Nicholas D. Hastie

Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts

Recent genome-wide association (GWA) studies of lipids have been conducted in samples ascertained for other phenotypes, particularly diabetes. Here we report the first GWA analysis of loci affecting total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides sampled randomly from 16 population-based cohorts and genotyped using mainly the Illumina HumanHap300-Duo platform. Our study included a total of 17,797–22,562 persons, aged 18–104 years and from geographic regions spanning from the Nordic countries to Southern Europe. We established 22 loci associated with serum lipid levels at a genome-wide significance level (P < 5 × 10−8), including 16 loci that were identified by previous GWA studies. The six newly identified loci in our cohort samples are ABCG5 (TC, P = 1.5 × 10−11; LDL, P = 2.6 × 10−10), TMEM57 (TC, P = 5.4 × 10−10), CTCF-PRMT8 region (HDL, P = 8.3 × 10−16), DNAH11 (LDL, P = 6.1 × 10−9), FADS3-FADS2 (TC, P = 1.5 × 10−10; LDL, P = 4.4 × 10−13) and MADD-FOLH1 region (HDL, P = 6 × 10−11). For three loci, effect sizes differed significantly by sex. Genetic risk scores based on lipid loci explain up to 4.8% of variation in lipids and were also associated with increased intima media thickness (P = 0.001) and coronary heart disease incidence (P = 0.04). The genetic risk score improves the screening of high-risk groups of dyslipidemia over classical risk factors.

SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout

Uric acid is the end product of purine metabolism in humans and great apes, which have lost hepatic uricase activity, leading to uniquely high serum uric acid concentrations (200–500 μM) compared with other mammals (3–120 μM). About 70% of daily urate disposal occurs via the kidneys, and in 5–25% of the human population, impaired renal excretion leads to hyperuricemia. About 10% of people with hyperuricemia develop gout, an inflammatory arthritis that results from deposition of monosodium urate crystals in the joint. We have identified genetic variants within a transporter gene, SLC2A9, that explain 1.7–5.3% of the variance in serum uric acid concentrations, following a genome-wide association scan in a Croatian population sample. SLC2A9 variants were also associated with low fractional excretion of uric acid and/or gout in UK, Croatian and German population samples. SLC2A9 is a known fructose transporter, and we now show that it has strong uric acid transport activity in Xenopus laevis oocytes.

Influence of PAX6 Gene Dosage on Development: Overexpression Causes Severe Eye Abnormalities

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. To investigate the role of gene dosage in more detail, we have generated yeast artificial chromosome transgenic mice carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. We provide evidence that not only reduced, but also increased levels of transcriptional regulators can cause developmental defects.

Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06–1.23 Mb upstream of SOX9, and microdeletions both ∼1.5 Mb centromeric and ∼1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

Accelerated evolution in the reactive centre regions of serine protease inhibitors.

The serine protease inhibitors (serpins) are a family of proteins that function to control the action of serine proteases in many diverse physiological processes. The functional region or reactive centre of these inhibitors is near the C-terminal end and is an exposed site that acts as a bait for the appropriate serine protease to recognize and covalently bind. The specificity of the inhibitor is determined, at least in part, by a single amino acid that resides in this region at the P1 position1. We show here that following a gene duplication event the reactive centres of three related rodent protease inhibitors have diverged from each other at unprecedented rates. This has resulted in proteins with different predicted specificities and we postulate that these changes were fixed by positive darwinian selection and that the most likely selective forces are extrinsic proteases, namely those used by parasites to facilitate their spread throughout the host.

The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature

Most internal organs are situated in a coelomic cavity and are covered by a mesothelium. During heart development, epicardial cells (a mesothelium) move to and over the heart, undergo epithelial-mesenchymal transition (EMT), and subsequently differentiate into endothelial and vascular smooth muscle cells. This is thought to be a unique process in blood vessel formation. Still, structural and developmental similarities between the heart and gut led us to test the hypothesis that a conserved or related mechanism may regulate blood vessel development to the gut, which, similar to the heart, is housed in a coelomic cavity. By using a combination of molecular genetics, vital dye fate mapping, organ culture and immunohistochemistry, we demonstrate that the serosal mesothelium is the major source of vasculogenic cells in developing mouse gut. Our studies show that the gut is initially devoid of a mesothelium but that serosal mesothelial cells expressing the Wilms tumor protein (Wt1) move to and over the gut. Subsequently, a subset of these cells undergoes EMT and migrates throughout the gut. Using Wt1-Cre genetic lineage marking of serosal cells and their progeny, we demonstrate that these cells differentiate to smooth muscle of all major blood vessels in the mesenteries and gut. Our data reveal a conserved mechanism in blood vessel formation to coelomic organs, and have major implications for our understanding of vertebrate organogenesis and vascular deficiencies of the gut.

Human telomeres: fusion and interstitial sites

The ends of human chromosomes have been shown recently to resemble those of simple organisms. With this in mind, we discuss the nature and possible significance of rare chromosome fusion events thought to involve telomeres, particularly those fusion events found in some tumours. Also we argue that interstitial telomere-like stretches may be particularly prone to recombination, breakage and fragility.

High throughput isolation and glycosylation analysis of IgG - variability and heritability of the IgG glycome in three isolated human populations

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individuals age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.

The European dimension for the mouse genome mutagenesis program

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source

Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes.