Proc. Indian natn Sci Acad. B69 No. 4 pp 625-640 (2003)

Molecular Basis of –s–s– the WWthe erner andanderner Hutchinson-Gilford Syndromes

JUNKO OSHIMA*, NANCY B HANSON and GEORGE M MARTIN Department of , University of W ashington, Seattle, WA 98195, USA

(Received on 17 July 2003; Accepted after r evision on 6 August 2003)

Segmental progeroid syndromes are members of a group of disorders in which affected individuals present various featur es suggestive of accelerated aging. The two best-known examples are the Werner syndro m e (WS; “ of the adult”) and the Hutchinson-Gilford Progeria syndrome (HGPS; “Progeria of child- hood”). The responsible for WS, WRN, was identified in 1996 and encodes a multifunctional nuclear protein with and domains. WS patients and cells isolated from the WS patients show various genomic instability phenotypes, including an incr eased incidence of . The WRN protein is thought to play a crucial role in optimizing the regulation of DNA repair processes. Recently, a novel r ecurr ent in the LMNA gene has been shown to be responsible for HGPS. LMNA encodes nuclear intermediate filaments, A and C; mutant lamins are thought to result in nuclear fragility. Ther e ar e at least six other disor ders caused by LMNA , most of which affect cells and tissues of mesenchymal origins, including atypical forms of WS. The pathophysiologies of these and certain other progeroid syndromes indicate an important role for DNA damage in the genesis of common age- related disorders. Key WWKey ords: WWords: erner syndrome, WRN, RecQ, Hutchinson-Gilford Progeria syndrome, LMNA, , Genomic instability, Aging, Human

Introduction of WS, previously based upon clinical criteria, can Segmental progeroid syndromes encompass a now be confirmed by molecular biological methods. group of disorders characterized by an apparent The most striking example of a segmental acceleration of , in that multiple progeroid syndrome with an onset in childhood is phenotypes associated with the last decades of life the Hutchinson-Gilford Progeria syndrome appear during early and mid-life. (HGPS), also known as Progeria. Three independ- The most striking example of an adult-onset ent groups have now shown that the syndrome is segmental progeroid syndrome is the Werner caused by a common mutation at the LMNA syndrome (WS; OMIM277700) (McKusick 1998). locus, which codes for nuclear intermediate WS was first r eported by Dr. Otto Werner in his filaments (De Sandre-Giovannoli et al. 2002, Cao medical doctoral thesis at Kiel University in 1908, et al. 2003, De Sandre-Giovannoli et al. 2003, but the gene responsible for WS, WRN, was not Eriksson et al. 2003). discovered until 1996 (Yu et al. 1996). The WRN Approximately 80-90% of cases with a clinical gene encodes a multifunctional nuclear protein diagnosis of WS have demonstrable mutations in the with both exonuclease and helicase activities. As a WRN gene. Our group has therefore been seeking result of the identification of WRN, the diagnosis mutations responsible for the remaining 10-20% of

Abbreviations: BAF, Barrier to autointegration factor; BER, ; DSB, double strand break; IR, Illegitimate recombination; HGPS, Hutchinson-Gilford Progeria Syndrome; HR, ; Lamin A/C, type A and C lamin; NER, Nucleotide excision repair; NHEJ, Non-homologous end joining; WS, . *Corr esponding Address: Email : picar [email protected]; Tel: (206) 616-4227; Fax: (206) 685-8356 626 Oshima et al. cases. To our surprise, we r ecently discover ed that increased up to 1000-fold for the case of acral mutant forms of LMNA, distinct from what has lentigenous . The ratio of carcinomas to been observed in HGPS, are responsible for a subset sarcomas is approximately 1:1, as opposed to 10:1 in of these “atypical” forms of WS. the general population. Thyroid carcinoma is the It is not known how mutations in the WRN and most commonly exhibited epithelial neoplasm. The LMNA cause progeroid features, much less most common sarcomas observed in WS patients why abnormalities of functionally different genes are soft tissue sarcomas, osteosarcomas, acral can sometimes lead to similar progeroid phenotypes lentigenous , and . The or whether these genes are involved at all in human occurr ence of multiple tumors is also common. A s aging pr ocesses. In this r eview, the curr ent many as five types of have been understanding of the pathogenesis of disabilities due observed in a single patient (Tsuchiya et al. 1991). to mutations at these two loci will be explored. In addition to the unusual types of , there are several other discrepancies between The WWThe erner Synmdrome W erner syndrome and usual normal aging. While Disease Course of Werner Syndr ome (WS)(WS)ome is typically observed, it is unusual in WS patients develop normally until the end of their that it especially affects the distal long bones. There first decade. The first symptom, often recognized are also unusual and characteristic osteolytic lesions retr ospectively, is short statur e r esulting fr om the lack of the distal joints of fingers. Deep, chronic ulcers of a growth spurt during the patient’s early teens. ar ound the ankles (malleoli and Achilles tendon) ar e Other clinical symptoms typically start toward the highly characteristic—almost pathogenomic. There end of the second decade of life and include gray is some controversy concern-ing the extent to which hair, alopecia (loss of hair), hoarseness or a high- the brain is affected. Central nervous system pitched voice, and degenerative changes of . complications associated with clearly These symptoms are typically followed in the third can occur, but while beta amyloid deposits have decade by bilateral ocular , type 2 been observed in patients bearing the APOE epsilon mellitus, , skin ulcers, and osteoporosis 4 susceptibility allele for dementia of the Alzheimer (Epstein 1966, Tollefsbol et al. 1984, Goto 1997). type (Leverenz et al. 1998), that disease is not part of WS patients exhibit several forms of arterio- the clinical picture (Martin et al. 1999). sclerosis (hardening of the arteries). Myocardial The fr equency of Werner syndrome can be infar ction, together with cancer, constitute the most expected to vary with the level of consanguinity in common causes of death, typically around the age populations, as it is caused by an autosomal recessive of forty-eight or about ten years after the typical mutation of WRN. In the Japanese population, the age of diagnosis. The most recent review by Goto frequency may range from about 1/20,000 to 1/ et al. (1996) shows that cancer is the most common 40,000 based upon the frequencies of detectible cause of death among Japanese WS patients. The heterozygous mutations (Satoh et al. 1999). The chronological onset of these symptoms is similar in prevalence in the US population is not known, but has all WS cases regardless of the type of underlying been estimated to be on the order of 1/200,000 genetic mutations (Epstein 1966, Tollefsbol et al. (Martin et al. 1999). WRN mutation heterozygotes do 1984, Goto 1997). not appear to be at incr eased risk for any Werner The mean age of cancer detection in WS syndrome-specific symptoms, but this question patients is 44 years of age, but there is a very wide requir es much more study. The male-to-female ratio range (25-64 years of age). A variety of benign and of WS patients is believed to be 1:1. In our malignant neoplasms have been observed International Registry of Werner Syndrome, females (Epstein 1966, Tollefsbol et al. 1984, Goto 1997). The are slightly over-represented, possibly due to a spectr um of cancers is unusual, however, in that clinical ascertainment bias. there are large numbers of sarcomas and some very Clinical Diagnosis of Werner Syndr omeomeome rare types of cancers (Goto et al. 1996). The overall The median age of WS diagnosis has been cancer risk is increased 30- to 50-fold over that of determined in two separate studies to be around the general population and tumor specific risks are thirty-eight years of age (Epstein 1966, Tollefsbol Molecular Basis of Progeroid Syndromes 627

