Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre- and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5' and 3' ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues in which it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action.

Juxtaposed regions of extensive and minimal linkage disequilibrium in human Xq25 and Xq28.

Linkage disequilibrium (LD), or the non-random association of alleles, is poorly understood in the human genome. Population genetic theory suggests that LD is determined by the age of the markers, population history, recombination rate, selection and genetic drift. Despite the uncertainties in determining the relative contributions of these factors, some groups have argued that LD is a simple function of distance between markers. Disease-gene mapping studies and a simulation study gave differing predictions on the degree of LD in isolated and general populations. In view of the discrepancies between theory and experimental observations, we constructed a high-density SNP map of the Xq25-Xq28 region and analysed the male genotypes and haplotypes across this region for LD in three populations. The populations included an outbred European sample (CEPH males) and isolated population samples from Finland and Sardinia. We found two extended regions of strong LD bracketed by regions with no evidence for LD in all three samples. Haplotype analysis showed a paucity of haplotypes in regions of strong LD. Our results suggest that, in this region of the X chromosome, LD is not a monotonic function of the distance between markers, but is more a property of the particular location in the human genome.

X-linked situs abnormalities result from mutations in ZIC3.

Vertebrates position unpaired organs of the chest and abdomen asymmetrically along the left-right (LR) body axis. Each structure comes to lie non-randomly with respect to the midline in an overall position designated situs solitus, exemplified in humans by placement of the heart, stomach and spleen consistently to the left. Aberrant LR axis development can lead to randomization of individual organ position (situs ambiguus) or to mirror-image reversal of all lateralized structures (situs inversus). Previously we mapped a locus for situs abnormalities in humans, HTX1, to Xq26.2 by linkage analysis in a single family (LR1) and by detection of a deletion in an unrelated situs ambiguus male (Family LR2; refs 2,3). From this chromosomal region we have positionally cloned ZIC3, a gene encoding a putative zinc-finger transcription factor. One frameshift, two missense and two nonsense mutations have been identified in familial and sporadic situs ambiguus. The frameshift allele is also associated with situs inversus among some heterozygous females, suggesting that ZIC3 functions in the earliest stages of LR-axis formation. ZIC3, which has not been previously implicated in vertebrate LR-axis development, is the first gene unequivocally associated with human situs abnormalities.

The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome.

In type I blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), eyelid abnormalities are associated with ovarian failure. Type II BPES shows only the eyelid defects, but both types map to chromosome 3q23. We have positionally cloned a novel, putative winged helix/forkhead transcription factor gene, FOXL2, that is mutated to produce truncated proteins in type I families and larger proteins in type II. Consistent with an involvement in those tissues, FOXL2 is selectively expressed in the mesenchyme of developing mouse eyelids and in adult ovarian follicles; in adult humans, it appears predominantly in the ovary. FOXL2 represents a candidate gene for the polled/intersex syndrome XX sex-reversal goat.

A YAC contig spanning the blepharophimosis-ptosis-epicanthus inversus syndrome and propionic acidemia loci.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant condition consisting of congenital dysplasia of the eyelids with a reduced horizontal diameter of the palpebral fissures, droopy eyelids and epicanthus inversus. Two clinical entities have been described: type I and type II. The former is distinguished by female infertility, whereas the latter presents without other symptoms. Both type I and type II were recently mapped on the long arm of chromosome 3 (3q22-q23), suggesting a common gene may be affected. The centromeric and the telomeric limits of this region are well defined between loci D3S1316 and D3S1615, which reside approximately 5 cM apart. Here, we present the construction of a YAC contig spanning the entire BPES locus using 17 polymorphic markers, 2 STS and 28 ESTs. This region of approximately 5 Mb was covered by 31 YACs, and was supported by detailed FISH analysis. In addition, we have precisely mapped the propionyl-CoA carboxylase beta polypeptide (PCCB), the gene mutated in propionic acidemia, within this contig. Apart from providing a framework for the identification of the BPES gene, this contig will also be useful for the future identification of defects and genes mapped to this region, and for developing template resources for genomic sequencing.

Recombination trapping: an in-vivo approach to recover cDNAs encoded in YACs.

We have developed an approach to identify and localize cDNAs encoded by YACs. In this scheme, a YAC truncation vector containing a cDNA library is used to interrupt the YAC by homologous recombination in yeast. This approach generates YACs truncated at the site of recombination between the cDNA and the cognate YAC sequence and thus localizes the gene in the YAC. This method results in the production of a large percentage of true recombinants identifying gene encoding regions of the genome. This approach is shown to identify an unique EST sequence from a YAC in Xp22, the recently described transketolase-like gene in a YAC from Xq28 and a putative kinesin-like gene in Xq13. This system should also be useful in the mapping of YACs by targeted integration. We have constructed a new telomere truncation vector, pGR8, which incorporates two selectable markers, HIS5 and LYS2. This vector overcomes problems of previous vectors including: incompatibility with most YAC libraries, vector homology with the YAC arms and high backgrounds resulting from the use of a single selectible marker. A third counterselection with 5-fluoroorotic acid (5FOA) against yeast clones retaining the URA3 gene was also employed to reduce background further. Therefore, this vector and approach should be useful to the transcriptional analysis of YAC maps of any genome.

