The Werner Syndrome Gene and Global Sequence Variation

Passarino, G.; Shen, P.; Kirk, J.B. Van; Lin, A.A.; Benedictis, G. De; Sforza, L.L. Cavalli; Oefner, P.J.; Underhill, P.A.

The Werner Syndrome Gene and Global Sequence Variation

Genomics, 2001

SHORT COMMUNICATION The Werner Syndrome Gene and Global Sequence Variation G. Passarino,* , † ,1 P. Shen,‡ J. B. Van Kirk,* A. A. Lin,* G. De Benedictis,† L. L. Cavalli Sforza,* P. J. Oefner,‡ and P. A. Underhill * * Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305; ‡Stanford Genome Technology Center, 855 California Avenue, Palo Alto, California 94304; and †Department of Cell Biology, University of Calabria, Arcavacata, 87030, Rende, Italy Received August 15, 2000; accepted September 29, 2000 We have identiﬁed a dense set of markers useful in association studies involving the Werner syndrome (WRN) gene. The homozygotic disruption of the WRN gene is the cause of Werner disease. In addition, this gene is likely to be involved in many complex traits, such as aging, or at least some of the traits and dis- eases related to age. To investigate the genetic varia- tion associated with the WRN gene, a sample of 93 individuals representing all the continents was ana- lyzed by denaturing high-performance liquid chroma- tography. A systematic survey of all 35 exons and ﬂanking regions identiﬁed 58 single-nucleotide poly- morphisms, 15 of which fall in the coding region and cause 11 missense mutations. The resulting global nu- cleotide diversity was 5.226  10 4 , with a slight differ- ence between coding and noncoding regions. © 2001 Academic Press Werner syndrome (WRN) is an autosomal recessive progeroid disorder (3, 13). Patients prematurely dis- play age-related conditions, such atherosclerosis, can- cer, and osteoporosis. The median life span of patients is 47 years (3). This disorder is caused by homozygotic null mutations in the WRN gene (15). This gene lies on the short arm of chromosome 8 and is composed of 35 exons. The gene encodes a 1432-amino-acid protein with a central domain homologous to the RecQ family of DNA helicases (15). In contrast to other known RecQ-like helicases, the WRN protein displays both exonuclease and helicase activities in vitro (5). All the Werner syndrome mutations implicated in disease in- volve a truncation of the protein or a shift of the read- ing frame. Different mutations have been found to cause the disease in Caucasian and Japanese patients (10, 15, 16). Beyond its role in Werner syndrome, the WRN pro- tein is likely to be a risk factor involved in common diseases (2). For example, it has been proposed that the 1367Cys/Arg mutation could cause susceptibility to myocardial infarction (17). B lymphocyte cell lines from clinically normal heterozygous carriers exhibit in vitro features intermediate between those of null and nor- mal homozygotes (9). It is possible that the WRN pro- tein is involved in normal aging (15, 16). This inference is suggested by symptoms of Werner syndrome pa- tients and also by the ﬁnding that the yeast sgs1 pro- tein, which is homologous to WRN helicases, inﬂuences aging (12). An intriguing cooperation has been found between telomerase and the WRN protein: cooperation to prevent the aging of Werner syndrome ﬁbroblasts (14). We have screened the WRN gene and the surround- ing genome region for single-nucleotide polymorphisms (SNPs). SNPs have proven valuable in the study of sequence variation in human genes (1, 4, 11). For ex- ample, SNPs facilitate the construction of dense, stable haplotypes for association and population afﬁnity stud- ies. They may also contribute directly to genetic risk for common diseases as indicated by the common dis- ease– common variant hypothesis (4). Thus, under- standing WRN gene variability might be useful in de- termining whether or not this gene plays a role in other complex traits. To that end, we have performed a SNP search across the WRN gene region. All 35 exons and their respective ﬂanking regions have been screened by denaturing high-performance liquid chromatography (DHPLC) (8) in a worldwide