The RIIIS/J inbred mouse strain is a model for type 1 von Willebrand disease (VWD), a common human bleeding disorder. Low von Willebrand factor (VWF) levels in RIIIS/J are due to a regulatory ...mutation, Mvwf1, which directs a tissue-specific switch in expression of a glycosyltransferase, B4GALNT2, from intestine to blood vessel. We recently found that Mvwf1 lies on a founder allele common among laboratory mouse strains. To investigate the evolutionary forces operating at B4galnt2, we conducted a survey of DNA sequence polymorphism and microsatellite variation spanning the B4galnt2 gene region in natural Mus musculus domesticus populations. Two divergent haplotypes segregate in these natural populations, one of which corresponds to the RIIIS/J sequence. Different local populations display dramatic differences in the frequency of these haplotypes, and reduced microsatellite variability near B4galnt2 within the RIIIS/J haplotype is consistent with the recent action of natural selection. The level and pattern of DNA sequence polymorphism in the 5′ flanking region of the gene significantly deviates from the neutral expectation and suggests that variation in B4galnt2 expression may be under balancing selection and/or arose from a recently introgressed allele that subsequently increased in frequency due to natural selection. However, coalescent simulations indicate that the heterogeneity in divergence between haplotypes is greater than expected under an introgression model. Analysis of a population where the RIIIS/J haplotype is in high frequency reveals an association between this haplotype, the B4galnt2 tissue-specific switch, and a significant decrease in plasma VWF levels. Given these observations, we propose that low VWF levels may represent a fitness cost that is offset by a yet unknown benefit of the B4galnt2 tissue-specific switch. Similar mechanisms may account for the variability in VWF levels and high prevalence of VWD in other mammals, including humans.
Recently, many statistical methods have been proposed to test for associations between rare genetic variants and complex traits. Most of these methods test for association by aggregating genetic ...variations within a predefined region, such as a gene. Although there is evidence that “aggregate” tests are more powerful than the single marker test, these tests generally ignore neutral variants and therefore are unable to identify specific variants driving the association with phenotype. We propose a novel aggregate rare‐variant test that explicitly models a fraction of variants as neutral, tests associations at the gene‐level, and infers the rare‐variants driving the association. Simulations show that in the practical scenario where there are many variants within a given region of the genome with only a fraction causal our approach has greater power compared to other popular tests such as the Sequence Kernel Association Test (SKAT), the Weighted Sum Statistic (WSS), and the collapsing method of Morris and Zeggini (MZ). Our algorithm leverages a fast variational Bayes approximate inference methodology to scale to exome‐wide analyses, a significant computational advantage over exact inference model selection methodologies. To demonstrate the efficacy of our methodology we test for associations between von Willebrand Factor (VWF) levels and VWF missense rare‐variants imputed from the National Heart, Lung, and Blood Institute's Exome Sequencing project into 2,487 African Americans within the VWF gene. Our method suggests that a relatively small fraction (∼10%) of the imputed rare missense variants within VWF are strongly associated with lower VWF levels in African Americans.
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Genetic variation in the VWF gene is associated with von Willebrand factor (VWF) and factor VIII (FVIII) levels in healthy individuals.
We hypothesized that VWF sequence variants ...associated with higher VWF or FVIII could impact the diagnosis of type 1 von Willebrand disease (VWD).
We examined VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF propeptide (VWFpp), and FVIII levels along with VWF gene sequencing in 256 healthy control and 97 type 1 VWD subjects as part of a cross‐sectional study.
We found several VWF sequence variants (VWF c.2880G>A and VWF c.2365A>G(;)c.2385T>C, found in linkage disequilibrium) associated with higher VWF and FVIII levels in healthy controls (P<.001 for both variants). In addition, these variants were significantly more common in controls than in subjects diagnosed with type 1 VWD and VWF:Ag <30 (P<.005). The decreased variant frequencies in type 1 VWD was not seen in other VWD types. VWF:Ag, VWF:RCo, and FVIII were not statistically different in type 1 VWD subjects who had these VWF variants compared to type 1 VWD patients without them. There was no difference in ABO blood group, VWF propeptide levels (excluding subjects with known VWF clearance defects), or bleeding score using the ISTH bleeding assessment tool.
These data suggest that certain VWF sequence variants associated with elevated FVIII and VWF levels may protect against reduced VWF levels. These findings were independent of other pathogenic sequence variants in VWF, suggesting a possible independent effect of c.2880G>A and c.2365A>G(;)c.2385T>C on VWF levels.
Mvwf1 is a cis-regulatory mutation previously identified in the RIIIS/J mouse strain that causes a unique tissue-specific switch in the expression of an N-acetylgalactosaminyltransferase, B4GALNT2, ...from intestinal epithelium to vascular endothelium. Vascular B4galnt2 expression results in aberrant glycosylation of von Willebrand Factor (VWF) and accelerated VWF clearance from plasma. We now report that 13 inbred mouse strains share the Mvwf1 tissue-specific switch and low VWF phenotype, including five wild-derived strains. Genomic sequencing identified a highly conserved 97-kb Mvwf1 haplotype block shared by these strains that encompasses a 30-kb region of high nucleotide sequence divergence from C57BL6/J flanking B4galnt2 exon 1. The analysis of a series of bacterial artificial chromosome (BAC) transgenes containing B4galnt2 derived from the RIIIS/J or C57BL6/J inbred mouse strains demonstrates that the corresponding sequences are sufficient to confer the vessel (RIIIS/J) or intestine (C57BL6/J)-specific expression patterns. Taken together, our data suggest that the region responsible for the Mvwf1 regulatory switch lies within an approximately 30-kb genomic interval upstream of the B4galnt2 gene. The observation that Mvwf1 is present in multiple wild-derived strains suggests that this locus may be retained in wild mouse populations due to positive selection. Similar selective pressures could contribute to the high prevalence of von Willebrand disease in humans.
