The Parkinson's disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release.

Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson's disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

The medico-legal helpline: A content analysis of postgraduate medical trainee advice calls.

CONTEXT: Available literature exploring medical liability and postgraduate medical education consistently posits that postgraduate trainees worry about their exposure to medico-legal liability. This assumption has formed the basis for research and curriculum development. OBJECTIVES: The aim of this study was to describe the encounters that lead physicians-in-training to seek external medico-legal guidance. We sought to provide empirical evidence on trends and themes related to medico-legal advice requests from physicians-in-training. METHODS: Our primary dataset consisted of records of calls from physicians-in-training to the medico-legal helpline of the Canadian Medical Protective Association (CMPA), a national mutual defence organisation providing medico-legal advice and liability protection for over 95% of Canada's physicians. We conducted a trend analysis of the frequency of calls for advice over 10 years from physician-in-training compared with non-trainee physicians. Furthermore, we performed a content analysis of calls made over the most recent 2 years (2016-2017) to elucidate the concerns that led to trainees seeking medico-legal advice. RESULTS: The 10-year trend analysis revealed that the annual growth in the number of physician-in-training advice calls (8.8%) exceeded other CMPA physician groups and was in excess of trainee population growth over the same period. The content analysis identified four core themes: managing confidential information, complex care situations, academic matters and patient safety incidents. CONCLUSIONS: Our findings indicate that trainees are asking questions about their medico-legal liability with increasing frequency. This study contributes new evidence on the issues that lead to trainees seeking help. We believe that understanding trainees' medico-legal advice requests will support medical educators to tailor quality improvement education to learners' needs.

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next-generation sequencing.

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A-related leukodystrophy, Peroxisomal biogenesis disorders, POLR3-related Leukodystrophy, Vanishing White Matter, and Pelizaeus-Merzbacher Disease. Despite the relative frequency of these conditions, carrier-screening laboratories regularly test only 20 of the 55 leukodystrophy-related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.

Characterizing reduced coverage regions through comparison of exome and genome sequencing data across 10 centers.

PURPOSE: As massively parallel sequencing is increasingly being used for clinical decision making, it has become critical to understand parameters that affect sequencing quality and to establish methods for measuring and reporting clinical sequencing standards. In this report, we propose a definition for reduced coverage regions and describe a set of standards for variant calling in clinical sequencing applications. METHODS: To enable sequencing centers to assess the regions of poor sequencing quality in their own data, we optimized and used a tool (ExCID) to identify reduced coverage loci within genes or regions of particular interest. We used this framework to examine sequencing data from 500 patients generated in 10 projects at sequencing centers in the National Human Genome Research Institute/National Cancer Institute Clinical Sequencing Exploratory Research Consortium. RESULTS: This approach identified reduced coverage regions in clinically relevant genes, including known clinically relevant loci that were uniquely missed at individual centers, in multiple centers, and in all centers. CONCLUSION: This report provides a process road map for clinical sequencing centers looking to perform similar analyses on their data.

Heterozygous deletion of FOXA2 segregates with disease in a family with heterotaxy, panhypopituitarism, and biliary atresia.

Biliary atresia (BA) is a pediatric cholangiopathy with unknown etiology occurring in isolated and syndromic forms. Laterality defects affecting the cardiovascular and gastrointestinal systems are the most common features present in syndromic BA. Most cases are sporadic, although reports of familial cases have led to the hypothesis of genetic susceptibility in some patients. We identified a child with BA, malrotation, and interrupted inferior vena cava whose father presented with situs inversus, polysplenia, panhypopituitarism, and mildly dysmorphic facial features. Chromosomal microarray analysis demonstrated a 277 kb heterozygous deletion on chromosome 20, which included a single gene, FOXA2, in the proband and her father. This deletion was confirmed to be de novo in the father. The proband and her father share a common diagnosis of heterotaxy, but they also each presented with a variety of other issues. Further genetic screening revealed that the proband carried an additional protein-altering polymorphism (rs1904589; p.His165Arg) in the NODAL gene that is not present in the father, and this variant has been shown to decrease expression of the gene. As FOXA2 can be a regulator of NODAL expression, we propose that haploinsufficiency for FOXA2 combined with a decreased expression of NODAL is the likely cause for syndromic BA in this proband.