sample of 93 individuals. To determine correctly the ancestral allele, and to un- derstand better the evolution of the gene, we have sequenced the equivalent regions in one chimpanzee. A total of 58 SNPs were identiﬁed over the 12,839 bp surveyed, encompassing 3558 bp of coding sequence. All of the polymorphisms that we have found are listed in Table 1. Thirty-four nucleotide changes are transi- tions, and 20 are transversions; of the remaining 4, 3 are single-nucleotide deletions while 1 is an insertion. Among the 15 nucleotide changes that occurred in the 1 To whom correspondence should be addressed at Department of Genetics, Room M304, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305. Telephone: (650) 723-6506. Fax: (650) 725-1534. E-mail: g.passarino@stanford.edu. All articles available online at http://www.idealibrary.com on Genomics 71, 118 –122 (2001) doi:10.1006/geno.2000.6405 118 0888-7543/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

TABLE 1 Fifty-eight Germline Sequence Variations Found in the WRN Gene in 93 Representatives of Worldwide Populations Position a Nucleotide change Amino acid change Global heterozygosity Geographic distribution Ex 1 20 A/G 0.107 Africa + Asia 70 C/G 0.043 Africa + Asia EX 2 326 A/G Lys32Arg b 0.010 Asia +26 C/T 0.010 Asia +56 C/T 0.010 Asia EX 4 -161 A/C 0.053 Africa + Asia -53 C/T 0.043 Africa + Asia -52 A/G 0.172 Africa + Asia 571 A/G Val114Ile b 0.172 Every continent +20 A/T 0.107 Europe + Asia + Africa EX 5 -85 T/C 0.010 Africa -81 T/C 0.043 Europe + Africa EX 6 744 T/C Syn c 0.333 Every continent 745 A/C Thr172Pro 0.010 Asia +58 G/T 0.010 Asia EX 7 -45 C/T 0.010 Asia -23 T/A 0.086 Africa + Asia + Europe 951 T/G Asn240Lys 0.010 Africa EX 8 +46 A/T 0.053 Asia EX 9 1379 T/G Leu383Trp 0.107 Africa + Asia 1386 A/G Syn 0.032 Asia 1392 A/G Met387Ile b,c 0.130 Africa + Asia + Oceania + Europe EX 10 +22 T/C 0.333 Every continent EX 11 -48 T/C 0.010 Asia -5 Ins T 0.010 Africa +25 T/A 0.064 Africa EX 12 +60 T/G 0.010 Asia EX 13 -3 C/A 0.182 Africa + Asia + Oceania + Europe EX 14 -130 Del G 0.043 Africa +24 A/T 0.150 Asia + Oceania + America + Europe EX 16 -98 T/A 0.010 Europe EX 19 -19 C/T 0.010 Asia 2402 A/T Gln724Leu 0.021 Asia + Europe EX 20 -30 G/A 0.032 Africa 2592 T/G Syn c 0.344 Every continent +51 A/G 0.032 Africa EX 21 -83 A/G 0.021 Africa -63 A/G 0.172 Every continent -42 C/T 0.333 Every continent EX 22 -95 A/G 0.010 Africa -67 G/T 0.419 Every continent +64 A/G 0.010 Asia EX 23 +36 G/T 0.096 Europe + Asia + Africa 119 SHORT COMMUNICATION

All articles available online at http://www.idealibrary.com on SHORT COM M UNICATION The Werner Syndrome Gene and Global Sequence Variation G. Passarino,* , † ,1 P. Shen,‡ J. B. Van Kirk,* A. A. Lin,* G. De Benedict is,† L. L. Cavalli Sf orza,* P. J. Oef ner,‡ and P. A. Underhill * * Department of Genetics, Stanford University School of M edicine, 300 Pasteur Drive, Stanford, California 94305; ‡ Stanford Genome Technology Center, 855 California Avenue, Palo Alto, California 94304; and † Department of Cell Biology, University of Calabria, Arcavacata, 87030, Rende, Italy Received August 15, 2000; accepted September 29, 2000 We have identified a dense set of markers useful in association studies involving the Werner syndrome (WRN) gene. The homozygotic disruption of the WRN gene is the cause of Werner disease. In addition, this gene is likely to be involved in many complex traits, such as aging, or at least some of the traits and diseases related to age. To investigate the genetic variation associated with the WRN gene, a sample of 93 individuals representing all the continents was analyzed by denaturing high-performance liquid chromatography. A systematic survey of all 35 exons and flanking regions identified 58 single-nucleotide polymorphisms, 15 of which fall in the coding region and cause 11 missense mutations. The resulting global nucleotide diversity was 5.226 3 10 24, with a slight difference between coding and noncoding regions. © 2001 Academic Press Werner syndrome (WRN) is an autosomal recessive progeroid disorder (3, 13). Patients prematurely