Common genetic variants do not associate with CAD in familial hypercholesterolemia.

In recent years, multiple loci dispersed on the genome have been shown to be associated with coronary artery disease (CAD). We investigated whether these common genetic variants also hold value for CAD prediction in a large cohort of patients with familial hypercholesterolemia (FH). We genotyped a total of 41 single-nucleotide polymorphisms (SNPs) in 1701 FH patients, of whom 482 patients (28.3%) had at least one coronary event during an average follow up of 66 years. The association of each SNP with event-free survival time was calculated with a Cox proportional hazard model. In the cardiovascular disease risk factor adjusted analysis, the most significant SNP was rs1122608:G>T in the SMARCA4 gene near the LDL-receptor (LDLR) gene, with a hazard ratio for CAD risk of 0.74 (95% CI 0.49-0.99; P-value 0.021). However, none of the SNPs reached the Bonferroni threshold. Of all the known CAD loci analyzed, the SMARCA4 locus near the LDLR had the strongest negative association with CAD in this high-risk FH cohort. The effect is contrary to what was expected. None of the other loci showed association with CAD.

SMIM1 underlies the Vel blood group and influences red blood cell traits.

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.

A GWAS sequence variant for platelet volume marks an alternative DNM3 promoter in megakaryocytes near a MEIS1 binding site.

We recently identified 68 genomic loci where common sequence variants are associated with platelet count and volume. Platelets are formed in the bone marrow by megakaryocytes, which are derived from hematopoietic stem cells by a process mainly controlled by transcription factors. The homeobox transcription factor MEIS1 is uniquely transcribed in megakaryocytes and not in the other lineage-committed blood cells. By ChIP-seq, we show that 5 of the 68 loci pinpoint a MEIS1 binding event within a group of 252 MK-overexpressed genes. In one such locus in DNM3, regulating platelet volume, the MEIS1 binding site falls within a region acting as an alternative promoter that is solely used in megakaryocytes, where allelic variation dictates different levels of a shorter transcript. The importance of dynamin activity to the latter stages of thrombopoiesis was confirmed by the observation that the inhibitor Dynasore reduced murine proplatelet for-mation in vitro.

Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exon-junction complex subunit RBM8A causes TAR syndrome.

The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 × 10(-228)) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.

Powerful identification of cis-regulatory SNPs in human primary monocytes using allele-specific gene expression.

A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.

Multiple loci influence erythrocyte phenotypes in the CHARGE Consortium.

Measurements of erythrocytes within the blood are important clinical traits and can indicate various hematological disorders. We report here genome-wide association studies (GWAS) for six erythrocyte traits, including hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). We performed an initial GWAS in cohorts of the CHARGE Consortium totaling 24,167 individuals of European ancestry and replication in additional independent cohorts of the HaemGen Consortium totaling 9,456 individuals. We identified 23 loci significantly associated with these traits in a meta-analysis of the discovery and replication cohorts (combined P values ranging from 5 x 10(-8) to 7 x 10(-86)). Our findings include loci previously associated with these traits (HBS1L-MYB, HFE, TMPRSS6, TFR2, SPTA1) as well as new associations (EPO, TFRC, SH2B3 and 15 other loci). This study has identified new determinants of erythrocyte traits, offering insight into common variants underlying variation in erythrocyte measures.

Evolutionary vignettes of natural killer cell receptors.

The discovery of novel immune receptors has led to a recent renaissance of research into the innate immune system, following decades of intense research of the adaptive immune system. Of particular interest has been the discovery of the natural killer (NK) cell receptors which, depending on type, interact with classical or non-classical MHC class I antigens of the adaptive immune system, thus functioning at the interface of innate and adaptive immunity. Here, we review recent progress with respect to two such families of NK receptors, the killer immunoglobulin-like receptors (KIRs) and the killer cell lectin-like receptors (KLRs), and attempt to trace their evolution across vertebrates.

Identification of the ancestral killer immunoglobulin-like receptor gene in primates.

