Marcy E. MacDonald, ~ Hamish S. Scott, z William L. Whaley, Thomas Pohl, 3 John J. Wasmuth, Hans Lehrach, 5 C. Phillip Morris, 2 Anne-Marie Frischauf, ...
Somatic Cell and Molecular Genetics, Vol. 17, No. 4, I 991, pp. 421-425
Brief Communication
Huntington Disease-Linked Locus D 4 S l l l Exposed as the C~-L-Iduronidase Gene Marcy E. MacDonald, ~ Hamish S. Scott, z William L. Whaley, ~ Thomas Pohl, 3 John J. Wasmuth, ~ Hans Lehrach, 5 C. Phillip Morris, 2 Anne-Marie Frischauf, 5 John J. Hopwood, ~ and James F. Gusella ~ Weurogenetics Laboratory, Massachusetts"General Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts' 02114; 2Lysosomal Diseases Research Unit, Depanment of Chemical Pathology, Adelaide Children's Hospital, North Adelaide, S.A. 5006, Australia; 3EMBL, P.O. Box 1022.40, D6900, HeiIdelberg, Germany; 4Department of Biological Chemistry, Universityof California, Irvine, California 92717; and Slmperial Cancer Research Fund, Lincoln's Inn Fields, London, WC2A 3PX, U.K. Received 8 March 1991
Abstraet--~x-L-Iduronidase (IDUA) has been intensively studied due to its causative rote in mucopolysaccharidosis type I (Hurler, Scheie and Hurler/Scheie syndromes). The recent cloning of a human IDUA cDNA has resulted in a reevaluation of the chromosomal location of this gene. Previously assigned to chromosome 22, IDUA now has been localized to 4p16.3, the region of chromosome 4 associated with Huntington's disease (HD). The existence of a battery of cloned DNA, physical map information, and genetic polymorphism data for this region has allowed the rapid fine mapping of lDUA within the terminal cytogenetic band of 4p. IDUA was found to be coincident with D4Sl11, an anonymous locus displaying a highly informative multiallele DNA polymorphism. This map location, 1.1 x 106 bp from the wlomere, makes IDUd the most distal cIoned gene assigned to 4p. However, it falls within a segment of 4p16.3 that has been eliminated fi'om the HD candidate region, excluding a role for IDUA in this disorder.
INTRODUCTION Huntington's disease (HD) is a lateonset disorder caused by an autosomal dominant defect mapping in 4p16.3 (1). The disease is characterized by progressive chorea, psychiatric disturbance, and intellectual decline due to loss of striatal neurons. Symptoms typically appear in middle age, and death ensues in 10-20 years, after an inexorable decline that currently cannot be prevented or delayed by therapy. Many cloned genes, including candidates such as the two GABA receptor subunits (GABRA2, GABRB1) (2), and dihydropteridine reductase (QDPR) (3) have been mapped on
chromosome 4p, but none has been assigned within the immediate vicinity of HD, which lies in the terminal cytogenetic subband, a region expected to contain 6 x 106 bp (4). An intensive effort has been undertaken to isolate the HD gene based on its chromosomal position and has produced detailed genetic and physical maps of the 4p telomeric region (5-9). In addition, much of the candidate region has been isolated as cloned DNA (9-11). To date, it has not been possible to place the liD gene in an unequivocal position on the physical map, due to the existence of contradictory recombination events in HD kindreds (5). Two candidate regions must be considered, one spanning
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the terminal 100,000 bp of the chromosome, and one extending at least 2.2 x 10 6 bp between the markers D4S126 and D4S168 in the central portion of 4p16.3 (9). Recently, Scott et al. (12) have presented evidence that the gene encoding ~-L-iduronidase (IDUA), previously assigned to chromosome 22 (13), in fact maps within 4p16.3. IDUA is required for the degradation of the glycosaminoglycans, heparan sulfate, and dermatan sulfate. Deficiency of IDUA results in the lysozomal storage disorder mucopotysaccharidosis type I (Hurler, Scheie and Hurler/Scheie syndromes), which is inherited as an autosomal recessive (14). Scott et al. (12) assigned IDUA to chromosome 4 by hybridization of a cloned IDUA cDNA to a panel of somatic cell hybrid lines and confirmed this finding by using a monoclonal antibody for human IDUA to specifically capture the human enzyme from the hybrid cells. The gene was regionally localized to 4p16.3 by in situ hybridization of both cDNA and genomic probes to metaphase chromosomes.
MacDonald et al.
