Faculty Bibliography

Reduced penetrance in genetic disorders may be either dependent or independent of the genetic background of gene carriers. Hirschsprung disease (HSCR) demonstrates a complex pattern of inheritance with approximately 50% of familial cases being heterozygous for mutations in the receptor tyrosine kinase RET. Even when identified, the penetrance of RET mutations is only 50-70%, gender-dependent, and varies with the extent of aganglionosis. We searched for additional susceptibility genes which, in conjunction with RET, lead to phenotypic expression by studying 12 multiplex HSCR families. Haplotype analysis and extensive mutation screening demonstrated three types of families: six families harboring severe RET mutations (group I); and the six remaining families, five of which are RET-linked families with no sequence alterations and one RET-unlinked family (group II). Although the presence of RET mutations in group I families is sufficient to explain HSCR inheritance, a genome scan reveals a new susceptibility locus on 9q31 exclusively in group II families. As such, the gene at 9q31 is a modifier of HSCR penetrance. These observations imply that identification of new susceptibility factors in a complex disease may depend on classification of families by mutational type at known susceptibility genes.

This review discusses the prospects for understanding the genetic basis of complex traits in humans. We take the view that work done on Drosophila melanogaster can serve as a model for understanding complex traits in humans, and the literature on this model system, as well as on humans, is reviewed. The prospects for success in understanding the genetic basis of complex traits depend, in part, on the nature of the forces acting on genetic variation. We suggest that different experimental approaches should be undertaken for traits caused by common genetic variants versus those arising from rare genetic variants.

As the human genome is sequenced and annotated, an important step in future genetic studies of complex traits and diseases will be the identification of relevant candidate genes. To enable such compilations, it would be useful to collate all necessary and available genetic information for each candidate gene. To this end, we have created a web tool (http://genome.cwru.edu/gist/gist.html+ ++) to allow the rapid cataloging of currently available genetic data. This tool, called GIST (or "Gene Information Search Tool"), allows an investigator to search the major genomic databases containing gene and marker information from a single query point. To prove the utility of GIST, a catalog of 150 hypertension candidate genes was created. This resource collates all available nucleotide and amino acid sequence data, expression data, chromosomal map location, and genetic marker interval for each gene, collected from on-line databases. These data can be used to guide genetic studies of hypertension.

There is considerable controversy regarding a putative association between schizophrenia and a biallelic BalI polymorphism in the first exon of the dopamine D3 receptor gene (DRD3), although meta-analyses of published data suggest an association. If such an association exists, it may be detectable at markers physically close to DRD3. Accordingly, we conducted a case-control association study using D3S1310, a short tandem repeat polymorphism located approximately 700 kb telomeric to DRD3 on chromosome 3q13.3. The subjects were Swedish patients with schizophrenia (DSM III-R criteria, n = 110) and screened adult controls (n = 83). A trend for a negative association with the 141 bp allele was detected (chi2 = 7.6, d.f. = 1, P = 0.006; odds ratio 0.46, 95% confidence intervals 0.26, 0.81). However, following corrections for multiple comparisons using subgroups (n = 15) the difference was not significant. Also, due to the risk for population stratification in case-control association studies the results must be treated as tentative. If replicated the results may lend further support for the proposition of an association between schizophrenia and DRD3 or a gene in close proximity to DRD3 on chromosome 3q.

Sequence variation in human genes is largely confined to single-nucleotide polymorphisms (SNPs) and is valuable in tests of association with common diseases and pharmacogenetic traits. We performed a systematic and comprehensive survey of molecular variation to assess the nature, pattern and frequency of SNPs in 75 candidate human genes for blood-pressure homeostasis and hypertension. We assayed 28 Mb (190 kb in 148 alleles) of genomic sequence, comprising the 5' and 3' untranslated regions (UTRs), introns and coding sequence of these genes, for sequence differences in individuals of African and Northern European descent using high-density variant detection arrays (VDAs). We identified 874 candidate human SNPs, of which 22% were confirmed by DNA sequencing to reveal a discordancy rate of 21% for VDA detection. The SNPs detected have an average minor allele frequency of 11%, and 387 are within the coding sequence (cSNPs). Of all cSNPs, 54% lead to a predicted change in the protein sequence, implying a high level of human protein diversity. These protein-altering SNPs are 38% of the total number of such SNPs expected, are more likely to be population-specific and are rarer in the human population, directly demonstrating the effects of natural selection on human genes. Overall, the degree of nucleotide polymorphism across these human genes, and orthologous great ape sequences, is highly variable and is correlated with the effects of functional conservation on gene sequences.

