Expert Panel Curation of 113 Primary Mitochondrial Disease Genes for the Leigh Syndrome Spectrum.

OBJECTIVE: Primary mitochondrial diseases (PMDs) are heterogeneous disorders caused by inherited mitochondrial dysfunction. Classically defined neuropathologically as subacute necrotizing encephalomyelopathy, Leigh syndrome spectrum (LSS) is the most frequent manifestation of PMD in children, but may also present in adults. A major challenge for accurate diagnosis of LSS in the genomic medicine era is establishing gene-disease relationships (GDRs) for this syndrome with >100 monogenic causes across both nuclear and mitochondrial genomes. METHODS: The Clinical Genome Resource (ClinGen) Mitochondrial Disease Gene Curation Expert Panel (GCEP), comprising 40 international PMD experts, met monthly for 4 years to review GDRs for LSS. The GCEP standardized gene curation for LSS by refining the phenotypic definition, modifying the ClinGen Gene-Disease Clinical Validity Curation Framework to improve interpretation for LSS, and establishing a scoring rubric for LSS. RESULTS: The GDR with LSS across the nuclear and mitochondrial genomes was classified as definitive for 31 of 114 GDRs curated (27%), moderate for 38 (33%), limited for 43 (38%), and disputed for 2 (2%). Ninety genes were associated with autosomal recessive inheritance, 16 were maternally inherited, 5 were autosomal dominant, and 3 were X-linked. INTERPRETATION: GDRs for LSS were established for genes across both nuclear and mitochondrial genomes. Establishing these GDRs will allow accurate variant interpretation, expedite genetic diagnosis of LSS, and facilitate precision medicine, multisystem organ surveillance, recurrence risk counseling, reproductive choice, natural history studies, and determination of eligibility for interventional clinical trials. ANN NEUROL 2023;94:696-712.

A clinical laboratory's experience using GeneMatcher-Building stronger gene-disease relationships.

The use of whole-genome sequencing (WGS) has accelerated the pace of gene discovery and highlighted the need for open and collaborative data sharing in the search for novel disease genes and variants. GeneMatcher (GM) is designed to facilitate connections between researchers, clinicians, health-care providers, and others to help in the identification of additional patients with variants in the same candidate disease genes. The Illumina Clinical Services Laboratory offers a WGS test for patients with suspected rare and undiagnosed genetic disease  and regularly submits potential candidate genes to GM to strengthen gene-disease relationships. We describe our experience with GM, including criteria for evaluation of candidate genes, and our workflow for the submission and review process. We have made 69 submissions, 36 of which are currently active. Ten percent of submissions have resulted in publications, with an additional 14 submissions part of ongoing collaborations and expected to result in a publication.

The Gene Curation Coalition: A global effort to harmonize gene-disease evidence resources.

PURPOSE: Several groups and resources provide information that pertains to the validity of gene-disease relationships used in genomic medicine and research; however, universal standards and terminologies to define the evidence base for the role of a gene in disease and a single harmonized resource were lacking. To tackle this issue, the Gene Curation Coalition (GenCC) was formed. METHODS: The GenCC drafted harmonized definitions for differing levels of gene-disease validity on the basis of existing resources, and performed a modified Delphi survey with 3 rounds to narrow the list of terms. The GenCC also developed a unified database to display curated gene-disease validity assertions from its members. RESULTS: On the basis of 241 survey responses from the genetics community, a consensus term set was chosen for grading gene-disease validity and database submissions. As of December 2021, the database contained 15,241 gene-disease assertions on 4569 unique genes from 12 submitters. When comparing submissions to the database from distinct sources, conflicts in assertions of gene-disease validity ranged from 5.3% to 13.4%. CONCLUSION: Terminology standardization, sharing of gene-disease validity classifications, and resolution of curation conflicts will facilitate collaborations across international curation efforts and in turn, improve consistency in genetic testing and variant interpretation.

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease.

