A whole exome sequencing study to identify rare variants in multiplex families with alcohol use disorder.

Background: Alcohol use disorder (AUD) runs in families and is accompanied by genetic variation. Some families exhibit an extreme susceptibility in which multiple cases are found and often with an early onset of the disorder. Large scale genome-wide association studies have identified several genes with impressive statistical probabilities. Most of these genes are common variants. Our goal was to perform exome sequencing in families characterized by multiple cases (multiplex families) to determine if rare variants might be segregating with disease status. Methods: A case-control approach was used to leverage the power of a large control sample of unrelated individuals (N = 8,983) with exome sequencing [Institute for Genomic Medicine (IGM)], for comparison with probands with AUD (N = 53) from families selected for AUD multiplex status. The probands were sequenced at IGM using similar protocols to those used for the archival controls. Specifically, the presence of a same-sex pair of adult siblings with AUD was the minimal criteria for inclusion. Using a gene-based collapsing analysis strategy, a search for qualifying variants within the sequence data was undertaken to identify ultra-rare non-synonymous variants. Results: We searched 18,666 protein coding genes to identify an excess of rare deleterious genetic variation using whole exome sequence data in the 53 AUD individuals from a total of 282 family members. To complete a case/control analysis of unrelated individuals, probands were compared to unrelated controls. Case enrichment for 16 genes with significance at 10-4 and one at 10-5 are plausible candidates for follow-up studies. Six genes were ultra rare [minor allele frequency (MAF) of 0.0005]: CDSN, CHRNA9, IFT43, TLR6, SELENBP1, and GMPPB. Eight genes with MAF of 0.001: ZNF514, OXGR1, DIEXF, TMX4, MTBP, PON2, CRHBP, and ANKRD46 were identified along with three protein-truncating variants associated with loss-of-function: AGTRAP, ANKRD46, and PPA1. Using an ancestry filtered control group (N = 2,814), nine genes were found; three were also significant in the comparison to the larger control group including CHRNA9 previously implicated in alcohol and nicotine dependence. Conclusion: This study implicates ultra-rare loss-of-function genes in AUD cases. Among the genes identified include those previously reported for nicotine and alcohol dependence (CHRNA9 and CRHBP).

Myosin Mutations and Sudden Sensorineural Hearing Loss: Results of Whole Exome Sequencing.

OBJECTIVE: Idiopathic sudden sensorineural hearing loss (ISSNHL) affects 66,000 patients per year in the United States. Genetic mutations have been associated with progressive hearing loss; however, genetic mutations associated with ISSNHL have not been identified. METHODS: A prospective cohort study of adults older than 18 years presenting with ISSNHL at a tertiary academic medical center. Whole exome sequencing (WES) was conducted using Genome Analysis Toolkit best practices. An automated diagnostic screen employing a variety of models for pathogenicity was conducted across all genes with no specific targets. Candidate pathogenic variants were reviewed by a team of geneticists and clinicians. Variants were crossed-referenced with 92 known hearing loss associated genes. RESULTS: Twenty-nine patients with SSNHL were screened using WES. The average age of patients was 53 ± 17.1 years, and most patients were White (62%) and men (55%). The mean pure tone average was 64.8 ± 31.3 dB for the affected ear. Using a 0.1% allele frequency screen, 12 (41%) cases had a mutation in any of the nine selected myosin genes. When we restrict to singletons (allele frequency = 0%), 21% (n = 6) of cases have qualifying variants, whereas only 3.8% (n = 481) of 12,577 healthy controls carry qualifying variants (p < 0.01). Most mutations (80%) were missense mutations. Of the novel mutations, one was a frameshift mutation, and two were a stop-gained function. Three were missense mutations. CONCLUSION: Myosin mutations may be associated with ISSNHL. However, larger population screening is needed to confirm the association of myosin mutation with ISSNHL and steroid responsiveness.

Risk Variants in the Exomes of Children With Critical Illness.

Importance: 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. Objective: To assess the association between loss-of-function (LOF) variants and pediatric critical illness. Design, Setting, and Participants: 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. Exposure: Critical illness. Main Outcomes and Measures: 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. Results: 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). Conclusions and Relevance: 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.

Whole exome sequencing reveals potentially pathogenic variants in a small subset of premenopausal women with idiopathic osteoporosis.

