Junko Oshima

Candidate gene for the chromosome 1 familial Alzheimer's disease locus

A candidate gene for the chromosome 1 Alzheimers disease (AD) locus was identified (STM2). The predicted amino acid sequence for STM2 is homologous to that of the recently cloned chromosome 14 AD gene (S182). A point mutation in STM2, resulting in the substitution of an isoleucine for an asparagine (N141l), was identified in affected people from Volga German AD kindreds. This N141l mutation occurs at an amino acid residue that is conserved in human S182 and in the mouse S182 homolog. The presence of missense mutations in AD subjects in two highly similar genes strongly supports the hypothesis that mutations in both are pathogenic.

Positional Cloning of the Werner's Syndrome Gene

Werners syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging. Early susceptibility to a number of major age-related diseases is a key feature of this disorder. The gene responsible for WS (known as WRN) was identified by positional cloning. The predicted protein is 1432 amino acids in length and shows significant similarity to DNA helicases. Four mutations in WS patients were identified. Two of the mutations are splice-junction mutations, with the predicted result being the exclusion of exons from the final messenger RNA. One of these mutations, which results in a frameshift and a predicted truncated protein, was found in the homozygous state in 60 percent of Japanese WS patients examined. The other two mutations are nonsense mutations. The identification of a mutated putative helicase as the gene product of the WS gene suggests that defective DNA metabolism is involved in the complex process of aging in WS patients.

The premature ageing syndrome protein, WRN, is a 3'-->5' exonuclease.

