Transcription factor Wilms' tumor 1 regulates developmental RNAs through 3' UTR interaction.

Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.

Recent genomic heritage in Scotland.

BACKGROUND: The Generation Scotland Scottish Family Health Study (GS:SFHS) includes 23,960 participants from across Scotland with records for many health-related traits and environmental covariates. Genotypes at ~700 K SNPs are currently available for 10,000 participants. The cohort was designed as a resource for genetic and health related research and the study of complex traits. In this study we developed a suite of analyses to disentangle the genomic differentiation within GS:SFHS individuals to describe and optimise the sample and methods for future analyses. RESULTS: We combined the genotypic information of GS:SFHS with 1092 individuals from the 1000 Genomes project and estimated their genomic relationships. Then, we performed Principal Component Analyses of the resulting relationships to investigate the genomic origin of different groups. We characterised two groups of individuals: those with a few sparse rare markers in the genome, and those with several large rare haplotypes which might represent relatively recent exogenous ancestors. We identified some individuals with likely Italian ancestry and a group with some potential African/Asian ancestry. An analysis of homozygosity in the GS:SFHS sample revealed a very similar pattern to other European populations. We also identified an individual carrying a chromosome 1 uniparental disomy. We found evidence of local geographic stratification within the population having impact on the genomic structure. CONCLUSIONS: These findings illuminate the history of the Scottish population and have implications for further analyses such as the study of the contributions of common and rare variants to trait heritabilities and the evaluation of genomic and phenotypic prediction of disease.

ErbB4 modulates tubular cell polarity and lumen diameter during kidney development.

ErbB4 receptor tyrosine kinase contributes to the development of the heart, the central nervous system, and the lactating mammary gland, but whether it has a role in the development of the kidney epithelium is unknown. Here, we found that expression of Erbb4 isoforms JM-a CYT-1 and JM-a CYT-2 was first detectable around embryonic day 13 in the mouse, mainly in the collecting ducts and both the proximal and distal tubules. In vitro, overexpression of a relevant ErbB4 isoform promoted proliferation and disturbed polarization of kidney epithelial cells when cultured as three-dimensional structures. We examined ErbB4 function in developing kidney tubules in vivo with Pax8-Cre-mediated conditional overexpression of Rosa26 locus-targeted ERBB4 and with conditional Erbb4 knock-out mice. The Pax8-Cre-driven ERBB4 overexpression enhanced proliferation in the collecting ducts, reduced the size of epithelial duct lumens, and promoted formation of cortical tubular cysts. These defects were associated with changes in the subcellular distribution of markers of epithelial cell polarity. Similarly, the Pax8-Cre-mediated Erbb4 knock-out mice manifested dysfunctional kidneys with larger duct lumens and epithelial cell mispolarization. Taken together, these data suggest that ErbB4 signaling modulates proliferation and polarization, cellular functions critical for the development of epithelial ducts in the kidney.

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.

A murine model of Denys-Drash syndrome reveals novel transcriptional targets of WT1 in podocytes.

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.

Genetic determinants of circulating sphingolipid concentrations in European populations.

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic beta-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08x10(-66). The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1-3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10(-4) or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases.

Common variants in the JAZF1 gene associated with height identified by linkage and genome-wide association analysis.

Genes for height have gained interest for decades, but only recently have candidate genes started to be identified. We have performed linkage analysis and genome-wide association for height in approximately 4000 individuals from five European populations. A total of five chromosomal regions showed suggestive linkage and in one of these regions, two SNPs (rs849140 and rs1635852) were associated with height (nominal P = 7.0 x 10(-8) and P = 9.6 x 10(-7), respectively). In total, five SNPs across the genome showed an association with height that reached the threshold of genome-wide significance (nominal P < 1.6 x 10(-7)). The association with height was replicated for two SNPs (rs1635852 and rs849140) using three independent studies (n = 31 077, n=1268 and n = 5746) with overall meta P-values of 9.4 x 10(-10) and 5.3 x 10(-8). These SNPs are located in the JAZF1 gene, which has recently been associated with type II diabetes, prostate and endometrial cancer. JAZF1 is a transcriptional repressor of NR2C2, which results in low IGF1 serum concentrations, perinatal and early postnatal hypoglycemia and growth retardation when knocked out in mice. Both the linkage and association analyses independently identified the JAZF1 region affecting human height. We have demonstrated, through replication in additional independent populations, the consistency of the effect of the JAZF1 SNPs on height. Since this gene also has a key function in the metabolism of growth, JAZF1 represents one of the strongest candidates influencing human height identified so far.