BackgroundHemophilia A (HA) and hemophilia B (HB) are rare inherited bleeding disorders. Although causative genetic variants are clinically relevant, in 2012 only 20% of US patients had been ...genotyped.ObjectivesMy Life, Our Future (MLOF) was a multisector cross‐sectional US initiative to improve our understanding of hemophilia through widespread genotyping.MethodsSubjects and potential genetic carriers were enrolled at US hemophilia treatment centers (HTCs). Bloodworks performed genotyping and returned results to providers. Clinical data were abstracted from the American Thrombosis and Hemostasis Network dataset. Community education was provided by the National Hemophilia Foundation.ResultsFrom 2013 to 2017, 107 HTCs enrolled 11 341 subjects (68.8% male, 31.2% female) for testing for HA (n = 8976), HB (n = 2358), HA/HB (n = 3), and hemophilia not otherwise specified (n = 4). Variants were detected in most male patients (98.2%% HA, 98.1% HB). 1914 unique variants were found (1482 F8, 431 F9); 744 were novel (610 F8, 134 F9). Inhibitor data were available for 6986 subjects (5583 HA; 1403 HB). In severe HA, genotypes with the highest inhibitor rates were large deletions (77/80), complex intron 22 inversions (9/17), and no variant found (7/14). In severe HB, the highest rates were large deletions (24/42). Inhibitors were reported in 27.3% of Black versus 16.2% of White patients.ConclusionsThe findings of MLOF are reported, the largest hemophilia genotyping project performed to date. The results support the need for comprehensive genetic approaches in hemophilia. This effort has contributed significantly towards better understanding variation in the F8 and F9 genes in hemophilia and risks of inhibitor formation.
Background. Hemophilia A is a rare X-linked bleeding disorder resulting from deficiency in coagulation factor VIII. Numerous genetic variants (>2000) affecting the F8 gene have been implicated as ...causative of hemophilia A, including structural variants (SVs) such as copy number variants (CNVs) and large intra-chromosomal inversions caused by recombination between distant regions with high homology to sequences within F8 intron 1 or intron 22. SVs detected in patients with hemophilia are associated with more severe disease, and different types of SVs may inform inhibitor risk. For the vast majority of patients, causative variants can be identified using targeted DNA sequencing of F8 coding regions and/or the use of methods which detect known SVs (e.g. inverse shifting PCR, long-range PCR, MLPA). However, these approaches fail to explain 1-3% of hemophilia A cases. We hypothesized that a dedicated structural variant analysis at the F8 locus using whole genome sequencing data could identify previously undetected deleterious F8 gene variants in unsolved cases of hemophilia A.
Methods. Cases were selected from the My Life, Our Future (MLOF) hemophilia study cohort recently whole genome sequenced by the NHLBI TOPMed program. In this study, we performed a custom SV analyses using whole genome sequencing (WGS) data from 11 cases of severe hemophilia A (factor VIII activity level < 1%) that remained genetically unexplained after exhausting all available laboratory testing methods. Two of the eleven unsolved severe hemophilia A cases (18%) were reported to have had an inhibitor.
Results. SV analyses of the F8 genomic region revealed previously undetected deletions and inversions in 6 out of the 11 cases. In these 6 samples, SV calls were supported by multiple sequencing reads (> 25 reads) and multiple types of read evidence (read depth, paired-end and/or split read evidence). Two deletions within intron 6 were detected in a single hemophilia A case, a finding which suggests F8 intron 6 may contain one or more regulatory elements critical for F8 expression. Three distinct large inversions predicted to disrupt the F8 structural gene were detected in five other cases; a 720Kb inversion with breakpoints in F8 intron 6 and SPRY3 intron 1 (n=1), a 20Mb inversion with breakpoints in F8 intron 1 and INTS6L intron 8 (n=1), and a 7.4Kb inversion with breakpoints in F8 intron 25 and the SMIM9 intron 1 (n=3). These events are novel in hemophilia and were also not present in the larger, sequenced My Life, Our Future dataset (N=2186), supporting these SVs as likely causative of severe hemophilia A. Both cases with inhibitors had the F8 intron 25-SMIM9 inversion.
Conclusions. This work demonstrates that dedicated analyses of WGS for SVs originating in non-coding regions can identify novel variants in previously unsolved cases of hemophilia A. We conclude that any genetic studies of diseases caused by loss-of-function variants should consider dedicated analyses for SVs. We predict additional deleterious SVs remain to be discovered in rare unexplained cases of hemophilia.
Konkle:BioMarin: Consultancy; Bioverativ: Research Funding; CSL Behring: Consultancy; Genentech: Consultancy; Spark: Consultancy, Research Funding; Pfizer: Research Funding; Gilead: Consultancy; Sangamo: Research Funding; Shire: Research Funding. Johnsen:CSL Behring: Consultancy; Octapharma: Consultancy.
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