Osteoporosis in premenopausal women with intact gonadal function and no known secondary cause of bone loss is termed idiopathic osteoporosis (IOP). Women with IOP diagnosed in adulthood have profound bone structural deficits and often report adult and childhood fractures, and family history of osteoporosis. Some have very low bone formation rates (BFR/BS) suggesting osteoblast dysfunction. These features led us to investigate potential genetic etiologies of bone fragility. In 75 IOP women (aged 20-49) with low trauma fractures and/or very low BMD who had undergone transiliac bone biopsies, we performed Whole Exome Sequencing (WES) using our variant analysis pipeline to select candidate rare and novel variants likely to affect known disease genes. We ran rare-variant burden analyses on all genes individually and on phenotypically-relevant gene sets. For particular genes implicated in osteoporosis, we also assessed the frequency of all (including common) variants in subjects versus 6540 non-comorbid female controls. The variant analysis pipeline identified 4 women with 4 heterozygous variants in LRP5 and PLS3 that were considered to contribute to osteoporosis. All 4 women had adult fractures, and 3 women also had multiple fractures, childhood fractures and a family history of osteoporosis. Two women presented during pregnancy/lactation. In an additional 4 subjects, 4 different relevant Variants of Uncertain Significance (VUS) were detected in the genes FKBP10, SLC34A3, and HGD. Of the subjects with VUS, 2 had multiple adult fractures, childhood fractures, and presented during pregnancy/lactation, and 2 had nephrolithiasis. BFR/BS varied among the 8 subjects with identified variants; BFR/BS was quite low in those with variants that are likely to have adverse effects on bone formation. The analysis pipeline did not discover candidate variants in COL1A1, COL1A2, WNT, or ALPL. Although we found several novel and rare variants in LRP5, cases did not have an increased burden of common LRP5 variants compared to controls. Cohort-wide collapsing analysis did not reveal any novel disease genes with genome-wide significance for qualifying variants between controls and our 75 cases. In summary, WES revealed likely pathogenic variants or relevant VUS in 8 (11%) of 75 women with IOP. Notably, the genetic variants identified were consistent with the affected women's diagnostic evaluations that revealed histological evidence of low BFR/BS or biochemical evidence of increased bone resorption and urinary calcium excretion. These results, and the fact that the majority of the women had no identifiable genetic etiology, also suggest that the pathogenesis of and mechanisms leading to osteoporosis in this cohort are heterogeneous. Future research is necessary to identify both new genetic and non-genetic etiologies of early-onset osteoporosis.

Genomic analysis of "microphenotypes" in epilepsy.

Large international consortia examining the genomic architecture of the epilepsies focus on large diagnostic subgroupings such as "all focal epilepsy" and "all genetic generalized epilepsy". In addition, phenotypic data are generally entered into these large discovery databases in a unidirectional manner at one point in time only. However, there are many smaller phenotypic subgroupings in epilepsy, many of which may have unique genomic risk factors. Such a subgrouping or "microphenotype" may be defined as an uncommon or rare phenotype that is well recognized by epileptologists and the epilepsy community, and which may or may not be formally recognized within the International League Against Epilepsy classification system. Here we examine the genetic structure of a number of such microphenotypes and report in particular on two interesting clinical phenotypes, Jeavons syndrome and pediatric status epilepticus. Although no single gene reached exome-wide statistical significance to be associated with any of the diagnostic categories, we observe enrichment of rare damaging variants in established epilepsy genes among Landau-Kleffner patients (GRIN2A) and pediatric status epilepticus patients (MECP2, SCN1A, SCN2A, SCN8A).

ATAV: a comprehensive platform for population-scale genomic analyses.

BACKGROUND: A common approach for sequencing studies is to do joint-calling and store variants of all samples in a single file. If new samples are continually added or controls are re-used for several studies, the cost and time required to perform joint-calling for each analysis can become prohibitive. RESULTS: We present ATAV, an analysis platform for large-scale whole-exome and whole-genome sequencing projects. ATAV stores variant and per site coverage data for all samples in a centralized database, which is efficiently queried by ATAV to support diagnostic analyses for trios and singletons, as well as rare-variant collapsing analyses for finding disease associations in complex diseases. Runtime logs ensure full reproducibility and the modularized ATAV framework makes it extensible to continuous development. Besides helping with the identification of disease-causing variants for a range of diseases, ATAV has also enabled the discovery of disease-genes by rare-variant collapsing on datasets containing more than 20,000 samples. Analyses to date have been performed on data of more than 110,000 individuals demonstrating the scalability of the framework. To allow users to easily access variant-level data directly from the database, we provide a web-based interface, the ATAV data browser ( http://atavdb.org/ ). Through this browser, summary-level data for more than 40,000 samples can be queried by the general public representing a mix of cases and controls of diverse ancestries. Users have access to phenotype categories of variant carriers, as well as predicted ancestry, gender, and quality metrics. In contrast to many other platforms, the data browser is able to show data of newly-added samples in real-time and therefore evolves rapidly as more and more samples are sequenced. CONCLUSIONS: Through ATAV, users have public access to one of the largest variant databases for patients sequenced at a tertiary care center and can look up any genes or variants of interest. Additionally, since the entire code is freely available on GitHub, ATAV can easily be deployed by other groups that wish to build their own platform, database, and user interface.

