Nonsynonymous Coding Variants Research Articles

Neurodevelopmental disorders caused by variants in TRPM3

Developmental and epileptic encephalopathies (DEE) are a broad and varied group of disorders that affect the brain and are characterized by epilepsy and comorbid intellectual disability (ID). These conditions have a broad spectrum of symptoms and can be caused by various underlying factors, including genetic mutations, infections, and other medical conditions. The exact cause of DEE remains largely unknown in the majority of cases. However, in around 25 % of patients, rare nonsynonymous coding variants in genes encoding ion channels, cell-surface receptors, and other neuronally expressed proteins are identified. This review focuses on a subgroup of DEE patients carrying variations in the gene encoding the Transient Receptor Potential Melastatin 3 (TRPM3) ion channel, where recent data indicate that gain-of-function of TRPM3 channel activity underlies a spectrum of dominant neurodevelopmental disorders.

Genome-Wide Association Analysis of Protein-Coding Variants in IgA Nephropathy.

Genome-wide association studies have identified nearly 20 IgA nephropathy susceptibility loci. However, most nonsynonymous coding variants, particularly ones that occur rarely or at a low frequency, have not been well investigated. The authors performed a chip-based association study of IgA nephropathy in 8529 patients with the disorder and 23,224 controls. They identified a rare variant in the gene encoding vascular endothelial growth factor A (VEGFA) that was significantly associated with a two-fold increased risk of IgA nephropathy, which was further confirmed by sequencing analysis. They also identified a novel common variant in PKD1L3 that was significantly associated with lower haptoglobin protein levels. This study, which was well-powered to detect low-frequency variants with moderate to large effect sizes, helps expand our understanding of the genetic basis of IgA nephropathy susceptibility. Genome-wide association studies have identified nearly 20 susceptibility loci for IgA nephropathy. However, most nonsynonymous coding variants, particularly those occurring rarely or at a low frequency, have not been well investigated. We performed a three-stage exome chip-based association study of coding variants in 8529 patients with IgA nephropathy and 23,224 controls, all of Han Chinese ancestry. Sequencing analysis was conducted to investigate rare coding variants that were not covered by the exome chip. We used molecular dynamic simulation to characterize the effects of mutations of VEGFA on the protein's structure and function. We also explored the relationship between the identified variants and the risk of disease progression. We discovered a novel rare nonsynonymous risk variant in VEGFA (odds ratio, 1.97; 95% confidence interval [95% CI], 1.61 to 2.41; P = 3.61×10 -11 ). Further sequencing of VEGFA revealed twice as many carriers of other rare variants in 2148 cases compared with 2732 controls. We also identified a common nonsynonymous risk variant in PKD1L3 (odds ratio, 1.16; 95% CI, 1.11 to 1.21; P = 1.43×10 -11 ), which was associated with lower haptoglobin protein levels. The rare VEGFA mutation could cause a conformational change and increase the binding affinity of VEGFA to its receptors. Furthermore, this variant was associated with the increased risk of kidney disease progression in IgA nephropathy (hazard ratio, 2.99; 95% CI, 1.09 to 8.21; P = 0.03). Our study identified two novel risk variants for IgA nephropathy in VEGFA and PKD1L3 and helps expand our understanding of the genetic basis of IgA nephropathy susceptibility.

Targeted resequencing of the 13q13 spondyloarthritis-linked locus identifies a rare variant in FREM2 possibly associated with familial spondyloarthritis.

