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Background: The Worm Study, ascertained from a multigeneration pedigree segregating a single amino acid deletion in SCN5A (c.4850_4852delTCT, p.(Phe1617del), rs749697698), is characterized by substantial phenotypic heterogeneity and overlap of sudden cardiac death, long-QT syndrome, cardiac conduction disease, Brugada syndrome, and isorhythmic atrioventricular dissociation. Linkage analysis for a synthetic trait derived from these phenotypes identified a single peak (logarithm of the odds [LOD] = 4.52) at the SCN5A/SCN10A/SCN11A locus on chromosome 3. Objective: This study explored the role of additional genetic variation in the chromosome 3 locus as a source of phenotypic heterogeneity in the Worm Study population. Methods: Genotypes underlying the linkage peak (n = 70) were characterized using microarrays. Haplotypes were determined using family-aware phasing and a population-specific reference panel. Variants with minor allele frequencies >0.10 were tested for association with cardiac conduction disease and isorhythmic dissociation using LAMP and logistic regression. Results: Only 1 haplotype carried the p.Phe1617del/rs749697698 deletion, suggesting relatively recent development (∼18 generations); this haplotype contained 5 other missense variants spanning SCN5A/SCN10A/SCN11A. Noncarrier haplotypes (n = 74) ranged in frequency from 0.5% to 5%. Although no variants were associated with cardiac conduction disease, a homozygous missense variant in SCN10A was associated with isorhythmic dissociation after correction for multiple comparisons (odds ratio 11.23; 95% confidence interval 2.76–23.39; P = 1.2 × 10 −4). This variant (rs12632942) was previously associated with PR interval. Conclusion: Our data suggest that other variants, alongside a pathogenic mutation, are associated with phenotypic heterogeneity. Single-mutation screening may be insufficient to predict electrical heart disease in patients and family members. In the Worm Study population, segregating a pathogenic SCN5A mutation, compound variation in the SCN5A/SCN10A/SCN11A locus determines arrhythmic outcome.

The human proteome is a crucial intermediate between complex diseases and their genetic and environmental components, and an important source of drug development targets and biomarkers. Here, we comprehensively assess the genetic architecture of 257 circulating protein biomarkers of cardiometabolic relevance through high-depth (22.5×) whole-genome sequencing (WGS) in 1328 individuals. We discover 131 independent sequence variant associations (P, The human proteome represents a crucial link between complex disease and genetic/environmental factors. Here, the authors investigate 257 cardiometabolic-relevant protein biomarkers in whole genome sequencing data from 1328 individuals, revealing the genetic architecture underlying biomarker variation.

Cardiometabolic diseases, such as type 2 diabetes and cardiovascular disease, have a high public health burden. Understanding the genetically determined regulation of proteins that are dysregulated in disease can help to dissect the complex biology underpinning them. Here, we perform a protein quantitative trait locus (pQTL) analysis of 248 serum proteins relevant to cardiometabolic processes in 2893 individuals. Meta-analyzing whole-genome sequencing (WGS) data from two Greek cohorts, MANOLIS (n = 1356; 22.5× WGS) and Pomak (n = 1537; 18.4× WGS), we detect 301 independently associated pQTL variants for 170 proteins, including 12 rare variants (minor allele frequency

Summary Osteoarthritis affects over 300 million people worldwide. Here, we conduct a genome-wide association study meta-analysis across 826,690 individuals (177,517 with osteoarthritis) and identify 100 independently associated risk variants across 11 osteoarthritis phenotypes, 52 of which have not been associated with the disease before. We report thumb and spine osteoarthritis risk variants and identify differences in genetic effects between weight-bearing and non-weight-bearing joints. We identify sex-specific and early age-at-onset osteoarthritis risk loci. We integrate functional genomics data from primary patient tissues (including articular cartilage, subchondral bone, and osteophytic cartilage) and identify high-confidence effector genes. We provide evidence for genetic correlation with phenotypes related to pain, the main disease symptom, and identify likely causal genes linked to neuronal processes. Our results provide insights into key molecular players in disease processes and highlight attractive drug targets to accelerate translation., Graphical abstract, Highlights • A multicohort study identifies 52 previously unknown osteoarthritis genetic risk variants • Similarities and differences in osteoarthritis genetic risk depend on joint sites • Osteoarthritis genetic components are associated with pain-related phenotypes • High-confidence effector genes highlight potential targets for drug intervention, A multicohort genome-wide association meta-analysis of osteoarthritis highlights the impact of joint site types on the features of genetic risk variants and the link between osteoarthritis genetics and pain-related phenotypes, pointing toward potential targets for therapeutic intervention.