Multiple Sp1 sites efficiently drive transcription of the TATA-less promoter of the human glypican 3 (GPC3) gene.

Simpson-Golabi-Behmel Syndrome (SGBS) is an X-linked disease characterized by pre- and postnatal overgrowth. Recently, we have shown that mutations in the glypican family gene, GPC3, cause SGBS. This gene is predominantly expressed in the same mesoderm-derived tissues that overgrow in its absence. To investigate the basis for promoter function, 3.3kb of GC-rich DNA 5' of the transcribed region were fused to a luciferase cDNA, transfected into Caco-2 and NT2 cells, and assayed for activity. Deletion analysis identified a 218-bp fragment upstream of the transcription start site that conferred more than 80% of maximal reporter gene activation. This fragment contains five putative Sp1 binding sites, three of which (centered at nt -14, -34, and -92) were active when assessed by DNaseI footprinting and gel shift/supershift assays. Additionally, Sp1 specifically transactivated transcription in Sp1-deficient Drosophila SL2 cells, demonstrating the functionality of Sp1 on the GPC3 promoter. A full-length promoter construct was also highly active in HeLa cells, which do not express endogenous GPC3. These results indicate that the GPC3 promoter is dependent on Sp1 for proper activation, but tissue-specific repression in non-expressing cells must involve either DNA that lies outside the region tested or auxiliary structural features of chromatin.

Glypican 3 and glypican 4 are juxtaposed in Xq26.1.

Recently, we have shown that mutations in the X-linked glypican 3 (GPC3) gene cause the Simpson-Golabi-Behmel overgrowth syndrome (SGBS; ). The next centromeric gene detected is another glypican, glypican 4 (GPC4), with its 5' end 120763bp downstream of the 3' terminus of GPC3. One recovered GPC4 cDNA with an open reading frame of 1668nt encodes a putative protein containing three heparan sulfate glycosylation signals and the 14 signature cysteines of the glypican family. This protein is 94.3% identical to mouse GPC4 and 26% identical to human GPC3. In contrast to GPC3, which produces a single transcript of 2.3kb and is stringently restricted in expression to predominantly mesoderm-derived tissues, Northern analyses show that GPC4 produces two transcripts, 3.4 and 4.6kb, which are very widely expressed (though at a much higher level in fetal lung and kidney). Interestingly, of 20 SGBS patients who showed deletions in GPC3, one was also deleted for part of GPC4. Thus, GPC4 is not required for human viability, even in the absence of GPC3. This patient shows a complex phenotype, including the unusual feature of hydrocephalus; but because an uncle with SGBS is less affected, it remains unclear whether the GPC4 deletion itself contributes to the phenotype.

Refinement of the background genetic map of Xq26-q27 and gene localisation for Börjeson-Forssman-Lehmann Syndrome.

A detailed map of genetic markers was constructed around the gene for the X-linked mental retardation syndrome of Börjeson-Forssman-Lehmann (BFLS). A multipoint linkage map of framework markers across Xq26-27, based on CEPH families, was integrated with the physical map, based on a YAC contig, to confirm marker order. The remaining genetic markers, which could not be ordered by linkage, were added to create the comprehensive genetic back-ground map, in the order determined by physical mapping, to determine genetic distances between adjacent markers. This background genetic map is applicable to the refinement of the regional localisation for any disease gene mapping to this region. The BFLS gene was localised using this background map in an extended version of the family described by Turner et al. [1989]. The regional localisation for BFLS extends between recombination events at DXS425 and DXS105, an interval of 24.6 cM on the background genetic map. The phenotypic findings commonly seen in the feet of affected males and obligate carrier females may represent a useful clinical indicator of carrier status in potential female carriers in the family. Recombination between DXS425 and DXS105 in a female with such characteristic feet suggests that the distal limit of the regional localisation for the BFLS gene might reasonably be reduced to DXS294 for the purpose of selecting candidate genes, reducing the interval for the BFLS gene to 15.5 cM. Positional candidate genes from the interval between DXS425 and DXS105 include the SOX3 gene, mapped between DXS51(52A) and DXS98(4D-8). SOX3 may have a role in regulating the development of the nervous system. The HMG-box region of this single exon gene was examined by PCR for a deletion and then sequenced. No deviation from normal was observed, excluding mutations in the conserved HMG-box region as the cause of BFLS in this family.

