Rare Coding Variants Research Articles

A deCIDEdly New Germline Mutation That Protects Against Liver Disease

Verweij N, Haas ME, Nielsen JB, et al. Germline mutations in CIDEB and protection against liver disease. N Engl J Med 2022;387:332–344. Li JZ, Ye J, Xue B, et al. Cideb regulates diet-induced obesity, liver steatosis, and insulin sensitivity by controlling lipogenesis and fatty acid oxidation. Diabetes 2007;56:2523–2532. Genetic and environmental factors mediate progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) and cirrhosis. However, specific genetic determinants driving this transition are not well-understood. Rapid, high-throughput whole-exome sequencing technology now affords the opportunity to identify rare disease-relevant coding variants in large patient cohorts. This is critical because no therapies approved by the US Food and Drug Administration exist to treat or prevent NASH and because such novel methods can reveal new NASH target candidates. Furthermore, the precise means by which to stratify high-risk populations toward early, aggressive metabolic therapy are currently lacking. Verweij et al applied whole-exome sequencing and genetic association analysis to over 500,000 patients (24,944 with liver disease) in the UK Biobank and Geisinger Health System MyCode cohorts to identify protein-coding variants associated with liver disease. They used a multistage approach to identify four gene variants associated with high transaminases: APOB, ABCB4, SLC30A10, and TM6SF2. Interestingly, 1 putative loss-of-function coding variant in Cell death-inducing DFF45-like effector B (CIDEB) associated with lower transaminases and milder histological NAFLD Activity Scoring. Three CIDE isoforms comprise a family of lipid droplet–associated proteins that regulate lipid droplet homeostasis. CIDEB is of particular interest, because CIDEB-deficient mouse models have lower plasma triglycerides and are protected from diet-induced obesity, insulin resistance, and hepatic steatosis. These new data now implicate pathophysiological functions for CIDEB in liver disease in people. The primary strengths of this study include its multistage analytical approach; applying exome sequencing to a large, well-characterized study population; and validation of the CIDEB function using a cell-based assay system. Subsequent work should validate these associations in non–European-predominant populations, define further insight into precisely how this CIDEB variant is protective, and determine if targeting this protein will indeed prove beneficial in liver disease. The data nevertheless advance the field by nominating CIDEB as a novel NAFLD and NASH target, by uncovering a potential risk allele by which to stratify patients for aggressive, early NASH-specific treatment, and by providing a novel genetic approach to identify targets in other diseases using large-cohort exome sequencing. Each of these advances represents exciting steps toward precision treatment of NAFLD, NASH, and beyond.

Gene Sequencing Identifies Perturbation in Nitric Oxide Signaling as a Nonlipid Molecular Subtype of Coronary Artery Disease.

A key goal of precision medicine is to disaggregate common, complex diseases into discrete molecular subtypes. Rare coding variants in the low-density lipoprotein receptor gene (LDLR) are identified in 1% to 2% of coronary artery disease (CAD) patients, defining a molecular subtype with risk driven by hypercholesterolemia. To search for additional subtypes, we compared the frequency of rare, predicted loss-of-function and damaging missense variants aggregated within a given gene in 41 081 CAD cases versus 217 115 controls. Rare variants in LDLR were most strongly associated with CAD, present in 1% of cases and associated with 4.4-fold increased CAD risk. A second subtype was characterized by variants in endothelial nitric oxide synthase gene (NOS3), a key enzyme regulating vascular tone, endothelial function, and platelet aggregation. A rare predicted loss-of-function or damaging missense variants in NOS3 was present in 0.6% of cases and associated with 2.42-fold increased risk of CAD (95% CI, 1.80-3.26; P=5.50×10-9). These variants were associated with higher systolic blood pressure (+3.25 mm Hg; [95% CI, 1.86-4.65]; P=5.00×10-6) and increased risk of hypertension (adjusted odds ratio 1.31; [95% CI, 1.14-1.51]; P=2.00×10-4) but not circulating cholesterol concentrations, suggesting that, beyond lipid pathways, nitric oxide synthesis is a key nonlipid driver of CAD risk. Beyond LDLR, we identified an additional nonlipid molecular subtype of CAD characterized by rare variants in the NOS3 gene.

Long-read HiFi sequencing of NUDT15: Phased full-gene haplotyping and pharmacogenomic allele discovery.

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

A Fast and Robust Strategy to Remove Variant-Level Artifacts in Alzheimer Disease Sequencing Project Data.

Background and ObjectivesExome sequencing (ES) and genome sequencing (GS) are expected to be critical to further elucidate the missing genetic heritability of Alzheimer disease (AD) risk by identifying rare coding and/or noncoding variants that contribute to AD pathogenesis. In the United States, the Alzheimer Disease Sequencing Project (ADSP) has taken a leading role in sequencing AD-related samples at scale, with the resultant data being made publicly available to researchers to generate new insights into the genetic etiology of AD. To achieve sufficient power, the ADSP has adapted a study design where subsets of larger AD cohorts are collected and sequenced across multiple centers, using a variety of sequencing platforms. This approach may lead to variable variant quality across sequencing centers and/or platforms. In this study, we sought to implement and evaluate filters that can be applied fast to robustly remove variant-level artifacts in the ADSP data.MethodsWe implemented a robust quality control procedure to handle ADSP data. We evaluated this procedure while performing exome-wide and genome-wide association analyses on AD risk using the latest ADSP whole ES (WES) and whole GS (WGS) data releases (NG00067.v5).ResultsWe observed that many variants displayed large variation in allele frequencies across sequencing centers/platforms and contributed to spurious association signals with AD risk. We also observed that sequencing platform/center adjustment in association models could not fully account for these spurious signals. To address this issue, we designed and implemented variant filters that could capture and remove these center-specific/platform-specific artifactual variants.DiscussionWe derived a fast and robust approach to filter variants that represent sequencing center-related or platform-related artifacts underlying spurious associations with AD risk in ADSP WES and WGS data. This approach will be important to support future robust genetic association studies on ADSP data, as well as other studies with similar designs.

Germline Mutations in CIDEB and Protection against Liver Disease

BackgroundExome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets.MethodsWe performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations.ResultsThe multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, −1.24 U per liter; 95% confidence interval [CI], −1.66 to −0.83; P=4.8×10−9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P=9.9×10−7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, −0.98; 95% CI, −1.54 to −0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets.ConclusionsRare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.)

TREM2 coding variants in Slovak Alzheimer's disease patients.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an important modulator of innate immune responses. In the human brain, TREM2 is primarily expressed on microglia and is involved in cell survival, phagocytosis, and regulation of inflammation. TREM2 dysfunction has been linked to the pathogenesis of various neurodegenerative diseases including Alzheimer's disease (AD). Rare coding variants of the TREM2 gene have been reported to modulate AD risk in several populations, however, data on their association with susceptibility to AD in the Slovak population have been missing. We have analyzed 10 non-synonymous coding variants located in TREM2 exon 2 by direct sequencing in 270 late-onset Alzheimer's disease (LOAD) patients and 331 controls. Four out of 10 TREM2 mutant variants have been identified in the analyzed groups, namely rs75932628 C > T (R47H), rs142232675 C > T (D87N), rs143332484 C > T (R62H), and rs2234253 G > T (T96K). R47H was found only in the AD group, while T96K was present only in the controls. Although no significant association between TREM2 coding variants and LOAD susceptibility has been detected, the observed odds ratio (OR) of 3.69 for R47H carriers suggests an increased risk of LOAD for this variant in the Slovak population. Moreover, we also found a higher OR for the rs143332484-T allele in APOEε4 non-carriers (1.99) when compared to APOEε4 carriers (0.62). Our results suggest an impact of specific TREM2 rare coding variants on AD risk in the Slovak population.

PO-629-03 IN-DEPTH ANALYSIS OF THE SCN5A LOCUS HIGHLIGHTS DISTINCT GENETIC ARCHITECTURES FOR BRUGADA SYNDROME IN DIFFERENT ANCESTRIES AND IDENTIFIES A NOVEL RARE ENHANCER VARIANT ASSOCIATED WITH DISEASE IN SOUTHEAST ASIAN PATIENTS