display age-related conditions, such atherosclerosis, cancer, and osteoporosis. The median life span of patients is 47 years (3). This disorder is caused by homozygotic null mutations in the WRN gene (15). This gene lies on the short arm of chromosome 8 and is composed of 35 exons. The gene encodes a 1432-amino-acid protein with a central domain homologous to the RecQ family of DNA helicases (15). In contrast to other known RecQ-like helicases, the WRN protein displays both exonuclease and helicase activities in vitro (5). All the Werner syndrome mutations implicated in disease involve a truncation of the protein or a shift of the reading frame. Different mutations have been found to cause the disease in Caucasian and Japanese patients (10, 15, 16). Beyond its role in Werner syndrome, the WRN pro1 To whom correspondence should be addressed at Department of Genetics, Room M304, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305. Telephone: (650) 723-6506. Fax: (650) 725-1534. E-mail: g.passarino@stanford.edu. Genomics 71, 118 –122 (2001) doi:10.1006/geno.2000.6405 0888-7543/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. tein is likely to be a risk factor involved in common diseases (2). For example, it has been proposed that the 1367Cys/Arg mutation could cause susceptibility to myocardial infarction (17). B lymphocyte cell lines from clinically normal heterozygous carriers exhibit in vitro features intermediate between those of null and normal homozygotes (9). It is possible that the WRN protein is involved in normal aging (15, 16). This inference is suggested by symptoms of Werner syndrome patients and also by the finding that the yeast sgs1 protein, which is homologous to WRN helicases, influences aging (12). An intriguing cooperation has been found between telomerase and the WRN protein: cooperation to prevent the aging of Werner syndrome fibroblasts (14). We have screened the WRN gene and the surrounding genome region for single-nucleotide polymorphisms (SNPs). SNPs have proven valuable in the study of sequence variation in human genes (1, 4, 11). For example, SNPs facilitate the construction of dense, stable haplotypes for association and population affinity studies. They may also contribute directly to genetic risk for common diseases as indicated by the common disease– common variant hypothesis (4). Thus, understanding WRN gene variability might be useful in determining whether or not this gene plays a role in other complex traits. To that end, we have performed a SNP search across the WRN gene region. All 35 exons and their respective flanking regions have been screened by denaturing high-performance liquid chromatography (DHPLC) (8) in a worldwide sample of 93 individuals. To determine correctly the ancestral allele, and to understand better the evolution of the gene, we have sequenced the equivalent regions in one chimpanzee. A total of 58 SNPs were identified over the 12,839 bp surveyed, encompassing 3558 bp of coding sequence. All of the polymorphisms that we have found are listed in Table 1. Thirty-four nucleotide changes are transitions, and 20 are transversions; of the remaining 4, 3 are single-nucleotide deletions while 1 is an insertion. Among the 15 nucleotide changes that occurred in the 118 119 SHORT COMMUNICATION TABLE 1 Fifty-eight Germline Sequence Variations Found in the WRN Gene in 93 Representatives of Worldwide Populations Position a Ex 1 20 70 EX 2 326 126 156 EX 4 2161 253 252 571 120 EX 5 285 281 EX 6 744 745 158 EX 7 245 223 951 EX 8 146 EX 9 1379 1386 1392 EX 10 122 EX 11 248 25 125 EX 12 160 EX 13 23 EX 14 2130 124 EX 16 298 EX 19 219 2402 EX 20 230 2592 151 EX 21 283 263 242 EX 22 295 267 164 EX 23 136 Nucleotide change Amino acid change A/G C/G Global heterozygosity Geographic distribution 0.107 0.043 Africa 1 Asia Africa 1 Asia 0.010 0.010 0.010 Asia Asia Asia 0.053 0.043 0.172 0.172 0.107 Africa 1 Asia Africa 1 Asia Africa 1 Asia Every continent Europe 1 Asia 1 Africa 0.010 0.043 Africa Europe 1 Africa 0.333 0.010 0.010 Every continent Asia Asia 0.010 0.086 0.010 Asia Africa 1 Asia 1 