The strong heritability of spondyloarthritis remains poorly explained, despite several large-scale association studies. A recent linkage analysis identified a new region linked to SpA on 13q13. Here we searched for variants potentially explaining this linkage signal by deep-sequencing of the region. Re-sequencing of the 1.4 Mb target interval was performed in 92 subjects from the 43 best-linked multicases families (71 spondyloarthritis and 21 unaffected relatives), using hybridization capture-based protocol (Illumina Nextera®). Variants of interest were then genotyped by TaqMan and high resolution melting to check their co-segregation with disease in the same families and to test their association with spondyloarthritis in an independent cohort of 1,091 unrelated cases and 399 controls. Expression of FREM2 was assessed by immunostaining. Of the 7,563 variants identified, 24 were non-synonymous coding single-nucleotide variants. Two of them were located in the FREM2 gene on a haplotype co-segregating with the disease, including one common variant (R1840W, minor allele frequency=0.11) and one rare variant (R727H, minor allele frequency=0.0001). In the case-control analysis, there was no significant association between R1840W and spondyloarthritis (P-value=0.21), whereas R727H was not detected in any of the genotyped individuals. Immunostaining experiments revealed that FREM2 is expressed in synovial membrane, cartilage and colon. Targeted re-sequencing of a spondyloarthritis-linked region allowed us to identify a rare non-synonymous coding variant in FREM2, co-segregating with spondyloarthritis in a large family. This gene is expressed in several tissues relevant to spondyloarthritis pathogenesis, supporting its putative implication in spondyloarthritis.

Pharmacogenomic Profile of Amazonian Amerindians.

Given the role of pharmacogenomics in the large variability observed in drug efficacy/safety, an assessment about the pharmacogenomic profile of patients prior to drug prescription or dose adjustment is paramount to improve adherence to treatment and prevent adverse drug reaction events. A population commonly underrepresented in pharmacogenomic studies is the Native American populations, which have a unique genetic profile due to a long process of geographic isolation and other genetic and evolutionary processes. Here, we describe the pharmacogenetic variability of Native American populations regarding 160 pharmacogenes involved in absorption, distribution, metabolism, and excretion processes and biological pathways of different therapies. Data were obtained through complete exome sequencing of individuals from 12 different Amerindian groups of the Brazilian Amazon. The study reports a total of 3311 variants; of this, 167 are exclusive to Amerindian populations, and 1183 are located in coding regions. Among these new variants, we found non-synonymous coding variants in the DPYD and the IFNL4 genes and variants with high allelic frequencies in intronic regions of the MTHFR, TYMS, GSTT1, and CYP2D6 genes. Additionally, 332 variants with either high or moderate (disruptive or non-disruptive impact in protein effectiveness, respectively) significance were found with a minimum of 1% frequency in the Amazonian Amerindian population. The data reported here serve as scientific basis for future design of specific treatment protocols for Amazonian Amerindian populations as well as for populations admixed with them, such as the Northern Brazilian population.

Identification of pharmacogenomic variants associated with oncology treatments in Brazilian Amazonian Amerindians.

e15082 Background: Adverse drug reactions are an important cause of morbidity and mortality commonly found in different therapeutic regimens, particularly in oncologic. About 20-30% of these reactions are due to the individual genetic variability of patients. Pharmacogenomics focuses on the identification of genetic variants that influence drug efficacy, response, and/or toxicity by changes in pharmacokinetics/pharmacodynamics. Pharmacogenomic investigations are known to have a population bias. There is a gap in the accumulated about pharmacogenomic variants in poorly studied populations, such as the Amerindian population from the Brazilian Amazon region. The objective of this work is to describe the pharmacogenetic variability regarding 160 pharmacogenes involved in pharmacokinetic processes and biological pathways of different therapies, including oncology treatments, based on data obtained through complete exome sequencing of 64 individuals from different Amerindian groups of the Brazilian Amazon. Methods: The present study was approved by the National Committee for Ethics in Research and the Research Ethics Committee of the UFPA Tropical Medicine Center, under CAAE number 20654313.6.0000.5172. All participants signed a free-informed consent. The DNA extraction was performed using phenol-chloroform. Libraries were prepared using the Nextera Rapid Capture Exome and SureSelect Human All Exon V6 kits. Bioinformatic analysis was performed by ViVa software. Results: Our results show a total of 3,311 variants found in the study subjects. Of this, 167 are exclusive variants found in the Amerindians. Among these new variants, we found a non-synonymous coding variant in the DPYD gene with an allelic frequency of 3%, and variants with high allelic frequencies in intronic regions, which may regulate gene expression: MTHFR (5.56%), TYMS (11.5%) GSTT1 (54.1%), and three variants in the CYP2D6 gene with frequencies of 54%, 62.3%, and 65.6%. The DPYD is a pivotal gene involved with the efficacy of fluoropyrimidine-based treatment; the MTHFR and TYMS genes also participate in the biological pathways of these drugs, responsible for variation in response rates. The GSTT1 is a gene associated with platinum-based treatments, such as carboplatin and cisplatin. And, finally, CYP2D6 is one of the main pharmacogene described, involved in several drug schemes, including tamoxifen and gefitinib, used to treat breast and lung cancer, respectively. Conclusions: Understanding the diversity of genetic markers in Amazonian Amerindian is crucial to the implementation of pharmacogenomic-guided oncology treatment protocols, since pharmacogenomic data validated in other ancestral groups may not be fully applicable in these populations due to their unique genetic profile. Keywords: Exome, Native American populations, fluoropyrimidine, platinum-based drugs, tamoxifen, gefitinib.