Replication of a GWAS signal in a Caucasian population implicates ADD3 in susceptibility to biliary atresia.

In the United States, biliary atresia (BA) is the most frequent indication for liver transplantation in pediatric patients. BA is a complex disease, with suspected environmental and genetic risk factors. A genome-wide association study in Chinese patients identified association to the 10q24.2 (hg18) genomic region. This signal was upstream of two genes, XPNPEP1 and ADD3, both expressed in intrahepatic bile ducts. We tested association to this region in 171 BA patients and 1,630 controls of European descent and found the strongest signal to be at rs7099604 (p = 2.5 × 10(-3)) in intron 1 of the ADD3 gene. Moreover, expression data suggest that ADD3, but not XPNPEP1, is differentially expressed in BA patients. The role of ADD3 in biliary development is unclear, but our findings suggest that this gene may be functionally relevant for the development of BA.

Evidence from human and zebrafish that GPC1 is a biliary atresia susceptibility gene.

BACKGROUND & AIMS: Biliary atresia (BA) is a progressive fibroinflammatory disorder of infants involving the extrahepatic and intrahepatic biliary tree. Its etiology is unclear but is believed to involve exposure of a genetically susceptible individual to certain environmental factors. BA occurs exclusively in the neonatal liver, so variants of genes expressed during hepatobiliary development could affect susceptibility. Genome-wide association studies previously identified a potential region of interest at 2q37. We continued these studies to narrow the region and identify BA susceptibility genes. METHODS: We searched for copy number variants that were increased among patients with BA (n = 61) compared with healthy individuals (controls; n = 5088). After identifying a candidate gene, we investigated expression patterns of orthologues in zebrafish liver and the effects of reducing expression, with morpholino antisense oligonucleotides, on biliary development, gene expression, and signal transduction. RESULTS: We observed a statistically significant increase in deletions at 2q37.3 in patients with BA that resulted in deletion of one copy of GPC1, which encodes glypican 1, a heparan sulfate proteoglycan that regulates Hedgehog signaling and inflammation. Knockdown of gpc1 in zebrafish led to developmental biliary defects. Exposure of the gpc1 morphants to cyclopamine, a Hedgehog antagonist, partially rescued the gpc1-knockdown phenotype. Injection of zebrafish with recombinant Sonic Hedgehog led to biliary defects similar to those of the gpc1 morphants. Liver samples from patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with samples from controls. CONCLUSIONS: Based on genetic analysis of patients with BA and zebrafish, GPC1 appears to be a BA susceptibility gene. These findings also support a role for Hedgehog signaling in the pathogenesis of BA.

DRAW+SneakPeek: analysis workflow and quality metric management for DNA-seq experiments.

SUMMARY: We report our new DRAW+SneakPeek software for DNA-seq analysis. DNA resequencing analysis workflow (DRAW) automates the workflow of processing raw sequence reads including quality control, read alignment and variant calling on high-performance computing facilities such as Amazon elastic compute cloud. SneakPeek provides an effective interface for reviewing dozens of quality metrics reported by DRAW, so users can assess the quality of data and diagnose problems in their sequencing procedures. Both DRAW and SneakPeek are freely available under the MIT license, and are available as Amazon machine images to be used directly on Amazon cloud with minimal installation. AVAILABILITY: DRAW+SneakPeek is released under the MIT license and is available for academic and nonprofit use for free. The information about source code, Amazon machine images and instructions on how to install and run DRAW+SneakPeek locally and on Amazon elastic compute cloud is available at the National Institute on Aging Genetics of Alzheimer's Disease Data Storage Site (http://www.niagads.org/) and Wang lab Web site (http://wanglab.pcbi.upenn.edu/).

PICALO: principal interaction component analysis for the identification of discrete technical, cell-type, and environmental factors that mediate eQTLs.

Expression quantitative trait loci (eQTL) offer insights into the regulatory mechanisms of trait-associated variants, but their effects often rely on contexts that are unknown or unmeasured. We introduce PICALO, a method for hidden variable inference of eQTL contexts. PICALO identifies and disentangles technical from biological context in heterogeneous blood and brain bulk eQTL datasets. These contexts are biologically informative and reproducible, outperforming cell counts or expression-based principal components. Furthermore, we show that RNA quality and cell type proportions interact with thousands of eQTLs. Knowledge of hidden eQTL contexts may aid in the inference of functional mechanisms underlying disease variants.

