Regional heritability mapping method helps explain missing heritability of blood lipid traits in isolated populations.

Single single-nucleotide polymorphism (SNP) genome-wide association studies (SSGWAS) may fail to identify loci with modest effects on a trait. The recently developed regional heritability mapping (RHM) method can potentially identify such loci. In this study, RHM was compared with the SSGWAS for blood lipid traits (high-density lipoprotein (HDL), low-density lipoprotein (LDL), plasma concentrations of total cholesterol (TC) and triglycerides (TG)). Data comprised 2246 adults from isolated populations genotyped using ∼300 000 SNP arrays. The results were compared with large meta-analyses of these traits for validation. Using RHM, two significant regions affecting HDL on chromosomes 15 and 16 and one affecting LDL on chromosome 19 were identified. These regions covered the most significant SNPs associated with HDL and LDL from the meta-analysis. The chromosome 19 region was identified in our data despite the fact that the most significant SNP in the meta-analysis (or any SNP tagging it) was not genotyped in our SNP array. The SSGWAS identified one SNP associated with HDL on chromosome 16 (the top meta-analysis SNP) and one on chromosome 10 (not reported by RHM or in the meta-analysis and hence possibly a false positive association). The results further confirm that RHM can have better power than SSGWAS in detecting causal regions including regions containing crucial ungenotyped variants. This study suggests that RHM can be a useful tool to explain some of the 'missing heritability' of complex trait variation.

Calcium/NFAT signalling promotes early nephrogenesis.

A number of Wnt genes are expressed during, and are known to be essential for, early kidney development. It is typically assumed that their products will act through the canonical ß-catenin signalling pathway. We have found evidence that suggests canonical Wnt signalling is not active in the early nephrogenic metanephric mesenchyme, but instead provide expressional and functional evidence that implicates the non-canonical Calcium/NFAT Wnt signalling pathway in nephrogenesis. Members of the NFAT (Nuclear Factor Activated in T cells) transcription factor gene family are expressed throughout murine kidney morphogenesis and NFATc3 is localised to the developing nephrons. Treatment of kidney rudiments with Cyclosporin A (CSA), an inhibitor of Calcium/NFAT signalling, decreases nephron formation--a phenotype similar to that in Wnt4(-/-) embryos. Treatment of Wnt4(-/-) kidneys with Ionomycin, an activator of the pathway, partially rescues the phenotype. We propose that the non-canonical Calcium/NFAT Wnt signalling pathway plays an important role in early mammalian renal development and is required for complete MET during nephrogenesis, potentially acting downstream of Wnt4.

Wt1 and retinoic acid signaling are essential for stellate cell development and liver morphogenesis.

Previous studies of knock-out mouse embryos have shown that the Wilms' tumor suppressor gene (Wt1) is indispensable for the development of kidneys, gonads, heart, adrenals and spleen. Using OPT (Optical Projection Tomography) we have found a new role for Wt1 in mouse liver development. In the absence of Wt1, the liver is reduced in size, and shows lobing abnormalities. In normal embryos, coelomic cells expressing Wt1, GATA-4, RALDH2 and RXRalpha delaminate from the surface of the liver, intermingle with the hepatoblasts and incorporate to the sinusoidal walls. Some of these cells express desmin, suggesting a contribution to the stellate cell population. Other cells, keeping high levels of RXRalpha immunoreactivity, are negative for stellate or smooth muscle cell markers. However, coelomic cells lining the liver of Wt1-null embryos show decreased or absent RALDH2 expression, the population of cells expressing high levels of RXRalpha is much reduced and the proliferation of hepatoblasts and RXRalpha-positive cells is significantly decreased. On the other hand, the expression of smooth muscle cell specific alpha-actin increases throughout the liver, suggesting an accelerated and probably anomalous differentiation of stellate cell progenitors. We describe a similar retardation of liver growth in RXRalpha-null mice as well as in chick embryos after inhibition of retinoic acid synthesis. We propose that Wt1 expression in cells delaminating from the coelomic epithelium is essential for the expansion of the progenitor population of liver stellate cells and for liver morphogenesis. Mechanistically, at least part of this effect is mediated via the retinoic acid signaling pathway.