et al. 1984, Goto 1997). By middle age, WS patients Possible: Either cataracts or dermatological develop gray hair; baldness; wrinkled, tight skin; alterations and any four others. regional loss of subcutaneous fat; ocular cataracts; Exclusion: Onset of signs and symptoms mellitus; osteoporosis; atheros - before (except stature, since clerosis; medical ; arteriolar sclerosis; current data on pre-adolescent growth of gonads; and certain types of cancers. patterns are inadequate.) It should be emphasized that, unlike HGPS, the Goto proposes the clinical diagnosis of Werner onset of WS-related symptoms occurs after syndrome if four of five of the following criteria adolescence (with the exception of short stature). are present (Goto 1997): The following diagnostic criteria have been 1. Consanguinity. proposed (Nakura et al. 1994). They were used 2. Characteristic “bird-like” appearance and prospectively for the successful mapping and body habitus. positional cloning of the WRN locus (Yu et al. 1996). 3. Premature senescence. Cardinal signs and symptoms (onset over 10 4. -like skin changes. years old): 5. Endocrine-metabolic disorders. 1. Cataracts (bilateral). An increased production of urinary hyaluronic 2. Characteristic dermatological pathology acid is observed in most cases of Werner syndro m e (tight skin, atrophic skin, pigmentary (Tollefsbol et al. 1984, Goto 1997). Historically, alterations, ulceration, hyperkeratosis, elevated urinary hyaluronic acid production was regional subcutaneous atrophy) and used to support a clinical diagnosis. Due to the typical facies (‘bird’ facies). availability of modern molecular testing, however, 3. Short stature. such measures ar e rarely used today. Likewise, an 4. Parental consanguinity (3rd cousin or increase in chromosomal aberrations (i.e., greater) or affected sibling. variegated translocation mosaicism) (Hoehn et al. 5. Premature graying and/or thinning of 1975) has been observed in cells of WS, but is not scalp hair. currently considered among the standard 6. (Elevated 24-hour urinary hyaluronic acid diagnostic criteria. Other standard laboratory test, when available). tests such as alteration in blood lipids, while informative, are not specific for WS. Further signs and symptoms: 1. Diabetes mellitus (Type 2). WRN Gene Product and WRN Mutations 2. Hypogonadism (secondary sexual The Wildtype WRN Protein underdevelopment, diminished , The WRN gene consists of 35 that encode a testicular or ovarian atrophy). multifunctional nuclear protein of 1,432 amino 3. Osteoporosis. acids (Yu et al. 1996). The central r egion of the 4. Osteosclerosis of distal phalanges of WRN protein contains the consensus domains of RecQ type (Gray et al. 1997) and the fingers and/or toes (x-ray diagnosis). N-terminal region contains exonuclease domains 5. Soft tissue calcification. (Huang et al. 1998) (figure 1). There is a nuclear 6. Evidence of premature localization signal at the C-terminal end of the (e.g. history of myocardial infarction). protein (Matsumoto et al. 1997). Between the 7. Mesenchymal neoplasms, rare neoplasms exonuclease and helicase domains, there is a highly or multiple neoplasms. acidic transactivation sequence (Balajee et al. 1999). 8. V oice changes (high pitched, squeaky or There are two consensus domains in the C-terminal hoarse voice). region whose functions have not been completely 9. Flat feet. elucidated: 1) a RecQ C-terminal conserved region Diagnosis: speculated to be involved in protein-protein Definite: All the car dinal signs and two others. interactions, and 2) a helicase RNaseD C-terminal Probable: The first three cardinal signs and any (HDRC) conserved region speculated to be two others. involved in protein-DNA interactions. 628 Oshima et al.

There are at least four non-synonymous WRN activities during DNA metabolism. Neither RecQL polymorphisms (Castro et al. 1999, Passarino et al. nor RecQ5 have yet been associated with any 2001), two of which show evidence of a role in human disease. altering the risks of certain age-related human WRNWRNWRN Mutations in WS Patients disorders. The first, Phe homozygosity at amino The mechanism by which WRN mutations cause the acid 1076, may be associated with reduced WS phenotype is not clear. All of the mutations longevity and an age-dependent risk of identified to date result in truncation of the WRN atherosclerosis when compared to the common protein and the loss of the nuclear localization wildtype allele (Leu) (Castro et al. 2000). The signal at the C-terminal region of the WRN protein second, A r g at amino acid 1367, has been (Moser et al. 1999). The known mutations associated with a decreased risk of myocardial correspond either to stop codons, insertions, or infarction when compared to the common allele deletions that result in frame shifts or splicing (Cys) (Ye et al. 1997). donor/acceptor site mutations, ultimately causing RecQ type helicases ar e A TP-dependent 3’?5’ one or more exons to be skipped (figure 2). helicases that belong to the DEAH (Asp-Glu-Ala- Dominant negative forms of WRN have not been His) subfamily of DNA and RNA helicases. identified in WS patients. Mutant mRNAs and the Originally identified as the RecQ of E. coli., RecQ resulting mutant proteins exhibit shorter half-lives helicases consist of seven canonical helicase motifs: than do the wild-type mRNA and WRN protein I (A TPase), Ia II, III, IV, V and VI. E. coli and yeast (Yamabe et al. 1997). Unlike the case for HGPS, have only one member of the RecQ helicase W erner syndrome is the only disease known to be whereas humans have at least five. Other human associated with mutations at the WRN locus. RecQ helicases include RecQL1, BLM (mutations at The specific cell type in which WS-associated which are responsible for the ), cancers develop may differ depending on the type RecQ4 (mutations at which are responsible for the of mutation in the WRN gene. Whereas papillary Rothmund-Thomson Syndrome; RTS) and RecQ5 carcinoma has been associated with an N-terminal (figure 1)(reviewed by Oshima (Martin et al. 2000, mutation, follicular carcinoma is more frequently Oshima 2000, Oshima 2000)). The unique feature of the WRN helicase is that it has the exonuclease domain on the same protein. E. coli RecQ helicase activity is associated with 3' →5' exonuclease activity (Phillips et al. 1988, Myers et al. 1995) raising the possibility that WRN evolved to ranscriptional Activation 1 78781 219219219 TTTranscriptional569569569 Helicase, Activation859859859 3'->5', ATP-DependentHRDC ConservedNuclear143214321432 LocalizationDomain efficiently coordinate exonuclease and helicase 3'->5'219219 and 5'->3' Exonuclease RecQ Conserved Domain