Werner syndrome (WS) is a human autosomal recessive disorder that causes the premature appearance of a partial array of disorders characteristic of old age1,2. These disorders include atherosclerosis, cancer, type 2 diabetes, osteoporosis, cataracts, wrinkled skin and grey hair, among other ailments. Cells cultured from WS subjects have a shortened replicative life span3,4 and elevated rates of chromosome translocations, large deletions and homologous recombination5,6. The gene defective in WS, WRN, encodes a large RecQ-like DNA helicase7 of 1432 aa. Defects in another human RecQ-like helicase, BLM, result in Bloom’s syndrome8 (BS), a genetic disorder that is quite different from WS. BS is manifested by short stature, neoplasia, immunodeficiency and high risk of cancer. Cells from BS subjects show an increase in sister chromatid exchanges. DNA helicases can function in replication, repair, recombination, transcription or RNA processing. As WRN and BLM share no obvious homology outside the helicase domain, the non-helicase domains probably determine in which process each RecQ-like helicase participates, which provides the basis for the disparate cellular and organismal phenotypes that result from defects in these proteins. Statistical sequence analyses showed subtle but significant similarities between WRN and several 3′→5′ exonucleases9,10. To test the prediction that WRN is an exonuclease, we produced tagged recombinant wild-type and mutant WRN proteins. Two mutants had amino-acid substitutions at either position 82 (D82A) or 84 (E84A), two of the five residues predicted to be critical for exonuclease activity9,10. A third mutant had a substitution at position 577 (K577M), which abolished WRN helicase activity11. The fourth mutant was an N-terminal fragment (aa 1–333; N333) containing the putative exonuclease domain, but lacking the helicase domain. A tagged 36-aa vector-derived peptide served as a negative control (mock). Purified WRN and mock proteins were incubated with doubled-stranded DNA substrates. Wild-type WRN degraded a 5′ labelled substrate to a series of smaller, labelled products (Fig. 1a), and a 3′ labelled substrate to a single labelled product that migrated as a mononucleotide (Fig. 1b). Thus, WRN degraded DNA with 3′→5′ directionality. Although mock and full-length WRN preparations contained low levels of a contaminating 5′→3′ exonuclease, as shown by release of the 5′ label as a mononucleotide (Figs 1a,​,2b),2b), 3′→5′ degradation was entirely dependent on WRN. Fig. 1 Exonuclease activity of wild-type WRN and the N333 fragment. 6×his-tagged proteins were purified from baculovirus-infected insect cells using either nuclear (WRN, D82A, E84A, K577M, mock control) or cytosolic (N333, mock control) extracts. WRN, ... Fig. 2 Helicase and exonuclease activities of wild-type and mutant WRN proteins. a, WRN, K577M, E84A, D82A or mock proteins (10 ng) were assayed for helicase activity by incubating 5′ 32P-labelled DNA substrate (0.4 pmol; Fig. 1a) in helicase assay buffer ... WRN exonuclease activity resided in the N terminus. N333, which was essentially free of contaminating 5′→3′ exonuclease, degraded 5′ and 3′ labelled substrates similarly to full-length WRN (Fig. 1c,d). When incubated with a 374-bp DNA fragment labelled at the 3′ end with 32P, and internally with 3H, N333 released most of both labels (Fig. 1e). Thus, the WRN exonuclease is capable of substantial DNA degradation. Consistent with 3′→5′ directionality, N333 released 32P from 3′ ends more rapidly than 3H from internal residues. In addition, gel-purified N333, which lacked contaminating nuclease activities, efficiently removed the 3′, but not the 5′, label when incubated with DNA substrates labelled at either the 3′ or the 5′ end (Fig. 1f). Genetic evidence for WRN exonuclease activity was obtained by introducing point mutations at critical amino acids in the exonuclease domain (D82A and E84A). These mutants retained the wild-type level of helicase activity (Fig. 2a), but had little or no 3′→5′ exonuclease activity, using either a 5′ (Fig. 2b) or 3′ (Fig. 2c) 32P-labelled substrate, and were indistinguishable from mock protein in this regard (Fig. 2d). The K577M mutant, in contrast, was devoid of helicase activity (Fig. 2a), as expected, but had 3′→5′ exonuclease activity comparable to that of wild-type WRN (Fig. 2b–d). Our data indicate that WRN is indeed a 3′→5′ exonuclease. This activity resides in the N terminus, and is physically and functionally separable from the helicase activity. The identification of an exonuclease activity in WRN clearly distinguishes it from other human RecQ-like helicases, and may help explain the differences between WS and BS. What are the functions of the WRN exonuclease in vivo? It may participate in recombination and DNA repair. Exonucleases are integral components of some recombination pathways12, and WRN appears to have a role in recombination5,6,13. The finding that WS cells are hypersensitive to the DNA damaging agent 4-nitroquinoline-1-oxide14 suggests a role for WRN in DNA repair. Finally, WRN is homologous to FFA-1 (replication focus-forming activity 1) in Xenopus laevis15, raising the possibility that WRN may also be involved in DNA replication. In this context, the WRN exonuclease may provide 3′→5′ proofreading function to DNA polymerases that lack such activity. Whatever the case, an understanding of the functions of WRN exonuclease and their relationships to the other functions of WRN will lead to new insights into the molecular and cellular basis for WS and a subset of age-associated pathologies.

LMNA mutations in atypical Werner's syndrome

BACKGROUND Werners syndrome is a progeroid syndrome caused by mutations at the WRN helicase locus. Some features of this disorder are also present in laminopathies caused by mutant LMNA encoding nuclear lamin A/C. Because of this similarity, we sequenced LMNA in individuals with atypical Werners syndrome (wild-type WRN). METHODS Of 129 index patients referred to our international registry for molecular diagnosis of Werners syndrome, 26 (20%) had wildtype WRN coding regions and were categorised as having atypical Werners syndrome on the basis of molecular criteria. We sequenced all exons of LMNA in these individuals. Mutations were confirmed at the mRNA level by RT-PCR sequencing. In one patient in whom an LMNA mutation was detected and fibroblasts were available, we established nuclear morphology and subnuclear localisation. FINDINGS In four (15%) of 26 patients with atypical Werners syndrome, we noted heterozygosity for novel missense mutations in LMNA, specifically A57P, R133L (in two people), and L140R. The mutations altered relatively conserved residues within lamin A/C. Fibroblasts from the patient with the L140R mutation had a substantially enhanced proportion of nuclei with altered morphology and mislocalised lamins. Individuals with atypical Werners syndrome with mutations in LMNA had a more severe phenotype than did those with the disorder due to mutant WRN. INTERPRETATION Our findings indicate that Werners syndrome is molecularly heterogeneous, and a subset of the disorder can be judged a laminopathy.