Brugada syndrome (BrS) is a cardiac arrhythmia disorder that can cause sudden death in young adults. Genetic variation in the Nav1.5 cardiac sodium channel encoding SCN5A gene is robustly associated with BrS, across both rare coding and common non-coding variants. Despite a markedly higher prevalence of BrS, the diagnostic yield from SCN5A genetic testing in east and southeast Asia has been reported to be much lower than other regions. To compare the contribution of different classes of SCN5A variants (by variant type and rarity) to BrS between northwest European and Thai populations in order to investigate the apparent paradox between high disease prevalence and low diagnostic yield in southeast Asia. Genome sequencing data from European (412 cases, 769 controls) and Thai (202 cases, 410 controls) samples was analysed. SCN5A coding variants were binned by gnomAD exomes (v2.1) filtering allele frequency (FAF) and case-control odds ratios (OR) were calculated for each bin and ancestry. Rare non-coding variants in promoter and enhancer regions of SCN5A and SCN10A were assessed for enrichment or depletion in BrS cohorts. Ultra-rare (FAF<0.00001) SCN5A coding variants are more prevalent and enriched in European (22.6% of cases, OR=22.1, 11.4-43.0) compared to Thai (5.9% of cases, OR=4.3, 1.6-11.5) BrS patients. In contrast, low frequency variants (FAF 0.00005-0.001) that are expected to have intermediate effect sizes are uniquely enriched in Thai samples, occurring in 9.9% of patients. 3/6 of these variants (p.R965C, p.A1428S, p.V2016M) are known to affect Nav1.5 function. A rare non-coding variant in the SCN5A RE5 enhancer region (GRCh37:3-38621871-A-C), affecting a base conserved across species, was highly and uniquely enriched in Thai BrS cases (4.5% vs 0.2%, OR=19.1, p=3e-04). Distinct SCN5A genetic architectures are observed in European and southeast Asian BrS patients. The increased prevalence of BrS in southeast Asia is not due to an increased population burden of ultra-rare and highly deleterious SCN5A coding variants (which are relatively depleted in case cohorts). Our data suggest that other SCN5A variant classes, including coding variants of intermediate effect size and rare non-coding enhancer variants, are genetic risk factors in Thai BrS cases.

Rare coding variants in ten genes confer substantial risk for schizophrenia.

Rare coding variation has historically provided the most direct connections between gene function and disease pathogenesis. By meta-analysing the whole exomes of 24,248 schizophrenia cases and 97,322 controls, we implicate ultra-rare coding variants (URVs) in 10 genes as conferring substantial risk for schizophrenia (odds ratios of 3-50, P < 2.14 × 10-6) and 32 genes at a false discovery rate of <5%. These genes have the greatest expression in central nervous system neurons and have diverse molecular functions that include the formation, structure and function of the synapse. The associations of theNMDA (N-methyl-D-aspartate) receptor subunit GRIN2A and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GRIA3 provide support for dysfunction of the glutamatergic system as a mechanistic hypothesis in the pathogenesis of schizophrenia. We observe an overlap of rare variant risk among schizophrenia, autism spectrum disorders1, epilepsy and severe neurodevelopmental disorders2, although different mutation types are implicated in some shared genes. Most genes described here, however, are not implicated in neurodevelopment. We demonstrate that genes prioritized from common variant analyses of schizophrenia are enriched in rare variant risk3, suggesting that common and rare genetic risk factors converge at least partially on the same underlying pathogenic biological processes. Even after excluding significantly associated genes, schizophrenia cases still carry a substantial excess of URVs, which indicates that more risk genes await discovery using this approach.

Mapping genomic loci implicates genes and synaptic biology in schizophrenia.

Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies.

Rare coding variants in RCN3 are associated with blood pressure

BackgroundWhile large genome-wide association studies have identified nearly one thousand loci associated with variation in blood pressure, rare variant identification is still a challenge. In family-based cohorts, genome-wide linkage scans have been successful in identifying rare genetic variants for blood pressure. This study aims to identify low frequency and rare genetic variants within previously reported linkage regions on chromosomes 1 and 19 in African American families from the Trans-Omics for Precision Medicine (TOPMed) program. Genetic association analyses weighted by linkage evidence were completed with whole genome sequencing data within and across TOPMed ancestral groups consisting of 60,388 individuals of European, African, East Asian, Hispanic, and Samoan ancestries.ResultsAssociations of low frequency and rare variants in RCN3 and multiple other genes were observed for blood pressure traits in TOPMed samples. The association of low frequency and rare coding variants in RCN3 was further replicated in UK Biobank samples (N = 403,522), and reached genome-wide significance for diastolic blood pressure (p = 2.01 × 10− 7).ConclusionsLow frequency and rare variants in RCN3 contributes blood pressure variation. This study demonstrates that focusing association analyses in linkage regions greatly reduces multiple-testing burden and improves power to identify novel rare variants associated with blood pressure traits.