Immune-mediated heparin-induced thrombocytopenia (HIT) is the clinically most important adverse drug reaction (ADR) in response to heparin therapy characterized by a prothrombotic state despite a decrease in platelet count. We conducted a genome-wide association study in 96 suspected HIT cases and 96 controls to explore the genetic predisposition for HIT within a case-control pharmacovigilance study followed by replication in additional 86 cases and 86 controls from the same study. One single nucleotide polymorphism (SNP, rs1433265, P = 6.5 x 10(-5), odds ratio (OR) 2.79) from 16 identified SNPs was successfully replicated (P = 1.5 x 10(-4), OR 2.77; combined data set P = 2.7 x 10(-8), OR 2.77) and remained the most strongly associated SNP after imputing locus genotypes. Fine mapping revealed a significantly associated risk-conferring haplotype (P = 4.9 x 10(-6), OR 2.41). In order to find rare variants contributing to the association signals, we applied a targeted resequencing approach in a subgroup of 73 HIT patients and 23 controls for the regions with the 16 most strongly HIT-associated SNPs. C-alpha testing was applied to test for the impact of rare variants and we detected two candidate genes, the discoidin domain receptor tyrosine kinase 1 (DDR1, P = 3.6 x 10(-2)) and the multiple C2 and transmembrane domain containing 2 (MCTP2, P = 4.5 x 10(-2)). For the genes interactor of little elongation complex ELL subunit 1 (ICE1) and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, 16 (ADAMTS16) nearby rs1433265, we identified several missense variants. Although replication in an independent population is warranted, these findings provide a basis for future studies aiming to identify and characterize genetic susceptibility factors for HIT. We identified and validated a HIT-associated locus on chromosome 5. Targeted NGS analysis for rare variants identifies DDR1 and MCTP2 as novel candidates. In addition, missense variants for ADAMTS16 and ICE1 were identified in the locus.

BACKGROUND: Heritable cardiac-sodium channel dysfunction is associated with various arrhythmia syndromes, some predisposing to ventricular fibrillation. Phenotypic diversity among carriers of identical-by-descent mutations is often remarkable, suggesting influences of genetic modifiers.OBJECTIVE: The purpose of this study was to identify a unique SCN5A-mutation founder population with mixed clinical phenotypes and sudden cardiac death, and to investigate the heritability of electromechanical traits besides the SCN5A-mutation effect.METHODS: The 16-generation founder population segregating SCN5A c.4850_4852delTCT, p.(Phe1617del), was comprehensively phenotyped. Variance component analysis was used to evaluate the mutation's effects and assess heritability.RESULTS: In 45 p.(Phe1617del) positives, the mutation associated strongly with QTc prolongation (472 ± 60 ms vs 423 ± 35 ms in 26 mutation negatives; P P P P = .006). p.(Phe1617del) was an important determinant of QTcbaseline, QTcmax, and EMW, explaining 18%, 28%, and 37%, respectively, of the trait’s variance. Significant heritability was observed for PQ interval (P = .003) after accounting for the p.(Phe1617del) effect.CONCLUSION: This SCN5A-p.(Phe1617del) founder population with phenotypic divergence and overlap reveals long-QT syndrome-related and arousal-evoked ventricular tachyarrhythmias with a female preponderance. Variance component analysis indicates additional genetic variance for PQ interval hidden in the genome, besides a dominant p. .(Phe1617del) effect on QTc and EMW.

