Faculty Bibliography

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.

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)

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.

Given the DNA fingerprints of two individuals with some bands being shared by both individuals, we define a new measure of the degree of similarity between the DNA profiles of two individuals. We use this measure to calculate the expected DNA similarity of two unrelated individuals of a randomly mating population; this similarity is due to chance only. Then, the expected similarity between two related individuals is obtained; this similarity is due to chance and relatedness. From these results, the degree of similarity due to relatedness alone may be calculated.

With the advent of the Human Genome Project, the ability to rapidly construct comprehensive and accurate linkage maps based on genetic marker data from family studies is an absolute necessity. In addition to their usefulness in localizing genes for both simple and complex disorders, linkage maps are invaluable tools for genetic counseling using linked marker genes. Several computer program packages are publicly available which aid in the construction of linkage maps by computing multipoint likelihoods for specified locus orders. However, these programs work in a step-by-step fashion, requiring intensive user-intervention and analysis at each step. Such a repetitive process is amenable to computerized automation. We have developed and tested an expert system computer program, MultiMap, for automated genetic linkage mapping. This program greatly reduces the amount of user-computer interaction, increasing the accuracy and speed with which a map can be constructed. In addition, because the total mapping time is greatly reduced through automation, it is now feasible to explore and compare various mapping heuristics and mapping criteria in order to develop the most appropriate approach, or set of approaches, for genetic linkage mapping. MultiMap need not be restricted to the construction of genetic maps, but could be adapted to aid in the automated construction of physical maps as well.

We report the cloning, sequencing, and mapping of three short sequence repeat polymorphisms due to tetranucleotide (TAAA)n repeats from human chromosome 21. These DNA markers (D21S221, D21S225, D21S226) have been cloned from the chromosome 21-specific plasmid library of J. C. Fuscoe, C. C. Collins, D. Pinkel, and J. W. Gray (1989, Genomics 5: 100-109) and were shown to be polymorphic by polymerase chain reaction amplification and polyacrylamide gel electrophoresis. Genotypes were determined in informative CEPH pedigrees and used in linkage analysis relative to other mapped markers on human chromosome 21. One of these markers, D21S221, is closely linked to the amyloid precursor protein gene (APP), which has been implicated in the etiology of familial Alzheimer disease in some families.