Homozygous and compound heterozygous mutations at the Werner syndrome locus
Homozygous and compound heterozygous mutations at the Werner syndrome locusJunko Oshima*, Chang-En Yu1, Charles Piussan2, Georg Klein3, Jörg Jabkowski3, Sevim Balci4, Tetsuro Miki5, Jun Nakura5, Toshio Ogihara5, James Ells6, Marilia de A. C. Smith7, Maria I. Melaragno7, Marco Fraccaro8, Susi Scappaticci8, John Matthews9, Samir Ouais10, Amy Jarzebowicz, Gerard D. Schellenberg1 and George M. Martin
Box 357470, Department of Pathology, University of Washington, Seattle, WA 98195, USA, 1Geriatrics Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle Division, Seattle, WA, USA, 2Pediatric Genetics, University of Amiens, Amiens, 80054, France, 3Dermatologische Abteilung, Allgem. offentliche Krankenhaus der Elisabethinen, Linz, 239 Austria, 4Department of Clinical Genetics, Hacettepe University Children's Hispital, Ankara, Turkey, 5Department of Geriatric Medicine, Osaka University, Osaka, 565, Japan, 6Internal Medicine Clinic, Nellis Air Force Base Hospital, Las Vegas, NV 89101, USA, 7Division of Genetics, Escola Paulista de Medicina, São Paulo, 04023, Brazil, 8Medical Genetics, University of Pavia, Pavia, Italy, 9Group Health, Fairfax, VA 22039, USA and 10Section of Endocrinology, Damascus City Hospital, Damascus, Syria
Received June 22, 1996;Revised and Accepted September 23, 1996
The Werner syndrome (WS) is a rare autosomal recessive progeroid disorder. The Werner syndrome gene (WRN) has recently been identified as a member of the helicase family. Four distinct mutations were previously reported in three Japanese and one Syrian WS pedigrees. The latter mutation was originally described as a 4 bp deletion spanning a spliced junction. It is now shown that this mutation results in a 4 bp deletion at the beginning of an exon. Nine new WRN mutations in 10 additional WS patients, both Japanese and Caucasian, are described. These include three compound heterozygotes (one Japanese and two Caucasian). The new mutations are located all across the coding region.
Werner syndrome (WS) is a rare autosomal recessive segmental progeroid syndrome (2 ). Patients exhibit not only an appearance of accelerated aging (premature graying, thinning of hair, skin atrophy and atrophy of subcutaneous fat), but also several disorders commonly associated with aging. These include bilateral cataracts, diabetes mellitus, osteoporosis, several forms of arteriosclerosis and a variety of benign and malignant neoplasms (3 ,4 ).
WS fibroblasts have very limited proliferative capacities as compared with age-matched controls (5 -7 ). A prolongation of the S phase has been demonstrated both in WS fibroblasts and lymphoblastoid cell lines (8 ). Cultured cells exhibit a propensity for chromosomal and intragenic mutations (9 -12 ). The rate of repair of X-ray- or UV-damaged DNA appears to be normal in WS fibroblasts (13 ).
WRN was initially mapped to chromosome 8p (14 ,15 ). Physical and genetic maps of the region were constructed (16 -19 ). WRN has recently been identified (GenBank accession number L76937) and four distinct WRN mutations were described (1 ). The WRN gene encodes a 1432 amino acid protein partially homologous to RecQ helicases (20 ). The WRN protein contains seven helicase motifs; two of them have been identified in all ATP-binding proteins (21 ).
DNA helicases have been implicated in a number of molecular processes. One of the most important functions of DNA helicases is the unwinding of DNA during DNA replication as a component in a replication complex (22 -24 ). Another function of helicase involves DNA repair. It has been hypothesized that some forms of nucleotide excision repair are coupled with transcription; mutant helicases responsible for the DNA instability syndromes may impair lesion recognition and/or lesion removal of the damaged nucleotides during transcription (25 -27 ). Examples include: ERCC2 helicase, which complements xeroderma pigmentosum B and its yeast homologue RAD3 (28 ,29 ); ERCC3, which complements xeroderma pigmentosum D and its yeast homologue RAD25 (30 -34 ); ERCC3 and ERCC6, which complement a Cockayne syndrome mutation (35 ,36 ). In Escherichia coli, the RecQ helicase is involved in the initial step of DNA repair by recombination (37 ).
Helicases are required for accurate chromosomal segregation. In yeast, precise chromosome segregation requires Sgs1, a eukaryotic homologue of RecQ (38 ).