PURPOSE: Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test. METHODS: We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases. RESULTS: We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs. CONCLUSION: Robust identification of CNVs by GS is possible within a clinical testing environment.

A genome-wide association study and biological pathway analysis of epilepsy prognosis in a prospective cohort of newly treated epilepsy.

We present the analysis of a prospective multicentre study to investigate genetic effects on the prognosis of newly treated epilepsy. Patients with a new clinical diagnosis of epilepsy requiring medication were recruited and followed up prospectively. The clinical outcome was defined as freedom from seizures for a minimum of 12 months in accordance with the consensus statement from the International League Against Epilepsy (ILAE). Genetic effects on remission of seizures after starting treatment were analysed with and without adjustment for significant clinical prognostic factors, and the results from each cohort were combined using a fixed-effects meta-analysis. After quality control (QC), we analysed 889 newly treated epilepsy patients using 472 450 genotyped and 6.9 × 10(6) imputed single-nucleotide polymorphisms. Suggestive evidence for association (defined as Pmeta < 5.0 × 10(-7)) with remission of seizures after starting treatment was observed at three loci: 6p12.2 (rs492146, Pmeta = 2.1 × 10(-7), OR[G] = 0.57), 9p23 (rs72700966, Pmeta = 3.1 × 10(-7), OR[C] = 2.70) and 15q13.2 (rs143536437, Pmeta = 3.2 × 10(-7), OR[C] = 1.92). Genes of biological interest at these loci include PTPRD and ARHGAP11B (encoding functions implicated in neuronal development) and GSTA4 (a phase II biotransformation enzyme). Pathway analysis using two independent methods implicated a number of pathways in the prognosis of epilepsy, including KEGG categories 'calcium signaling pathway' and 'phosphatidylinositol signaling pathway'. Through a series of power curves, we conclude that it is unlikely any single common variant explains >4.4% of the variation in the outcome of newly treated epilepsy.

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls.

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.

Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome.

Gain-of-function mutations in CACNA1C, encoding the L-type Ca(2+) channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS.

Genome-wide methylation analyses of primary human leukocyte subsets identifies functionally important cell-type-specific hypomethylated regions.

DNA methylation is an important mechanism by which gene transcription and hence cellular function are regulated. Here, we provide detailed functional genome-wide methylome maps of 5 primary peripheral blood leukocyte subsets including T cells, B cells, monocytes/macrophages, and neutrophils obtained from healthy individuals. A comparison of these methylomes revealed highly specific cell-lineage and cell-subset methylation profiles. DNA hypomethylation is known to be permissive for gene expression and we identified cell-subset-specific hypomethylated regions (HMRs) that strongly correlate with gene transcription levels suggesting these HMRs may regulate corresponding cell functions. Single-nucleotide polymorphisms associated with immune-mediated disease in genome-wide association studies preferentially localized to these cell-specific regulatory HMRs, offering insight into pathogenesis by highlighting cell subsets in which specific epigenetic changes may drive disease. Our data provide a valuable reference tool for researchers aiming to investigate the role of DNA methylation in regulating primary leukocyte function in health and immune-mediated disease.

A genetic study of Wilson's disease in the United Kingdom.

