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.

Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript.

The technique of whole-genome polymerase chain reaction was used to study the DNA binding properties of the product of the wt1 gene. The zinc finger region of this gene is alternatively spliced such that the major transcript encodes a protein with three extra amino acids between the third and fourth fingers. The minor form of the protein binds specifically to DNA. It is now shown that the major form of wt1 messenger RNA encodes a protein that binds to DNA with a specificity that differs from that of the minor form. Therefore, alternative splicing within the DNA binding domain of a transcription factor can generate proteins with distinct DNA binding specificities and probably different physiological targets.

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.

Wilms' tumour gene and function.

The Wilms' tumour gene, WT1, encodes a protein with four zinc fingers that is probably a transcription factor. In humans, WT1 mutations can lead to childhood kidney tumours and to developmental defects of the kidney and gonad. The WT1 gene may have a role in the mesenchyme to epithelial switch in a range of mesodermally derived tissues. Furthermore, growth-factor genes may be targets for repression by the WT1 protein during development. WT1 is the first example of a tumour-suppressor gene with a specific developmental role.

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.

Wilms' tumour: reconciling genetics and biology.

Wilms' tumour, a paediatric malignancy of the kidney, is a striking example of the relationship between aberrant development and cancer. Several different genetic loci have been implicated in the aetiology of the tumour; genomic imprinting also plays a role. One Wilms' tumour predisposition gene (WT1), encoding a zinc finger protein, is expressed in a limited set of tissues, including developing nephrons and gonads. The biology and genetics of Wilms' tumour underline the developmental relationship between kidneys and gonads.

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.

A genetic locus closely linked to a protease inhibitor gene complex controls the level of multiple RNA transcripts.

The two major protease inhibitors in mouse plasma are alpha 1-protease inhibitor (alpha 1-PI), putative inhibitor of neutrophil elastase, and contrapsin, an inhibitor in vitro of trypsinlike proteases. We have shown by nucleotide sequence analysis that these two inhibitors are related (R. E. Hill, P. H. Shaw, P. A. Boyd, H. Baumann, and N. D. Hastie, Nature (London) 311:175-177, 1984). Here, we show that the contrapsin and alpha 1-PI genes are members of two different multigene families, each containing at least three genes in mice and rats. We established the chromosomal locations of these genes by analyzing the segregation of restriction fragment length polymorphisms in recombinant inbred mouse strains. These experiments show that the multiple genes in each family are clustered and that the two gene families are closely linked on chromosome 12. Thus the genes for contrapsin and alpha 1-PI are likely to have evolved by duplication of a common ancestral gene. The contrapsin multigene family codes for multiple mRNA transcripts in the liver. There is a genetic difference among inbred mouse strains in the regulation of two of these transcripts. In some inbred strains the transcripts are synthesized constitutively; in others they are induced by inflammation. We mapped in recombinant inbred strains the regulatory locus responsible for this genetic variation and found it is linked to the contrapsin multigene family, which suggests a cis-acting regulatory element. We also found that the contrapsin and the alpha 1-PI multigene families have acquired very different regulatory responses since the time of the gene duplication event.

Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

We used mitotic chromosomes isolated from a human EJ bladder carcinoma cell line for morphological transformation of mouse C127 cells. These chromosome-mediated transformants were analyzed for cotransfer of markers syntenic with c-Ha-ras-1 on human chromosome 11. We also used cloned, dispersed human DNA repeats, in a general mapping strategy, to quantitate the amounts and molecular state of human DNA transferred along with the activated c-Ha-ras-1 gene. In situ hybridization was used to visualize the physical state of the transfected human chromatin. The combined use of these various techniques revealed the occurrence of both chromosomal and DNA rearrangements. However, our analysis also demonstrated that, in general, very substantial lengths of DNA are transferred intact. Closely linked markers are likely to cosegregate. Therefore, these transformants should be invaluable sources for the complete molecular cloning of isolated fragments of the short arm of human chromosome 11.

Most highly repeated dispersed DNA families in the mouse genome.

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.

The evolution of WT1 sequence and expression pattern in the vertebrates.