Resolving the heterogeneity of diaphragmatic mesenchyme: a novel mouse model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common developmental defect with considerable mortality and morbidity. Formation of the diaphragm is a complex process that involves several cell types, each with different developmental origins. Owing to this complexity, the aetiology of CDH is not well understood. The pleuroperitoneal folds (PPFs) and the posthepatic mesenchymal plate (PHMP) are transient structures that are essential during diaphragm development. Using several mouse models, including lineage tracing, we demonstrate the heterogeneous nature of the cells that make up the PPFs. The conditional deletion of Wilms tumor 1 homolog (Wt1) in the non-muscle mesenchyme of the PPFs results in CDH. We show that the fusion of the PPFs and the PHMP to form a continuous band of tissue involves movements of cells from both sources. The PPFs of mutant mice fail to fuse with the PHMP and exhibit increased RALDH2 (also known as ALDH1A2) expression. However, no changes in the expression of genes (including Snai1, Snai2, Cdh1 and Vim) implicated in epithelial-to-mesenchymal transition are observed. Additionally, the mutant PPFs lack migrating myoblasts and muscle connective tissue fibroblasts (TCF4+/GATA4+), suggesting possible interactions between these cell types. Our study demonstrates the importance of the non-muscle mesenchyme in development of the diaphragm.

A meta-analysis of genome-wide data from five European isolates reveals an association of COL22A1, SYT1, and GABRR2 with serum creatinine level.

BACKGROUND: Serum creatinine (S CR) is the most important biomarker for a quick and non-invasive assessment of kidney function in population-based surveys. A substantial proportion of the inter-individual variability in S CR level is explicable by genetic factors. METHODS: We performed a meta-analysis of genome-wide association studies of S CR undertaken in five population isolates ('discovery cohorts'), all of which are part of the European Special Population Network (EUROSPAN) project. Genes showing the strongest evidence for an association with SCR (candidate loci) were replicated in two additional population-based samples ('replication cohorts'). RESULTS: After the discovery meta-analysis, 29 loci were selected for replication. Association between SCR level and polymorphisms in the collagen type XXII alpha 1 (COL22A1) gene, on chromosome 8, and in the synaptotagmin-1 (SYT1) gene, on chromosome 12, were successfully replicated in the replication cohorts (p value = 1.0 x 10(-6) and 1.7 x 10(-4), respectively). Evidence of association was also found for polymorphisms in a locus including the gamma-aminobutyric acid receptor rho-2 (GABRR2) gene and the ubiquitin-conjugating enzyme E2-J1 (UBE2J1) gene (replication p value = 3.6 x 10(-3)). Previously reported findings, associating glomerular filtration rate with SNPs in the uromodulin (UMOD) gene and in the schroom family member 3 (SCHROOM3) gene were also replicated. CONCLUSIONS: While confirming earlier results, our study provides new insights in the understanding of the genetic basis of serum creatinine regulatory processes. In particular, the association with the genes SYT1 and GABRR2 corroborate previous findings that highlighted a possible role of the neurotransmitters GABAA receptors in the regulation of the glomerular basement membrane and a possible interaction between GABAA receptors and synaptotagmin-I at the podocyte level.

Genome-wide linkage analysis of serum creatinine in three isolated European populations.

There is increasing evidence for a role of genetic predisposition in the etiology of kidney disease, but linkage scans have been poorly replicated. Here we performed a genome-wide linkage analysis of serum creatinine on 2859 individuals from isolated villages in South Tyrol (Italy), Rucphen (The Netherlands) and Vis Island (Croatia), populations that have been stable and permanently resident in their region. Linkage of serum creatinine levels to loci on chromosomes 7p14, 9p21, 11p15, 15q15-21, 16p13, and 18p11 was successfully replicated in at least one discovery population or in the pooled analysis. A novel locus was found on chromosome 10p11. Linkage to chromosome 22q13, independent of diabetes and hypertension, was detected over a region containing the non-muscle myosin heavy chain type II isoform A (MYH9) gene (LOD score=3.52). In non-diabetic individuals, serum creatinine was associated with this gene in two of the three populations and in meta-analysis (SNP rs11089788, P-value=0.0089). In populations sharing a homogeneous environment and genetic background, heritability of serum creatinine was higher than in outbred populations, with consequent detection of a larger number of loci than reported before. Our finding of a replicated association of serum creatinine with the MYH9 gene, recently linked to pathological renal conditions in African Americans, suggests that this gene may also influence kidney function in healthy Europeans.