Genetic analysis for type 1 diabetes genes in juvenile dermatomyositis unveils genetic disease overlap.

JDM is a serious autoimmune and complex genetic disease. Another autoimmune genetic disease, type 1 diabetes (T1D), has been observed for significantly increased prevalence in families with JDM, while increased JDM risk has also been observed in T1D cases. This study aimed to study whether these two autoimmune diseases, JDM and T1D, share common genetic susceptibility. From 169 JDM families, 121 unrelated cases with European ancestry (EA) were identified by genome-wide genotyping, principal component analysis and identical-by-descent (IBD) analysis. T1D genetic risk score (GRS) were calculated in these cases and were compared with 361 EA T1D cases and 1943 non-diabetes EA controls. A total of 113 cases of the 121 unrelated European cases were sequenced by whole exome sequencing. We observed increased T1D GRS in JDM cases (P = 9.42E-05). Using whole exome sequencing, we uncovered the T1D genes, phospholipase B1, cystic fibrosis transmembrane conductance regulator, tyrosine hydroxylase, CD6 molecule, perforin 1 and dynein axonemal heavy chain 2, potentially associated with JDM by the burden test of rare functional coding variants. Novel mechanisms of JDM related to these T1D genes are suggested by this study, which may imply novel therapeutic targets for JDM and warrant further study.

Abstract IA-40: CDKN2A germline rare coding variants and risk of pancreatic cancer in minority populations

Abstract Performing genetic epidemiology studies in underrepresented populations is challenging, but is extremely important in order to understand how cancer impacts differing populations, This can then allow more tailored and nuanced management of persons at risk. Our group at Mayo Clinic encountered some interesting preliminary findings regarding CDKN2A, but given small numbers of subjects in our registry, we needed to partner with other collaborators to have enough statistical power to make sense of the findings and generate a publishable result. This talk will focus on the means of doing this, the importance of team-based science across centers, and future opportunities. Background: Pathogenic germline mutations in the CDKN2A tumor suppressor gene are rare and associated with highly penetrant familial melanoma and pancreatic cancer in non-Hispanic whites. WeTo date, the prevalence and impact of CDKN2A rare coding variants (RCV) in racial minority groups remain poorly characterized. We examined the role of CDKN2A RCVs on the risk of pancreatic cancer among minority subjects. Methods: We performed a case-control study of germline mutations in CDKN2A among patients with incident pancreatic cancer and controls, focused on underrepresented minority populations. We sequenced CDKN2A in 220 African American pancreatic cancer cases, 900 noncancer African American controls, and 183 Nigerian controls. RCV frequencies were determined for each group and compared with that of 1,537 Non-Hispanic White patients with pancreatic cancer. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for both a case–case comparison of RCV frequencies in African Americans versus Non-Hispanic Whites, and case–control comparison between AA cases versus noncancer AA controls plus Nigerian controls. Smaller sets of Hispanic and Native American cases and controls also were sequenced. Race/ethnicity categorization was from self-report. Results: One novel missense RCV and one novel frameshift RCV were found among AA patients: 400G&amp;gt;A and 258_278del. RCV carrier status was associated with increased risk of pancreatic cancer among AA cases (11/220; OR, 3.3; 95% CI, 1.5–7.1; P ¼ 0.004) compared with African American and Nigerian controls (17/1,083). Further, African American cases had higher frequency of RCVs: 5.0% (OR, 13.4;95%CI, 4.9–36.7; P &amp;lt; 0.001) compared with Non-Hispanic White cases (0.4%). Conclusions: CDKN2A RCVs are more common in African Americans than in Non-Hispanic White patients with pancreatic cancer and associated with moderately increased pancreatic cancer risk among African Americans. Citation Format: Robert McWilliams. CDKN2A germline rare coding variants and risk of pancreatic cancer in minority populations [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA-40.

Rare coding variants in DNA damage repair genes associated with timing of natural menopause.