Helicase domain I Ia II III VI V Human RecQL 649aa mutation Human BLM 1,417aa Insertion/ Exonuclease domain I II III Splicing mutation Human WRN 1,432aa Human RecQ4/RTS 1,208aa Figure 2.2.Figure Functional domains and disease mutations of the WRN gene product. Arabic numerals above Human RecQ5 410aa the rectangle indicate the segments of amino acid S. cerevisiae Sgs1 1,477aa numbers of the WRN protein. Roman numerals indicate exonuclease or helicase domains. The 1,328aa S. pombe Rqh1 functions of the RecQ conserved domain (green) and E.coli RecQ 610aa the helicase RNaseD C-terminal (HRDC) conserved domain (purple) have not yet been determined. WS Figure 1. Human RecQ helicases. The yellow mutations are grouped by type below the main figure. rectangles represent the five human RecQ helicases. The green symbol indicates the most frequent WRN Helicase domains are shown in red. Exonuclease mutation among Caucasians. Note that all the domains, shown in blue, are present only in the identified WS mutations eliminate the nuclear WRN protein. localization signal at the C-terminal region. Molecular Basis of Progeroid Syndromes 629

observed in association with C-terminal mutations sequence (Fry et al. 1999). However, in a process (Ishikawa et al. 1999). This finding is inconsistent which is stabilized by human r eplication pr otein A, with the assumption that all identified mutations WRN protein does appear to unwind up to 23 kb of within WRN result in loss of the nuclear localization a PCR-generated repeat sequence to signal of WRN protein, and thereby act as null single-stranded DNA(Ohsugi et al. 2000). WRN mutations. Further studies may reveal additional exonuclease also preferentially digests single correlations between specific genotypes and strands in complex DNA structures, such as double- phenotypes. stranded DNA with mismatched ends or bubbles Enzymatic Activities of WRN Protein (Oshima 2000, Shen et al. 2000). WRN helicase unwinds double-stranded DNA and Biochemical and cell biological studies suggest DNA-RNA hybrids, but its catalytic activities may that WRN protein is involved in DNA r epair, not be limited to those substrates. Several unusual recombination, replication, and as well DNA substrates have been tested as potential as complex functions such as DNA repair during physiological targets for WRN helicase activity replication (Oshima 2000). (figure 3). WRN protein is able to efficiently Roles of WRN Protein in Regulation of unwind double-stranded DNA with mismatch Recombination “bubbles.” It also unwinds G4 quartets made by The repair of double-strand breaks in eukaryotic two hairpin loops (G’2 biomolecular tetraplex) of cells occurs through one of two different d(CGG)n (Fry et al. 1999). Though its presence has mechanisms: 1) homologous recombination (HR), not been demonstrated in vivo , G4 quartets can or 2) illegitimate recombination (IR), also called potentially be formed from two GC-rich regions of nonhomologous end-joining (NHEJ). The latter unwound single-stranded DNA (or RNA) during process is error-prone as it requires little or no replication, r epair, r ecombination or transcription. D N A homology. The E. coli RecQ helicase has been Another interesting structure is the recombin- shown to initiate or to disrupt recombinational ation intermediate, or a-structure. WRN protein is intermediates (Harmon et al. 1998). Combined with able to promote branch migration of Holliday the variegated translocation mosaicism seen in WS junctions (Constantinou et al. 2000). In fact, the cells, a number of findings suggest a model in WRN helicase appears to more effectively dissociate which WRN protein participates both in the a-structure than the simple DNA duplex, as homologous recombination and in illegitimate assessed by the length of the migration. Double- recombination by modulating the structures of stranded DNA with mismatched tails is also a recombination intermediates (Saintigny et al. 2002). preferred substrate for WRN helicase (Suzuki et al. Several investigators have observed a prolonged 1997). These structures can be formed both during S-phase during the active . This may be recombination and replication, such as break- due to the mechanistic similarity of DNA strand- induced DNA replication or repair of a stalled processing in DNA replication, recombination/ replication. Recombinant WRN protein does not double-strand break r epair, and transcription unwind a G’2 tetraplex containing a telomere repeat (Kodadek 1998).

branch migration 40 mer gDNA 5'3' 10 10 M 13 Linear duplex Recombination Intermediates (α-structures) Tail (Suzuki et al. 1997) G'2 tetraplex Holliday junction (Fry et al. 1999) (Constantinou et al., 2000)

Figure 3.3.Figure Preferred substrates of WRN helicase. Double-stranded DNA with tail (left), G4 structure (center) and alpha structure during recombination (right) are shown.

Molecular Basis of Progeroid Syndromes 631

was higher than the control value of 2.8 aberrations in the growth hormone cascade do not years. Approximately 20% of the fathers appear to be primarily responsible for HGPS. were about 20 years older than mothers. Genetic regulation of general metabolism in HGPS Advanced paternal age is often seen in cells is similar to control cells. This includes the rate autosomal dominant mutations. of decline of thyroid hormone induction of (Na + 2. The lack of evidence of an increase in the K) A TPase in cell membranes during replicative frequency of parental consanguinity argues senescence (Guernsey et al. 1986) and replicative against an autosomal recessive inheritance. senescence-dependent declines of ornithine In an argument against autosomal recessive decarboxylase activity (Chen et al. 1986). inheritance, DeBusk (1972) reported that Markedly increased urinary hyaluronic acid the parents were related only in 3 out of 19 (HA) (Tokunaga et al. 1978, Kieras et al. 1986) has then-reported cases. Brown reported no been claimed to be characteristic to HGPS. A 17-fold consanguinity in 50 cases he examined mean increase of HA was reported in HGPS patients (Brown 2003). (Zebrower et al. 1986). These findings have recently 3. A report of monozygotic twins been challenged, however (Gordon et al. 2003). concordant for HGPS with 14 normal sibs V arious components of the extracellular matrix ar e also is consistent with more recent data in expressed differently in HGPS fibroblasts compared to controls. Elastin and type IV collagen were autosomal dominant inheritance (Brown markedly increased in early passage HGPS 1979). fibroblasts; this is observed in senescent normal Based on these findings, sporadic dominant fibroblasts (Sephel et al. 1988, Giro et al. 1993); there mutations of the fertilizing sperm or ovum were is variation for these phenotypes among HGPS suggested as causes of HGPS (Badame 1989). Rare fibroblasts, however (Colige et al. 1991). The instances of HGPS sibling cases (Maciel 1988, secretions of elastin and fibronectin were said to be Khalifa 1989) have been considered to be due to increased (Maquart et al. 1988). Unlike control cell germline mosaicism in the parents. lines, stimulation of elastin production by serum or Cell Biological Studies of HGPS Cells transforming growth factor (TGF)-beta in HGPS Primary fibroblasts isolated from HGPS have cells was only modestly increased, suggesting that diminished replicative capacities (Colige et al. 1991, elastin is near -maximally expressed. An increase in Saito 1991), but the limitation may not be as the expression levels of metalloproteinases, consistently severe as is observed in the WS collagens and stromelysin, and a decrease in tissue (Martin et al. 1970). The cumulative population inhibitor of metalloproteinase-1 (TIMP-1) gene doublings (CPD) of HGPS fibroblasts is limited to expression while undergoing replicative senescence approximately fifteen, while CPDs of control in HGPS fibroblasts was similar to that of senescent fibr oblasts ar e appr oximately forty. The r eplicative normal fibr oblasts (Millis et al. 1992). A marked senescence of HGPS cells appears to be associated increase of glycoprotein gp200 has been noted with an accelerated rate of loss of (Clark et al. 1993). This may reflect the cutaneous (Allsopp et al. 1992). and vascular alterations in HGPS patients. Another Several studies have shown altered growth- glyco-protein, decorin, was remarkably decreased related in HGPS. Reduced insulin at the transcriptional level in HGPS fibroblasts, but receptor gene expression has been shown in HGPS only in one of two patients (Beavan et al. 1993). We lymphoblastoid cell lines (LCLs) (Briata et al. 1991). can probably conclude that the overall expression Elevated c-myc, but not c-erbB or csrc, expression pattern of extracellular matrix components in HGPS has been reported (Nakamura et al. 1988) although fibroblasts is similar to senescent normal fibroblasts the significance is not known. Somatomedin-C and is more likely to be a consequence rather than a binding to its r eceptor, or the cellular r esponse to cause of the HGPS phenotype. somatomedin-C in HGPS cells, was similar to Genomic Instability in HGPS Cells contr ol cells (Conover et al. 1985). Although there There is some evidence to suggest genomic is resistance to growth hormone in HGPS patients, instability in cells derived from HGPS patients 632 Oshima et al.

(Goldstein et al. 1985). Unscheduled DNA α-helical coiled coil domains Globular synthesis following UV irradiation in HGPS cells Flexible Heptad Head Linker Globular has been independently reported by at least three domain repeat tail domain groups as being reduced (Stefanini et al. 1986, Lipman et al. 1989, Wang et al. 1990). The reduction of UV-induced, unscheduled DNA synthesis was G 608 G Common HGPS (1,2,3) 30-50% in HGPS cells as compared to control cells. G 608 S AG10801, HGPS (1,3) This suggests defective nucleotide excision repair E 578 V AG04110, WS, 13yo (5) R133L R 527 C HGPS (1) (NER) in HGPS. Lack of induction of NER prior to WS (4) L140R R 471 C HGPS (1) unscheduled DNA synthesis has been reported WS (4) (Lipman et al. 1989). E145K AG 10677, atypical HGPS, 19 yo (3) In contrast, the cell cycle-mediated regulation (1) Cao and Hegele 2003 (2) De Sandre-Giovannoli et al. 2003. of two-base excision repair (BER) enzymes, uracil (3) Eriksson et al. 2003 DNA glycosylase and hypoxanthine DNA (4) Chen et al. 2003. (5) Oshima unpublished. glycosylase, appear to be normal (Cool et al. 1990) Figure 5. Functional domains of lamin A and the sites of mutations identified in atypical WS and HGPS. The indicating that regulation of BER, at least, seems to diagram shows the functional and structural domains be intact. The results of assays for the sensitivity to of lamin A. Only mutations identified in atypical WS gamma rays and rejoining of double-strand DNA and HGPS are shown. breaks of HGPS cells following gamma irradiation 1998). Like other intermediate filaments, nuclear have been controversial (Little et al. 1975, Rainbow lamins possess a central a-helical coiled-coil rod et al. 1977, Arlett et al. 1980). The specific activities domain, flanked by globular N-terminal head and of DNA polymerase alpha, beta, and gamma appear C-terminal tail domains (figure 5). Hydrophobic to be normal in HGPS as well (Bertazzoni et al. repeats within the central rod domain promote 1978). Cytogenetic studies of HGPS have not formation of the a-helical coiled-coil dimer and shown consistent abnormalities (Gahan et al. 1984). charged residues along the surface of the coiled- Sister chromatid exchange frequencies did not coil dimer promote interactions between rod differ between HGPS and controls (Darlington et al. dimers, thus producing a complex assembly of the 1981). Transcriptional rates and transcriptional filaments (Stuurman et al. 1998). fidelity also appear to be normal (O’Brien et al. 1995). While lamin B is expressed in all cell types, lamin It is clear that we require much more research in A/C is developmentally regulated. Mouse order to assess the significance of genomic instability development can be divided into three phases: 1) in HGPS cells. A glaring deficiency is the lack of germ layer formation, 2) organogenesis, and 3) investigations of forward mutation rates in cultured tissue dif fer entiation. Lamin A/C expression starts somatic cells. Such studies have demonstrated 10- to during the third phase in the trunk, head and 100-fold increases in mutation rates in WS cells appendages (Rober et al. 1989). Lamin A / C (Fukuchi et al. 1989). expression does not appear until well after birth, The LMNA Gene Product and LMNA Mutations when it appears in the simple epithelia of lung, liver, Biology of Lamins kidney and intestine. In general, lamin A/C is The LMNA gene encodes type A and type C lamins expressed in differentiated cell types. In humans, (lamin A/C). These gene products r esult fr o m lamin A is most pr ominent in well-dif fer entiated alternative splicing (Fisher et al. 1986). Type A epithelial cells. Relatively undifferentiated cells and lamin (lamin A) is first translated as a 664 amino- proliferating epithelial cells show reduced acid pr elamin A; the 18 amino acid C-terminal is expressions of lamin A (Br oers et al. 1997). then cleaved to generate mature lamin A. Lamin A Potential Role of Lamins in Gene Regulation and lamin B (encoded by LMNB1 and LMNB2) are In addition to its structural role, the nuclear the major components of nuclear lamina, a 20~50nm envelope might be involved in the global pattern of mesh network which lines the inner nuclear gene expression. There is considerable circum- membrane of the (Stuurman et al. stantial evidence to support this hypothesis. Some Molecular Basis of Progeroid Syndromes 633