BACKGROUND: Killer Immunoglobulin-like Receptors (KIR) are essential immuno-surveillance molecules. They are expressed on natural killer and T cells, and interact with human leukocyte antigens. KIR genes are highly polymorphic and contribute vital variability to our immune system. Numerous KIR genes, belonging to five distinct lineages, have been identified in all primates examined thus far and shown to be rapidly evolving. Since few KIR remain orthologous between species, with only one of them, KIR2DL4, shown to be common to human, apes and monkeys, the evolution of the KIR gene family in primates remains unclear. RESULTS: Using comparative analyses, we have identified the ancestral KIR lineage (provisionally named KIR3DL0) in primates. We show KIR3DL0 to be highly conserved with the identification of orthologues in human (Homo sapiens), common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), rhesus monkey (Macaca mulatta) and common marmoset (Callithrix jacchus). We predict KIR3DL0 to encode a functional molecule in all primates by demonstrating expression in human, chimpanzee and rhesus monkey. Using the rhesus monkey as a model, we further show the expression profile to be typical of KIR by quantitative measurement of KIR3DL0 from an enriched population of natural killer cells. CONCLUSION: One reason why KIR3DL0 may have escaped discovery for so long is that, in human, it maps in between two related leukocyte immunoglobulin-like receptor clusters outside the known KIR gene cluster on Chromosome 19. Based on genomic, cDNA, expression and phylogenetic data, we report a novel lineage of immunoglobulin receptors belonging to the KIR family, which is highly conserved throughout 50 million years of primate evolution.

Identification of a single killer immunoglobulin-like receptor (KIR) gene in the porcine leukocyte receptor complex on chromosome 6q.

Human killer immunoglobulin-like receptors (KIR) are expressed on natural killer (NK) cells and are involved in their immunoreactivity. While KIR with a long cytoplasmic tail deliver an inhibitory signal when bound to their respective major histocompatibility complex class I ligands, KIR with a short cytoplasmic tail can activate NK responses. The expansion of the KIR gene family originally appeared to be a phenomenon restricted to primates (human, apes, and monkeys) in comparison to rodents, which via convergent evolution have numerous C-type lectin-like Ly49 molecules that function analogously. Further studies have shown that multiple KIR are also present in cow and horse. In this study, we have identified by comparative genomics the first and possibly only KIR gene, named KIR2DL1, in the domesticated pig (Sus scrofa) allowing further evolutionary comparisons to be made. It encodes a protein with two extracellular immunoglobulin domains (D0 + D2), and a long cytoplasmic tail containing two inhibitory motifs. We have mapped the pig KIR2DL1 gene to chromosome 6q. Flanked by LILRa, LILRb, and LILRc, members of the leukocyte immunoglobulin-like receptor (LILR) family, on the centromeric end, and FCAR, NCR1, NALP7, NALP2, and GP6 on the telomeric end, pig demonstrates conservation of synteny with the human leukocyte receptor complex (LRC). Both the porcine KIR and LILR genes have diverged sufficiently to no longer be clearly orthologous with known human LRC family members.

A genome-wide survey of Major Histocompatibility Complex (MHC) genes and their paralogues in zebrafish.

BACKGROUND: The genomic organisation of the Major Histocompatibility Complex (MHC) varies greatly between different vertebrates. In mammals, the classical MHC consists of a large number of linked genes (e.g. greater than 200 in humans) with predominantly immune function. In some birds, it consists of only a small number of linked MHC core genes (e.g. smaller than 20 in chickens) forming a minimal essential MHC and, in fish, the MHC consists of a so far unknown number of genes including non-linked MHC core genes. Here we report a survey of MHC genes and their paralogues in the zebrafish genome. RESULTS: Using sequence similarity searches against the zebrafish draft genome assembly (Zv4, September 2004), 149 putative MHC gene loci and their paralogues have been identified. Of these, 41 map to chromosome 19 while the remaining loci are spread across essentially all chromosomes. Despite the fragmentation, a set of MHC core genes involved in peptide transport, loading and presentation are still found in a single linkage group. CONCLUSION: The results extend the linkage information of MHC core genes on zebrafish chromosome 19 and show the distribution of the remaining MHC genes and their paralogues to be genome-wide. Although based on a draft genome assembly, this survey demonstrates an essentially fragmented MHC in zebrafish.

