Physical mapping of the rippling muscle disease locus

Rippling muscle disease (RMD) is an autosomal dominant disorder characterized by electrically silent, percussion-induced muscular contractions. We previously reported the localization of a gene for RMD to 1q41-q42 by genome-wide linkage analysis in a large family from Oregon. This RMD gene was initially found to be contained within a 12-cM interval with a maximum multipoint lod score of 3.56. A YAC/BAC contig was assembled by STS content mapping and database searches spanning the nonrecombinant interval containing the RMD gene (RMD1). The physical map, in conjunction with recent mapping information from various other sources, clarified the order of genetic markers in this region and necessitated redefinition of the RMD genetic interval by linkage analysis with the newly ordered markers. Polymorphisms that mapped to the YACs in this contig were genotyped in this family and used to provide statistical support for narrowing of the critical genetic interval to 3 cM, corresponding to a maximum possible physical distance of 4.0 Mb. In addition, recombination breakpoint mapping supported the evidence that RMD1 must reside within this interval between markers D1S446 and D1S2680. ESTs (82) were mapped to the YACs spanning the region known to contain the RMD1 gene, and of these, 9 become strong positional candidates. The physical and refined genetic maps of this RMD locus set the stage for isolation of the responsible gene and elucidation of a novel patho-mechanism of calcium homeostasis in skeletal muscle.     

Refining the DFNB17 interval in consanguineous Indian families

We previously mapped the DFNB17 locus to a 3-4 cM interval on human chromosome 7q31 in a large consanguineous Indian family with congenital profound sensorineural hearing loss. To further refine this interval, 30 new highly polymorphic markers and 8 SNPs were analyzed against the pedigree. Re-analysis in the original DFNB 17 family and additional data from a second unrelated consanguineous family with congenital deafness found to map to the interval, limited the area of shared homozygosity-by-descent (HBD) to approximately 4 megabase (Mb) between markers D7S2453 and D7S525. Nineteen known genes and over 20 other cDNAs have been identified in the refined DFNB 17 interval, including the SLC26A4 gene. We have analyzed 4 other cochlear-expressed genes that map to the DFNB17 interval as candidate genes. Analysis of coding and splice site regions of these cochlear expressed genes did not reveal any disease causing mutations. Further study of other candidate genes is currently underway.     

Genomic structures of SCN2A and SCN3A - candidate genes for deafness at the DFNA16 locus

DFNA16 is a form of autosomal dominant non-syndromic hearing loss (ADNSHL) characterized by fluctuating progressive hearing impairment. Earlier, we mapped the deafness-causing gene to chromosome 2q23-24.3. In this paper, we describe fine mapping results using additional markers tightly linked to the DFNA16 candidate region. Critical recombinants at markers D2S354 and D2S124 define a 3.5-cM interval that contains the DFNA16 gene. Positional candidate genes include two members of the voltage-gated sodium channel family, the type 2 alpha subunit (SCN2A) and the type 3 alpha subunit (SCN3A). After showing that SCN2A is expressed in human fetal cochlea, we determined its genomic structure to facilitate mutation screening in our DFNA16 kindred. We also determined the genomic structure of SCN3A. These two genes are oriented head-to-head, with their 5' ends separated by approximately 40 kb; their homology is 82% at the nucleotide level, and 85% for identities and 90% for positives at the amino acid level. They share similar genomic structures and have alternative splice isoforms that are developmentally regulated and highly conserved between species. Although no DFNA16-causing mutations were found in either gene, haplotype analysis with polymorphic markers in SCN2A introns further narrowed the candidate gene interval to the region flanked by D2S354 and STS SHGC-82894.     

Assignment of 48 ESTs to Chromosome 13 Band q14.3 and Expression Pattern for ESTs Located in the Core Region Deleted in B-CLL

An expression map containing 48 ESTs was constructed to identify a tumor-suppressor gene involved in B-cell chronic lymphocytic leukemia (B-CLL), which was previously assigned to chromosome band 13q14.3 close to genetic markers D13S25 and D13S319. Thirty-nine of these 48 ESTs, together with 11 additional ones listed in databases, were initially assigned to chromosome 13q14 between markers D13S168 and D13S176. Nine others have recently been located in the D13S319 region. Our results indicate that 48 of the 59 ESTs analyzed belong to a YAC contig of chromosome 13 band q14, and 22 are contained on YAC 933e9, which encompasses the B-CLL critical region. Ten of these 22 ESTs were accurately assigned on a PAC, BAC, and cosmid contig encompassing the smallest minimal deletion area described so far in B-CLL, and 20 were tested for their expression on 27 normal or tumor tissues. One EST appears to be a likely candidate for the tumor-suppressor gene involved in B-CLL.     

A sequence-ready 840-kb PAC contig spanning the candidate tumor suppressor locus DBC1 on human chromosome 9q32-q33.

A putative tumor suppressor locus involved in bladder cancer has been mapped to human chromosome 9q32-q33 and designated DBC1. Our previous microsatellite-based deletion mapping study indicated that DBC1 was localized between D9S1848 and AFMA239XA9. We have constructed an 840-kb sequence-ready contig composed of bacteriophage P1-derived artificial chromosomes (PACs), which encompasses DBC1. Clones were initially identified by screening a PAC library with markers localized to the region by physical mapping, and subsequently PAC end probes were used to complete the contig. This contig contains a minimum tiling path of six PAC clones between D9S1848 and AFMA239XA9. Three expressed sequence tags (ESTs) were mapped to the DBC1 region by screening 24 ESTs mapped to the surrounding area by radiation hybrids. One represented the gene for DBCCR1, a known candidate for DBC1, and the other two were novel. This contig and preliminary expression map form the basis for the identification of the bladder cancer tumor suppressor gene in this region.