Given the several potential roles of the WRN protein, a careful delineation of spontaneous mutations at this locus could facilitate the characterizations of its functions. We report nine WRN gene mutations from 10 WS patients, three of which were compound heterozygotes. Two other mutations found in three patients have been previously reported (1 ). These various mutations involve sequences throughout the coding region.
Four Japanese and eight non-Japanese WS patients were selected from our International Registry. Six of them (AUS, KO, MIM3, SEP, TUR, UH) were classified as `definite WS' and three (LGS, OW, SUG) as `probable WS'. Clinical and laboratory data for members of BLS, KUN and SYR remain incomplete, but the affected subjects had been diagnosed as WS by the submitting physicians.
Three new mutations were found in regions N-terminal with respect to the helicase consensus motifs. The point mutation at nt 1336, CGA (Arg) to TGA (Stp), was found as a homozygous mutation in one Caucasian (LGS) and two consanguineous Japanese (OW, KO) WS subjects and as a heterozygous mutation in one Japanese WS subject (KUN). LGS denied consanguinity; non-consanguinity was supported by haplotype data (19 ). A single nucleotide deletion at 1194-1196, AAA to AA, was seen as a heterozygous mutation in AUS. This mutation would create a frameshift which ends at 1406-1408 TGA (Stp). A four nucleotide insertion (ATCT) between 1509 and 1520 was homozygous in MIM3. This frameshift mutation would terminate at 1535-1537 TGA (Stp).
Three mutations were found within or just 3' to the helicase motifs in two Caucasian patients. One (SEP) mutation was a 105 bp insertion between 2319 and 2320. The insertion results in a termination codon, creating a truncated protein that excludes helicase domains III and the subsequent C terminus of the WRN protein. A second mutation was a deletion of nucleotide 2320-3056 seen in SUG as a heterozygous mutation, terminating at nt 3081-3083 TGA (Stp). The third mutation was a heterozygous termination mutation found in SUG, located 30 amino acids after the last helicase motif.
Three new mutations were found in regions C-terminal to the helicase motifs. A Japanese patient, IB, was homozygous for an A deletion at nt 3677. The mutated protein stops at nt 3713-3715 TAG (Stp). BLS (French) and TUR (Turkish) patients shared the same mutation at nt 3724, CGA (Gln) to TGA (Stp), which was previously found in the Japanese SY family (1 ). A 74 bp deletion of nt 3541-3614 was seen as a heterozygous mutation in a Japanese WS, KUN. This deletion results in a termination at 3720-3722 TAG (Stp). A 113 bp deletion of nt 3691-3803, which would result in a termination at nt 3816-3818 TGA (Stp), was found as a heterozygous deletion in the Caucasian WS, AUS.
These mutations were confirmed by sequencing of genomic PCR products, using the primers from the intron sequences of WRN (39 ). A summary of the newly discovered mutations is given in Figure 1 .
In our original report of the positional cloning of the WRN locus, four distinct homozygous mutations in the 3' region of the WRN gene were described (1 ). Using the present RT-PCR strategy mutations were readily found in various locations within the gene. The biochemical consequences of these mutations are not known.
All of the WRN mutations we have found to date either create a stop codon mutation or cause frameshifts that lead to premature terminations. We have not yet found an amino acid substitution in WRN that seems to be responsible for the pathogenesis of WS. It is quite possible that the various truncated WRN proteins may be rapidly degraded, resulting in comparable null mutations and comparable phenotypes. Such altered mRNAs are thought to be degraded via a specific pathway (40 ). In preliminary experiments, we do observe evidence for reduced levels of WRN mRNA expression in WS LCLs with four different mutations.
Identical mutations were found across a variety of ethnic groups, raising the question of potential mutationally susceptible sequences. Although the total number of mutations so far found in the WRN protein is not extensive, candidate sequences for such susceptibility would include nt 3677-3920, nt 1336-1395 and nt 2319-2320.
Three instances of compound heterozygous mutations were found: KUN (Japanese), AUS (Caucasian) and SUG (Caucasian). There have been numerous reports of compound heterozygotic mutations in `disease genes' (41 ,42 ). However, comparatively few compound heterozygotes have been reported in the genomic instability syndromes. Given the comparatively low prevalence of consanguinity in the USA, clinicians should therefore be alert to the diagnosis of WS in the absence of a history of consanguinity. Our experience suggests that WS is underdiagnosed in the USA.