Genetic Control of Splicing at SIRPG Modulates Risk of Type 1 Diabetes.

Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes, where it acts by engaging its ligand, CD47. SIRPG, which encodes SIRPγ, contains a nonsynonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of SIRPG transcript isoforms. All of these transcript isoforms produced protein upon transient expression in vitro. However, CRISPR/Cas9 targeting of one of the alternatively spliced exons in SIRPG eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild-type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling, and had significantly increased levels of cell-surface CD47. In primary CD4+ and CD8+ T cells, cell-surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that SIRPG is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.

Whole Genome Sequencing Uncovers a Rare Germline Variant in ATM Associated with Familial Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are sporadic diseases characterized by a somatic driver mutation in the JAK2, CALR or MPL gene. Although it is generally considered a sporadic disease, approximately 10% of MPN cases display familial clustering, and there is a 5 to 7-fold increased risk of developing an MPN among first degree relatives of MPN patients. In contrast to other myeloid malignancies, investigation of large pedigrees with familial clustering of MPN has failed to identify high-risk predisposition genes relevant to the general MPN population. Genome wide association studies (GWAS) have identified common, low penetrance risk alleles for MPN predisposition in multiple genes including JAK2, TERT, TET2, ATM and SH2B3. In order to identify novel germline predisposition variants in MPN, an unbiased whole genome sequencing (WGS) approach was utilized to examine genomic structure and germline variations in a cohort of individuals with familial MPN.Methods: The study cohort was comprised of 67 individuals with familial MPN enrolled in a prospective research registry at Johns Hopkins Hospital. Familial MPN was defined as a diagnosis of MPN in an individual with a family history of MPN or related myeloid malignancy (myelodysplastic syndrome and chronic myelomonocytic leukemia) in a first or second degree relative. Neutrophil genomic DNA was subjected to WGS using Illumina HiSeq platform and sequenced to 60x depth. We performed germline variant calling using HaplotypeCaller and following the GATK best practices. The variants detected were further enriched for germline by allele frequency 40-60% or >90% and presence in the gnomAD database. Non-synonymous coding variants that occurred in the study cohort at a statistically higher frequency that in the general population (gnomAD) were selected for further analysis. Prediction of variant deleteriousness was assessed by 4 algorithms (Provean, SIFT, Polyphen-2, CADD).Results: Filtering of 32,788 non-synonymous, likely germline variants produced 148 that occurred at a higher frequency in our cohort than in the general population (p < 0.01, Fisher's exact test). Of these, 29 were predicted to be pathogenic in 3 out of 4 algorithms. Five unrelated individuals were found to harbor a heterozygous p.Leu2307Phe variant in the ATM gene (chr11:108326169 C>T). The clinical characteristics of these individuals are presented in Table 1. The structure prediction of ATM indicates that Ser2306 is a potential phosphorylation site of protein kinase A, suggesting that the methionine-aromatic bond between M2026 and the mutated F2307 may block the phosphorylation of S2306 (Figure 1).Conclusions: We identified a rare ATM germline variant (chr11:108326169 C>T; p.Leu2307Phe) present in 5 individuals with familial MPN. ATM is involved in DNA damage repair and important in the maintenance of genomic integrity. Heterozygous germline variants in ATM are known to predispose to multiple cancer types, including breast, prostate, pancreatic and melanoma. Further, common polymorphisms in ATM have been found to be associated with the MPN phenotype via GWAS. Our data suggests that this variant may impact ATM activation and its function in DNA damage repair, and functional studies are in progress. These data implicate a rare germline ATM variant as a novel risk factor for development of MPN. [Display omitted] DisclosuresHourigan: Sellas: Research Funding.

