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We have constructed a physical map covering over 4 Mb of human chromosome 8q24.1 and used this map to refine the locations of the genes responsible for Langer-Giedion syndrome. The map is composed of overlapping YAC clones that were identified and ordered in relation to sequence tagged sites mapped to the Langer-Giedion chromosomal region on somatic cell hybrids. The minimal region of overlap of Langer-Giedion syndrome deletions, previously identified by analysis of 15 patients, was placed on the map by analysis of 2 patients whose deletions define the endpoints. The chromosome 8 breakpoint of a balanced t(8;9)(q24.11;q33.3) translocation from a patient with trichorhinophalangeal syndrome (TRPS I) was found to be located just within the proximal end of the minimal deletion region. A deletion of 8q24.11-q24.3 in a patient with multiple exostoses was found to overlap the distal end of the LGS deletion region, indicating that the EXT1 gene is distal to the TRPS1 gene and supporting the hypothesis that Langer-Giedion syndrome is due to loss of functional copies of both the TRPS1 and the EXT1 genes.

Microcell hybrids containing various regions of human chromosome 8 were formed by microcell-mediated transfer of neo-tagged chromosome 8 into the cells derived from severe combined immunodeficiency (SCID) mouse. Thus, 110 cosmid markers were isolated from SV40-transformed SCID fibroblast cell line (SCVA) containing a p12-q11.1 region of human chromosome 8 and were assigned to eight regions in 8p12-q11.1, using a microcell-hybrid panel. For positional cloning of a human gene that restores the DNA-repair defect in a mouse with SCID on 8p11.1-q11.1 (SCID region), we constructed a yeast artificial chromosome (YAC) contig of about 4.5 Mb. Overlapping YACs were further aligned by restriction mapping, using rare-cutting restriction endonucleases. The cosmids and YAC contig should facilitate isolation of the SCID gene and other genes, such as the Werner syndrome-responsible gene in or near this region.

We have developed a panel of radiation hybrids containing fragments of chromosome 8 as the only human material. The human chromosome content of each cell line was determined relative to an ordered map of sequence tagged sites (STSs) specific to chromosome 8. Between one and four fragments of chromosome 8 were identified in each cell line, with an average of 25% of the STSs retained in each line. Subclones of one radiation hybrid were examined to determine whether all cells within a line are homogeneous with respect to chromosome 8 sequence content. There was considerable variability between subclones, with retention rates for individual STSs ranging from 5 to 100% in different clones. Furthermore, a gradient of retention of sequences along the length of one large chromosome fragment was found, suggesting that sequence loss involved deletions from one end of the fragment at early stages in the establishment of the cell line. We have also made use of the radiation hybrids to develop novel sequence tagged sites for the pericentromeric region of chromosome 8.

We have characterized alphoid repeat clones derived from a chromosome 8 library. These clones are specific for human chromosome 8, as demonstrated by use of a somatic cell hybrid mapping panel and by in situ hybridization. Hybridization of the clones to Hin dIII digests of human genomic DNA reveals a complex pattern of fragments ranging in size from 1.3 to >20 kb. One clone, which corresponds in size to the most prevalent genomic Hin dIII fragment, appears to represent a major higher order repeat in the chromosome 8 centromere. The DNA sequence of this clone reveals a dimeric organization of alphoid monomers. Restriction analysis of two other clones indicates that they are derivatives of this same repeat unit. The chromosome 8 alphoid clones hybridize to Eco RI fragments of genomic DNA ranging up to 1000 kb in length and reveal a high degree of polymorphism between chromosomes. Distribution of higher order repeat units across the centromere was examined by two-dimensional gel electrophoresis. Repeat units of the same size class tended to cluster together in restricted regions of centromeric DNA.

Hereditary predisposition to multiple exostoses is a genetically heterogeneous disease. Recently, we have reported the identification of the EXT1 gene on human chromosome 8. We have now isolated a cDNA clone from a human adult lung cDNA library and have determined the genomic organization and promoter structure of the EXT1 gene. The gene is composed of 11 exons, ranging from 90 to 1735 bp, and spans approximately 350 kb of genomic DNA. Sequence analysis of the promoter region revealed the presence of a CpG island containing GC and CAAT boxes, but no TATA box. Such a promoter is characteristic for housekeeping genes. This finding is in good agreement with the ubiquitous expression of the EXT1 gene.

We have developed an integrated physical map for human chromosome 8q23-q24.1 based on STS content analysis of somatic cell hybrids and YAC contigs. Among the 63 STSs used are markers from multiple genetic maps and several previously unmapped polymorphisms, thus integrating genetic markers from different sources into comprehensive, ordered sets based on their positions in the physical map. These maps will facilitate the isolation of genes causing human inherited diseases that map to these chromosomal regions.