Single haplotype analysis demonstrates rapid evolution of the killer immunoglobulin-like receptor (KIR) loci in primates.

The human killer immunoglobulin-like receptors (KIR) are encoded within the Leukocyte Receptor Complex (LRC) on chromosome 19q13.4. Here we report the comparative genomic analysis of single KIR haplotypes in two other primates. In the common chimpanzee (Pan troglodytes), seven KIR genes (ptKIRnewI, ptKIRnewII, ptKIR2DL5, ptKIRnewIII, ptKIR3DP1, ptKIR2DL4, ptKIR3DL1/2) have been identified, and five KIR genes (mmKIRnewI, mmKIR1D, mmKIR2DL4, mmKIR3DL10, mmKIR3DL1) are present in the haplotype sequenced for the rhesus macaque (Macaca mulatta). Additional cDNA analysis confirms the genes predicted from the genomic sequence and reveals the presence of a fifth novel KIR gene (mmKIRnewII) in the second haplotype of the rhesus macaque. While all known human haplotypes contain both activating and inhibitory KIR genes, only inhibitory KIR genes (characterized by long cytoplasmic tails) were found by in silico and cDNA analyses in the two primate haplotypes studied here. Comparison of the two human and the two non-human primate haplotypes demonstrates rapid diversification of the KIR gene family members, many of which have diverged in a species-specific manner. An analysis of the intronic regions of the two non-human primates reveals the presence of ancient repeat elements, which are indicative of the duplication events that have taken place since the last common ancestor.

Characterisation of a gene cluster in Fugu rubripes containing the complement component C4 gene.

In this study, we describe the characterisation of the complement component C4 gene in Fugu rubripes. The Fugu C4 gene, orthologous to the tetrapod C4 gene, encompasses a genomic span of 9702 base pairs and contains 41 exons, encoding the typical C4 three-chain polypeptide. The gene encodes a protein containing 1703 amino acids. The Fugu C4 protein demonstrates the presence of 25 conserved cysteine residues, as well as conservation of the functionally important thioester site. Complete sequencing of one cosmid and sequence scans from a cluster of 18 overlapping BAC clones, centering around the C4 gene, have identified the short-range linkage with five orthologous human genes mapping to the Major Histocompatibility Complex (MHC) including: tenascin X (TNX); cytochrome P450, subfamily XXIA, polypeptide 2 (CYP21A2); allograft inflammatory factor 1 (AIF1) and casein kinase 2, beta polypeptide (CSNK2B), all found in the MHC class III region; and retinoid X receptor, beta (RXRB),which resides in the MHC extended class II region. To date, this syntenic association of the Fugu C4 and other MHC class III region genes has not been observed in other teleost fish. Data from the recent whole-genome shotgun assemblies reveal the Fugu MHC-related cluster of genes to be flanked predominantly by genes mapping to human chromosomes 7 and 19. All of the six identified Fugu MHC-related genes have been characterised at the genomic level.

Fugu orthologues of human major histocompatibility complex genes: a genome survey.

The major histocompatibility complex (MHC) region in fish has been subjected to piecemeal analysis centering on the in-depth characterization of single genes. The emphasis has been on those genes proven to be involved in the immune response such as the class I and class II antigen presenting genes and the complement genes. The Fugu genome data presents the opportunity to examine the short-range linkage of potentially all the human MHC orthologues and examine conserved synteny with the human and, to a more limited extent, zebrafish genomes. Analysis confirms the existence of a limited MHC locus in Fugu comprising the MHC class Ia genes and associated class II region genes involved in class I antigen presentation. Identification of additional human MHC orthologues indicates the completely dispersed nature of this region in fish, with a maximum of six MHC genes maintained within close proximity in any one contig. The majority of the other genes are present in the genome data as either singletons or pairs. Comparison with zebrafish substantiates previously observed linkages between class III region orthologues and hints at an ancient conserved class III region.