Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Cosmid Contig Construction and Gene Identification in Human Xq28

J.E. Parrish[1], E.E. Eichler[1], Y. Gu[1], B.A. Oostra[2], A.J.M.H. Verkerk[2], J. Reynolds[3], C.S. Richards[1], A.S. Spikes[1], L.G. Shaffer[1] and D.L. Nelson[1]
[1]Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas; [2]Department of Clinical Genetics, Erasmus University, Rotterdam, The Netherlands; [3]Shodair Hospital, Helena, Montana.

Efforts to identify and characterize genes in human Xq28 have led to the construction of cosmid- based physical maps of several regions using clones derived from the Lawrence Livermore National Laboratories flow sorted X chromosome-specific cosmid library. Contigs have been seeded using cDNA clones; walking with cosmid ends has provided physical linkage between several genes, particularly in the gene-dense region between the opsin genes and G6PD. This region is fully covered by a contig of about 300 kb; a novel muscle gene has been identified and is currently being tested as a candidate for Emery-Dreifuss Muscular Dystrophy. Two other contigs of 150-200 kb each have been constructed in the LlCAM-HCF region and in the ALD region. An EST, DXSl012E, has also been used to seed a contig between the opsin genes and HCF. We are currently attempting to close the gaps between these four contigs. Further proximal, scattered cosmids have been identified using polymorphic markers and repeat sequences. Over 30 cosmids have been isolated for most markers known in the interval between FRAXA and IDS in efforts to support the long-range sequencing efforts of Dr. R. A. Gibbs. Efforts to identify potential transcripts at the FRAXE locus are ongoing and aided by the long-range sequence effort, however cosmids spanning FRAXE and FRAXA CGG repeats have not been found in this library.

A fourth large contig has been established in the proximal portion of Xq28. The original cosmid was identified by hybridization to a novel cDNA with homology to FMRl, the gene involved with Fragile X syndrome. We have characterized a GCC repeat in this cosmid which represents FRAXF, the third folate-sensitive fragile site to be defined in Xq27.3-q28. A 5kb Eco RI fragment of the cosmid detects a fragment increased by 900 base pairs in a mentally retarded male exhibiting 28% fragile site expression. By DNA analysis, this patient demonstrates normal alleles at both FRAXA and FRAXE. The mother of the proband carries an allele expanded to a lesser extent, in addition to a normal allele. The proband's DNA at this locus is methylated at three sites (SacII, EagI, and BssHII) within the CpG island. Additional normal and retarded family members were tested both for fragile site expression and for expansion and methylation at this locus; the expansion and methylation are found only in individuals expressing the fragile site, suggesting that this clone does represent the fragile site. Inheritance of the fragile site does not show a direct correlation with the mental impairment in this pedigree.

The site of variation was localized to within 300 base pairs. By sequence analysis, the plasmid subclone contains (CGG)8. PCR primers were designed to amplify across the repeat. Expanded alleles amplify poorly or not at all, which is consistent with difficulties observed with FRAXA. Alleles in the normal population vary from 6 to 29 repeats. Fluorescence in situ hybridization studies have been carried out using cosmids which overlap in the region of the fragile site; the results demonstrate unequivocally that this clone spans the fragile site in the patient mentioned above. Efforts to define the gene content of the region are under way, in order to determine the potential involvement of this repeat in regulation of genes in its vicinity. A cDNA has been isolated which lies just distal to the fragile site. Sequence analysis is currently under way; preliminary results suggest that this clone represents a member of the MAGE gene family of tumor antigens. Screening of the cosmid library with this cDNA produced a total of 70 positive cosmids, which likely represent the entire cluster (three known MAGE genes, plus the novel gene cloned in this effort). Current efforts involve the characterization of this genomic region, including the structure of the MAGE gene family and analysis of expression of the MAGE genes in individuals with expansions at FRAXF.