Serine biosynthesis defect due to haploinsufficiency of PHGDH causes retinal disease.

Macular telangiectasia type 2 (MacTel) is a progressive, late-onset retinal degenerative disease linked to decreased serum levels of serine that elevate circulating levels of a toxic ceramide species, deoxysphingolipids (deoxySLs); however, causal genetic variants that reduce serine levels in patients have not been identified. Here we identify rare, functional variants in the gene encoding the rate-limiting serine biosynthetic enzyme, phosphoglycerate dehydrogenase (PHGDH), as the single locus accounting for a significant fraction of MacTel. Under a dominant collapsing analysis model of a genome-wide enrichment analysis of rare variants predicted to impact protein function in 793 MacTel cases and 17,610 matched controls, the PHGDH gene achieves genome-wide significance (P = 1.2 × 10-13) with variants explaining ~3.2% of affected individuals. We further show that the resulting functional defects in PHGDH cause decreased serine biosynthesis and accumulation of deoxySLs in retinal pigmented epithelial cells. PHGDH is a significant locus for MacTel that explains the typical disease phenotype and suggests a number of potential treatment options.

Causal Genetic Variants in Stillbirth.

BACKGROUND: In the majority of cases, the cause of stillbirth remains unknown despite detailed clinical and laboratory evaluation. Approximately 10 to 20% of stillbirths are attributed to chromosomal abnormalities. However, the causal nature of single-nucleotide variants and small insertions and deletions in exomes has been understudied. METHODS: We generated exome sequencing data for 246 stillborn cases and followed established guidelines to identify causal variants in disease-associated genes. These genes included those that have been associated with stillbirth and strong candidate genes. We also evaluated the contribution of 18,653 genes in case-control analyses stratified according to the degree of depletion of functional variation (described here as "intolerance" to variation). RESULTS: We identified molecular diagnoses in 15 of 246 cases of stillbirth (6.1%) involving seven genes that have been implicated in stillbirth and six disease genes that are good candidates for phenotypic expansion. Among the cases we evaluated, we also found an enrichment of loss-of-function variants in genes that are intolerant to such variation in the human population (odds ratio, 2.15; 95% confidence interval [CI], 1.46 to 3.06). Loss-of-function variants in intolerant genes were concentrated in genes that have not been associated with human disease (odds ratio, 2.22; 95% CI, 1.41 to 3.34), findings that differ from those in two postnatal clinical populations that were also evaluated in this study. CONCLUSIONS: Our findings establish the diagnostic utility of clinical exome sequencing to evaluate the role of small genomic changes in stillbirth. The strength of the novel risk signal (as generated through the stratified analysis) was similar to that in known disease genes, which indicates that the genetic cause of stillbirth remains largely unknown. (Funded by the Institute for Genomic Medicine.).

Rare-variant collapsing analyses for complex traits: guidelines and applications.

The first phase of genome-wide association studies (GWAS) assessed the role of common variation in human disease. Advances optimizing and economizing high-throughput sequencing have enabled a second phase of association studies that assess the contribution of rare variation to complex disease in all protein-coding genes. Unlike the early microarray-based studies, sequencing-based studies catalogue the full range of genetic variation, including the evolutionarily youngest forms. Although the experience with common variants helped establish relevant standards for genome-wide studies, the analysis of rare variation introduces several challenges that require novel analysis approaches.

A framework for the investigation of rare genetic disorders in neuropsychiatry.

De novo and inherited rare genetic disorders (RGDs) are a major cause of human morbidity, frequently involving neuropsychiatric symptoms. Recent advances in genomic technologies and data sharing have revolutionized the identification and diagnosis of RGDs, presenting an opportunity to elucidate the mechanisms underlying neuropsychiatric disorders by investigating the pathophysiology of high-penetrance genetic risk factors. Here we seek out the best path forward for achieving these goals. We think future research will require consistent approaches across multiple RGDs and developmental stages, involving both the characterization of shared neuropsychiatric dimensions in humans and the identification of neurobiological commonalities in model systems. A coordinated and concerted effort across patients, families, researchers, clinicians and institutions, including rapid and broad sharing of data, is now needed to translate these discoveries into urgently needed therapies.