The human proteome is a crucial intermediate between complex diseases and their genetic and environmental components, and an important source of drug development targets and biomarkers. Here, we conduct high-depth (22.5x) whole-genome sequencing (WGS) in 1,328 individuals to fully assess the genetic architecture of 257 circulating protein biomarkers of cardiometabolic relevance. We discover 132 independent sequence variant associations ( P

Recent genome-wide association studies (GWAS) have confirmed known risk mutations for venous thromboembolism (VTE) and identified a number of novel susceptibility loci in adults. Here we present a GWAS in 212 nuclear families with pediatric VTE followed by targeted next-generation sequencing (NGS) to identify causative mutations contributing to the association. Three single nucleotide polymorphisms (SNPs) exceeded the threshold for genome-wide significance as determined by permutation testing using 100 000 bootstrap permutations (P

Background— Recently, we reported a gene network of ADAMTS (A Disintegrin-like and Metalloprotease with Thrombospondin motifs) genes as central component of the genetic risk contributing to pediatric stroke. ADAMTS13 is a prime example for such a key component as it cleaves von Willebrand factor multimers, reduces platelet adhesion and aggregation, and downregulates thrombus formation and inflammation. Methods and Results— We characterized the genetic architecture of ADAMTS13 through targeted next-generation sequencing of 48 affected children and their unaffected siblings and identified in total 241 variants (single nucleotide polymorphisms or insertions/deletions) in the ADAMTS13 gene. From these, based on significance in the sibship disequilibrium test ( P ADAMTS13 gene for genotyping in 270 trios and subsequent association analyses. Transmission disequilibrium testing was performed for affection status and ADAMTS13 activity levels using PLINK and FBAT, respectively. Ten single nucleotide polymorphisms were significantly associated with pediatric stroke ( P P P =0.0004 and P =0.0092). The resulting protective haplotype H1.1. (T:U 95.5: 144.4; P =0.0016) is associated with increased ADAMTS13 levels (age-adjusted P =0.0108). Haplotype association using a sliding window approach assigns this association to the ADAMTS13 von Willebrand factor–binding domain ( P =1.2×10 −4 ). Conclusions— Our data provide a link between the genetic architecture of ADAMTS13 , ADAMTS13 levels, and stroke susceptibility. Altogether, these studies render ADAMTS13 an attractive candidate for functional studies and may contribute to personalized diagnosis and treatment options in future.

Gene and protein expression profiles of iris biopsies, aqueous humor (AqH), and sera in patients with juvenile idiopathic arthritis-associated uveitis (JIAU) in comparison to control patients with primary open-angle glaucoma (POAG) and HLA-B27-positive acute anterior uveitis (AAU) were investigated. Via RNA Sequencing (RNA-Seq) and mass spectrometry-based protein expression analyses 136 genes and 56 proteins could be identified as being significantly differentially expressed (DE) between the JIAU and POAG group. Gene expression of different immunoglobulin (Ig) components as well as of the B cell-associated factors ID3, ID1, and EBF1 was significantly upregulated in the JIAU group as compared to POAG patients. qRT-PCR analysis showed a significantly higher gene expression of the B cell-related genes CD19, CD20, CD27, CD138, and MZB1 in the JIAU group. At the protein level, a significantly higher expression of Ig components in JIAU than in POAG was confirmed. The B cell-associated protein MZB1 showed a higher expression in JIAU patients than in POAG which was confirmed by western blot analysis. Using bead-based immunoassay analysis we were able to detect a significantly higher concentration of the B cell-activating and survival factors BAFF, APRIL, and IL-6 in the AqH of JIAU and AAU patients than in POAG patients. The intraocularly upregulated B cell-specific genes and proteins in iris tissue suggest that B cells participate in the immunopathology of JIAU. The intracameral environment in JIAU may facilitate local effector and survival functions of B cells, leading to disease course typical for anterior uveitis.

