Faculty Bibliography

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.

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.

Schizophrenia is a common disorder characterized by psychotic symptoms; diagnostic criteria have been established. Family, twin and adoption studies suggest that both genetic and environmental factors influence susceptibility (heritability is approximately 71%; ref. 2), however, little is known about the aetiology of schizophrenia. Clinical and family studies suggest aetiological heterogeneity. Previously, we reported that regions on chromosomes 22, 3 and 8 may be associated with susceptibility to schizophrenia, and collaborations provided some support for regions on chromosomes 8 and 22 (refs 9-13). We present here a genome-wide scan for schizophrenia susceptibility loci (SSL) using 452 microsatellite markers on 54 multiplex pedigrees. Non-parametric linkage (NPL) analysis provided significant evidence for an SSL on chromosome 13q32 (NPL score=4.18; P=0.00002), and suggestive evidence for another SSL on chromosome 8p21-22 (NPL=3.64; P=0.0001). Parametric linkage analysis provided additional support for these SSL. Linkage evidence at chromosome 8 is weaker than that at chromosome 13, so it is more probable that chromosome 8 may be a false positive linkage. Additional putative SSL were noted on chromosomes 14q13 (NPL=2.57; P=0.005), 7q11 (NPL=2.50, P=0.007) and 22q11 (NPL=2.42, P=0.009). Verification of suggestive SSL on chromosomes 13q and 8p was attempted in a follow-up sample of 51 multiplex pedigrees. This analysis confirmed the SSL in 13q14-q33 (NPL=2.36, P=0.007) and supported the SSL in 8p22-p21 (NPL=1.95, P=0.023).

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.

The Human Genome Project has successfully completed all the major goals in its current 5-year plan, covering the period 1993-98. A new plan, for 1998-2003, is presented, in which human DNA sequencing will be the major emphasis. An ambitious schedule has been set to complete the full sequence by the end of 2003, 2 years ahead of previous projections. In the course of completing the sequence, a "working draft" of the human sequence will be produced by the end of 2001. The plan also includes goals for sequencing technology development; for studying human genome sequence variation; for developing technology for functional genomics; for completing the sequence of Caenorhabditis elegans and Drosophila melanogaster and starting the mouse genome; for studying the ethical, legal, and social implications of genome research; for bioinformatics and computational studies; and for training of genome scientists.

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.