Evolution of the male-determining gene SRY within the cat family Felidae.

In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 5' flank (997 bp) and 3' flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (omega = 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids.

DAX-1, an "antitestis" gene.

The DAX-1 gene has been involved in the dosage sensitive sex reversal (DSS) phenotype, a male-to-female sex-reversal syndrome due to the duplication of a small region of human chromosome Xp21. Dax-1 and Sry have been shown to act antagonistically in the mouse system, where increasing expression of the former leads to female development and increasing activity of the latter to male development. Although these data strongly implicate DAX-1 in sex determination, the mouse and human proteins appear to behave differently. Absence of DAX-1 is responsible for adrenal hypoplasia congenita, a human inherited disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Unlike human patients, Dax-1-deficient XY mice have normal levels of corticotropins and adrenal hormones but are sterile. Dax-1-deficient females are fertile. The DAX-1 protein, an unusual member of the nuclear hormone receptor, may act as a transcriptional repressor. It has been shown to both repress transcriptional activators by direct protein-protein interactions and to bind DNA hairpin structures and repress target genes.

No evidence for association of multiple sclerosis with the complement factors C6 and C7.

Four genome screens in multiple sclerosis have been completed and each has identified evidence for linkage in the pericentromeric region of chromosome 5. This region encodes a number of candidate genes including those for the complement components C6, C7 and C9. We have used a multiplexed oligoligation assay (OLA) to test single nucleotide polymorphisms (SNPs) from the C6 and C7 genes for evidence of association with multiple sclerosis in our sibling pair families. There was no statistically significant difference in the allele frequencies of these polymorphisms in the index cases from our families when compared with locally derived controls. No evidence for transmission distortion was seen with any of the polymorphisms, or with the haplotype built from the three SNPs from the C7 gene. Despite offering themselves as potential candidates these complement genes appear not to confer susceptibility to multiple sclerosis.

A screen of candidates from peaks of linkage: evidence for the involvement of myeloperoxidase in multiple sclerosis.

We tested 11 microsatellite markers for evidence of transmission distortion in 744 trio families with multiple sclerosis. Ten of the markers lie within or near to candidate genes selected on the basis that they map within the regions of potential linkage identified in our previously reported linkage genome screen, while the eleventh is an anonymous marker which had previously shown modest evidence for transmission distortion in our sibling pair families. Only the marker related to the myeloperoxidase (MPO) gene revealed tentative evidence for linkage disequilibrium and further work on this gene is clearly needed in order to resolve the status of this region in conferring susceptibility to multiple sclerosis.

DAX-1, an 'antitestis' gene.

The DAX-1 gene has been involved in the dosage sensitive sex reversal (DSS) phenotype, a male-to-female sex-reversal syndrome due to the duplication of a small region of human chromosome Xp21. Dax-1 and Sry have been shown to act antagonistically in the mouse system, where increasing expression of the former leads to female development and increasing activity of the latter to male development. Although these data strongly implicate DAX-1 in sex determination, the mouse and human proteins appear to behave differently. Absence of DAX-1 is responsible for adrenal hypoplasia congenita, a human inherited disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Unlike human patients, Dax-1-deficient XY mice have normal levels of corticotropins and adrenal hormones but are sterile. Dax-1-deficient females are fertile. The DAX-1 protein, an unusual member of the nuclear hormone receptor, may act as a transcriptional repressor. It has been shown to both repress transcriptional activators by direct protein-protein interactions and to bind DNA hairpin structures and repress target genes.

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Exploring the dense mapping of a region of potential linkage in complex disease: an example in multiple sclerosis.

In 1996 we reported the results of a genome screen in multiple sclerosis, in which potential linkage was identified in a total of twenty regions, including the centromeric region of chromosome 5. In order to investigate the efficiency of typing dense arrays of markers in regions of potential linkage, we have typed an additional nineteen microsatellite markers from this chromosome 5 region (D5S623 - D5S428) in the same sibling pair families. The mean additional information extracted per marker typed declined with increasing map density, while inaccuracies in the mapping and the density of genotyping errors increased. Our empirical results suggest that, in linkage-based experiments, there is a limit to the benefits that are gained from typing additional markers in the same families. Increasing map density up to the 2.5-5 cM level efficiently extracts valuable extra information; however, beyond this level efficiency declines while the confounding effects of mapping and genotyping errors accumulate. We, therefore, recommend that extra markers typed in linkage studies be limited to this level of resolution. Mapping regions beyond this density should only be initiated when searching for linkage disequilibrium.

DNA microarrays in drug discovery and development.

DNA microarrays can be used to measure the expression patterns of thousands of genes in parallel, generating clues to gene function that can help to identify appropriate targets for therapeutic intervention. They can also be used to monitor changes in gene expression in response to drug treatments. Here, we discuss the different ways in which microarray analysis is likely to affect drug discovery.

Evidence that allelic variants of the spinocerebellar ataxia type 2 gene influence susceptibility to multiple sclerosis.

Expanded CAG trinucleotide repeats are known to be responsible for five of the autosomal dominant spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6, and SCA7). We have typed each of these repeats in 226 multiple sclerosis sibling pair families. No expanded repeats were seen, indicating an absence of SCA phenocopies in clinically defined familial multiple sclerosis. However, transmission disequilibrium testing for these repeats demonstrated significant excess transmission of the 22 repeat length allele of the SCA2 gene (P=4. 4E-06) in multiple sclerosis patients. This observation is consistent with pleiotropic effects of the SCA2 gene, with a non-dynamic mutation/polymorphism contributing epistatically to susceptibility in multiple sclerosis.

Deletion of long-range regulatory elements upstream of SOX9 causes campomelic dysplasia.

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9. Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5' to SOX9 are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9, we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9. We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5' to SOX9.

Characterization of whole genome radiation hybrid mapping resources for non-mammalian vertebrates.

Radiation hybrid panels are already available for genome mapping in human and mouse. In this study we have used two model organisms (chicken and zebrafish) to show that hybrid panels that contain a full complement of the donor genome can be generated by fusion to hamster cells. The quality of the resulting hybrids has been assessed using PCR and FISH. We confirmed the utility of our panels by establishing the percentage of donor DNA present in the hybrids. Our hybrid resources will allow inexpensive gene mapping and we expect that this technology can be transferred to many other species. Such successes are providing the basis for a new era of mapping tools, in the form of whole genome radiation hybrid panels, and are opening new possibilities for systematic genome analysis in the animal genetics community.

HLA typing in the United Kingdom multiple sclerosis genome screen.

The United Kingdom multiple sclerosis genome screen demonstrated a peak maximum lod score of 2.8 in the HLA region, together with statistically significant excess transmission of the 121-base pair (bp) allele of the tumour necrosis factor-a marker. In order to determine whether this association is independent of the established HLA association, or simply a consequence of the 121-bp allele being part of the same haplotype, we HLA-DR and -DQ typed the 227 sibling-pair families used in the original screen. The expected associations of multiple sclerosis with the DR15 (p=8.7E-18), DQ6 (p=2.0E-09) and DR51 (p=2.8E-16) phenotypes were confirmed, and excess transmission of the DRB1*1501 and DQB1*0602 alleles was demonstrated. Combining HLA typing with the original microsatellite data demonstrated extensive linkage disequilibrium between the 121-bp allele and the 1501-0602 haplotype. Outside this extended haplotype (121-1501-0602), none of the alleles demonstrated significant transmission distortion. Having established the importance of this extended haplotype, we reanalysed the entire genome screen data after excluding those sibling pairs sharing the extended haplotype (n=27). Conditioning the full genome screen data on the basis of identity by state sharing showed that some potential linkage regions identified in the original screen clustered in families, in which the extended haplotype was shared (1p, 2p and 17q), whereas others grouped with those in which it was not (5cen, 7p and Xq). This suggests complexity in the genetics of multiple sclerosis.

Inheritance of susceptibility to multiple sclerosis.

In recent years, epidemiological evidence supporting the genetic basis of multiple sclerosis has been extended and whole-genome linkage screening has advanced the mapping of the involved genes. Understanding of the known HLA associations has also improved and many candidate genes have been studied.