Lessons from human progeroid syndromes

A number of human genes have been identified in which mutations can lead to the accelerated emergence of features of senescence. Studies of these genes, and of the functions of their protein products, may lead to a clearer understanding of the nature of senescence, and could provide clues for ways in which ageing might be retarded.

An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants

Abstract Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to this genotoxic agent, which are intermediate to those of wild-type and homozygous mutants. 4NQO is shown to induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the question of an enhanced sensitivity of the relatively prevalent heterozygous carriers to certain environmental genotoxic agents.

Accumulation of Werner protein at DNA double-strand breaks in human cells

Werner syndrome is an autosomal recessive accelerated-aging disorder caused by a defect in the WRN gene, which encodes a member of the RecQ family of DNA helicases with an exonuclease activity. In vitro experiments have suggested that WRN functions in several DNA repair processes, but the actual functions of WRN in living cells remain unknown. Here, we analyzed the kinetics of the intranuclear mobilization of WRN protein in response to a variety of types of DNA damage produced locally in the nucleus of human cells. A striking accumulation of WRN was observed at laser-induced double-strand breaks, but not at single-strand breaks or oxidative base damage. The accumulation of WRN at double-strand breaks was rapid, persisted for many hours, and occurred in the absence of several known interacting proteins including polymerase β, poly(ADP-ribose) polymerase 1 (PARP1), Ku80, DNA-dependent protein kinase (DNA-PKcs), NBS1 and histone H2AX. Abolition of helicase activity or deletion of the exonuclease domain had no effect on accumulation, whereas the presence of the HRDC (helicase and RNaseD C-terminal) domain was necessary and sufficient for the accumulation. Our data suggest that WRN functions mainly at DNA double-strand breaks and structures resembling double-strand breaks in living cells, and that an autonomous accumulation through the HRDC domain is the initial response of WRN to the double-strand breaks.

WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair.

Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ‐like helicase and is thought to participate in DNA double‐strand break (DSB) repair by non‐homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non‐homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild‐type (wt) WRN. WRN possesses both 3′ → 5′ exonuclease and 3′ → 5′ helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN–/–) complemented with either wtWRN, exonuclease‐defective WRN (E–), helicase‐defective WRN (H–) or exonuclease/helicase‐defective WRN (E–H–). The single E– and H– mutants each partially complemented the NHEJ abnormality of WRN–/– cells. Strikingly, the E–H– double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E– and H– single mutants increased HR to levels higher than those restored by either E–H– or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events.

Association of a polymorphic variant of the Werner helicase gene with myocardial infarction in a Japanese population

The Werner syndrome (WS) is a rare autosomal recessive progeroid syndrome characterized by the premature onset of multiple age-related disorders, including atherosclerosis, cancer, non-insulin-dependent diabetes mellitus (NIDDM), ocular cataracts and osteoporosis [Epstein et al., 1966]. The major cause of death (at a median age of 47) is myocardial infarction (MI) [Epstein et al., 1966]. The WS mutation involves a member (WRN) of the RecQ family of helicases and may perturb DNA replication, repair, recombination, transcription, or chromosomal segregation [Yu et al., 1996]. We now report data on 149 MI cases and age-matched controls suggesting that a polymorphic WRN variant is associated with increased risk for MI. Based on our data, homozygosity for a cysteine at amino acid 1367 (the most prevalent genotype) predicts a 2.78 times greater risk of MI (95% confidence intervals: 1.23 to 6.86). The variant was not significantly associated with NIDDM. The two alleles (cysteine vs. arginine) could influence helicase activity, turnover, macromolecular interactions or, alternatively, could be markers for haplotypes influencing WRN regulation or reflecting gene action at linked loci. However, given the caveats implicit in genetic association studies, it is imperative that the present results be replicated in independent populations.

WRN mutations in Werner syndrome

Werner syndrome (WS) is one of a group of human genetic diseases that have recently been linked to deficits in cellular helicase function. We review the spectrum of WS‐associated WRN mutations, the organization and potential functions of the WRN protein, and potential mechanistic links between the loss of WRN function and pathogenesis of the WS clinical and cellular phenotypes. Hum Mutat 13:271–279, 1999.