Europe Africa 0.053 Asia 0.107 0.032 0.130 Africa 1 Asia Asia Africa 1 Asia 1 Oceania 1 Europe T/C 0.333 Every continent T/C Ins T T/A 0.010 0.010 0.064 Asia Africa Africa T/G 0.010 Asia C/A 0.182 Africa 1 Asia 1 Oceania 1 Europe Del G A/T 0.043 0.150 Africa Asia 1 Oceania 1 America 1 Europe T/A 0.010 Europe 0.010 0.021 Asia Asia 1 Europe 0.032 0.344 0.032 Africa Every continent Africa A/G A/G C/T 0.021 0.172 0.333 Africa Every continent Every continent A/G G/T A/G 0.010 0.419 0.010 Africa Every continent Asia G/T 0.096 Europe 1 Asia 1 Africa A/G C/T C/T A/C C/T A/G A/G A/T Lys32Arg b Val114Ile b T/C T/C T/C A/C G/T C/T T/A T/G Syn c Thr172Pro Asn240Lys A/T T/G A/G A/G C/T A/T G/A T/G A/G Leu383Trp Syn Met387Ile b,c Gln724Leu Syn c 120 SHORT COMMUNICATION TABLE 1—Continued Position a EX 24 2205 2191 EX 25 274 16 17 EX 26 3453 EX 27 126 EX 29 23 EX 30 126 EX 32 4036 EX 33 234 EX 34 4314 4330 EX 35 4447 4544 Nucleotide change Amino acid change Global heterozygosity Geographic distribution T/G C/T 0.247 0.483 Every continent Every continent Del G C/T A/G 0.129 0.397 0.354 Every continent Every continent Every continent 0.387 Every continent C/T 0.311 Every continent T/C 0.010 Africa Del C 0.010 Africa 0.010 Africa 0.032 Africa T/G A/G Phe1074Leu c Gly1269Glu C/T C/T C/T Syn c Cys1367Arg d 0.290 0.118 Every continent Asia 1 Oceania 1 America 1 Europe C/T A/G Arg1406Stop 0.021 0.010 Asia Africa Note. Primers and PCR and DHPLC conditions are available upon request. a Polymorphic sites inside the exons are given according to the GenBank Accession No. L76937. Intronic polymorphic sites are reported with respect to their upstream (1) or downstream (2) position to the closest exon. b Conservative amino acid changes. c Observed by Castro et al. (2). d Observed by Ye et al. (17). e 93 individuals from five continents were examined. The ethnic affiliations of these subjects were as follows: Africa (18): 2 Pygmies from the Central African Republic, 5 Pygmies from Congo-Kinshasa, 4 Lissongo, 3 San from Namibia, 2 Ethiopians, 1 Sudanese, 1 Mandenka from Senegal. Asia (41): 2 Bedouine, 2 Druze, 2 Palestinian, 2 Sephardic Jews, 1 Iranian, 1 Iraqi, 10 Pakistani, 1 Tamil, 1 Laotian, 3 Cambodian, 1 Jakut, 8 Chinese, 2 aboriginal Taiwanese, 4 Japanese, 1 Korean. Europe (16): 1 Adigey, 1 Russian, 2 Georgian, 7 Caucasian Americans of central European ancestry, 2 French, 2 Italian, 1 Dane. America (10): 2 Amerindians from Brazil, 2 Amerindians from Colombia, 2 Mayans, 1 Quechua, 1 Muskogee from Oklahoma, 1 Pima from New Mexico, 1 Navaho from Arizona. Oceania (8): 2 New Guinean, 2 Australian, 1 Micronesian, 2 Melanesian, 1 Samoan. coding region, 10 caused amino acid substitutions, 1 caused premature termination of the protein, and 4 were synonymous. Four mutations, three of which result in an amino acid change, occur in the exonuclease region (472–951, corresponding to amino acids 80 –249). Of the three nonsynonymous mutations, two are extremely rare, while one (A571G) is common worldwide. By contrast, in the helicase motifs, only one synonymous variation (T2592G, corresponding to Leu 787) was found in helicase motif IV. A nonsynonymous polymorphism, A2402T (Gln724Leu), is located close to motif III. A comparison of human with chimpanzee WRN protein confirms the functional constraints on these motifs (Table 2). Another important region of the WRN protein comprises amino acids 1370 –1375. This region is essential for the correct cellular localization of the protein (7). No mutations were detected in this region of the gene, but several interesting polymorphisms were observed in its vicinity. These include the Cys1367Arg polymorphism, which has been reported to be associated with resistance to myocardial infarction (17) and is proposed to interfere with the nuclear localization signal (2). In our ascertainment set, this mutation has been found in every continent but Africa, where only the ancestral cysteine allele is present. The nonsense mutation at nucleotide position 4447 that results in a truncated protein of 1405 amino acids (27 amino acids shorter than normal) was