Apr 14, 1987 - cpm/400 cm2 of membrane was used for each hy- .... Sixty-six of 72 unrelated per- ..... Anvret, I. Kanazawa, J. Gusella, and M. Conneally. 1986.
Am. J. Hum. Genet. 42:125-131, 1988
A Polymorphic DNA Marker That Represents a Conserved Expressed Sequence in the Region of the Huntington Disease Gene Michael R. Hayden,* Jeffrey Hewitt,* John J. Wasmuth,t Jan J. Kastelein,* Sylvie Langlois,* Michael Conneally,4 Jonathan Haines,4 Barbara Smith,t Chantal Hilbert* and Denis Allard* *Department of Medical Genetics, University of British Columbia, Vancouver; tDepartment of Biological Chemistry, University of California, Irvine; and tDepartment of Medical Genetics, University of Indiana
Summary A polymorphic marker (D4S62) that is genetically closely linked to D4S10 and is in the region of the gene for Huntington disease is described. A four-allele polymorphism is detected when HindI-digested DNA is hybridized with D4S62. D4S62 maps, by Southern blot analysis using somatic-cell hybrids, to 4pl6.1 closer to the centromere than does D4S10. The use of the polymorphisms detected by D4S62 increases the informativeness of markers close to the gene for Huntington disease and will be useful for preclinical diagnosis. D4S62 detects transcripts of -6,000 nucleotides in rat, mouse, and monkey liver and brain. This represents the first demonstration of conserved expressed sequences close to the gene for Huntington disease.
Introduction
Huntington disease (HD) is a progressive neurodegenerative disorder characterized by chorea, intellectual dysfunction, and personality change (Hayden 1981). The mutant gene is inherited as an autosomal dominant trait with complete penetrance. Little is known concerning the primary genetic defect in this disorder. However, the report of a closely linked polymorphic marker (D4S10) to the gene for HD (Gusella et al. 1983) has allowed preliminary localization of the mutant gene to 4p16 (Magenis et al. 1986; Wang et al. 1986). No evidence for nonallelic heterogeneity has been demonstrated after investigation of more than 50 families of different ancestries (Haines et al. 1986). The finding of a closely linked polymorphic DNA marker for HD has led to the establishment of programs aimed at preclinical detection of HD in Received April 14, 1987; revision received June 23, 1987. Address for correspondence and reprints: Michael R. Hayden, M.D., Ph.D., The University of British Columbia, Department of Medical Genetics, F168-2211 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 2B5. X 1988 by The American Society of Human Genetics. All rights reserved. 0002-9297/88/4201-0017$02.00
Canada, the United States, and other countries. Major drawbacks for clinical use include an estimate of recombination of 4% between D4S10 and the gene for HD and an -10% failure to detect heterozygosity for the polymorphic sites within and around D4S10 (Hewitt et al. 1986). Thus there is a need for new DNA markers that would either be closer to or flanking the gene for HD and that would increase the informativeness in this region. We report here a polymorphic DNA marker that represents an expressed sequence that is both linked to D4S10 and in the region of the gene for HD. Material and Methods A DNA probe was initially isolated from an African green monkey genomic library (Thayer et al.
1987). Somatic-cell hybrid analyses and in situ hybridization studies had previously mapped this probe to the terminal region of chromosome 4 (Thayer et al., in press). This DNA clone has been named D4S62 by the Human Gene Mapping Center at Yale. Polymorphism Studies DNA was prepared from freshly taken whole blood of 10 unrelated persons of Caucasian descent 125
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as described elsewhere (Hayden et al. 1987a). Fivemicrogram aliquots were digested with one of 35 restriction endonucleases. The resulting DNA fragments were electrophoresed through a 1% agarose gel and transferred to nylon membranes by means of Southern blotting. The membranes were prehybridized in S x SSPE (pH 7.5), 10 x Denhardt's (0.2% BSA, 0.2% PVP360, and 0.2% Ficoll), 0.05 mg sheared denatured salmon-sperm DNA/ml, 0.3% SDS, and incubated for several hours at 65 C. Membranes were hybridized overnight at 65 C in 5 x SSPE, 5 x Denhardt's, 0.5 mg salmon-sperm DNA/ml, and 0.1% SDS, using inserts from subclones of D4S10 as probes. Approximately 20 ng of D4S62 was labeled with 32p (Amersham, Canada) to 108 cpm by means of the random oligonucleotide primer method (Feinberg and Vogelstein 1983). A minimum of 1 x 107 cpm/400 cm2 of membrane was used for each hybridization. Membranes were washed twice in 0.5 x SSPE, 0.1% SDS for 15 min at 65 C and then once in 0.1 x SSPE, 0.1% SDS for 15 min and exposed to Kodak XAR-5 film with intensifying screens at - 70 C for 1-4 days. A polymorphism was detected with HincII (Hayden et al. 1987b). To determine the relationship between the gene for HD, D4S62, and D4S10, DNA samples from persons in eight large affected families including one of Russian-Mennonite (family 1), one of Norwegian (family 2), and one of black descent (family 3) were analyzed. DNA was digested separately with HincII, HindIII, EcoRI, and BgAI, transferred to nylon membranes, and hybridized with D4S62 and subclones of D4S10 (R7 and pK082). Linkage Analysis The computer programs LIPED and LINKAGE were used to analyze all data for the individual polymorphisms. Offspring of affected parents were assigned a probability of having inherited the gene based on an age-at-onset function described elsewhere (Conneally et al. 1984). Linkage disequilibrium between specific alleles at the three loci was examined by determining whether the observed haplotype frequencies deviated significantly from the values that would be expected on the basis of random association of alleles. Northem Blot Analysis To determine whether D4S62 codes for a tran-
scribed sequence, nitrocellulose membranes containing Poly (A +) mRNA from rat, mouse, and monkey
liver and brain were hybridized with D4S62 (Sutcliffe et al. 1984). Hybrid-Cell Mapping Panels
The interspecific Chinese hamster ovary-human cell hybrids used in this study were constructed as described elsewhere (Wasmuth et al. 1986). Hybrid HHW 416 retains an intact chromosome 4 exclusively (Carlock et al. 1986) (lanes 1, figs. 1, 2). Hybrid HHW 661 retains as its only known DNA a derivative chromosome 5 in which the p15.1-pter segment of chromosome 5 has been replaced with the plS.1-pter segment of chromosome 4 (Wasmuth et al. 1986) (lanes 2, figs. 1, 2). Hybrids HHW 847 and HHW 878 retain abnormal chromosomes 4 from individuals with either an unbalanced translocation or
1 2 3 45 16.3 z 16.2 go a 16.1 15.3 15.2 15.1 14 13
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Southern blot analysis of EcoRI-digested DNA Figure I from somatic-cell hybrids retaining exclusively all (lane 1) or parts of chromosome 4 (lanes 2-4) after hybridization with D4S62.
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