The Werner syndrome locus (WRN) is located at 8p11-p12. To facilitate eventual cloning of the WRN gene, a 10,000-rad radiation-reduced hybrid (RH) cell panel was generated to map genetic markers, sequence-tagged sites (STSs), and genes in this region. A hamster cell line carrying an intact human chromosome 8 was fused with another hamster cell line. Two sets of hybrid cell panels from 2 separate fusions were generated; each panel consisted of 50 independent clones; 33 and 34 cell lines from the 2 fusions retained human chromosome material as determined by inter-Alu PCR. The combined panel was genotyped for 52 markers spanning the entire chromosome, including 10 genes, 29 anonymous polymorphic loci, and 13 STSs. Seventeen of these markers have not been previously described. Markers near the centromere were retained at a higher frequency than more distal markers. Fluorescence in situ hybridization was also used to localize and order a subset of the markers. An RH map of the WRN region was constructed using a maximum likelihood method, giving the following most likely order: D8S131 - D8S339(GSR) - D8S124 - D8S278 - D8S259 - (D8S71) - D8S283 - D8S87 - D8S105 - D8S135(FGFR1) - D8S135PB - D8S255 - ANK1. A geneticmore » map of 15 short tandem repeat polymorphic loci in the WRN region was also constructed. The marker orders from the genetic and RH maps were consistent. In addition, an integrated map of 24 loci in the WRN region was generated using information from both genetic and RH mapping methods. A 1000:1 framework map for 6 loci (LPL-D8S136-D8S137-D8S87-FGFR1-ANK1) was determined by genetic mapping, and the resulting locus order was fixed during analysis of the RH genotype data. The resulting integrated map contained more markers than could confidently be ordered by either genetic or RH mapping alone. 64 refs., 5 figs., 6 tab.« less

In an attempt to identify C/EBP-like transcription factors expressed in the prostate, a cDNA homologous to the mouse C/EBP delta (CRP3) and the rat CELF gene was isolated. A genomic clone containing the entire C/EBP delta gene was isolated using a cDNA fragment as a probe. The gene was characterized by restriction mapping and sequence analysis. By fluorescence in situ hybridization, using the biotinylated genomic clone as a probe, the C/EBP delta gene was assigned to the pericentromeric region of human chromosome 8, most probably to 8q11. This chromosomal localization was confirmed by analysis of a panel of human x hamster somatic cell hybrid DNA samples with a C/EBP delta-specific STS. As a result, the C/EBP delta gene could be positioned between the PLAT and the MOS loci.

A linkage map consisting of 21 dinucleotide repeat polymorphisms, 1 tetranucleotide repeat polymorphism, and 3 RFLPs was constructed for human chromosome 8. The map spanned most of the chromosome length from near pter to q23-q24 on the distal portion of the long arm. The total 186 cM length of the female map was over two times the 84 cM length of the male map. Cytogenetic mapping of the polymorphisms using a panel of hybrids containing rearranged chromosomes was completely consistent with the linkage map. Special effort was made to remove as many genotyping errors, including parental phase errors, as possible. Removal of errors, in agreement with recent theoretical predictions, led to reduction of the total length of the sex-equal map by 10% from 145 to 130 cM.

Linkage mapping in a large, seven-generation family with type 2 autosomal dominant retinitis pigmentosa (ADRP) demonstrates linkage between the disease locus (RP1) and DNA markers on the short arm of human chromosome 8. Five markers were most informative for mapping ADRP in this family using two-point linkage analysis. The markers, their maximum lod scores, and recombination distances were ANK1 (ankyrin)--2.0 at 16%; D8S5 (TL11)--5.3 at 17%; D8S87 [a(CA)n repeat]--7.2 at 14%; LPL (lipoprotein lipase)--1.5 at 26%; and PLAT (plasminigen activator, tissue)--10.6 at 7%. Multipoint linkage analysis, using a simplified pedigree structure for the family (which contains 192 individuals and two inbreeding loops), gave a maximum lod score of 12.2 for RP1 at a distance 8.1 cM proximal to PLAT in the pericentric region of the chromosome. Based on linkage data from the CEPH (Paris) reference families and physical mapping information from a somatic cell hybrid panel of chromosome 8 fragments, the most likely order for four of these five loci and the diseases locus is 8pter-LPL-D8S5-D8S87-PLAT-RP1. (The precise location of ANK1 relative to PLAT in this map is not established). The most likely location for RP1 is in the pericentric region of the chromosome. Recently, several families with ADRP with tight linkage to the rhodopsin locus at 3q21-q24 were reported and a number of specific rhodopsin mutations in families with ADRP have since been reported. In other ADRP families, including the one in this study, linkage to rhodopsin has been excluded. Thus mutations at two different loci, at least, have been shown to cause ADRP. There is no remarkable clinical disparity in the expression of disease caused by these different loci.

We have obtained lymphoblastoid cell lines from three patients with Langer-Giedion syndrome who have over-lapping deletions in 8q24.1. To isolate the deletion chromosomes from their normal homologs, patient cell lines were fused with hamster cells and hybrid cells were selected for retention of human chromosome 8. These hybrid cell lines were screened for the presence of chromosome 8 by fluorescence in situ hybridization and by Southern blot hybridization. We have hybridized 31 recombinant DNA clones derived from the 8q22-qter region to Southern blots of the hybrid cell lines; 8 were found to lie within the deletion of at least one patient. One clone identified sequences that were missing from one copy of chromosome 8 in all three patients. These clones help to further define the deletions in these patients and will serve as starting points for detailed characterization of the region.

We have characterized a panel of somatic cell hybrids that carry fragments of human chromosome 8 and used this panel for the regional localization of anonymous clones derived from a chromosome 8 library. The hybrid panel includes 11 cell lines, which were characterized by Southern blot hybridization with chromosome 8-specific probes of known map location and by fluorescent in situ hybridization with a probe derived from a chromosome 8 library. The chromosome fragments in the hybrid cell lines divide the chromosome into 10 intervals. Using this mapping panel, we have mapped 56 newly derived anonymous clones to regions of chromosome 8. We have also obtained physical map locations for 7 loci from the genetic map of chromosome 8, thus aligning the genetic and physical maps of the chromosome.