Adaptive and Innate Immunity Are Key Drivers of Age at Onset of Multiple Sclerosis.

Background and Objectives: Multiple sclerosis (MS) age at onset (AAO) is a clinical predictor of long-term disease outcomes, independent of disease duration. Little is known about the genetic and biological mechanisms underlying age of first symptoms. We conducted a genome-wide association study (GWAS) to investigate associations between individual genetic variation and the MS AAO phenotype. Methods: The study population was comprised participants with MS in 6 clinical trials: ADVANCE (N = 655; relapsing-remitting [RR] MS), ASCEND (N = 555; secondary-progressive [SP] MS), DECIDE (N = 1,017; RRMS), OPERA1 (N = 581; RRMS), OPERA2 (N = 577; RRMS), and ORATORIO (N = 529; primary-progressive [PP] MS). Altogether, 3,905 persons with MS of European ancestry were analyzed. GWAS were conducted for MS AAO in each trial using linear additive models controlling for sex and 10 principal components. Resultant summary statistics across the 6 trials were then meta-analyzed, for a total of 8.3 × 10-6 single nucleotide polymorphisms (SNPs) across all trials after quality control and filtering for heterogeneity. Gene-based tests of associations, pathway enrichment analyses, and Mendelian randomization analyses for select exposures were also performed. Results: Four lead SNPs within 2 loci were identified (p < 5 × 10-8), including a) 3 SNPs in the major histocompatibility complex and their effects were independent of HLA-DRB1*15:01 and b) a LOC105375167 variant on chromosome 7. At the gene level, the top association was HLA-C (p = 1.2 × 10-7), which plays an important role in antiviral immunity. Functional annotation revealed the enrichment of pathways related to T-cell receptor signaling, autoimmunity, and the complement cascade. Mendelian randomization analyses suggested a link between both earlier age at puberty and shorter telomere length and earlier AAO, while there was no evidence for a role for either body mass index or vitamin D levels. Discussion: Two genetic loci associated with MS AAO were identified, and functional annotation demonstrated an enrichment of genes involved in adaptive and complement immunity. There was also evidence supporting a link with age at puberty and telomere length. The findings suggest that AAO in MS is multifactorial, and the factors driving onset of symptoms overlap with those influencing MS risk.

Whole-exome sequencing in UK Biobank reveals rare genetic architecture for depression.

Nearly two hundred common-variant depression risk loci have been identified by genome-wide association studies (GWAS). However, the impact of rare coding variants on depression remains poorly understood. Here, we present whole-exome sequencing analyses of depression with seven different definitions based on survey, questionnaire, and electronic health records in 320,356 UK Biobank participants. We showed that the burden of rare damaging coding variants in loss-of-function intolerant genes is significantly associated with risk of depression with various definitions. We compared the rare and common genetic architecture across depression definitions by genetic correlation and showed different genetic relationships between definitions across common and rare variants. In addition, we demonstrated that the effects of rare damaging coding variant burden and polygenic risk score on depression risk are additive. The gene set burden analyses revealed overlapping rare genetic variant components with developmental disorder, autism, and schizophrenia. Our study provides insights into the contribution of rare coding variants, separately and in conjunction with common variants, on depression with various definitions and their genetic relationships with neurodevelopmental disorders.

Cross-ancestry atlas of gene, isoform, and splicing regulation in the developing human brain.

Neuropsychiatric genome-wide association studies (GWASs), including those for autism spectrum disorder and schizophrenia, show strong enrichment for regulatory elements in the developing brain. However, prioritizing risk genes and mechanisms is challenging without a unified regulatory atlas. Across 672 diverse developing human brains, we identified 15,752 genes harboring gene, isoform, and/or splicing quantitative trait loci, mapping 3739 to cellular contexts. Gene expression heritability drops during development, likely reflecting both increasing cellular heterogeneity and the intrinsic properties of neuronal maturation. Isoform-level regulation, particularly in the second trimester, mediated the largest proportion of GWAS heritability. Through colocalization, we prioritized mechanisms for about 60% of GWAS loci across five disorders, exceeding adult brain findings. Finally, we contextualized results within gene and isoform coexpression networks, revealing the comprehensive landscape of transcriptome regulation in development and disease.