SummaryThe age of menopause is associated with fertility and disease risk, and its genetic control is of great interest. We use whole-exome sequences from 132,370 women in the UK Biobank to test for associations between rare damaging variants and age at natural menopause. Rare damaging variants in five genes are significantly associated with menopause: CHEK2 (p = 3.3 × 10−51), DCLRE1A (p = 8.4 × 10−13), and HELB (p = 5.7 × 10−7) with later menopause and TOP3A (p = 7.6 × 10−8) and CLPB (p = 8.1 × 10−7) with earlier menopause. Two additional genes are suggestive: RAD54L (p = 2.4 × 10−6) with later menopause and HROB (p = 2.9 × 10−6) with earlier menopause. In a follow-up analysis of repeated questionnaires in women who were initially premenopausal, CHEK2, TOP3A, and RAD54L genotypes are associated with subsequent menopause. Consistent with previous genome-wide association studies (GWASs), six of the seven genes are involved in the DNA damage repair pathway. Phenome-wide scans across 398,569 men and women revealed that in addition to known associations with cancers and blood cell counts, rare variants in CHEK2 are also associated with increased risk for uterine fibroids, polycystic ovary syndrome, and prostate hypertrophy; these associations are not shared with higher-penetrance breast cancer genes. Causal mediation analysis suggests that approximately 8% of the breast cancer risk conferred by CHEK2 pathogenic variants after menopause is mediated through delayed menopause.

Rare coding variants in 35 genes associate with circulating lipid levels—A multi-ancestry analysis of 170,000 exomes

Large-scale gene sequencing studies for complex traits have the potential to identify causal genes with therapeutic implications. We performed gene-based association testing of blood lipid levels with rare (minor allele frequency < 1%) predicted damaging coding variation by using sequence data from >170,000 individuals from multiple ancestries: 97,493 European, 30,025 South Asian, 16,507 African, 16,440 Hispanic/Latino, 10,420 East Asian, and 1,182 Samoan. We identified 35 genes associated with circulating lipid levels; some of these genes have not been previously associated with lipid levels when using rare coding variation from population-based samples. We prioritize 32 genes in array-based genome-wide association study (GWAS) loci based on aggregations of rare coding variants; three (EVI5, SH2B3, and PLIN1) had no prior association of rare coding variants with lipid levels. Most of our associated genes showed evidence of association among multiple ancestries. Finally, we observed an enrichment of gene-based associations for low-density lipoprotein cholesterol drug target genes and for genes closest to GWAS index single-nucleotide polymorphisms (SNPs). Our results demonstrate that gene-based associations can be beneficial for drug target development and provide evidence that the gene closest to the array-based GWAS index SNP is often the functional gene for blood lipid levels.

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.

Beyond Trinucleotide Repeat Expansion in Fragile X Syndrome: Rare Coding and Noncoding Variants in FMR1 and Associated Phenotypes.

FMR1 (FMRP translational regulator 1) variants other than repeat expansion are known to cause disease phenotypes but can be overlooked if they are not accounted for in genetic testing strategies. We collected and reanalyzed the evidence for pathogenicity of FMR1 coding, noncoding, and copy number variants published to date. There is a spectrum of disease-causing FMR1 variation, with clinical and functional evidence supporting pathogenicity of five splicing, five missense, one in-frame deletion, one nonsense, and four frameshift variants. In addition, FMR1 deletions occur in both mosaic full mutation patients and as constitutional pathogenic alleles. De novo deletions arise not only from full mutation alleles but also alleles with normal-sized CGG repeats in several patients, suggesting that the CGG repeat region may be prone to genomic instability even in the absence of repeat expansion. We conclude that clinical tests for potentially FMR1-related indications such as intellectual disability should include methods capable of detecting small coding, noncoding, and copy number variants.

Functional characterization of novel rare CYP2A6 variants and potential implications for clinical outcomes.