of the lamina-associated proteins have a so-called this case is not available (Brown et al. 2001). Another “LEM domain” which interacts with BAF (barrier mutation, a leucine (CTG) to arginine (CGG) to autointegration factor), an abundant chromatin- substitution at amino acid 140, was seen in the associated protein (Shumaker et al. 2001). Both N O RW A Y pedigr ee. All thr ee mutations were human and mouse studies indicate that the lamina heterozygous mutations ( figure 6). exerts a profound influence on the organization of Unlike classical WS, the symptomatic onset of heterochromatin in the nucleus (Fidzianska et al. atypical WS due to LMNA mutation is in the early 1998, Sullivan et al. 1999). As heter ochr omatin is teens. In the POR TU index case, poor growth was generally transcriptionally silent, changes in first noted at age 4. The cases of interest were heterochromatin structure could lead to changes in initially referred to us because of a general the gene expression patterns with potentially “prematurely aged” appearance, short stature, and deleterious ef fects. As a r esult, lamins may have a other features suggesting WS. Cardiac symptoms, direct effect on gene expression. osteoporosis, and muscular atrophy Transcriptional r egulators such as the appear to have been common features. Progression retinoblastoma gene product, a tumour suppressor, of these age-related disorders may be more have been shown to interact with lamin A in vitro. accelerated than in classical WS cases. Lamin A also interacts with ster ol response W e also found a thir d new mutation, which element binding protein, a transcriptional factor alters alanine (GCA) to proline (CCA) at amino acid that is involved in adipocyte differentiation (Lloyd 57 in Registry IRAN1010. This patient presented et al. 2002) . with features of the mandibuloacral dysplasia, LMNA Mutations in a typical WS and HGPS including sloping shoulders, small jaw and relatively The International Registry of Werner Syndro m e short fingers. The A57P mutation r esides at the N- has 127 WS index cases and 72 family members terminal region of an a-helical domain. Since the from many parts of the world. WS referrals are by structure of the proline residue is incompatible with primary care physicians or specialists who suspect a WS diagnosis. Among 127 index cases, 29 cases were found to not have mutations at the WRN locus. In collaboration with Dr. Abhimanyu Garg at NORWAY 1010 the Southwestern Medical Center, University of *** PORTU8010 Texas at Dallas, we sequenced all 12 exons of LMNA with previously published primers (Brown et al. 2001), and identified three new mutations in the LMNA gene (Chen et al. 2003). A base substitution in 2, which alters amino acid 133, arginine (CGG) to leucine (CTG), was seen in two pedigrees (ATLAN and PORTU). In the P O RTU pedigree, family samples were available and we did not find this mutation in any of the family members, indicating that R133L mutation is a de novo mutation. In the A TLAN pedigree, the father of the patient was clinically diagnosed with WS and died at age 49 as a result of insulin-resistant diabetes mellitus with pr ogr essive neur opathy, angiopathy and retinopathy, suggesting that the R133L mutation was G A T C inherited. An unaffected br other did not have this Figure 6 Figure 6 LMNA mutations identified in atypical WS. mutation. A similar mutation, R133P, has been Sequencing analysis shows a heterozygous Arg reported in one case of Emery-Dreifuss muscular (CGG)→eu (CTG) substitution at amino acid 133 and a dystr ophy, though a detailed clinical description of Leu (CTG)→Arg (CGG) substitution at amino acid 140. 634 Oshima et al.