NOTCH3 p.Arg1231Cys is markedly enriched in South Asians and associated with stroke.

The genetic factors of stroke in South Asians are largely unexplored. Exome-wide sequencing and association analysis (ExWAS) in 75 K Pakistanis identified NM_000435.3(NOTCH3):c.3691 C > T, encoding the missense amino acid substitution p.Arg1231Cys, enriched in South Asians (alternate allele frequency = 0.58% compared to 0.019% in Western Europeans), and associated with subcortical hemorrhagic stroke [odds ratio (OR) = 3.39, 95% confidence interval (CI) = [2.26, 5.10], p = 3.87 × 10-9), and all strokes (OR [CI] = 2.30 [1.77, 3.01], p = 7.79 × 10-10). NOTCH3 p.Arg231Cys was strongly associated with white matter hyperintensity on MRI in United Kingdom Biobank (UKB) participants (effect [95% CI] in SD units = 1.1 [0.61, 1.5], p = 3.0 × 10-6). The variant is attributable for approximately 2.0% of hemorrhagic strokes and 1.1% of all strokes in South Asians. These findings highlight the value of diversity in genetic studies and have major implications for genomic medicine and therapeutic development in South Asian populations.

Homozygosity for the V37I GJB2 mutation in fifteen probands with mild to moderate sensorineural hearing impairment: further confirmation of pathogenicity and haplotype analysis in Asian populations.

Hearing impairment affects 1 in 650 newborns, making it the most common congenital sensory impairment. Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing impairment cases. Mutations in GJB2 account for a significant number of ARNSHI (and up to 50% of documented recessive (e.g., more than 1 affected sibling) hearing impairment in some populations). Mutations in the GJB2 gene are amongst the most common causes of hearing impairment in populations of various ethnic backgrounds. Two mutations of this gene, 35delG and 167delT, account for the majority of reported mutations in Caucasian populations, especially those of Mediterranean and Ashkenazi Jewish background. The 235delC mutation is most prevalent in East Asian populations. Some mutations are of less well-characterized significance. The V37I missense mutation, common in Asian populations, was initially described as a polymorphism and later as a potentially pathogenic mutation. We report here on 15 unrelated individuals with ARNSHI and homozygosity for the V37I GJB2 missense mutation. Nine individuals are of Chinese ancestry, two are of unspecified Asian descent, one is of Japanese descent, one individual is of Vietnamese ancestry, one of Philippine background and one of Italian and Cuban/Caucasian background. Homozygosity for the V37I GJB2 mutation may be a more common pathogenic missense mutation in Asian populations, resulting in mild to moderate sensorineural hearing impairment. We report a presumed haplotype block specific to East Asian individuals with the V37I mutation encompassing the GJB2 gene that may account for the high prevalence in East Asian populations.

PECONPI: a novel software for uncovering pathogenic copy number variations in non-syndromic sensorineural hearing loss and other genetically heterogeneous disorders.

This report describes an algorithm developed to predict the pathogenicity of copy number variants (CNVs) in large sample cohorts. CNVs (genomic deletions and duplications) are found in healthy individuals and in individuals with genetic diagnoses, and differentiation of these two classes of CNVs can be challenging and usually requires extensive manual curation. We have developed PECONPI, an algorithm to assess the pathogenicity of CNVs based on gene content and CNV frequency. This software was applied to a large cohort of patients with genetically heterogeneous non-syndromic hearing loss to score and rank each CNV based on its relative pathogenicity. Of 636 individuals tested, we identified the likely underlying etiology of the hearing loss in 14 (2%) of the patients (1 with a homozygous deletion, 7 with a deletion of a known hearing loss gene and a point mutation on the trans allele and 6 with a deletion larger than 1 Mb). We also identified two probands with smaller deletions encompassing genes that may be functionally related to their hearing loss. The ability of PECONPI to determine the pathogenicity of CNVs was tested on a second genetically heterogeneous cohort with congenital heart defects (CHDs). It successfully identified a likely etiology in 6 of 355 individuals (2%). We believe this tool is useful for researchers with large genetically heterogeneous cohorts to help identify known pathogenic causes and novel disease genes.