Estimating human inbreeding coefficients: comparison of genealogical and marker heterozygosity approaches.

We have used genealogies and genomic polymorphisms to estimate individual inbreeding coefficients (F) in 50 subjects with an expected range (based on recent genealogies) of F from 0.0 to 0.0625. The estimates were based on two approaches, using genotypes respectively from 410 microsatellite markers (410-STR panel) and from 10,000 SNPs (10K-SNP panel). The latter was performed in a sub-sample of 15 individuals. We concluded that for both marker panels measures of inbreeding based on the excess of homozygosity over Hardy-Weinberg expectation were not closely correlated with 4-5 generation genealogical F-values. For the 10K-SNP panel we found two alternative measures which correlated more closely with F, based respectively on standard errors and on paired homozygosity of nearby SNPs over distances of 2-4 cM. We propose an empirical method for estimating standard errors and hence individual F-values, based on the variation between individual autosomes. This method could provide useful estimates of average F-values for groups of individuals in population-based studies of the effects of inbreeding/homozygosity on quantitative traits.

Gonadal effects of a mouse Denys-Drash syndrome mutation.

Gonadal effects of the Denys-Drash syndrome (DDS) mutation Wt1(tmT396 )were examined in chimaeric and heterozygous mice. Since the only heterozygote was 41,XXY, Sertoli cell function was assessed by comparison with age-matched control XXY testes. Control XXY Sertoli cells showed immuno-expression of WT1 and androgen receptor (AR) indistinguishable from wild-type (40,XY), but expressed anti-Mullerian hormone (AMH). In contrast, DDS Sertoli cells showed only faint immuno-expression of WT1 and did not express AR or AMH. While XY<-->XY DDS chimaeras were male, XX<-->XY chimaeras were predominantly female. In the rare XX<-->XY DDS males the Sertoli cell lineage was largely derived from Wt1 mutant XY cells. We conclude that DDS mutant cells can form Sertoli cells, that the dominant mutation does not cause male sex reversal in mice but distorts the sex ratio of XX<-->XY chimaeras, and that there may be a link between WT1, AMH and AR expression by Sertoli cells in vivo.

Extent of linkage disequilibrium in a Sardinian sub-isolate: sampling and methodological considerations.

The extent of linkage disequilibrium (LD) is an important factor when designing experiments for mapping disease or trait loci using LD mapping methods. It depends on the population history and hence is a characteristic of each population. Here, we have assessed the extent of LD in a sub-isolate of the general Sardinian population (775 members of one village) using 22 polymorphic markers on chromosome 19. We found high levels of disequilibrium that extended to 8 cM, when based on D', and 11 cM when based on the significance level of the allelic association. The fact that conclusions based on both methods are similar suggests that the estimates are quite robust. We have also shown, through a simple resampling technique, that small sample sizes can overestimate both the mean value of D' and its variance up to a factor of about 2 and 16, respectively, when the number of diplotypes (the pair of haplotypes that compose the genotype) decreased from 186 to 26. We evaluated the effect on D' of the depth of the pedigree available when using phased founders, and compared the estimates with those obtained when using unphased founders, and also the effect of grouping alleles on the value of D' and the significance level. Owing to the high sampling variance of LD, we recommend the use of at least 200 unrelated individuals when characterizing the extent of LD.

Complex genetic diseases: controversy over the Croesus code.

The polarization of views on how best to exploit new information from the Human Genome Project for medicine reflects our ignorance of the genetic architecture underlying common diseases: are susceptibility alleles common or rare, neutral or deleterious, few or many? Single-nucleotide polymorphism (SNP) technology is almost in place to dissect such diseases and to create a personalized medicine, but success is critically dependent on the biology and "Nature to be commanded must be obeyed" (Francis Bacon, 1620, Novum Organum).

Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice.