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.

A KCNK16 mutation causing TALK-1 gain of function is associated with maturity-onset diabetes of the young.

Maturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders of impaired pancreatic β cell function. The mechanisms underlying MODY include β cell KATP channel dysfunction (e.g., KCNJ11 [MODY13] or ABCC8 [MODY12] mutations); however, no other β cell channelopathies have been associated with MODY to date. Here, we have identified a nonsynonymous coding variant in KCNK16 (NM_001135105: c.341T>C, p.Leu114Pro) segregating with MODY. KCNK16 is the most abundant and β cell–restricted K+ channel transcript, encoding the two-pore-domain K+ channel TALK-1. Whole-cell K+ currents demonstrated a large gain of function with TALK-1 Leu114Pro compared with TALK-1 WT, due to greater single-channel activity. Glucose-stimulated membrane potential depolarization and Ca2+ influx were inhibited in mouse islets expressing TALK-1 Leu114Pro with less endoplasmic reticulum Ca2+ storage. TALK-1 Leu114Pro significantly blunted glucose-stimulated insulin secretion compared with TALK-1 WT in mouse and human islets. These data suggest that KCNK16 is a previously unreported gene for MODY.

A Chinese patient with developmental and epileptic encephalopathies (DEE) carrying a TRPM3 gene mutation: a paediatric case report

BackgroundDevelopmental and epileptic encephalopathies (DEEs) are a heterogeneous group of chronic encephalopathies characterized by epilepsy with comorbid intellectual disability that are frequently associated with de novo nonsynonymous coding variants in ion channels, cell-surface receptors, and other neuronally expressed genes. Mutations in TRPM3 were identified as the cause of DEE. We report a novel patient with DEE carrying a de novo missense mutation in TRPM3, p.(S1202T); this missense mutation has never been reported.Case presentationA 7-year and 2-month-old Chinese patient who had recurrent polymorphic seizures was clinically diagnosed with DEE. A de novo missense mutation in TRPM3, which has not yet been reported, was identified in this case. The patient had a clinical phenotype consistent with previous reports.ConclusionsThese findings could expand the spectrum of TRPM3 mutations and might also support that de novo substitutions of TRPM3 are a cause of DEE.

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans.

Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans. In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting. Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men. These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.

A zebrafish functional genomics model to investigate the role of human A20 variants in vivo

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.

ATRAID regulates the action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.

De novo substitutions of TRPM3 cause intellectual disability and epilepsy

The developmental and epileptic encephalopathies (DEE) are a heterogeneous group of chronic encephalopathies frequently associated with rare de novo nonsynonymous coding variants in neuronally expressed genes. Here, we describe eight probands with a DEE phenotype comprising intellectual disability, epilepsy, and hypotonia. Exome trio analysis showed de novo variants in TRPM3, encoding a brain-expressed transient receptor potential channel, in each. Seven probands were identically heterozygous for a recurrent substitution, p.(Val837Met), in TRPM3’s S4–S5 linker region, a conserved domain proposed to undergo conformational change during gated channel opening. The eighth individual was heterozygous for a proline substitution, p.(Pro937Gln), at the boundary between TRPM3’s flexible pore-forming loop and an adjacent alpha-helix. General-population truncating variants and microdeletions occur throughout TRPM3, suggesting a pathomechanism other than simple haploinsufficiency. We conclude that de novo variants in TRPM3 are a cause of intellectual disability and epilepsy.

