Faculty Bibliography

We used single-strand conformation polymorphism (SSCP) to detect DNA polymorphisms in the 3' untranslated (3'UT) region of the gene for cystathionine beta-synthase (CBS). A polymorphism due to a T-to-C substitution at nucleotide 549 of the 3'UT region with heterozygosity of 46% has been identified. Genotypes for this polymorphism have been obtained in all of the informative CEPH families, and CBS has been placed in the linkage map of human chromosome 21.

DNA polymorphisms can be used to place loci and phenotypes on the linkage maps of human chromosomes. In an effort to localize genes on the linkage map of human chromosome 21 better, we examined their 3' untranslated (3'UT) regions for the presence of polymorphisms. We amplified the 3'UT region of 17 genes of chromosome 21 by the polymerase chain reaction and subjected the product to single-stranded conformation analysis (SSCA). We have found eight polymorphisms in the 3'UT region of genes. The total area examined was 8144 nucleotides and therefore the variability detected by this method was 1 in 1018 nucleotides. This is not different from the estimated variability of DNA sequences based on restriction analysis. Sequence analysis revealed that all polymorphisms found are due to single nucleotide substitutions. Additional polymorphisms were identified in the last intron of BCEI gene and in the 3'-flanking region of the S100B gene. We conclude that the 3'UT region of genes is a relatively rich source of polymorphisms and that SSCA is an effective method of detecting the normal sequence variation in the human genome.

A genetic linkage map of human chromosome 21q (HC21q) containing 43 markers genotyped by the polymerase chain reaction in the CEPH pedigrees is presented. The markers placed on this map are highly polymorphic with an average heterozygosity of 61%. The average interval size of the markers localized at 1000:1 odds is 2.5 cM. The map has a total length of 65.5 cM, with male and female lengths of 47.7 and 83.3 cM, respectively. The genotypes used in the construction of this map were subjected to rigorous error checking, which is reflected in the shorter map length compared to previous maps; the estimated error rate in genotyping is less than 0.04%. As noted in previous linkage maps there is increased recombination in females on proximal HC 21q and in the male in a region near the telomere. This map of HC 21 represents a highly informative and dense meiotic linkage map and will be useful in linking disease phenotypes to loci on this chromosome.

We have constructed a chromosome 13 somatic cell hybrid map using seven cell lines: PGMEA6, a hybrid containing the entire chromosome 13, and six hybrids containing various deletions of chromosome 13 (BARF7, PPF22, KBF11, KSF39, CF25, and CF27). We have mapped 80 markers that define 10 regions of chromosome 13 with respect to 10 breakpoints in the mapping panel; these regions range in size from 4 to 24 Mb, with an average size of 8 Mb. The 80 markers sublocalized on our mapping panel include 10 Alu-PCR clones, 6 of which were converted to sequence-tagged sites; 40 (CA)n repeat-containing clones, 27 of which are microsatellite PCR markers; 8 (AAAG)n repeat-containing PCR markers, 1 two-allele PCR marker, 4 genes or expressed sequences, and 17 anonymous DNA probes. This low-resolution physical map can be used as a backbone map for more refined physical mapping using radiation hybrids or yeast artificial chromosomes.

For the positional cloning of genes and other novel types of genetic experiments, in humans and other organisms, there is a crucial need for techniques with which genome-wide high-resolution ordered clone maps can be rapidly constructed. Current best methods, such as sequence-tagged site (STS) content mapping, entail a large number of experiments and, in practice, require large low-resolution yeast artificial chromosome (YAC) clones and very many STSs. In this paper, we introduce a new approach, inner product mapping (IPM), that overcomes these limitations. IPM uses radiation hybrids (RHs) to provide localizing signatures for YACs. Two independent data tables that compare YACs against RHs and RHs against STSs are obtained; these tables are combined to produce a computed map of the YACs against ordered STSs. IPM maps each YAC independently, requires relatively few RH comparisons to map a YAC, and can work with small (or large) YACs and few (or many) STSs. This paper describes IPM and presents computer simulations supporting the efficiency of IPM over that of competing methods.

Twelve polymorphic (CA)n microsatellites were isolated from a flow-sorted chromosome 13 genomic library. These, and two others that have been previously described, were genotyped in 41 families from the CEPH (Centre d'Etude Polymorphisme Humain, Paris), and a primary linkage map with considerable support for order (odds > 10,000:1) was constructed. Two RFLP-based markers, COL4A1 and D13S52, with heterozygosities above 0.67 and an RFLP-based centromeric marker at D13Z1 were included in this map which extends from 13cen to 13q34. The heterozygosity of all of the PCR-based markers is above 60%. The total map spans a genetic distance of 144 cM, extending from D13Z1 to D13S52 with a single maximum intermarker recombination distance of 35 cM. All other intermarker recombination distances are 18 cM or less. Marker order was confirmed by sublocalizing many of the microsatellite containing clones on a panel of rodent-human somatic cell hybrids with deletions and rearrangements of chromosome 13. One spontaneous new mutation for these 14 (CA)n repeat markers was identified from a total of 8006 gametes, giving an overall observed spontaneous mutation rate of 0.00012 per locus per gamete. An integrated map of chromosome 13q was constructed with the microsatellite markers described here and previously genotyped RFLP-based markers. This sex-average map spans 209 cM with an average distance between unique map locations of 4.5 cM; the maximum intermarker distance was 14 cM.

We describe a highly polymorphic (GT)n repeat with 14 alleles that is closely linked to the amyloid precursor protein (APP) gene on human chromosome 21. This marker, D21S210, will be useful for studies of linkage of disorders such as Alzheimer disease to the APP gene.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders caused by a deficiency of paternal (PWS) or maternal (AS) contributions for chromosome 15 by either deletion or uniparental disomy (UPD). To further study the molecular mechanisms involved in these disorders and to improve molecular diagnostic methods, we have isolated three dinucleotide repeat markers in the PWS/AS critical region. An Alu-CA PCR method was used to isolate CA-repeat markers directly from yeast artificial chromosome (YAC) clones identified by probes IR4-3R (D15S11), LS6-1 (D15S113), and GABAA receptor B3 (GABRB3). Three markers with 6-11 alleles and 73-83% heterozygosities were identified and analyzed by multiplex PCR. Gene-centromere mapping was performed on a panel of ovarian teratomas of known meiotic origin, and showed the most proximal marker, IR4-3R, to be 13 cM (95% confidence limits: 7-19 cM) from the centromere of chromosome 15. Molecular diagnostic studies were performed on 20 PWS and 9 AS patients. In 17 patients with deletions, the parental origin of deletion was determined. Ten PWS patients were shown to have maternal heterodisomy. Since these markers are only 13 cM from the centromere, heterodisomy indicates that maternal meiosis I nondisjunction is involved in the origin of UPD. In contrast, two paternal disomy cases of AS showed isodisomy for all markers tested along the length of chromosome 15. This suggests a paternal meiosis II nondisjunction event (without crossing over) or, more likely, monosomic conception (due to maternal nondisjunction) followed by chromosome duplication.(ABSTRACT TRUNCATED AT 250 WORDS)