found in one individual each from Iran and Pakistan. Analysis of 30 additional individuals from Pakistan yielded one more heterozygous individual. The total nucleotide diversity estimate was 5.226 3 10 24, and, as previously reported (1, 4), it is the highest in Africa. No difference was noticed between the nucleotide diversity estimates obtained for coding and noncoding regions. As to the polymorphisms falling in the coding sequence, 11 of 15 were nonsynonymous, 3 of which involved conservative amino acid changes (326A . G, 571A . G, and 1392A . G). These results are in good agreement with those obtained by Cargill et 121 SHORT COMMUNICATION TABLE 2 Exonic Sequence Differences between the Werner Gene in Human and That in Chimpanzee Nucleotide Position 4/5 88/89 337 423 447 571 762 1259 1313 1456 1481 1680 1743 1817 2275 2301 2355 2592 2633 2918 3188 3317 3355–56 3367 3468 3527 3655 3661 3771 4083 4359 4544 Human Chimp C C T G T A A T T T C G G A T T G T T T GC A G G C G A C G A TG insertion G insertion T G G A C C G G C C T T A G C G A C C A AT G A C G A C T A G Amino acid Human Chimp Not coding Not coding Arg Trp Phe Leu Ser Arg Val Ile Leu Leu Glu Ala Asp Gly Ser Ala Ile Thr Asn Asn Pro Leu Trp Leu Asp Asn Leu Leu Ser Ser Leu Leu Ser Asn Met Thr Leu Pro Val Glu Ala Ile Lys Glu Ser Ser Ser Thr Gln Gln Val Ile Thr Thr Leu Leu Gly Ser Not coding plest explanation is that these two alleles survived a bottleneck that reduced the variability of our species. This interpretation is bolstered by the fact that the constant population infinite sites model does not fit the data and by the Tajima test. The latter gave a negative value (21.1), suggesting population expansion as the HKA test (P 5 0.1) failed to reject neutrality (details on these statistical methods are in Ref. 11). Intriguingly, BRCA1, which codes for another helicase (the impairment of which causes breast cancer), does not comply with the Luria and Delbrück/Lea and Coulson expectation either (unpublished data). The polymorphisms that we have discovered are valuable for constructing a dense SNP map of this region of the genome. In addition, these polymorphisms will be helpful in constructing haplotypes and studying associations between different alleles of the WRN gene and complex traits. In particular, it would be useful to test in diverse populations whether the WRN gene is involved in the normal aging process or does Werner syndrome merely mimic aging by affecting age-related traits and diseases? In this case, it would be useful to see which functions or pathways are involved in specific aging features or in overall aging (6). ACKNOWLEDGMENTS We thank the DNA donors and the investigators who provided the samples: L. Excoffier, M. E. Ibrahim, T. Jenkins, J. Kidd, A. Langaney, S. Q. Mehdi, and P. Parham. We are grateful to R. Hyman for his helpful comments. The study was funded by an Ellison Medical Foundation grant and by NIH Grant 1R01-HG01932. REFERENCES Note. Position refers to sequence deposited under GenBank Accession No. L76937. al. (1) and Halushka et al. (4) on 106 and 75 genes, respectively. Other studies, however, have shown a twofold lower nucleotide diversity estimate for the coding regions (11). We also performed a comparison of the number of mutants for each allele observed in the WRN gene with values expected according to the Luria and Delbrück/Lea and Coulson hypothesis and the constant population infinite sites model. The first, which was initially elaborated to explain the growth of bacteria, proved to be useful in modeling demographic growth data from humans (11; and unpublished results). This is probably due to the fact that humans underwent a recent population expansion. As a consequence, most of the variability in the human genome seems to be due to low-frequency polymorphisms (11). The WRN gene does not comply with the distribution expected by Luria and Delbrück/Lea and Coulson (P 5 1.7 3 10 211). This is due to the fact that several polymorphisms have high heterozygosity and appear to be in linkage, suggesting that two alleles with an ancient coalescence time are present in the human population. The sim- 1. 