The hybrids cell lines used in the regional mapping panel for 4p have been described previously (20, 21). Eye3 Fa6 (22) and GM10888 (23) are human x hamster hybrid lines that contain an intact human chromosome 22. The former also possesses fragments of several other chromosomes but neither of these hybrids contains chromosome 4. DNA was prepared from all ceil lines as previously described (24). RESULTS AND DISCUSSION
To confirm the assignment of IDUA to chromosome 4 and to provide a finer regional localization, we employed a regional mapping panel of human x hamster somatic cell hybrids that dissects 4p16 into three regions (Fig. 1). A cloned 1.5-kb single-copy genomic fragment from the IDUA locus (plD89) was hybridized to a Southern blot containing HindIII-digested DNAs from the cell panel. As expected, the probe showed strong hybridization with the hamster parent line due to evolutionary conservation oflDUA. In addition to the 13-kb hamster fragment, a human fragment of approximately 20 kb was MATERIALS AND METHODS observed in some of the hybrid lines, includCloned DNAs and Cell Lines. Probe ing HHW416, which contains only human p157.9 contains a 450-bp PstI-Sau3A frag- chromosome 4. Notably, the human fragment subcloned into pGEM3 from the ment was not detected in two hybrids, cosmid A157.1, which overlaps the NotI- Eye3Fa6 and GM10888, containing chromolinking clone 157 (15) defining D4Slll. The some 22 as the only intact human chromoA157.1 cosmid was isolated from a library some. Rather, IDUA mapped within the most constructed in pcos2 (16) by Sau3AI-partial terminal segment of chromosome 4 defined digestion of genomic DNA from cell hybrid by the regional panel, the distal portion of HHW693, containing the regions 4pter- 4p16.3. We previously reported a detailed 4p15.1 and 5p15.1-5cen as its only human pulsed-field gel map of this distal region, material (17). pID89 contains a 1.5-kb PstI along with the position of numerous cosmid fragment derived from a genomic X phage clones and genetic markers (7, 9). The region clone isolated from a human library using the spans about 2.2 x 106 bp of DNA and IDUA cDNA (12). Agarose gel electrophore- contains one unmapped segment. We hybridized pID89 to our collection sis, DNA transfer, hybridization, and autoraof cosmids from 4p16 and observed specific diography were performed as described (18), hybridization with the cosmid A157.1 (9, 15). except that probes were labeled by the This cosmid also contains p157.9, a singleoligonucleotide priming method of Feinberg copy probe that defines the anonymous locus and Vogelstein (19).
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D 4 S l l l is IDUA
Fig. 1. Regional localization of IDUA in 4p16.3. (A) ttybridization of pID89 to hybrid cell DNAs digested with HindIII. Lanes: 1, human genomic DNA; 2, HHW416, a hybrid containing only human chromosome 4; 3, GM10888, containing only human chromosome 22; 4, Eye3 Fa6, containing chromosome 22 and fragments of 15 and 19; 5, HHW661, containing a t(4;5) translocation (see panel B); 6, HHW599, containing only chromosome 5; 7, HI.V842, containing chromosome 5 and a interstitial deletion chromosome 4 (see panel B); 8, HHW847, containing several human chromosomes and a t(4;21) chromosome (see panel B); 9, tsH1, the hamster parent of the HttW series and H64; 10, H64, containing chromosome 5 and a terminally deleted chromosome 4 (see panel B); 11, human genomic DNA. (B) The regions of chromosome 4p present in each hybrid cell of the regional mapping panel are shown schematically, Presence or absence oflDUA, indicated by the 20-kb HindlII band revealed by pID89 (see panel A), is given by + or - , respectively.
D 4 S I l l and detects a highly informative multiallele DNA polymorphism (25). The placement of both the plD89 probe and p157.9 on the restriction map of this cosmid is shown relative to the map of distal 4p16.3 in Fig. 2. The probes are located within 4 kb of each other on the same small NotI fragment. The 65-kDa o~-L-iduronidase polypeptide (26) is encoded by a contig of DNA spanning 4.2 kb. The p157.9 segment is within 2 kb of the 4.2-kb contig. Transcription of the IDUA locus is toward the telomere. IDUA is located 1.1 x 106 bp from the tip of 4p, making it the most telomeric cloned gene assigned to this chromosome arm. However, its position at the D 4 S l l l locus places it outside of either of the two regions that could contain the HD mutation. IDUA is located within a segment of 4p that is hemizygous in Wolf-Hirschhorn syndrome, a congenital anomaly involving severe mental retardation (27). However, deletion of one
copy of IDUA is unlikely to contribute to the Wolf-Hirschhorn phenotype given the absence of significant clinical features in heterozygous parents and siblings of Hurler patients (28). The chromosome segment that contains IDUA has been anatyzed extensively for DNA potymorphism, and numerous multiatlele markers have already been described (8, 25, 29). Consequently these markers are now applicable to genetic linkage analysis and carrier prediction in mucopolysaccharidosis type I and to the assessment of allelic heterogeneity and identification of potential independent mutations in this disorder. ACKNOWLEDGMENTS We are grateful to our colleagues in the Hereditary Disease Foundation HD Collaborative Research Group for their helpful discussions. This work was supported by NINDS grants NS16367 (Huntington's Dis-
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MacDonald et a|.
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Fig. 2. Positions of p157.9 and pID89 in cosmid A157.1, relative to the long-range restriction map of 4p16.3. A restriction map of cosmid A157.1 for the enzymes EcoRI (E), XhoI (X), and NotI (N) was prepared by agarose gel fractionation of partially digested cosmid DNA treated with X terminase and specifically labeled via the cos ends (30). The positions of p157.9 and pID89 was determined by hybridization of the probes to restriction digests with these enzymes. The direction of transcription of the IDUA gene was deduced from the IDUA cDNA (12) and a more detailed mapping of a genomic phage clone spanning this region (Scott et al., in preparation). The position of A157.1 on the pl~eviouslypublished long-range restriction map is shown for comparison with the HD candidate regions, given by the solid black arrows under the map. HD has been excluded from the central region between D4S168 and D4S142 that includes IDUA.
ease Center Without Walls), NS22031, and HG00169; by grants from the Adelaide Children's Hospital Research Foundation; the W.M. Keck Foundation; the Joan and William Schreyer Research Fund to Cure Huntington's Disease; the Hereditary Disease Foundation; the Huntington's Disease Society of America; and by a Program Grant from the National Health and Medical Research Council of Australia.
5. 6. 7. 8. 9.
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D4SH1 is IDUA
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