Unraveling the genetic susceptibility of complex diseases such as chronic kidney disease remains challenging. Here, we used inbred rat models of kidney damage associated with elevated blood pressure for the comprehensive analysis of a major albuminuria susceptibility locus detected in these models. We characterized its genomic architecture by congenic substitution mapping, targeted next-generation sequencing, and compartment-specific RNA sequencing analysis in isolated glomeruli. This led to prioritization of transmembrane protein Tmem63c as a novel potential target. Tmem63c is differentially expressed in glomeruli of allele-specific rat models during onset of albuminuria. Patients with focal segmental glomerulosclerosis exhibited specific TMEM63C loss in podocytes. Functional analysis in zebrafish revealed a role for tmem63c in mediating the glomerular filtration barrier function. Our data demonstrate that integrative analysis of the genomic architecture of a complex trait locus is a powerful tool for identification of new targets such as Tmem63c for further translational investigation., eLife digest The human kidneys filter the entire volume of the blood about 300 times each day. This ability depends on specialized cells, known as podocytes, which wrap around some of the blood vessels in the kidney. These cells control which molecules leave the blood based on their size. Normally large molecules like proteins are blocked, while smaller molecules including waste products, toxins, excess water and salts pass through into the urine. If this filtration system is damaged, by high blood pressure, for example, it can lead to chronic kidney disease. A hallmark of this disease, often called CKD for short, is high levels of the protein albumin in the urine. Previous studies involving rats with high blood pressure have found several regions of the genome that contribute to high levels of albumin in the urine, including one on chromosome 6. However, this region contains several genes and it was unclear which genes affected the condition. Schulz et al. set out to narrow down the list and find specific genes that might contribute to elevated albumin in the urine of rats with high blood pressure. This search identified the gene for a protein called TMEM63c as a likely candidate. This protein spans the outer membrane of podocyte cells. Analysis of kidney biopsies showed that patients with chronic kidney disease also had low levels of this protein in their podocytes. Further experiments, this time in zebrafish, showed that reducing the activity of the gene for tmem63c led to damaged podocytes and a leakier filter in the kidneys. The results suggest that this gene plays an important role in the integrity of the kidneys filtration barrier. It is possible that faulty versions of this gene are behind some cases of chronic kidney disease. If this proves to be the case, a better understanding of the role of this gene may lead to new treatments for the condition.

BACKGROUND The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients’ lymphocytes was reduced by JAK inhibitors. CONCLUSIONS STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.)

We present a technique to find threshold values that allows the user to separate signal from noise in fluorescence grey-level images. It can be classified as a purely comparative method based upon the amount of “Mutual Information” between two or more florescence images, and we apply it to stacks of such images produced using the newly-developed MELK technology. Our results are compared to results obtained by another research group using a quite different (completely independent and more technology-based) approach; and also to results obtained using Otsu's Thresholding Method, yet another completely distinct approach invented to separate foreground and background in a grey-level image, based on minimising “intra-class variance” [9,10]. The remarkably good agreement found suggests that our proposed comparative information based method not only accounts for the biological mechanisms governing cellular protein networks very well, but also (and probably much more importantly) shows that cells actually organise the spatial structure of their protein networks in a highly non-random fashion as might be expected – and thereby try to optimise their “mutual information content”, and thus most probably their efficiency.

In this note, we present a new method that allows us to determine threshold values for separating presence and absence of proteins in a stack of fluorescence images describing a spatial distribution of proteins across a biological object (like a slice of nervous tissue, a sample of blood cells, etc.). We apply this method to stacks of fluorescence images and find that the resulting threshold values are almost identical with threshold values found using completely independent methods based on technological and biological aspects of the images in question.

In this note, we present a new method that allows us to determine threshold values for separating presence and absence of proteins in a stack of fluorescence images describing a spatial distribution of proteins across a biological object (like a slice of nervous tissue, a sample of blood cells etc.). This method is based on the so-called Multi-Information Function which is closely related to the Mutual-Information Function and the Kullback-Leibler distance. We apply this method to stacks of fluorescence images and find that the resulting threshold values are almost identical with threshold values found using completely independent methods based on technological and biological aspects of the images in question.