25-OR: Investigating a Candidate Causal Allele in Type 2 Diabetes Susceptibility Gene PAM as a Cause of Neonatal Diabetes Mellitus

Peptidylglycine α-amidating monooxygenase (PAM) catalyses the amidation of glycine-extended peptides, thereby maximising their biological potency. We recently showed that coding alleles in PAM mediate type 2 diabetes (T2D) risk through alterations to PAM protein activity and expression in the β cell. Identifying alleles along a continuum of functional severity informs on the relationship between protein perturbation and clinical effect. We hypothesised that rare, loss of function PAM alleles could cause severe β cell dysfunction presenting as neonatal diabetes. Sequencing of 15 probands presenting with diabetes at ≤ 6 months of age and their unaffected immediate family, identified a novel de novo nonsynonymous coding variant (p.R36S) in PAM in a single proband. We investigated the impact of the variant allele on amidating activity, protein stability and localisation as a measure of PAM function and the consequences on β cell viability. Recombinant R36S-PAM exhibited normal amidating activity in vitro which was consistent with PAM activity measured in patient serum (WT: 391pmol/μl/hr vs. R36S: 305pmol/μl/hr). In human β cells (EndoC-βh1) the secreted luminal R36S-PAM isoform displayed reduced protein expression (-78%, p=0.004) compared to WT-PAM, which could be rescued by inhibiting the proteosomal pathway (+120% of WT); whilst the non-secreted membrane-integral isoform was retained in the endoplasmic reticulum (ER). We observed that overexpression of R36S-PAM elevated levels of ER stress (p-IRE1: +196%, p=0.02; CHOP: +114%, p=0.051) and pro-apoptotic markers (ASK1: +145%, caspase-3: +154%) compared with WT-PAM. We have identified a novel, de novo coding variant, in a gene with a proven role in T2D risk, in a single patient with neonatal diabetes. Functional studies provide evidence for protein mislocalisation and instability and support a role for this allele in ER stress and β cell dysfunction consistent with neonatal diabetes pathogenesis. Disclosure S. Sengupta: None. L.L. Bonnycastle: None. B. Hastoy: None. A. Grotz: None. M.M. Umapathysivam: None. A. Raimondo: None. A. Swift: None. P.S. Chines: None. A. Clark: None. H. Huopio: None. F.S. Collins: None. M. Laakso: None. A.L. Gloyn: Consultant; Spouse/Partner; Eli Lilly and Company, Merck &amp; Co., Inc. Consultant; Self; Merck &amp; Co., Inc. Consultant; Spouse/Partner; Novo Nordisk A/S, Pfizer Inc. Research Support; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Other Relationship; Self; Diabetes UK, European Foundation for the Study of Diabetes. Funding UK Wellcome Trust

A rare autism-associated MINT2/APBA2 mutation disrupts neurexin trafficking and synaptic function

MINT2/APBA2 is a synaptic adaptor protein involved in excitatory synaptic transmission. Several nonsynonymous coding variants in MINT2 have been identified in autism spectrum disorders (ASDs); however, these rare variants have not been examined functionally and the pathogenic mechanisms are unknown. Here, we examined the synaptic effects of rat Mint2 N723S mutation (equivalent to autism-linked human MINT2 N722S mutation) which targets a conserved asparagine residue in the second PDZ domain of Mint2 that binds to neurexin-1α (Nrxn1α), a presynaptic cell-adhesion protein implicated in ASDs. We show the N723S mutation impairs Nrxn1α stabilization and trafficking to the membrane while binding to Nrxn1α remains unaffected. Using time-lapse imaging in primary mouse neurons, we found that the N723S mutant had more immobile puncta at neuronal processes compared to Mint2 wild type. We therefore, reasoned that the N723S mutant may alter the co-transport of Nrxn1α at axonal processes to presynaptic terminals. Indeed, we found the N723S mutation affected Nrxn1α localization at presynaptic terminals which correlated with a decrease in Nrxn-mediated synaptogenesis and miniature event frequency in excitatory synapses. Together, our data reveal Mint2 N723S leads to neuronal dysfunction, in part due to alterations in Nrxn1α surface trafficking and synaptic function of Mint2.

Functional architecture of low-frequency variants highlights strength of negative selection across coding and non-coding annotations.