Congenital hydrocephalus is an etiologically diverse, poorly understood, but relatively common birth defect. Most human cases are sporadic with familial forms showing considerable phenotypic and etiologic heterogeneity. We have studied the autosomal recessive mouse mutation congenital hydrocephalus ( ch ) to identify candidate human hydrocephalus genes and their modifiers. ch mice have a congenital, lethal hydrocephalus in association with multiple developmental defects, notably skeletal defects, in tissues derived from the cephalic neural crest. We utilized positional cloning methods to map ch in the vicinity of D13Mit294 and confirm that the ch phenotype is caused by homozygosity for a nonsense mutation in a gene encoding a winged helix/forkhead transcription factor ( Mf1 ). Based on linked genetic markers, we performed detailed phenotypic characterization of mutant homozygotes and heterozygotes to demonstrate the pleiotropic effects of the mutant gene. Surprisingly, ch heterozygotes have the glaucoma-related distinct phenotype of multiple anterior segment defects resembling Axenfeld-Rieger anomaly. We also localized a second member of this gene family ( Hfh1 ), a candidate for other developmental defects, approximately 470 kb proximal to Mf1.

Hirschsprung disease (HSCR) is a multigenic neurocristopathy clinically recognized by aganglionosis of the distal gastrointestinal tract. Patients presenting with aganglionosis in association with hypopigmentation are classified as Waardenburg syndrome type 4 (Waardenburg-Shah, WS4). Variability in the disease phenotype of WS4 patients with equivalent mutations suggests the influence of genetic modifier loci in this disorder. Sox10(Dom)/+ mice exhibit variability of aganglionosis and hypopigmentation influenced by genetic background similar to that observed in WS4 patients. We have constructed Sox10(Dom)/+ congenic lines to segregate loci that modify the neural crest defects in these mice. Consistent with previous studies, increased lethality of Sox10(Dom)/+ animals resulted from a C57BL/6J locus(i). However, we also observed an increase in hypopigmentation in conjunction with a C3HeB/FeJLe-a/a locus(i). Linkage analysis localized a hypopigmentation modifier of the Dom phenotype to mouse chromosome 10 in close proximity to a previously reported modifier of hypopigmentation for the endothelin receptor B mouse model of WS4. To evaluate further the role of SOX10 in development and disease, we have performed comparative genomic analyses. An essential role for this gene in neural crest development is supported by zoo blot hybridizations that reveal extensive conservation throughout vertebrate evolution and by similar Northern blot expression profiles between mouse and man. Comparative sequence analysis of the mouse and human SOX10 gene have defined the exon-intron boundaries of SOX10 and facilitated mutation analysis leading to the identification of two new SOX10 mutations in individuals with WS4. Structural analysis of the HMG DNA-binding domain was performed to evaluate the effect of human mutations in this region.

To facilitate the identification of the gene responsible for Clouston hidrotic ectodermal dysplasia (HED), we used a chromosome 13-specific radiation hybrid panel to map 54 loci in the HED candidate region. The marker retention data were analyzed using RHMAP version 3. The 54 markers have an average retention frequency of 31.6% with decreasing retention as a function of distance from the centromere. Two-point analysis identified three linkage groups with a threshold lod score of 4.00; one linkage group consisted of 49 loci including the centromeric marker D13Z1 and the telomeric flanking marker for the HED candidate region D13S143. Assuming a centromeric retention model, multipoint maximum likelihood analysis of these 49 loci except D13Z1 provided a 1000:1 framework map ordering 29 loci with 21 unique map positions and approximately 2000 times more likely than the next order. Loci that could not be ordered with this level of support were positioned within a range of adjacent intervals. This map spans 347 cR9000, has an average resolution of 17.3 cR9000, and includes 3 genes (TUBA2, GJbeta2, and FGF-9), 18 ESTs, 19 polymorphic loci, and 8 single-copy DNA segments. Comparison of our RH map to a YAC contig showed an inconsistency in order involving a reversed interval of 6 loci. Fiber-FISH and FISH on interphase nuclei analyses with PACs isolated from this region supported our order. We also describe the isolation of 8 new chromosome 13q polymorphic (CA)n markers that have an average PIC value of 0.67. These data and mapping reagents will facilitate the isolation of disease genes from this region.