Makoto Goto

Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome

Rothmund-Thomson syndrome (RTS; also known as poikiloderma congenitale) is a rare, autosomal recessive genetic disorder characterized by abnormalities in skin and skeleton, juvenile cataracts, premature ageing and a predisposition to neoplasia. Cytogenetic studies indicate that cells from affected patients show genomic instability often associated with chromosomal rearrangements causing an acquired somatic mosaicism. The gene(s) responsible for RTS remains unknown. The genes responsible for Werner and Bloom syndromes (WRN and BLM, respectively) have been identified as homologues of Escherichia coli RecQ, which encodes a DNA helicase that unwinds double-stranded DNA into single-stranded DNAs. Other eukaryotic homologues thus far identified are human RECQL (Refs 13, 14), Saccharomyces cerevisiae SGS1 (Refs 15,16) and Schizosaccharomyces pombe rqh1+ (ref. 17). We recently cloned two new human helicase genes, RECQL4 at 8q24.3 and RECQL5 at 17q25, which encode members of the RecQ helicase family. Here, we report that three RTS patients carried two types of compound heterozygous mutations in RECQL4. The fact that the mutated alleles were inherited from the parents in one affected family and were not found in ethnically matched controls suggests that mutation of RECQL4 at human chromosome 8q24.3 is responsible for at least some cases of RTS.

Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51.

Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S‐phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear.

Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner's syndrome patients transformed by Epstein – Barr virus

The characteristics of B-lymphoblastoid cell strains transformed by Epstein – Barr virus (EBV) from normal individuals and Werners syndrome (WRN) patients were compared. We continuously passaged cell strains from 28 WRN patients and 20 normal individuals for about 2 years corresponding to over 160 population doubling levels (PDLs). First, the WRN mutation significantly suppressed the immortalization: all the 28 cell strains from WRN patients, as well as 15 out of 20 cell strains from normal individuals, died out before 160 PDLs mostly without developing a significant telomerase activity. The remaining five cell strains from normal individuals became moderately/strongly telomerase-positive and, three of them were apparently immortalized with an infinitively proliferating activity. Second, the monitoring of the telomere length of both normal and WRN cell strains during the culture period suggests that the WRN gene mutation causes abnormal dynamics of the telomere: (1) a significant proportion of WRN cell strains showed drastic shortening or lengthening of telomere lengths during cell passages compared with normal cell strains, and (2) WRN cell strains terminated their life-span at a wide range of telomere length (between 3.5 and 18.5 Kbp), whereas normal cell strains terminated within a narrow telomere length range (between 5.5 and 9 Kbp). The chromosomal aberration characteristic of WRN cells, including translocation was confirmed in our experiment. We discussed the correlation between the chromosomal instability, abnormal telomere dynamics and inability of immortalization of the WRN B-lymphobloastoid cell strains.

Mutation and haplotype analyses of the Werner's syndrome gene based on its genomic structure : genetic epidemiology in the Japanese population

Abstract The correlation between mutations in the Werner’s syndrome (WRN) gene and the haplotypes of surrounding markers was studied in Japanese patients. We have elucidated the genomic structure of WRN helicase, and found five additional mutations, designated mutations 6–10. Mutations 4 and 6 were found to be the two major mutations in this population; these mutations comprised 50.8% and 17.5%, respectively, of the total in a sample of 126 apparently unrelated chromosomes. Almost all the patients homozygous for mutation 4 shared a haplotype around the WRN gene, consistent with the view that they are derived from a single ancestor. This important advantage demonstrated in the identification of the WRN gene suggests that the Japanese present a unique population for the cloning of other disease genes. The conserved haplotype was observed across 19 loci, extending a distance estimated to be more than 1.4 Mbp around the WRN gene. This haplotype is rare among random Japanese individuals. Unexpectedly, all the nine patients homozygous for mutation 6 shared a haplotype that was identical to this haplotype at 18 of these 19 markers. These results suggest that mutations 4 and 6 arose independently in almost identical rare haplotypes. The remaining mutations (1, 5, 7, 8, 9, and 10) occurred rarely, and were each associated with different haplotypes.

Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex

Werner syndrome (WS) is a rare autosmomal recessive genetic disorder causing premature aging. The gene (WRN) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a nucleolar localization signal and shows intranuclear trafficking between the nucleolus and the nucleoplasm. WRN is recruited into the nucleolus when rRNA transcription is reactivated in quiescent cells. Inhibition of mRNA transcription with α-amanitin has no effect on nucleolar localization of WRN whereas inhibition of rRNA transcription with actinomycin D releases WRN from nucleoli, suggesting that nucleolar WRN is closely related to rRNA transcription by RNA polymerase I (RPI). A possible function of WRN on rRNA transcription through interaction with RPI is supported by the results described here showing that WRN is co-immunoprecipitated with an RPI subunit, RPA40. Here we show that WS fibroblasts are characterized by a decreased level of rRNA transcription compared with wild-type cells, and that the decreased level of rRNA transcription in WS fibroblasts recovers when wild-type WRN is exogenously expressed. By contrast, exogenously expressed mutant-type WRN lacking an ability to migrate into the nucleolus fails to stimulate rRNA transcription. These results suggest that WRN promotes rRNA transcription as a component of an RPI-associated complex in the nucleolus.

Prevalence of Werner's syndrome heterozygotes in Japan

1Worldwide, 1200 patients have been reported from 1904 to 1996; 845 from Japan. Patients are distributed all over Japan. 2 But how widely distributed is the mutated WRN gene in the general population? We previously analysed the mutation in 63 patients with Werner’s syndrome, and found that among 126 chromosomes, 65 (51·6%) chromosomes had mutation 4 and 22 (17·5%) had mutation 6, 3 and the rest had other mutations. We focused on mutations 4 and 6. DNA extracted from the blood of 1000 apparently healthy, unlinked anonymous volunteers in Kanagawa prefecture of Japan were analysed for mutations 4 and 6 by a method developed by us. 4

Reconsideration of senescence, immortalization and telomere maintenance of Epstein–Barr virus-transformed human B-lymphoblastoid cell lines

We review recent data on senescence and immortalization of human B-lymphoblastoid cell lines (LCLs) transformed by the Epstein-Barr virus (EBV). Although EBV-transformed LCLs are generally believed to be immortalized, a series of recent studies, including ours, provided strong evidence that they are mostly mortal and have non-malignant properties, except for a small proportion of LCLs that are immortalized by developing a strong telomerase activity. A large proportion of mortal LCLs have exceptionally long lifespans. Some of them have a lifespan over 150 population-doubling levels, keeping a relatively constant telomere length in spite of the absence of a detectable telomerase activity, suggesting that they maintain telomeres by a pathway other than that using telomerase. Here we propose a model of an alternative pathway to maintain telomeres of such long-lived mortal LCLs by exploiting extra-chromosomal telomere repeat DNA, which was recently found by us.

Immunological diagnosis of Werner syndrome by down-regulated and truncated gene products

Although immunological methods are widely used to diagnose various infectious diseases, they have rarely been employed to detect genetic diseases. In this study, we have established an immunoblot analysis system for the diagnosis of Werner syndrome (WS), a recessive genetic disorder causing premature aging and an enhanced risk of rare cancers. The method uses an immunoblot technique with specific monoclonal antibodies to WS gene product, and B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus; these cell lines express an increased level of normal WS gene product DNA helicase. The method clearly distinguishes normal from patient LCLs containing any of the mutation types found so far in Japan, primarily because of the drastically reduced levels of mutated gene products, and secondarily because of the truncated product sizes. A comparison of this immunological diagnosis with the symptom-based clinical diagnosis has narrowed down the criteria of symptoms essential for WS diagnosis. This procedure is compatible with, and has some advantage over, the genetic method, because WS patients can be diagnosed without determining the mutated gene sequences. The method exemplified in WS may also be applied to detect some other genetic diseases.

Atypical Osteosarcomas in Werner Syndrome (Adult Progeria)

Werner syndrome (WS), adult progeria, is more common in Japan than elsewhere. It predisposes to osteosarcoma (OS) and five other rare tumors. To determine if and how OS is atypical in this genetic disorder, we studied the characteristics of ten Japanese cases with respect to clinical features, pathology, and radiographs, and compared them with a hospital series of 36 skeletal OS with the same atypical age‐range, 35–57 years. The anatomic sites were also atypical: seven ankle/foot, two radius and one patella compared with only one at the ankle in the hospital series. The osteoblastic cell‐type was about equally frequent in both series, but, among others than the three major subtypes, there was only one in WS as compared with 14 (39%) in the hospital series. The types of mutations were sought in five WS cases with OS. One showed no mutation at any of the ten known loci for Japanese, two were of type 4/4 and two of type 6/6. The mutations 4 and 6 have been found in 66% of alleles of WS cases in Japan. The increased frequency and unusual age and site distributions of OS in WS may be due to increased susceptibility, related to later‐life leg ulcers, and weight‐bearing on spindly ankles weakened by severe loss of lower limb subcutaneous tissue.