Physical Mapping of the Rippling Muscle Disease LocusDietrich A. Stephan 1 and Eric P. HoffmanDepartment of Molecular Genetics and Biochemistry, Department of Human Genetics, Department of Pediatrics,and Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261Received August 24, 1998; accepted November 19, 1998R ippling muscle disease (R MD) is an autosomaldominant disorder characterized by electrically si-lent, percussion-induced muscular contractions. Wepreviously reported the localization of a gene for R MDto 1q41–q42 by genome-wide linkage analysis in alarge family from Oregon. T his R MD gene was initiallyfound to be contained within a 12-cM interval with amaximum multipoint lod score of 3.56. A YAC/BAC con-tig was assembled by ST S content mapping and data-base searches spanning the nonrecombinant intervalcontaining the R MD gene (R MD1). T he physical map,in conjunction with recent mapping information fromvarious other sources, clarified the order of geneticmarkers in this region and necessitated redefinition ofthe R MD genetic interval by linkage analysis with thenewly ordered markers. Polymorphisms that mappedto the YACs in this contig were genotyped in this fam-ily and used to provide statistical support for narrow-ing of the critical genetic interval to 3 cM, correspond-ing to a maximum possible physical distance of 4.0 Mb.In addition, recombination breakpoint mapping sup-ported the evidence that R MD1 must reside within thisinterval between markers D1S446 and D1S2680. E ST s(82) were mapped to the YACs spanning the regionknown to contain the R MD1 gene, and of these, 9 be-come strong positional candidates. T he physical andrefined genetic maps of this R MD locus set the stagefor isolation of the responsible gene and elucidation ofa novel patho-mechanism of calcium homeostasis inskeletal muscle. © 1999 Academic PressINTRODUCTIONRippling muscle disease (RMD; MIM 600332) is anautosomal dominant trait that was first described byTorbergsen (1975). This disorder is characterized bypercussion-induced muscular contractions that resem-ble myotonia or myokymia (spontaneous involuntarymuscular contractions). The contractions are distin-guished from myotonic and myokymic contractions inthat they arecompletely electrically silent as measuredby electromyography (Ricker et al., 1989). Muscle con-traction is a complex signal transduction cascade ini-tiated by electrical depolarization of the myofiberplasma membrane and culminating in cross-bridge cy-cling of the actin and myosin fibrils as a result ofincreased intracellular calcium levels (Rios andPizarro, 1991). Individual RMD muscle fibers (myofi-bers) can be seen to contract as a result of calciumrelease by the sarcoplasmic reticulum (SR) in the ab-sence of muscle plasma membrane depolarization(Burns et al., 1994). In addition, contraction of individ-ual myofibers can stimulate neighboring fibers to con-tract, causing “rippling” across the entire musclegroup. This intercellular communication has not beenpreviously described in myofibers and may be a crucialcomponent in synchronization of contraction of themany myofibers in a large muscle group. The RMDphenotype is most likely caused by a defect at somepoint in this signal transduction cascade. The elucida-tion of the etiological gene would shed light on thisunique disorder as well as the mechanics of this com-plex pathway.We have previously mapped a gene for RMD to1q41–q42 by genome-wide linkage analysis in the larg-est RMD family reported todate (Stephan et al., 1994).RMD is genetically heterogeneous (several large RMDfamilies are not linked to the chromosome 1 locus),indicating that multiple sites of genetic/biochemicaldefect cause a similar muscle phenotype (Stephan etal., 1994). Herewereport theconstruction of a physicalmap of the locus for the first RMD gene. We alsonarrow the critical genetic interval containing theRMD gene in this family from 12 to3 cM. This physicalmap and narrowed genetic interval set the stage foridentification of the gene defective in this intriguingdisorder.MATERIALS AND METHODSI. DNA extraction from patients. Whole blood was collected fromRMD families in EDTA tubes. Genomic DNA was prepared fromnucleated cells (Miller et al., 1988).II. YAC/BAC DNA isolation for PCR. Single yeast colonies werecollected from YPD plates and used to inoculate 5 ml of AHC selec-1To whom correspondence should be addressed at current address:National Human Genome Research Institute, National Institutes ofHealth, 36 Convent Drive3D05, Bethesda, MD 20892. Telephone: (301)435-5746. Fax: (301) 435-5465. E-mail: dstephan@nhgri.nih.gov.Genomics 55, 268–274 (1999)Article ID geno.1998.5689, available online at http://www.idealibrary.com on2680888-7543/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.
