Faculty Bibliography

The laboratory rat, Rattus novegicus, is a major model system for physiological and pathophysiological studies, and since 1966 more than 422,000 publications describe biological studies on the rat (NCBI/Medline). The rat is becoming an increasingly important genetic model for the study of specific diseases, as well as retaining its role as a major preclinical model system for pharmaceutical development. The initial genetic linkage map of the rat contained 432 genetic markers (Jacob et al. 1995) out of 1171 developed due to the relatively low polymorphism rate of the mapping cross used (SHR x BN) when compared to the interspecific crosses in the mouse. While the rat genome project continues to localize additional markers on the linkage map, and as of 11/97 more than 3,200 loci have been mapped. Current map construction is using two different crosses (SHRSP x BN and FHH x ACI) rather than the initial mapping cross. Consequently there is a need to provide integration among the different maps. We set out to develop an integrated map, as well as increase the number of markers on the rat genetic map. The crosses available for this analysis included the original mapping cross SHR x BN reciprocal F2 intercross (448 markers), a GH x BN intercross (205 markers), a SS/Mcw x BN intercross (235 markers), and a FHH/Eur x ACI/Hsd intercross (276 markers), which is also one of the new mapping crosses. Forty-six animals from each cross were genotyped with markers polymorphic for that cross. The maps appear to cover the vast majority of the rat genome. The availability of these additional markers should facilitate more complete whole genome scans in a greater number of strains and provide additional markers in specific genomic regions of interest.

Congenital aganglionic megacolon, commonly known as Hirschsprung disease (HSCR), is the most frequent cause of congenital bowel obstruction. Germline mutations in the RET receptor tyrosine kinase have been shown to cause HSCR. Knockout mice for RET and for its ligand, glial cell line-derived neurotrophic factor (GDNF), exhibit both complete intestinal aganglionosis and renal defects. Recently, GDNF and GFRA1 (GDNF family receptor, also known as GDNFR-alpha), its GPI-linked coreceptor, were demonstrated to be components of a functional ligand for RET. Moreover, GDNF has been implicated in rare cases of HSCR. We have mapped GFRA1 to human chromosome 10q25, isolated human and mouse genomic clones, determined the gene's intron-exon boundaries, isolated a highly polymorphic microsatellite marker adjacent to exon 7, and scanned for GFRA1 mutations in a large panel of HSCR patients. No evidence of linkage was detected in HSCR kindreds, and no sequence variants were found to be in significant excess in patients. These data suggest that GFRA1'S role in enteric neurogenesis in humans remains to be elucidated and that RET signaling in the gut may take place via alternate pathways, such as the recently described GDNF-related molecule neurturin and its GFRA1-like coreceptor, GFRA2.

Genetic studies of complex hereditary disorders require for their mapping the determination of genotypes at several hundred polymorphic loci in several hundred families. Because only a minority of markers are expected to show linkage and association in family data, a simple screen of genetic markers to identify those showing linkage in pooled DNA samples can greatly facilitate gene identification. All studies involving pooled DNA samples require the comparison of allele frequencies in appropriate family samples and subsamples. We have tested the accuracy of allele frequency estimates, in various DNA samples, by pooling DNA from multiple individuals prior to PCR amplification. We have used the ABI 377 automated DNA sequencer and GENESCAN software for quantifying total amplification using a 5' fluorescently labeled forward PCR primer and relative peak heights to estimate allele frequencies in pooled DNA samples. In these studies, we have genotyped 11 microsatellite markers in two separate DNA pools, and an additional four markers in a third DNA pool, and compared the estimated allele frequencies with those determined by direct genotyping. In addition, we have evaluated whether pooled DNA samples can be used to accurately assess allele frequencies on transmitted and untransmitted chromosomes, in a collection of families for fine-structure gene mapping using allelic association. Our studies show that accurate, quantitative data on allele frequencies, suitable for identifying markers for complex disorders, can be identified from pooled DNA samples. This approach, being independent of the number of samples comprising a pool, promises to drastically reduce the labor and cost of genotyping in the initial identification of disease loci. Additional applications of DNA pooling are discussed. These developments suggest that new statistical methods for analyzing pooled DNA data are required.

Non-syndromic recessive deafness (NSRD) is the most common form of prelingual hereditary hearing loss. To date, 10 autosomal NSRD loci (DFNBs) have been identified by genetic mapping; at least three times as many additional loci are expected to be identified. We have performed linkage analyses in two inter-related inbred kindreds, comprised of >50 affecteds, from a single Israeli-Arab village segregating NSRD. Genetic mapping by two-point and multi-point linkage analysis in 10 candidate regions identified the segregating gene to be on human chromosome 13q11 (DFNB1). Haplotype analysis, using eight microsatellite markers spanning 15 cM in 13q11, suggested the segregation of two different mutations in this kindred: affected individuals were homozygotes for either haplotype or compound heterozygotes. The gene for the connexin 26 gap junction protein, recently shown to be mutant in both dominant and recessive deafness, maps to this locus. We identified two distinct mutations, W77R and Gdel35, both of which likely inactivate connexin 26. The Gdel35 change likely occurs at a mutational hotspot within the connexin 26 gene. The recombination of marker alleles at the polymorphisms studied in 13q11, at known map distances from the mutations, allowed us to estimate the age of the mutations to be 3-5 generations (75-125 years). This study independently confirms the identity of connexin 26 as an NSRD gene. Importantly, we demonstrate that in small populations with high rates of consanguinity, as compared with large outbred populations, recessive mutations may have very recent origin and show allelic diversity.

To contribute to the development of the transcript map of human chromosome 21 and to the understanding of the pathogenesis of Down syndrome, we have used exon trapping to identify portions of genes from pools of HC21-specific cosmids. More than 550 potential exons have been isolated to date. One such trapped exon, hmc37a09 (GenBank Accession No. X88106), was identical to a region of a human EST, L12425 (GenBank Accession No. D31072). Its predicted amino acid sequence was homologous to the homeodomain region of homeobox-containing genes. Using the trapped sequence and the EST as probes to screen human fetal brain and kidney cDNA libraries, we have cloned the corresponding full-length cDNA. This novel gene encodes a homeodomain-containing polypeptide of 436 amino acids. The most closely related sequence is that of the mouse Meis1, a PBX-like homeobox gene. The homeodomain of the novel gene is closely related to those of the mammalian PBX family and the plant Knotted1 family (involved in plant development). This gene is named PKNOX1 by the Human Nomenclature Committee. By PCR amplification, hybridization, and genetic linkage analysis using a (GT)n polymorphism in the 3'UTR, we have precisely localized PKNOX1 to chromosome 21q22.3 between markers D21S212 and D21S25 on YAC350F7. PKNOX1 is expressed in many human tissues tested by Northern blot analysis. The involvement of the PKNOX1 gene in Down syndrome and/or monogenic disorders associated with dysfunction of this gene is presently unknown. Targeted disruption of the PKNOX1 homolog in mice will enhance our understanding of its biological function in normal mammalian development.