Rare genetic variants impact muscle strength.

Muscle strength is highly heritable and predictive for multiple adverse health outcomes including mortality. Here, we present a rare protein-coding variant association study in 340,319 individuals for hand grip strength, a proxy measure of muscle strength. We show that the exome-wide burden of rare protein-truncating and damaging missense variants is associated with a reduction in hand grip strength. We identify six significant hand grip strength genes, KDM5B, OBSCN, GIGYF1, TTN, RB1CC1, and EIF3J. In the example of the titin (TTN) locus we demonstrate a convergence of rare with common variant association signals and uncover genetic relationships between reduced hand grip strength and disease. Finally, we identify shared mechanisms between brain and muscle function and uncover additive effects between rare and common genetic variation on muscle strength.

The impact of rare protein coding genetic variation on adult cognitive function.

Compelling evidence suggests that human cognitive function is strongly influenced by genetics. Here, we conduct a large-scale exome study to examine whether rare protein-coding variants impact cognitive function in the adult population (n = 485,930). We identify eight genes (ADGRB2, KDM5B, GIGYF1, ANKRD12, SLC8A1, RC3H2, CACNA1A and BCAS3) that are associated with adult cognitive function through rare coding variants with large effects. Rare genetic architecture for cognitive function partially overlaps with that of neurodevelopmental disorders. In the case of KDM5B we show how the genetic dosage of one of these genes may determine the variability of cognitive, behavioral and molecular traits in mice and humans. We further provide evidence that rare and common variants overlap in association signals and contribute additively to cognitive function. Our study introduces the relevance of rare coding variants for cognitive function and unveils high-impact monogenic contributions to how cognitive function is distributed in the normal adult population.

Brain expression quantitative trait locus and network analyses reveal downstream effects and putative drivers for brain-related diseases.

Identification of therapeutic targets from genome-wide association studies (GWAS) requires insights into downstream functional consequences. We harmonized 8,613 RNA-sequencing samples from 14 brain datasets to create the MetaBrain resource and performed cis- and trans-expression quantitative trait locus (eQTL) meta-analyses in multiple brain region- and ancestry-specific datasets (n ≤ 2,759). Many of the 16,169 cortex cis-eQTLs were tissue-dependent when compared with blood cis-eQTLs. We inferred brain cell types for 3,549 cis-eQTLs by interaction analysis. We prioritized 186 cis-eQTLs for 31 brain-related traits using Mendelian randomization and co-localization including 40 cis-eQTLs with an inferred cell type, such as a neuron-specific cis-eQTL (CYP24A1) for multiple sclerosis. We further describe 737 trans-eQTLs for 526 unique variants and 108 unique genes. We used brain-specific gene-co-regulation networks to link GWAS loci and prioritize additional genes for five central nervous system diseases. This study represents a valuable resource for post-GWAS research on central nervous system diseases.

Cross-ancestry, cell-type-informed atlas of gene, isoform, and splicing regulation in the developing human brain.

Genomic regulatory elements active in the developing human brain are notably enriched in genetic risk for neuropsychiatric disorders, including autism spectrum disorder (ASD), schizophrenia, and bipolar disorder. However, prioritizing the specific risk genes and candidate molecular mechanisms underlying these genetic enrichments has been hindered by the lack of a single unified large-scale gene regulatory atlas of human brain development. Here, we uniformly process and systematically characterize gene, isoform, and splicing quantitative trait loci (xQTLs) in 672 fetal brain samples from unique subjects across multiple ancestral populations. We identify 15,752 genes harboring a significant xQTL and map 3,739 eQTLs to a specific cellular context. We observe a striking drop in gene expression and splicing heritability as the human brain develops. Isoform-level regulation, particularly in the second trimester, mediates the greatest proportion of heritability across multiple psychiatric GWAS, compared with eQTLs. Via colocalization and TWAS, we prioritize biological mechanisms for ~60% of GWAS loci across five neuropsychiatric disorders, nearly two-fold that observed in the adult brain. Finally, we build a comprehensive set of developmentally regulated gene and isoform co-expression networks capturing unique genetic enrichments across disorders. Together, this work provides a comprehensive view of genetic regulation across human brain development as well as the stage-and cell type-informed mechanistic underpinnings of neuropsychiatric disorders.