Beth McGee

pak2a mutations cause cerebral hemorrhage in redhead zebrafish

The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhdmi149), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function.

Expression of the blood-group-related glycosyltransferase B4galnt2 influences the intestinal microbiota in mice

Glycans on mucosal surfaces have an important role in host–microbe interactions. The locus encoding the blood-group-related glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2) is subject to strong selective forces in natural house-mouse populations that contain a common allelic variant that confers loss of B4galnt2 gene expression in the gastrointestinal (GI) tract. We reasoned that altered glycan-dependent intestinal host–microbe interactions may underlie these signatures of selection. To determine whether B4galnt2 influences the intestinal microbial ecology, we profiled the microbiota of wild-type and B4galnt2-deficient siblings throughout the GI tract using 16S rRNA gene pyrosequencing. This revealed both distinct communities at different anatomic sites and significant changes in composition with respect to genotype, indicating a previously unappreciated role of B4galnt2 in host–microbial homeostasis. Among the numerous B4galnt2-dependent differences identified in the abundance of specific bacterial taxa, we unexpectedly detected a difference in the pathogenic genus, Helicobacter, suggesting Helicobacter spp. also interact with B4galnt2 glycans. In contrast to other glycosyltransferases, we found that the host intestinal B4galnt2 expression is not dependent on presence of the microbiota. Given the long-term maintenance of alleles influencing B4galnt2 expression by natural selection and the GI phenotypes presented here, we suggest that variation in B4galnt2 GI expression may alter susceptibility to GI diseases such as infectious gastroenteritis.

Selection on cis-regulatory variation at B4galnt2 and its influence on von Willebrand factor in house mice.

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.

Genome-wide studies of von Willebrand factor propeptide identify loci contributing to variation in propeptide levels and von Willebrand factor clearance.

Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome‐wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates.

Genetic variants in ADAMTS13 as well as smoking are major determinants of plasma ADAMTS13 levels

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) in circulating blood, limiting the size of VWF multimers and regulating VWF activity. Abnormal regulation of VWF contributes to bleeding and to thrombotic disorders. ADAMTS13 levels in plasma are highly variable among healthy individuals, although the heritability and the genetic determinants of this variation are unclear. We performed genome-wide association studies of plasma ADAMTS13 concentrations in 3244 individuals from 2 independent cohorts of healthy individuals. The heritability of ADAMTS13 levels was between 59.1% (all individuals) and 83.5% (siblings only), whereas tobacco smoking was associated with a decrease in plasma ADAMTS13 levels. Meta-analysis identified common variants near the ADAMTS13 locus on chromosome 9q34.2 that were significantly associated with ADAMTS13 levels and collectively explained 20.0% of the variance. The top single nucleotide polymorphism (SNP), rs28673647, resides in an intron of ADAMTS13 (β, 6.7%; P = 1.3E-52). Conditional analysis revealed 3 additional independent signals represented by rs3739893 (β, -22.3%; P = 1.2E-30) and rs3124762 (β, 3.5%; P = 8.9E-9) close to ADAMTS13 and rs4075970 (β, 2.4%; P = 6.8E-9) on 21q22.3. Linkage analysis also identified the region around ADAMTS13 (9q34.2) as the top signal (LOD 3.5), consistent with our SNP association analyses. Two nonsynonymous ADAMTS13 variants in the top 2 independent linkage disequilibrium blocks (Q448E and A732V) were identified and characterized in vitro. This study uncovered specific common genetic polymorphisms that are key genetic determinants of the variation in plasma ADAMTS13 levels in healthy individuals.

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo

Significance In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type II, a disease of abnormal red blood cell development, while SEC23A deficiency results in cranio-lenticulo-sutural-dysplasia, a disease characterized by bone abnormalities due to defective collagen secretion (but no red blood cell defect). In this study, we show that SEC23A and SEC23B overlap in function, and that the disparate phenotypes of SEC23A/SEC23B deficiency within and across species are likely due to evolutionary shifts in gene-expression programs, rather than distinct functions of the SEC23 paralogs. Our studies provide a rationale for increased SEC23A or SEC23B expression as a therapeutic strategy for congenital dyserythropoietic anemia type II or cranio-lenticulo-sutural-dysplasia, respectively. Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.