2. 3. 4. 5. 6. Cargill, M., Altshuler, D., Ireland, J., Sklar, P., Ardlie, K., Patil, N., Shaw, N., Lane, C. R., Lim, E. P., Kalyanaraman, N., Nemesh, J., Ziaugra, L., Friedland, L., Rolfe, A., Warrington, J., Lipshutz, R., Daley, G. Q., and Lander, E. S. (1999). Characterization of single-nucleotide polymorphisms in coding regions of human genes. Nat. Genet. 22: 231–238. Castro, E., Ogburn, C. E., Hunt, K. E., Tilvis, R., Louhija, J., Penttinen, R., Erkkola, R., Panduro, A., Riestra, R., Piussan, C., Deeb, S. S., Wang, L., Edland, S. D., Martin, G. M., and Oshima, J. (1999). Polymorphisms at the Werner locus. I. Newly identified polymorphisms, ethnic variability of 1367Cys/Arg, and its stability in a population of Finnish centenarians. Am. J. Med. Genet. 82: 399 – 403. 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 relationship to the natural aging process. Medicine 45: 177–221. Halushka, M. K., Fan, J. B., Bentley, K., Hsie, L., Shen, N., Weder, A., Cooper, R., Lipshutz, R., and Chakravarti, A. (1999). Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat. Genet. 22: 239 –247. Huang, S., Li, B., Gray, M. D., Oshima, J., Mian, I. S., and Campisi, J. (1998). The premature ageing syndrome protein, WRN, is a 39 3 59 exonuclease. Nat. Genet. 20: 114 –116. Martin, G. M. (1997). The Werner mutation: Does it lead to a “public” or “private” mechanism of aging? Mol. Med. 3: 356 – 358. 122 7. 8. 9. 10. 11. SHORT COMMUNICATION Matsumoto, T., Shimamoto, A., Goto, M., and Furuichi, Y. (1997). Impaired nuclear localization of defective DNA helicases in Werner’s syndrome. Nat Genet. 16: 335–336. Oefner, P. J., and Underhill, P. A. (1998). DNA mutation detection using high performance liquid chromatography (DHPLC). “Current Protocols in Human Genetics,” Suppl. 19, pp. 7.10.1– 7.10.12, Wiley, New York. Ogburn, C. E., Oshima, J., Poot, M., Chen, R., Hunt, K. E., Gollahon, K. A., Rabinovitch, P. S., and Martin, G. M. (1997). An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants. Hum. Genet. 101: 121–125. Oshima, J., Yu, C. E., Piussan, C., Klein, G., Jabkowski, J., Balci, S., Miki, T., Nakura, J., Ogihara, T., Ells, J., Smith, M., Melaragno, M. I., Fraccaro, M., Scappaticci, S., Matthews, J., Ouais, S., Jarzebowicz, A., Schellenberg, G. D., and Martin, G. M. (1996). Homozygous and compound heterozygous mutations at the Werner syndrome locus. Hum. Mol. Genet. 5: 1909 – 1913. Shen, P., Wang, F., Underhill, P. A., Franco, C., Yang, W. H., Roxas, A., Sung, R., Lin, A. A., Hyman, R. W., Vollrath, D., Davis, R. W., Cavalli-Sforza, L. L., and Oefner, P. J. (2000). Population genetic implications from sequence variation in four Y chromosome genes. Proc. Natl. Acad. Sci. USA 97: 7354 – 7359. 12. 13. 14. 15. 16. 17. Sinclair, D. A., Mills, K., and Guarente, L. (1997). Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants. Science 277: 1313–1316. Werner, C. W. O. (1904). “Über Kataract in Verbindung mit Sclerodermie,” Schmidt & Klarnig, Kiel, Germany. Wyllie, F. S., Jones, C. J., Skinner, J. W., Haughton, M. F., Wallis, C., Wynford-Thomas, D., Faragher, R. G., and Kipling, D. (2000). Telomerase prevents the accelerated cell ageing of Werner syndrome fibroblasts. Nat. Genet. 24: 16 –17. Yu, C. E., Oshima, J., Fu, Y. H., Wijsman, E. M., Hisama, F., 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. Yu, C. E., Oshima, J., Wijsman, E. M., Nakura, J., Miki, T., Piussan, C., Matthews, S., Fu, Y. H., Mulligan, J., Martin, G. M., and Schellenberg, G. D. (1997). Mutations in the consensus helicase domains of the Werner syndrome gene. Werner’s Syndrome Collaborative Group. Am. J. Hum. Genet. 60: 330 – 341. Ye, L., Miki, T., Nakura, J., Oshima, J., Kamino, K., Rakugi, H., Ikegami, H., Higaki, J., Edland, S. D., Martin, G. M., and Ogihara, T. (1997). Association of a polymorphic variant of the Werner helicase gene with myocardial infarction in a Japanese population. Am. J. Med. Genet. 68: 494 – 498.

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The Werner Syndrome Gene and Global Sequence Variation