The incubation period (IP) and the neuropathology of transmissible spongiform encephalopathies (TSEs) have been extensively used to distinguish prion isolates (or strains) inoculated into panels of inbred mouse strains. Such studies have shown that the bovine spongiform encephalopathy (BSE) agent is indistinguishable from the agent causing variant Creutzfeldt-Jakob disease (vCJD), but differs from isolates of sporadic CJD, reinforcing the idea that the vCJD epidemic in Britain results from consumption of contaminated beef products. We present a mouse model for genetic and environmental factors that modify the incubation period of BSE cross-species transmission. We have used two mouse strains that carry the same prion protein (PrP) allele, but display a 100-day difference in their mean IP following intracerebral inoculation with primary BSE isolate. We report genetic effects on IP that map to four chromosomal regions, and in addition we find significant factors of host environment, namely the age of the host's mother, the age of the host at infection, and an X-cytoplasm interaction in the host.

Cross-talk in kidney development.

As in most organs, the emerging theme in kidney development is the importance of cross-talk between several tissues and cell lineages to allow morphogenesis to proceed in a complex but highly regulated way. Over the past few years, knock-out and transgenic analyses in mice and evolutionary comparison with non-mammalian species have been particularly instrumental in identifying molecules with crucial functions for tissue-tissue interactions. The transcription factors Wt1 and Eya1, the signalling molecules Gdnf and LIF and the receptors c-Ret and GdnfRalpha have been demonstrated to fulfil fundamental roles in the first step of metanephric induction, the outgrowth of the ureter. Signalling by members of the Wnt, BMP and FGF families, regulated by transcription factors such as Pax2, mediates nephrogenesis by adjusting the balance between the ureteric bud epithelium, stromal and nephrogenic tissues. The stromal tissue, neglected for many years, has been shown to serve important functions in regulating the growth of nephrons. Finally, we have also begun to gain insight into the molecular events underlying patterning of the nephron into distinct functional units including glomerulus, proximal and distal tubule.

Identification of WTAP, a novel Wilms' tumour 1-associating protein.

The Wilms' tumour suppressor gene WT1 is essential for the normal development of the genitourinary system. It appears to play a role in both transcriptional and post-transcriptional regulation of certain cellular genes. However, the mechanisms behind WT1 function are not clearly understood despite the identification of numerous potential target genes and the isolation of several WT1-binding proteins. This study therefore sets out to identify other WT1-associating proteins to help to unravel how WT1 interacts with the cellular machinery. We report the identification of a novel human WT1-associating protein, WTAP, which was isolated using the yeast two-hybrid system. Both in vitro and in vivo assays have shown that the interaction between WTAP and WT1 is specific and occurs endogenously in cells. The mouse homologue of WTAP was isolated and found to be >90% conserved at the nucleotide and protein levels. The human and mouse genes were mapped using fluorescence in situ hybridization to regions in chromosomes 6 (which is thought to harbour a tumour suppressor gene) and 17, respectively. The expression pattern of WTAP was investigated and shown to be ubiquitous, perhaps reflecting a housekeeping role. WTAP is a nuclear protein, which like WT1 localizes throughout the nucleoplasm as well as in speckles and partially co-localizes with splicing factors. Although the significance of this interaction is not yet known, WTAP promises to be an interesting WT1-binding partner.

Did nucleotides or amino acids drive evolutionary conservation of the WT1 +/-KTS alternative splice?

Evolutionary comparisons frequently pinpoint crucial parts of a protein but, even within coding regions, nucleotides can do more than determine amino acid sequence. One highly conserved feature of the Wilms' tumour suppressor gene, WT1, is the potential, following alternative pre-mRNA splicing, to insert three amino acids (KTS) between the third and fourth zinc fingers. The nucleotides at this position simultaneously define amino acids and the alternative splice site. At the protein level this insertion influences DNA binding affinity and specificity, protein-protein interactions and subnuclear localization. Mutations within the +/-KTS splice junction lead to severe urogenital developmental abnormalities such as Frasier syndrome, indicating that the isoform ratio is critical for wild-type function. Using a series of site-directed mutations in both the genomic and cDNA context, the nucleotide-amino acid relationship was investigated. Mutational analysis within the cDNA suggests that the precise amino acids inserted may not be critical, but rather the disruption of the zinc finger structure alone may be sufficient to generate proteins with different in vitro properties. However, analysis within the genomic context suggests that the precise structure of the splice junction is crucial in retaining the balance between the isoforms, and this may account for the high nucleo-tide conservation of this unusual gene structure from fish to mammals.