WT1 is a Wilms' tumour predisposition gene, encoding a protein with four C-terminal Kruppel-type zinc fingers, which is also a major regulator of kidney and gonadal development. To pinpoint key regulatory domains involved in development and evolution of the vertebrate genitourinary system, we have isolated WT1 orthologues from all gnathostome classes. Partial nucleotide sequence from chick, alligator, Xenopus laevis and zebrafish reveals extensive conservation. Both the zinc fingers and the transregulatory domain exhibit a high level of similarity in all the species examined. However, of the two alternatively spliced regions only one, the three amino acid KTS insertion between zinc fingers 3 and 4, is found in species other than mammals. The 17 amino acid insertion at the C-terminal end of the transregulatory domain is present only in mammals. Residues with reported human pathological mutations are also unaltered across species, underlining their structural significance. Studies in chick and alligator reveal that the mammalian intermediate mesoderm expression pattern is conserved in birds and reptiles. A wider role in mesodermal differentiation is suggested by expression in some paraxial and lateral mesoderm derivatives.

Equivalent expression of paternally and maternally inherited WT1 alleles in normal fetal tissue and Wilms' tumours.

Observations of non-random maternal 11p allele loss in Wilms' tumour (WT) have implied the possible involvement of an imprinted 11p locus in WT aetiology. A proposed 11p13 Wilms' tumour gene, WT1, has recently been isolated and encodes a zinc finger DNA-binding protein, the 3' untranslated region of which contains a polymorphic dinucleotide repeat (CA repeat) motif. We have exploited this transcribed CA repeat to examine the allelic expression pattern of WT1 and thereby determine whether transcriptional imprinting of this gene occurs. DNA and reverse-transcribed RNA from tumours and normal tissue were subjected to the polymerase chain reaction (PCR) using radiolabelled primers flanking the CA repeat. The gene was seen to be expressed from both of the constitutive alleles in 9-week human fetal kidney, all informative Wilm's tumours and neonatal kidney tissue adjacent to the tumours. In one tumour, known to be heterozygous for a point mutation in zinc finger 2, direct sequencing confirmed that both mutant and wild-type transcripts were being expressed. These results demonstrate that this gene is not subject to transcriptional imprinting in tumours or normal fetal kidney.

The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo.

In the developing mouse, the Wilms' tumour gene, WT1, is first expressed in the intermediate mesenchyme lateral to the coelomic cavity (13 somite, early 9 dpc embryo). A few hours later, it is present around all the cavity and in the urogenital ridge (the earliest mesonephric tubules) and the differentiating heart mesothelium. By 11 dpc, expression is in the uninduced metanephric mesenchyme and in the presumptive motor neurons of the spinal cord. By 12.5 dpc, WT1 expression has increased in the induced mesenchyme of the kidney and a day later is particularly marked in the nephrogenic condensations. At 13.5 dpc, WT1 is briefly expressed in some differentiating body-wall musculature, while two days later, there is a small domain of expression in the roof of the fourth ventricle of the brain. By day 20, however, expression has become restricted to the kidney glomeruli. RNA-PCR analysis on 12.5 dpc embryos and on adult tissues shows that WT1 is weakly expressed in both eye and tongue. The expression pattern in human embryos (28-70 days) is very similar to that in the equivalent mouse stages (10-15 dpc). The results indicate that WT1 is mainly present in mesodermally derived tissues, although exceptions are ectodermally derived spinal cord and brain. The data indicate that WT1 plays a role in mediating some cases of the mesenchyme-to-epithelial transition, but its expression elsewhere argues that it has other tissue-specific roles in 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.

Repression of promoters for the mouse insulin-like growth factor II-encoding gene (Igf-2) by products of the Wilms' tumour suppressor gene wt1.

We have investigated actions of the Wilms' tumour suppressor zinc finger transcription factor, WT1, on promoters of the mouse insulin-like growth factor II-encoding gene (Igf-2). Two variant forms of WT1 repressed the two major Igf-2 promoters (P2 and P3) in transient transfection assays. WT1-binding sites were characterised in both these promoters and in the transcribed region downstream from P2, exon 2. In each of these regions, there was a pair of WT1-binding sites, and mutational analysis of the exon-2 sites indicated that both were required for full repression. Cooperative binding of WT1 to these sites might explain the dominant-negative mutations of WT1 observed in some Wilms' tumours and Denys-Drash syndrome cases.