WS patients were from an International Registry of Werner Syndrome (George M. Martin, MD, Junko Oshima, MD, PhD, Amy Jarzebowicz, BS). Diagnostic criteria were previously described (18 ). This study was approved by the University of Washington Institutional Review Board.
Five [mu]g of poly(A) RNA, isolated from total RNA, using Oligotex (Qiagen Inc.) was reverse-transcribed with random hexamers in 100 [mu]l reaction volume with GeneAmp RNA PCR kit (Perkin Elmer Cetus). Two [mu]l of the RT product were amplified in a 50 [mu]l PCR reaction buffer containing 5 units Taq DNA polymerase, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 50 [mu]M each of dGTP, dATP, dTTP and dCTP. The cycle program was typically: 94oC for 5 min, then 94oC for 45 s, 55oC for 45 s, 72oC for 3.5 min with 2 s increase per cycle for 35 cycles, followed by 72oC for 10 min. Five [mu]l aliquots of the first amplification products were subjected to a nested second amplification in 100 [mu]l reaction volumes. The primer sequences for RT-PCR are listed in Table 1 . The secondary PCR products were separated on 1% agarose/1*TBE (100 mM Tris-HCl pH 8.0, 90 mM boric acid and 1 mM ethylenediaminetetraacetic acid) to estimate the concentrations of DNA before sequencing.
RT-PCR products were sequenced using a T7 sequence PCR product sequencing kit (UBS, Amersham Life Science, Inc.). Seven [mu]l of PCR product was pretreated with 15 U of exonuclease I and 1.5 U of shrimp alkaline phosphatase at 37oC for 15 min followed by inactivation of the enzymes at 80oC for 15 min, then mixed with 100 ng of sequencing primers. The sequencing reaction followed the manufacturer's instructions.
The sequencing gel contained 6.6% LongRanger polyacrylamide (J. T. Baker Inc.), 6 M urea and 1.2* TBE. The running buffer contained 0.6* TBE. The gel was run at 55 W, dried and exposed overnight to Biomax MR film (Eastman Kodak Co.).
We thank Dr Goberdhan P. Dimri for a human cDNA library, an important contribution leading to the original positional cloning of WRN. We also thank Annette Smith, Charles E. Ogburn, Thao Dang, Susan Fredell, Ellen Nemens and Deanne Sparlin for their technical support. This work was supported by National Institute on Aging grants P1 AG08303 (GMM), R37 AG08303 (GMM), T32 AG00057 (GMM), RO1 AG12019 (GDS) and CNPq and FAPESP, Brazil (MIM).
1 Yu, C.E., Oshima, J., Fu, Y.W., Hisama, F., Wijsman, E.M., Alisch, R., Matthews, S., Nakura, J., Miki, T., Ouais, S., Martin, G.M., Mulligan, J. and Schellenberg, G.D. (1996) Positional cloning of the Werner's syndrome gene. Science, 272, 258-262.MEDLINE Abstract
2 Martin, G.M. (1978) Genetic syndromes in man with potential relevance to the pathology of aging. Birth Defects, 14, 5-39.MEDLINE Abstract
3 Epstein, C.J., Martin, G.M., Schultz, A.L. and Motulsky, A.G. (1966) Werner's syndrome: a review of its symptomatology, natural history, pathologic features, genetics and relationships to the natural aging process. Medicine, 45, 172-221.
4 Tollefsbol, T.O. and Cohen, H.J. (1984) Werner's syndrome: an underdiagnosed disorder resembling premature aging. Age, 7, 75-88.