CYP2A6 activity, phenotyped by the nicotine metabolite ratio (NMR), is a predictor of several smoking behaviors, including cessation and smoking‐related disease risk. The heritability of the NMR is 60–80%, yet weighted genetic risk scores (wGRSs) based on common variants explain only 30–35%. Rare variants (minor allele frequency <1%) are hypothesized to explain some of this missing heritability. We present two targeted sequencing studies where rare protein‐coding variants are functionally characterized in vivo, in silico, and in vitro to examine this hypothesis. In a smoking cessation trial, 1687 individuals were sequenced; characterization measures included the in vivo NMR, in vitro protein expression, and metabolic activity measured from recombinant proteins. In a human liver bank, 312 human liver samples were sequenced; measures included RNA expression, protein expression, and metabolic activity from extracted liver tissue. In total, 38 of 47 rare coding variants identified were novel; characterizations ranged from gain‐of‐function to loss‐of‐function. On a population level, the portion of NMR variation explained by the rare coding variants was small (~1%). However, upon incorporation, the accuracy of the wGRS was improved for individuals with rare protein‐coding variants (i.e., the residuals were reduced), and approximately one‐third of these individuals (12/39) were re‐assigned from normal to slow metabolizer status. Rare coding variants can alter an individual’s CYP2A6 activity; their integration into wGRSs through precise functional characterization is necessary to accurately assess clinical outcomes and achieve precision medicine for all. Investigation into noncoding variants is warranted to further explain the missing heritability in the NMR.

Rare genetic coding variants associated with human longevity and protection against age-related diseases.

Extreme longevity in humans has a strong genetic component, but whether this involves genetic variation in the same longevity pathways as found in model organisms is unclear. Using whole-exome sequences of a large cohort of Ashkenazi Jewish centenarians to examine enrichment for rare coding variants, we found most longevity-associated rare coding variants converge upon conserved insulin/insulin-like growth factor 1 signaling and AMP-activating protein kinase signaling pathways. Centenarians have a number of pathogenic rare coding variants similar to control individuals, suggesting that rare variants detected in the conserved longevity pathways are protective against age-related pathology. Indeed, we detected a pro-longevity effect of rare coding variants in the Wnt signaling pathway on individuals harboring the known common risk allele APOE4. The genetic component of extreme human longevity constitutes, at least in part, rare coding variants in pathways that protect against aging, including those that control longevity in model organisms.

Aortic root aortopathy in bicuspid aortic valve associated with high genetic risk

BackgroundThe bicuspid aortic valve (BAV) is prone to ascending aortic dilatation (AAD) involving both the tubular segment and the aortic root. The genetic factor was proposed as one of the most important mechanisms for AAD. We hypothesized that the rare genetic variants mainly contribute to the pathogenesis of aortic roots in affected individuals.MethodsThe diameter of aortic root or ascending aorta ≥ 40 mm was counted as AAD. The targeted next-generation sequencing of 13 BAV-associated genes were performed on a continuous cohort of 96 unrelated BAV patients. The rare variants with allele frequency < 0.05% were selected and analyzed. Variants frequency was compared against the Exome aggregation consortium database. The pathogenicity of the genetic variants was evaluated according to the American College of Medical Genetics and Genomics guidelines.ResultsA total of 27 rare nonsynonymous coding variants involving 9 genes were identified in 25 individuals. The burden analysis revealed that variants in GATA5, GATA6, and NOTCH1 were significantly associated with BAV. Eighty percent of the pathogenic variants were detected in root group. The detection rate of rare variants was higher in root dilatation group (71.4%) compared with normal aorta (29.0%) and tubular dilatation groups (29.6%) (P = 0.018). The rare variant was identified as the independent risk factor of root dilatation [P = 0.014, hazard ratio = 23.9, 95% confidence interval (1.9–302.9)].ConclusionsOur results presented a broad genetic spectrum in BAV patients. The rare variants of BAV genes contribute the most to the root phenotype among BAV patients.

Gene expression atlas of energy balance brain regions.

Energy balance is controlled by interconnected brain regions in the hypothalamus, brainstem, cortex, and limbic system. Gene expression signatures of these regions can help elucidate the pathophysiology underlying obesity. RNA sequencing was conducted on P56 C57BL/6NTac male mice and E14.5 C57BL/6NTac embryo punch biopsies in 16 obesity-relevant brain regions. The expression of 190 known obesity-associated genes (monogenic, rare, and low-frequency coding variants; GWAS; syndromic) was analyzed in each anatomical region. Genes associated with these genetic categories of obesity had localized expression patterns across brain regions. Known monogenic obesity causal genes were highly enriched in the arcuate nucleus of the hypothalamus and developing hypothalamus. The obesity-associated genes clustered into distinct “modules” of similar expression profile, and these were distinct from expression modules formed by similar analysis with genes known to be associated with other disease phenotypes (type 1 and type 2 diabetes, autism, breast cancer) in the same energy balance–relevant brain regions.