Quantifying the increase in average human heterozygosity due to urbanisation.

The human population is undergoing a major transition from a historical metapopulation structure of relatively isolated small communities to an outbred structure. This process is predicted to increase average individual genome-wide heterozygosity (h) and could have effects on health. We attempted to quantify this increase in mean h. We initially sampled 1001 examinees from a metapopulation of nine isolated villages on five Dalmatian islands (Croatia). Village populations had high levels of genetic differentiation, endogamy and consanguinity. We then selected 166 individuals with highly specific personal genetic histories to form six subsamples, which could be ranked a priori by their predicted level of outbreeding. The measure h was then estimated in the 166 examinees by genotyping 1184 STR/indel markers and using two different computation methods. Compared to the value of mean h in the least outbred sample, values of h in the remaining samples increased successively with predicted outbreeding by 0.023, 0.038, 0.058, 0.067 and 0.079 (P<0.0001), where these values are measured on the same scale as the inbreeding coefficient (but opposite sign). We have shown that urbanisation was associated with an average increase in h of up to 0.08-0.10 in this Croatian metapopulation, regardless of the method used. Similar levels of differentiation have been described in many populations. Therefore, changes in the level of heterozygosity across the genome of this magnitude may be common during isolate break-up in humans and could have significant health effects through the established genetic mechanism of hybrid vigour/heterosis.

Regional variation in health is predominantly driven by lifestyle rather than genetics.

Regional differences in health-related phenotypes have been detected between and within countries. In Scotland, regions differ for a variety of health-related traits and display differences in mean lifespan of up to 7.5 years. Both genetics and lifestyle differences are potential causes of this variation. Using data on obesity-related traits of ~11,000 Scottish individuals with genome-wide genetic information and records of lifestyle and socioeconomic factors, we explored causes of regional variation by using models that incorporate genetic and environmental information jointly. We found that variation between individuals within regions showed substantial influence of both genetic variation and family environment. Regional variation for most obesity traits was associated with lifestyle and socioeconomic variables, such as smoking, diet and deprivation which are potentially modifiable. There was limited evidence that regional differences were of genetic origin. This has important implications for healthcare policies, suggesting that inequalities can be tackled with appropriate social and economic interventions.Health-related traits are known to vary geographically. Here, Amador and colleagues show that regional variation of obesity-related traits in a Scottish population is influenced more by lifestyle differences than it is by genetic differences.

3000 years of solitude: extreme differentiation in the island isolates of Dalmatia, Croatia.

Communities with increased shared ancestry represent invaluable tools for genetic studies of complex traits. "1001 Dalmatians" research program collects biomedical information for genetic epidemiological research from multiple small isolated populations ('metapopulation') in the islands of Dalmatia, Croatia. Random samples of 100 individuals from 10 small island settlements (n<2000 inhabitants) were collected in 2002 and 2003. These island communities were carefully chosen to represent a wide range of distinct and well-documented demographic histories. Here, we analysed their genetic make-up using 26 short tandem repeat (STR) markers, at least 5 cM apart. We found a very high level of differentiation between most of these island communities based on Wright's fixation indexes, even within the same island. The model-based clustering algorithm, implemented in STRUCTURE, defined six clusters with very distinct genetic signatures, four of which corresponded to single villages. The extent of background LD, assessed with eight linked markers on Xq13-21, paralleled the extent of differentiation and was also very high in most of the populations under study. For each population, demographic history was characterised and 12 "demographic history" variables were tentatively defined. Following stepwise regression, the demographic history variable that most significantly predicted the extent of LD was the proportion of locally born grandparents. Strong isolation and endogamy are likely to be the main forces maintaining this highly structured overall population.

Modification and amplification of yeast artificial chromosomes.