Common variant heritability has been widely reported to be concentrated in variants within cell-type-specific noncoding functional annotations, but little is known about low-frequency variant functional architectures. We partitioned the heritability of both low-frequency (0.5%≤MAF<5%) and common (MAF≥5%) variants in 40 UK Biobank traits across a broad set of functional annotations. We determined that non-synonymous coding variants explain 17±1% of low-frequency variant heritability ( ) versus 2.1±0.2% of common variant heritability ( ). Cell-type-specific noncoding annotations that were significantly enriched for of corresponding traits were similarly enriched for for most traits, but more enriched for brain-related annotations and traits. For example, H3K4me3 marks in brain dorsolateral prefrontal cortex explain 57±12% of vs. 12±2% of for neuroticism. Forward simulations confirmed that low-frequency variant enrichment depends on the mean selection coefficient of causal variants in the annotation, and can be used to predict effect size variance of causal rare variants (MAF<0.5%).

Evaluation of novel Parkinson's disease candidate genes in the Chinese population.

Recent whole-exome sequencing studies in European patients with Parkinson's disease (PD) have identified potential risk variants across 33 novel PD candidate genes. We aim to determine if these reported candidate genes are similarly implicated in Asians by assessing common, rare, and novel nonsynonymous coding variants by sequencing all 33 genes in 198 Chinese samples and genotyping coding variants in an independent set of 9756 Chinese samples. We carried out further targeted sequencing of CD36 in an additional 576 Chinese and Korean samples. We found that only 8 of 43 reported risk variants were polymorphic in our Chinese samples. We identified several heterozygotes for rare loss-of-function mutations, including the reported CD36 p.Gln74Ter variant, in both cases and controls. We also observed 2 potential compound heterozygotes among PD cases for rare loss-of-function mutations in CD36 and SSPO. The other reported variants were common in East Asians and not associated with PD, completely absent, or only found in controls. Therefore, the 33 reported candidate genes and associated variants are unlikely to confer significant PD risk in the East Asian population.

Targeted next-generation sequencing analysis in couples at increased risk for autosomal recessive disorders

BackgroundMany of the genetic childhood disorders leading to death in the pre- or neonatal period or during early childhood follow autosomal recessive modes of inheritance and bear specific challenges for genetic counseling and prenatal diagnostics. Parents are carriers but clinically unaffected, and diseases are rare but have recurrence risks of 25% in the same family. Often, affected children (or fetuses) die before a genetic diagnosis can be established, post-mortem analysis and phenotypic descriptions are insufficient and DNA from affected fetuses or children is not available for later analysis. A genetic diagnosis showing biallelic causative mutations is, however, the requirement for targeted carrier testing in parents and prenatal and preimplantation genetic diagnosis in further pregnancies.MethodsWe undertook targeted next-generation sequencing (NGS) for carrier screening of autosomal recessive lethal disorders in 8 consanguineous and 5 non-consanguineous couples with one or more affected children. We searched for heterozygous variants (non-synonymous coding or splice variants) in parents’ DNA, using a set of 430 genes known to be causative for rare autosomal recessive diseases with poor prognosis, and then filtering for variants present in genes overlapping in both partners. Putative pathogenic variants were tested for cosegregation in affected fetuses or children where material was available.ResultsThe diagnosis for the premature death in children was established in 5 of the 13 couples. Out of the 8 couples in which no causative diagnosis could be established 4 consented to undergo further analysis, in two of those a potentially causative variant in a novel candidate gene was identified.ConclusionsFor the families in whom causative variants could be identified, these may now be used for prenatal and preimplantation genetic diagnostics. Our data show that NGS based gene panel sequencing of selected genes involved in lethal autosomal recessive disorders is an effective tool for carrier screening in parents and for the identification of recessive gene defects and offers the possibility of prenatal and preimplantation genetic diagnosis in further pregnancies in families that have experienced deaths in early childhood and /or multiple abortions.

Screening of GLE1 mutations in Chinese amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is a lethal neurological disease primarily involving the spinal cord, brainstem, and corticospinal tract. Recently, mutations in the GLE1 gene were reported in Caucasian ALS patients. To inquire whether Chinese ALS patients carried causal mutations in the gene, we screened all 16 coding exons of GLE1 with Sanger sequencing in a Han Chinese cohort of 250 ALS cases. No nonsynonymous coding variants were detected. Our results suggest that pathogenic variants in the GLE1 gene are rare in Chinese ALS patients.