Previous studies have failed to identify mutations in the Wilson's disease gene ATP7B in a significant number of clinically diagnosed cases. This has led to concerns about genetic heterogeneity for this condition but also suggested the presence of unusual mutational mechanisms. We now present our findings in 181 patients from the United Kingdom with clinically and biochemically confirmed Wilson's disease. A total of 116 different ATP7B mutations were detected, 32 of which are novel. The overall mutation detection frequency was 98%. The likelihood of mutations in genes other than ATP7B causing a Wilson's disease phenotype is therefore very low. We report the first cases with Wilson's disease due to segmental uniparental isodisomy as well as three patients with three ATP7B mutations and three families with Wilson's disease in two consecutive generations. We determined the genetic prevalence of Wilson's disease in the United Kingdom by sequencing the entire coding region and adjacent splice sites of ATP7B in 1000 control subjects. The frequency of all single nucleotide variants with in silico evidence of pathogenicity (Class 1 variant) was 0.056 or 0.040 if only those single nucleotide variants that had previously been reported as mutations in patients with Wilson's disease were included in the analysis (Class 2 variant). The frequency of heterozygote, putative or definite disease-associated ATP7B mutations was therefore considerably higher than the previously reported occurrence of 1:90 (or 0.011) for heterozygote ATP7B mutation carriers in the general population (P < 2.2 × 10(-16) for Class 1 variants or P < 5 × 10(-11) for Class 2 variants only). Subsequent exclusion of four Class 2 variants without additional in silico evidence of pathogenicity led to a further reduction of the mutation frequency to 0.024. Using this most conservative approach, the calculated frequency of individuals predicted to carry two mutant pathogenic ATP7B alleles is 1:7026 and thus still considerably higher than the typically reported prevalence of Wilson's disease of 1:30 000 (P = 0.00093). Our study provides strong evidence for monogenic inheritance of Wilson's disease. It also has major implications for ATP7B analysis in clinical practice, namely the need to consider unusual genetic mechanisms such as uniparental disomy or the possible presence of three ATP7B mutations. The marked discrepancy between the genetic prevalence and the number of clinically diagnosed cases of Wilson's disease may be due to both reduced penetrance of ATP7B mutations and failure to diagnose patients with this eminently treatable disorder.

A framework for the evaluation and reporting of incidental findings in clinical genomic testing.

Currently, there are no widely accepted recommendations in the genomics field guiding the return of incidental findings (IFs), defined here as unexpected results that are unrelated to the indication for testing. Consequently, reporting policies for IFs among laboratories offering genomic testing are variable and may lack transparency. Herein we describe a framework developed to guide the evaluation and return of IFs encountered in probands undergoing clinical genome sequencing (cGS). The framework prioritizes clinical significance and actionability of IFs and follows a stepwise approach with stopping points at which IFs may be recommended for return or not. Over 18 months, implementation of the framework in a clinical laboratory facilitated the return of actionable IFs in 37 of 720 (5.1%) individuals referred for cGS, which is reduced to 3.1% if glucose-6-phosphate dehydrogenase (G6PD) deficiency is excluded. This framework can serve as a model to standardize reporting of IFs identified during genomic testing.

Determining the characteristics of genetic disorders that predict inclusion in newborn genomic sequencing programs.

Over 30 international research studies and commercial laboratories are exploring the use of genomic sequencing to screen apparently healthy newborns for genetic disorders. These programs have individualized processes for determining which genes and genetic disorders are queried and reported in newborns. We compared lists of genes from 26 research and commercial newborn screening programs and found substantial heterogeneity among the genes included. A total of 1,750 genes were included in at least one newborn genome sequencing program, but only 74 genes were included on >80% of gene lists, 16 of which are not associated with conditions on the Recommended Uniform Screening Panel. We used a linear regression model to explore factors related to the inclusion of individual genes across programs, finding that a high evidence base as well as treatment efficacy were two of the most important factors for inclusion. We applied a machine learning model to predict how suitable a gene is for newborn sequencing. As knowledge about and treatments for genetic disorders expand, this model provides a dynamic tool to reassess genes for newborn screening implementation. This study highlights the complex landscape of gene list curation among genomic newborn screening programs and proposes an empirical path forward for determining the genes and disorders of highest priority for newborn screening programs.

De novo variants in GABRA4 are associated with a neurological phenotype including developmental delay, behavioral abnormalities and epilepsy.