Presence of WT1, the Wilm's tumor suppressor gene product, in nuclear poly(A)(+) ribonucleoprotein.

The tumor suppressor gene WT1 encodes a zinc finger protein, which consists of four C-terminal C(2)-H(2) zinc fingers of the Krüppel type, and at the N terminus a Q/P-rich trans-regulatory domain, both characteristic of transcription factors. However, recent findings suggest that WT1 may also be involved in a post-transcriptional process. Specifically, WT1 isoforms containing the alternatively spliced exon 9 (+lysine-threonine-serine (KTS)) preferentially associate with nuclear speckles and co-immunoprecipitate splicing antigens (Larsson, S. H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V., and Hastie, N. D. (1995) Cell 81, 391-401); furthermore, WT1 has been shown to interact with the ubiquitous splicing factor U2AF65 (Davies, R. C., Calvo, C., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217-3225) and binds to RNA in vitro (Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D., Little, M., Holmes, G., Todorov, I., and Ward, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7562-7566; Bardeesy, N., and Pelletier, J. (1998) Nucleic Acids Res. 26, 1784-1792). To extend these findings, we have fractionated nuclear extracts to see if particles containing WT1 have the properties of ribonucleoprotein (RNP). In summary, WT1 is enriched by oligo(dT) chromatography, as are U2AF65, the U5 small nuclear RNP-associated protein p116 and hnRNP A1. Gel filtration and sedimentation profiles suggest that WT1 is present in RNase-sensitive particles, >2 MDa in size, peaking at approximately 60 S, and approximately 1.27 g/cm(3) on Nycodenz. Similar results were obtained from two cell lines expressing WT1, fetal kidneys (day E17), and transiently transfected cells, suggesting that the presence of WT1 protein in nuclear poly(A)(+) RNP is a general aspect of WT1 function.

YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis.

The Wilms' Tumour gene WT1 has important functions during development. Knock-out mice were shown to have defects in the urogenital system and to die at embryonic day E13.5, probably due to heart failure. Using a lacZ reporter gene inserted into a YAC construct, we demonstrate that WT1 is expressed in the early proepicardium, the epicardium and the subepicardial mesenchymal cells (SEMC). Lack of WT1 leads to severe defects in the epicardial layer and a concomitant absence of SEMCs, which explains the pericardial bleeding and subsequent embryonic death observed in Wt1 null embryos. We further show that a human-derived WT1 YAC construct is able to completely rescue heart defects, but only partially rescues defects in the urogenital system. Analysis of the observed hypoplastic kidneys demonstrate a continuous requirement for WT1 during nephrogenesis, in particular, in the formation of mature glomeruli. Finally, we show that the development of adrenal glands is also severely affected in partially rescued embryos. These data demonstrate a variety of new functions for WT1 and suggest a general requirement for this protein in the formation of organs derived from the intermediate mesoderm.

A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys-Drash syndrome.

The Wilms tumor-suppressor gene, WT1, plays a key role in urogenital development, and WT1 dysfunction is implicated in both neoplastic (Wilms tumor, mesothelioma, leukemias, and breast cancer) and nonneoplastic (glomerulosclerosis) disease. The analysis of diseases linked specifically with WT1 mutations, such as Denys-Drash syndrome (DDS), can provide valuable insight concerning the role of WT1 in development and disease. DDS is a rare childhood disease characterized by a nephropathy involving mesangial sclerosis, XY pseudohermaphroditism, and/or Wilms tumor (WT). DDS patients are constitutionally heterozygous for exonic point mutations in WT1, which include mutations predicted to truncate the protein within the C-terminal zinc finger (ZF) region. We report that heterozygosity for a targeted murine Wt1 allele, Wt1(tmT396), which truncates ZF3 at codon 396, induces mesangial sclerosis characteristic of DDS in adult heterozygous and chimeric mice. Male genital defects also were evident and there was a single case of Wilms tumor in which the transcript of the nontargeted allele showed an exon 9 skipping event, implying a causal link between Wt1 dysfunction and Wilms tumorigenesis in mice. However, the mutant WT1(tmT396) protein accounted for only 5% of WT1 in both heterozygous embryonic stem cells and the WT. This has implications regarding the mechanism by which the mutant allele exerts its effect.

WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes.

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms' tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both -KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.

The Wilms' tumor suppressor WT1: approaches to gene function.

Occurring with a frequency of 1 in 10,000 live births, Wilms' tumor is one of the most common solid tumors of children. The genetic basis of this tumor is highly complex and several loci have been shown to be associated with tumor formation. Thus far, however, WT1 is the only gene that has been isolated and proven to carry mutations within Wilms' tumors. During the last few years, a wealth of experiments has been carried out to address the function of WT1 as a tumor suppressor and developmental regulator. This review focuses on studies addressing WT1 function; new approaches to understand WT1 function in vivo and present transgenic data in which WT1 was driven ectopically using a CMV promoter are discussed. Our results suggest that ubiquitous expression of WT1 is not compatible with embryonic development.

YAC transgenic analysis reveals Wilms' tumour 1 gene activity in the proliferating coelomic epithelium, developing diaphragm and limb.

Wilms' Tumour 1 gene (WT1) is required for the correct development of the urogenital system. To examine its regulation and expression, we created several transgenic mouse lines containing a beta-galactosidase reporter driven by the human WT1 promoter. A 5 kb promoter weakly recapitulated a subset of the endogenous Wt1 expression pattern. In contrast, 470 and 280 kb YAC transgenes reproduced the correct pattern with high activity and highlighted new expression sites. Wt1 is expressed in the septum transversum revealing how its mutation causes diaphragmatic defects. Wt1 expression in the limb demarcates a zone between chondrogenic and apoptotic domains. Finally, Wt1 is expressed in mesenchymal cells derived from the coelomic epithelium. Based upon these and further data we discuss a Wt1 role in epithelial<-->mesenchymal transitions.

Regulation of the Wilms' tumor gene during spermatogenesis.

Spermatogenesis is the process by which male germ cells develop and mature, a pathway that includes a transition from a mitotic to a meiotic cell cycle. Throughout this pathway, the germ cells are in close contact with their nurturing cells, the Sertoli cells. Sertoli-germ cell interactions are difficult to study in mammals due to the complex cellular organization of their seminiferous tubules. The urodele amphibian testis, however, provides a unique system to study the process of germ cell maturation; it is organized in a gradient-like cystic structure, in which synchronized germ cells can be found within the same cyst. The Wilms' tumor gene (WT1) has been shown to be an essential gene for the formation of the gonads in mice, and it has been implicated in a variety of differentiation processes. The WT1 gene is thus a good candidate for the study of the differentiation processes involved in the maturation of the male germ cells. By using a probe for the urodele WT1 homologue in in situ hybridization studies, as well as an antibody against the WT1 protein in immunohistochemistry studies, we determined that WT1 gene expression in Sertoli cells depends on the stage of maturation of the associated germ cell. Thus, WT1 mRNA was detected only in Sertoli cells of cysts that contained early spermatogonia. No mRNA expression was observed in cysts containing late spermatogonia, germ cells undergoing meiosis, or germ cells going through spermiogenesis. Immunohistochemistry studies confirmed that WT1 protein was strongly expressed in Sertoli cells associated with early spermatogonia but not in late ones. The protein was also found in Sertoli cells associated with germ cells that undergo the subsequent stages of meiosis and spermiogenesis. These results suggest that WT1 could be involved in the regulation by Sertoli cells of germ cell maturation and possibly in the progression from a mitotic to a meiotic cell cycle.