5 Martin, G.M., Sprague, C.A. and Epstein, C.J. (1970) Replicative life-span of cultivated human cells. Effects of donor's age, tissue and genotype. Lab. Invest., 23, 86-92.MEDLINE Abstract
6 Salk, D., Bryant, E., Hoehn, H., Johnston, P. and Martin, G.M. (1985) Growth characteristics of Werner syndrome cells in vitro. Adv. Exp. Med. Biol., 190, 305-311.MEDLINE Abstract
7 Kill, I.R., Faragher, R.G., Lawrence, K. and Shall, S. (1994) The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture. J. Cell Sci., 107, 571-579.MEDLINE Abstract
8 Poot, M., Hoehn, H., Runger, T.M. and Martin, G.M. (1992) Impaired S-phase transit of Werner syndrome cells expressed in lymphoblastoid cell lines. Exp. Cell. Res., 202, 267-273.MEDLINE Abstract
9 Hoehn, H., Bryant, E.M., Au, K., Norwood, T.H., Boman, H. and Martin, G.M. (1975) Variegated translocation mosaicism in human skin fibroblast culture. Cytogenet. Cell. Genet., 15, 282-298.MEDLINE Abstract
10 Fukuchi, K., Martin, G.M. and Monnet, R.J. (1989) Mutator phenotype of Werner syndrome is characterized by extensive deletion. Proc. Natl Acad. Sci. USA, 86, 5893-5897.MEDLINE Abstract
11 Fukuchi, K., Tayama, K., Kumahara, Y., Murano, K., Pride, A.B., Martin, G.M., Monnet, R.J. (1990) Increased frequency of 6-thioguanine-resistant peripheral blood lymphocytes in Werner syndrome patients. Hum. Genet., 84, 249-252.MEDLINE Abstract
12 Runger, T.M., Bauer, C., Dekant, B., Moller, K., Sobotta, P., Czerny, C., Poot, M. and Martin, G.M. (1994) Hypermutable ligation of plasmid DNA ends in cells from patients with Werner syndrome. J. Invest. Dermatol., 102, 45-48.MEDLINE Abstract
13 Fujiwara, Y., Higashikawa, T. and Tatsumi, M. (1977) A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture. J. Cell. Physiol., 92, 365-374.MEDLINE Abstract
14 Goto, M., Rubenstein, M., Weber, J., Woods, K. and Drayna, D. (1992) Genetic linkage of Werner's syndrome to five markers on chromosome 8. Nature, 355, 735-758.MEDLINE Abstract
15 Schellenberg, G.D., Martin, G.M., Wijsman, E.M., Nakura, J., Miki, T. and Ogihara, T. (1992) Homozygosity mapping and Werner syndrome. Lancet, 339, 1002.MEDLINE Abstract
16 Yu, C.E., Oshima, J., Goddard, K.A.B., Miki, T., Nakura, J., Ogihara, T., Fraccaro, M., Puissan, C., Martin, G.M., Schellenberg, G.D. and Wijsman, E.M. (1994) Linkage disequilibrium and haplotype analysis of chromosome 8p11.1-21.1 markers and Werner syndrome. Am. J. Hum. Genet., 55, 356-364.MEDLINE Abstract
17 Oshima, J., Yu, C.E., Boehnke, M., Weber, J.L., Edelhoff, S., Wagner, M.J., Wells, D.E., Wood, S., Disteshe, D.M., Martin, G.M. and Schellenberg, G.D. (1994) Integrated mapping analysis of the Werner syndrome region of chromosome 8. Genomics, 23, 100-113.MEDLINE Abstract
18 Nakura, J., Wijsman, E.J., Miki, T., Kamino, K., Yu, C.E., Oshima, J., Fukuchi, K.I., Weber, J.L., Puissan, C., Melaragno, M.I., Epstein, C.J., Scappaticci, S., Fraccaro, M., Matusmura, T., Murano, S., Yoshida, S., Fujiwara, Y., Saida, T., Ogihara, T., Martin, G.M. and Schellenberg, G.D. (1994) Homozygosity mapping of the Werner syndrome locus (WRN). Genomics, 23, 600-608.MEDLINE Abstract
19 Goddard, K.A.B., Yu, C.E., Oshima, J., Martin, G.M., Schellenberg, G.D. and Wijsman, E.M. (1996) Towards localization of the Werner syndrome gene by linkage disequilibrium and ancestral haplotyping: lessons learned from analysis of 35 chromosome 8p11.1-21.1 markers. Am. J. Hum. Genet., 58, 1286-1302.MEDLINE Abstract
20 Schmid, S.R. and Linder, P. (1992) D-E-A-D protein family of putative RNA helicases. Mol. Microbiol., 6, 283-292.MEDLINE Abstract
21 Gorbalenya, A.E., Koonin, E.V., Donchenko, A.P. and Blinov, V.M. (1989) Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genome. Nucleic Acids Res., 17, 4713-4730.MEDLINE Abstract
22 Lohman, T.M. (1993) Helicase-catalyzed DNA unwinding. J. Biol. Chem., 268, 2269-2272.MEDLINE Abstract
23 Conaway, R.C. and Lehman, L.R. (1982) A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. Proc. Natl Acad. Sci. USA, 79, 2523-2527.MEDLINE Abstract
24 Dodson, M.S. and Lehman, I.R. (1991) Association of DNA helicase and primase activities with subassembly of the herpes simples virus 1 helicase-primase composed of the UL5 and UL52 gene products. Proc. Natl Acad. Sci. USA, 88, 1105-1109.MEDLINE Abstract
