available software package GenABEL which is based on a sliding window approach. We provide the proof of principle by conducting a genome-wide CDH analysis of red hair color, a trait known to be influenced by multiple loss-of-function alleles, in a total of 7,732 Dutch individuals with hair color ascertained. The association signals at the MC1R gene locus from CDH were uniformly more significant than traditional GWA analyses (the most significant P for CDH = 3.11×10−142 vs. P for rs258322 = 1.33×10−66). The CDH test will contribute towards finding rare LOF variants in GWAS and sequencing studies.