Nine out of 19 genes encoding GABAA receptor subunits have been linked to monogenic syndromes characterized by seizures and developmental disorders. Previously, we reported the de novo variant p.(Thr300Ile) in GABRA4 in a patient with epilepsy and neurodevelopmental abnormalities. However, no new cases have been reported since then. Through an international collaboration, we collected molecular and phenotype data of individuals carrying de novo variants in GABRA4. Patients and their parents were investigated either by exome or genome sequencing, followed by targeted Sanger sequencing in some cases. All variants within the transmembrane domain, including the previously reported p.(Thr300Ile) variant, were characterized in silico and analyzed by molecular dynamics (MD) simulation studies. We identified three novel de novo missense variants in GABRA4 (NM_000809.4): c.797 C > T, p.(Pro266Leu), c.899 C > A, p.(Thr300Asn), and c.634 G > A, p.(Val212Ile). The p.(Thr300Asn) variant impacts the same codon as the previously reported variant p.(Thr300Ile) and likely arose post-zygotically as evidenced by sequencing oral mucosal cells. Overlapping phenotypes among affected individuals included developmental delay (4/4), epileptiform EEG abnormalities (3/4), attention deficits (3/4), seizures (2/4), autistic features (2/4) and structural brain abnormalities (2/4). MD simulations of the three variants within the transmembrane domain of the receptor indicate that sub-microsecond scale dynamics differ between wild-type and mutated subunits. Taken together, our findings further corroborate an association between GABRA4 and a neurological phenotype including variable neurodevelopmental, behavioral and epileptic abnormalities.

Development of a comprehensive cardiovascular disease genetic risk assessment test.

Background: Despite monogenic and polygenic contributions to cardiovascular disease (CVD), genetic testing is not widely adopted, and current tests are limited by the breadth of surveyed conditions and interpretation burden. Methods: We developed a comprehensive clinical genome CVD test with semi-automated interpretation. Monogenic conditions and risk alleles were selected based on the strength of disease association and evidence for increased disease risk, respectively. Non-CVD secondary findings genes, pharmacogenomic (PGx) variants and CVD polygenic risk scores (PRS) were assessed for inclusion. Test performance was modeled using 2,594 genomes from the 1000 Genomes Project, and further investigated in 20 previously tested individuals. Results: The CVD genome test is composed of a panel of 215 CVD gene-disease pairs, 35 non-CVD secondary findings genes, 4 risk alleles or genotypes, 10 PGx genes and a PRS for coronary artery disease. Modeling of test performance using samples from the 1000 Genomes Project revealed ~6% of individuals with a monogenic finding in a CVD-associated gene, 6% with a risk allele finding, ~1% with a non-CVD secondary finding, and 93% with CVD-associated PGx variants. Assessment of blinded clinical samples showed complete concordance with prior testing. An average of 4 variants were reviewed per case, with interpretation and reporting time ranging from 9-96 min. Conclusions: A genome sequencing based CVD genetic risk assessment can provide comprehensive genetic disease and genetic risk information to patients with CVD. The semi-automated and limited interpretation burden suggest that this testing approach could be scaled to support population-level initiatives.

Target-enrichment strategies for next-generation sequencing.

We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.

Non dominant-negative KCNJ2 gene mutations leading to Andersen-Tawil syndrome with an isolated cardiac phenotype.

Andersen-Tawil syndrome (ATS) is characterized by dysmorphic features, periodic paralyses and abnormal ventricular repolarization. After genotyping a large set of patients with congenital long-QT syndrome, we identified two novel, heterozygous KCNJ2 mutations (p.N318S, p.W322C) located in the C-terminus of the Kir2.1 subunit. These mutations have a different localization than classical ATS mutations which are mostly located at a potential interaction face with the slide helix or at the interface between the C-termini. Mutation carriers were without the key features of ATS, causing an isolated cardiac phenotype. While the N318S mutants regularly reached the plasma membrane, W322C mutants primarily resided in late endosomes. Co-expression of N318S or W322C with wild-type Kir2.1 reduced current amplitudes only by 20-25 %. This mild loss-of-function for the heteromeric channels resulted from defective channel trafficking (W322C) or gating (N318S). Strikingly, and in contrast to the majority of ATS mutations, neither mutant caused a dominant-negative suppression of wild-type Kir2.1, Kir2.2 and Kir2.3 currents. Thus, a mild reduction of native Kir2.x currents by non dominant-negative mutants may cause ATS with an isolated cardiac phenotype.