Simpson-Golabi-Behmel syndrome: genotype/phenotype analysis of 18 affected males from 7 unrelated families.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth disorder recently shown to be caused by mutations in the heparan sulfate proteoglycan GPC3 [Pilia et al., Nat Genet; 12:241-247 1996]. We have used Southern blot analysis and polymerase chain reaction amplification of intra-exonic sequences to identify four new GPC3 mutations and further characterize three previously reported SGBS mutations. De novo GPC3 mutations were identified in 2 families. In general, the mutations were unique deletions ranging from less than 0.1 kb to more than 300 kb in length with no evidence of a mutational hot spot discerned. The lack of correlation between the phenotype of 18 affected males from these 7 families and the location and size of the GPC3 gene mutations suggest that SGBS is caused by a nonfunctional GPC3 protein.

BLOCK-based PCR markers to find gene family members in human and comparative genome analysis.

Degenerate primer pairs that include consensus sequences of evolutionary conserved portions of protein families (BLOCKs or ancient conserved regions) can be used to screen by polymerase chain reaction (PCR) for cognate cDNAs and YACs through much of phylogeny. Nine such primer pairs were developed, and five with sites on human chromosomes 7 or X were shown to identify YACs from chromosome-specific locations, including a candidate for a new zinc finger gene in Xq28. When linked to contig-based genomic maps, such BLOCK-based PCR assays may provide a route to recover the members and study the development of families containing up to 40% of genes, in genomes as diverse as humans, nematodes, and yeast.

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis.

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking the hprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and including hprt. We used "bubble" PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream of hprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream of hprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay, hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.

Isochores and CpG islands in YAC contigs in human Xq26.1-qter.

GC levels were assessed at 37 loci across 30 Mb of Xq26.1-qter, a region physically mapped in overlapping yeast artificial chromosome clones. In 8 Mb of R band Xq26, GC is relatively high (up to 44%) in the proximal 4 Mb and relatively low (40-41%) in the distal 4 Mb. Consistently low GC values (38-41%) are observed in G band Xq27. In contrast, further toward the telomere in Xq28, the GC level rises progressively to reach 52% at 2 to 4 Mb from the end of the chromosome; this region is delimited by low GC loci. Across these regions of Xq, the content of rare-cutter restriction enzyme sites containing CpG, including "CpG islands" in the most completely mapped Xq26-27.1 region, is correlated with GC level. Isochore mapping can thus provide one index of putative gene content across mapped regions.

Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26.

A successful test is reported to generate long-range contiguous coverage of DNA from a human cytogenetic band in overlapping yeast artificial chromosomes (YACs). Seed YACs in band Xq26 were recovered from a targeted library of clones from Xq24-q28 with 14 probes, including probes for the hypoxanthine guanine phosphoribosyltransferase- and coagulation factor IX-encoding genes and nine probes used in linkage mapping. Neighboring YACs were then identified by 25 "walking" steps with end-clones, and the content of 71 probes in cognate YACs was verified by further hybridization analyses. The resultant contig extends across 8 million base pairs, including most of band Xq26, with an order of markers consistent with linkage data. YAC-based mapping, thus, permits steps toward a fully integrated physical and genetic map and is probably adequate to sustain most of the human genome project.

YAC/STS map of 9 Mb of Xq26 at 100-kb resolution, localizing 6 ESTs, 6 genes, and 32 genetic markers.

To facilitate functional analysis of the Xq26 region, the physical map has been extended across 9 Mb with 192 YACs and markers including 90 STSs (sequence-tagged sites) and 50 hybridization probes. Six genes and six ESTs are localized. In addition, 32 markers that detect polymorphism permit an integration of physical with genetic linkage data. The localizations of eight uncloned disease genes are thereby delimited on the physical map. The data also suggest a possible gradient of recombination across the cytogenetic band, with little or no recombination reported in the centromeric 3.5-4 Mb.

Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes.

We have developed a way to fit yeast artificial chromosomes (YACs) with markers that permit the selection of stably transformed mammalian cells, and have determined the fate and expression of such YACs containing the genes for human ribosomal RNA (rDNA) or glucose-6-phosphate dehydrogenase (G6PD). The YACs in the yeast cell are "retrofitted" with selectable markers by homologous recombination with the URA3 gene of one vector arm. The DNA fragment introduced contains a LYS2 marker selective in yeast and a thymidine kinase (TK) marker selective in TK-deficient cells, bracketed by portions of the URA3 sequence that disrupt the endogenous gene during the recombination event. Analyses of transformed L-M TK- mouse cells showed that YACs containing rDNA or G6PD were incorporated in essentially intact form into the mammalian cell DNA. For G6PD, a single copy of the transfected YAC was found in each of two transformants analyzed and was fully expressed, producing the expected human isozyme as well as the heterodimer composed of the human gene product and the endogenous mouse gene product.