The development of yeast artificial chromosome (YAC) technology permits cloning of DNA segments that can be thousands of kilobases in size. This facilitates techniques such as the generation of transgenic animals as a YAC clone can, in most cases, carry an entire gene with all its regulatory elements. Similar to the endogenous yeast chromosomes, YAC DNA is replicated in yeast at one copy per cell. This presents problems when attempting to isolate sufficiently concentrated YAC DNA for pronuclear microinjection. To overcome this problem, we have constructed a YAC amplification vector (pYAM4) that concentrates YAC DNA up to three to eight copies per cell, thus ensuring that more concentrated YAC DNA can be purified. To simplify the analyses of transgene expression in transgenic animals, we have also developed a series of YAC modification vectors (pYIV1, 2, 3, and 4) that can be used to introduce a lacZ reporter gene into YAC DNA by homologous recombination. The protocols described in this chapter with the use of these vectors have led to generation of up to 10% of YAC transgenic mice born after microinjection.

Wt1, the mesothelium and the origins and heterogeneity of visceral fat progenitors.

One major gap in adipocyte biology has been a lack of understanding of the developmental origins of the different visceral white adipose tissue (WAT) depots and subcutaneous WAT. In a recent study we showed that most visceral WAT but no subcutaneous WAT arises from cells expressing the Wilms' tumor 1 (Wt1) gene late in mouse gestation.(1) Wt1 continues to be expressed in visceral WAT progenitors into adult life. We also showed that visceral WAT is lined by a mesothelium and provided evidence that this structure is the source of adipocytes. Our study also adds to the growing body of evidence that there is heterogeneity in the visceral progenitors, such that there are Wt1-expressing and non-expressing subsets, the relative proportions of which vary between depots. This raises the enticing prospect that the adipocytes arising from these progenitor subsets may have different properties and our preliminary data support this notion. Finally, evidence from our study and one from Spiegelman's group(2) suggests that Wt1 is not just a marker but regulates visceral WAT identity and the progenitor population. We discuss the implications of this work and some of the questions and future directions that arise from it.

In vivo imaging of the tumor and its associated microenvironment using combined CARS / 2-photon microscopy.

The use of confocal and multi-photon microscopy for intra-vital cancer imaging has impacted on our understanding of cancer cell behavior and interaction with the surrounding tumor microenvironment in vivo. However, many studies to-date rely on the use fluorescent dyes or genetically encoded probes that enable visualization of a structure or cell population of interest, but do not illuminate the complexity of the surrounding tumor microenvironment. Here, we show that multi-modal microscopy combining 2-photon fluorescence with CARS can begin to address this deficit, enabling detailed imaging of the tumor niche without the need for additional labeling. This can be performed on live tumor-bearing animals through optical observation windows, permitting real-time and longitudinal imaging of dynamic processes within the tumor niche.

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.

Linkage and genome-wide association analysis of obesity-related phenotypes: association of weight with the MGAT1 gene.

As major risk-factors for diabetes and cardiovascular diseases, the genetic contribution to obesity-related traits has been of interest for decades. Recently, a limited number of common genetic variants, which have replicated in different populations, have been identified. One approach to increase the statistical power in genetic mapping studies is to focus on populations with increased levels of linkage disequilibrium (LD) and reduced genetic diversity. We have performed joint linkage and genome-wide association analyses for weight and BMI in 3,448 (linkage) and 3,925 (association) partly overlapping healthy individuals from five European populations. A total of four chromosomal regions (two for weight and two for BMI) showed suggestive linkage (lod >2.69) either in one of the populations or in the joint data. At the genome-wide level (nominal P < 1.6 x 10(-7), Bonferroni-adjusted P < 0.05) one single-nucleotide polymorphism (SNP) (rs12517906) (nominal P = 7.3 x 10(-8)) was associated with weight, whereas none with BMI. The SNP associated with weight is located close to MGAT1. The monoacylglycerol acyltransferase (MGAT) enzyme family is known to be involved in dietary fat absorption. There was no overlap between the linkage regions and the associated SNPs. Our results show that genetic effects influencing weight and BMI are shared across diverse European populations, even though some of these populations have experienced recent population bottlenecks and/or been affected by genetic drift. The analysis enabled us to identify a new candidate gene, MGAT1, associated with weight in women.

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 x 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 x 10(-11); LDL, P = 2.6 x 10(-10)), TMEM57 (TC, P = 5.4 x 10(-10)), CTCF-PRMT8 region (HDL, P = 8.3 x 10(-16)), DNAH11 (LDL, P = 6.1 x 10(-9)), FADS3-FADS2 (TC, P = 1.5 x 10(-10); LDL, P = 4.4 x 10(-13)) and MADD-FOLH1 region (HDL, P = 6 x 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.