Comprehensive sequence analysis of nine Usher syndrome genes in the UK National Collaborative Usher Study.

BACKGROUND: Usher syndrome (USH) is an autosomal recessive disorder comprising retinitis pigmentosa, hearing loss and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous with three distinctive clinical types (I-III) and nine Usher genes identified. This study is a comprehensive clinical and genetic analysis of 172 Usher patients and evaluates the contribution of digenic inheritance. METHODS: The genes MYO7A, USH1C, CDH23, PCDH15, USH1G, USH2A, GPR98, WHRN, CLRN1 and the candidate gene SLC4A7 were sequenced in 172 UK Usher patients, regardless of clinical type. RESULTS: No subject had definite mutations (nonsense, frameshift or consensus splice site mutations) in two different USH genes. Novel missense variants were classified UV1-4 (unclassified variant): UV4 is 'probably pathogenic', based on control frequency <0.23%, identification in trans to a pathogenic/probably pathogenic mutation and segregation with USH in only one family; and UV3 ('likely pathogenic') as above, but no information on phase. Overall 79% of identified pathogenic/UV4/UV3 variants were truncating and 21% were missense changes. MYO7A accounted for 53.2%, and USH1C for 14.9% of USH1 families (USH1C:c.496+1G>A being the most common USH1 mutation in the cohort). USH2A was responsible for 79.3% of USH2 families and GPR98 for only 6.6%. No mutations were found in USH1G, WHRN or SLC4A7. CONCLUSIONS: One or two pathogenic/likely pathogenic variants were identified in 86% of cases. No convincing cases of digenic inheritance were found. It is concluded that digenic inheritance does not make a significant contribution to Usher syndrome; the observation of multiple variants in different genes is likely to reflect polymorphic variation, rather than digenic effects.

Reactive gene curation to support interpretation and reporting of a clinical genome test for rare disease: Experience from over 1,000 cases.

Current standards in clinical genetics recognize the need to establish the validity of gene-disease relationships as a first step in the interpretation of sequence variants. We describe our experience incorporating the ClinGen Gene-Disease Clinical Validity framework in our interpretation and reporting workflow for a clinical genome sequencing (cGS) test for individuals with rare and undiagnosed genetic diseases. This "reactive" gene curation is completed upon identification of candidate variants during active case analysis and within the test turn-around time by focusing on the most impactful evidence and taking advantage of the broad applicability of the framework to cover a wide range of disease areas. We demonstrate that reactive gene curation can be successfully implemented in support of cGS in a clinical laboratory environment, enabling robust clinical decision making and allowing all variants to be fully and appropriately considered and their clinical significance confidently interpreted.

Exploration of signals of positive selection derived from genotype-based human genome scans using re-sequencing data.

We have investigated whether regions of the genome showing signs of positive selection in scans based on haplotype structure also show evidence of positive selection when sequence-based tests are applied, whether the target of selection can be localized more precisely, and whether such extra evidence can lead to increased biological insights. We used two tools: simulations under neutrality or selection, and experimental investigation of two regions identified by the HapMap2 project as putatively selected in human populations. Simulations suggested that neutral and selected regions should be readily distinguished and that it should be possible to localize the selected variant to within 40 kb at least half of the time. Re-sequencing of two ~300 kb regions (chr4:158Mb and chr10:22Mb) lacking known targets of selection in HapMap CHB individuals provided strong evidence for positive selection within each and suggested the micro-RNA gene hsa-miR-548c as the best candidate target in one region, and changes in regulation of the sperm protein gene SPAG6 in the other.

Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways.

Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3' untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca(2+) levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.