25 Hanawalt, P.C. (1994) Transcription-coupled repair and human disease. Science, 266,1957-1958.MEDLINE Abstract
26 Drapkin, R. and Reinberg, D. (1992) The essential twist. Nature, 369, 523-533.
27 Bootsma, D., Weeda, G., Vermeulen, W., van Vuuren, H., Troelstra, C., van der Spek, P. and Hoeijmakers, J. (1995) Nucleotide excision repair syndrome: molecular basis and clinical symptoms. Phil. Trans. R. Soc. London B. Biol. Sci., 347, 75-81.MEDLINE Abstract
28 Weber, C.A., Salazer, E.P., Stewart, S.A. and Thompson, L.H. (1990) ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3. EMBO J., 9, 1437-1447.MEDLINE Abstract
29 Guzder, S.N., Qiu, H., Sommers, C.H., Sung, P., Prakash, L. and Prakash, S. (1994a) DNA repair gene RAD3 of S. cerevisiae is essential for transcription by RNA polymerase II. Nature, 367, 91-94.MEDLINE Abstract
30 Weeda, G., van Ham, R.C.A., Masurel, R., Westerveld, A., Odijk, H., de Wit, J., Bootsma, D., van del Eb, A.J. and Hoeijmakers, J.H.J. (1990a) Molecular cloning and biological characterization of the human excision repair gene ERCC-3. Mol. Cell. Biol., 10, 2570-2581.MEDLINE Abstract
31 Flejter, W.L., McDaniel, L.D., Johns, D., Friedberg, E.C. and Schults, R.A. (1992) Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of human ERCC2 DNA repair gene. Proc. Natl Acad. Sci. USA, 89, 261-265.MEDLINE Abstract
32 Sung, P., Bailly, V., Weber, C., Thompson, L.H., Prakash, L. and Prakash, S. (1993) Human xeroderma pigmentosum group D gene encodes a DNA helicase. Nature, 365, 852-855.MEDLINE Abstract
33 Guzder, S.N., Sung, P., Bailly, V., Prakash, L. and Prakash, S. (1994) RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. Nature, 369, 578-581.MEDLINE Abstract
34 Ma, L., Westbroek, A. Jochemsen, A.G., Weeda, G., Bosch, A., Bootsma, D., Hoeijmakers, J.H. and van der Eb, A.J. (1994) Mutational analysis of ECRR3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function. Mol. Cell. Biol., 14, 4126-4136.MEDLINE Abstract
35 Weeda, G., van Ham, R.C.A., Vermeulen, W., Bootsma, D., van der Eb, A.J. and Hoeijmakers, J.H.J. (1990) A presumed DNA helicase encoded by ERRC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome. Cell, 62, 777-791.MEDLINE Abstract
36 Troelstra, C., van Gool, A., de Wit, J., Vermeulen, W., Bootsma, D. and Hoeijmaker, J.H.J. (1992) ECCR6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active gene. Cell, 71, 939-953.MEDLINE Abstract
37 West, S.C. (1994) The processing of recombination intermediates: mechanistic insights from studies of bacterial protein. Cell, 76, 9-15.MEDLINE Abstract
38 Watt, P.M., Louis, E.J., Borts, R.H., Hickson, I.D. (1995) Sgs1: A eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation. Cell, 81, 253-260.MEDLINE Abstract
39 Yu, C.E., Oshima, J., Wijsman, E. M., Nakura, J., Miki, T., Puissan, C., Matthews, S., Martin, G.M., Schellenberg, G.D. and the Werner's Syndrome Collaborative Group. Mutations in the consensus helicase domains of the Werner's syndrome gene (submitted).
40 Jacobson, A. and Peltz, S.W. (1996) Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu. Rev. Biochem., 65, 693-739.MEDLINE Abstract
41 Satokata, I., Tanaka, K. and Okada, Y. (1992) Molecular basis of group A xeroderma pigmentosum: a missense mutation and two deletions located in a zinc finger consensus sequence of the XPAC gene. Hum. Genet., 88, 603-607.MEDLINE Abstract
42 Sato, M., Nishigori, C., Yagi, T. and Tanabe, H. (1996) Aberrant splicing and truncated-protein expression due to a newly identified XPA gene mutation. Mutat. Res., 362, 199-208.MEDLINE Abstract
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