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.
The mutation in the SYR pedigree was previously reported as a 4 bp deletion at the intron-exon boundary, 2 bp from the putative intron and 2 bp from the contiguous exon (gtagACAGACC at the DNA level). This was expected to cause an in-frame deletion of the exon. Our RT-PCR protocol, however, showed a deletion of 4 bp, ACAG, from the beginning of this exon. The ACAG deletion would result in a termination at nt 3971–3973 TAG (Stp).
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 underdiag-nosed in the USA.
Materials and Methods
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 µg of poly(A) RNA, isolated from total RNA, using Oligotex (Qiagen Inc.) was reverse-transcribed with random hexamers in 100 µl reaction volume with GeneAmp RNA PCR kit (Perkin Elmer Cetus). Two µl of the RT product were amplified in a 50 µl PCR reaction buffer containing 5 units Taq DNA polymerase, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 50 µM each of dGTP, dATP, dTTP and dCTP. The cycle program was typically: 94°C for 5 min, then 94°C for 45 s, 55°C for 45 s, 72°C for 3.5 min with 2 s increase per cycle for 35 cycles, followed by 72°C for 10 min. Five µl aliquots of the first amplification products were subjected to a nested second amplification in 100 µl reaction volumes. The primer sequences for RT-PCR are listed in Table 1. The secondary PCR products were separated on 1% aga-rose/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.
Direct sequencing of PCR products
RT-PCR products were sequenced using a T7 sequence PCR product sequencing kit (UBS, Amersham Life Science, Inc.). Seven µl of PCR product was pretreated with 15 U of exonuclease I and 1.5 U of shrimp alkaline phosphatase at 37°C for 15 min followed by inactivation of the enzymes at 80°C 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).
Werner syndrome gene
lymphoblastoid cell line
reverse transcription-polymerase chain reaction
polymerase chain reaction.