A et al. 2003, De Sandre-Giovannoli et al. 2003, Eriksson et al. 2003). R T-PCR spanning exons 10 though 12 showed AG01972(HG)* * the expected bands in all samples and an additional smaller band only in HGPS patients (figure 7B). Sequencing of the smaller RT-PCR products revealed that the G608G (GGC →GGT) mutation generated a cryptic splicing site which results in an in-frame deletion of amino acid 607 through 656 (D G A T C mutation)(figure 7C). This deletion eliminates the pr oteolytic site that converts pr elamin A to matur e lamin A. Sequencing showed that the sequence of the thir d position of amino acid 608 was C and T, B indicating that the mutation created an incomplete cryptic splicing site. AG3259AAG3262A (HG)AG3506 (Control)AG3508 (HG) (Control) Other Caused by LMNA Mutations LMNA mutations are responsible for at least six disorders: Emery-Dreifuss muscular dystrophy (Bonne et al. 1999, Raffaele Di Barletta et al. 2000, Brown et al. 2001), dilated cardiomyopathy type 1A (Fatkin et al. 1999, Brodsky et al. 2000), limb-girdle wt LMNA muscular dystrophy type 1B (Muchir et al. 2000), ∆ mutant LMNA familial partial lipodystrophy (Cao et al. 2000, Muchir et al. 2000, Speckman et al. 2000, Garg et al. C 2002), Char cot-Marie-Tooth disease type 2 (Speckman et al. 2000), mandibuloacral dysplasia (Novelli et al. 2002) and HGPS (De Sandre- Giovannoli et al. 2002, Cao et al. 2003, De Sandre- Giovannoli et al. 2003, Eriksson et al. 2003, Genschel et al. 2000, Burke et al. 2002). As noted above, we have identified heterozygous LMNA mutations in a subset of “atypical” Werner syndr ome cases. These cases had some clinical featur es of Werner Figure 7.7.Figure LMNA mutation identified in HGPS. (A). syndrome but no detectable mutations in the WRN Sequencing analysis showing C →T mutation in exon helicase gene (Oshima, in press). 12. Due to the compression, C in the wildtype allele Most of the mutations found in autosomal was seen only faintly. (B). RT-PCR products showing smaller products only in HGPS patients. (C). Diagram Emery-Dreifuss muscular dystr ophy, familial of cryptic splicing sites generated by the G →T mutation. partial lipodystr ophy, and dilated car diomyopathy the a-helix, the Ala to Pro substitution is expected to are heterozygous missense mutations and have cause misfolding of lamin A/C. been extensively mapped throughout the LMNA Sequencing of genomic PCR products from the exons (Burke et al. 2002). LMNA mutations in limb- HGPS pedigrees showed a heterozygous mutation girdle muscular dystrophy with atrio-ventricular con- at G608G (GGC→GGT) in LMNA exon 12 in the duction disturbance are also heterozygous missense patients but not in the normal family members or mutations. Many of these are predicted to cause unrelated controls (figure 7A). This is consistent failur e of lamina assembly. A homozygous mutation with the three independent reports describing found in autosomal r ecessive Charcot-Marie-Tooth LMNA mutations in HGPS. All but one r esides in disease type 2 (Arg to Cys substitution at amino acid the C-terminal region of the protein (figure 5) (Cao 298) resides at a rod domain, which likely perturbs Molecular Basis of Progeroid Syndromes 635

lateral interaction of lamin A (De Sandre-Giovannoli 2. Altered chromatin silencing: many lamina et al. 2002). A homozygous mutation had been associated pr oteins bind to BAF, an previously been identified in Italian mandibuloacral abundant chromatin associated protein. dysplasia patients (Arg to His substitution at amino Though the function of BAF is not well- acid 527) (Novelli et al. 2002). understood, abnormal lamin protein could alter global transcriptional regulation PostulaPostulaPostulated Disease Mechanism of LMNA (Fidzianska et al. 1998, Sullivan et al. 1999, MutationsMutationsMutations Shumaker et al. 2001). Disease mutations of laminopathies are predicted to cause misfolding of lamin A/C or a failur e to 3. Mislocalization of nuclear pr oteins. Lamins A dimerize and assemble the nuclear lamina, resulting and C act to regulate a number of critical in nuclear structural abnormalities and nuclear nuclear components whose activities may be fragility (Burke et al. 2002). The major fraction of important for the cell growth (Markiewicz lamin A is localized along the inner surface of the et al. 2002). nuclear envelope and co-localizes with lamin B. Some Future Directions of Research on Progeroid of the lamin A mutants show a dramatically Syndromes abnormal intranuclear localization of lamins. In one WS and HGPS share at least one probable common study, four out of fifteen LMNA mutants (N195K, denominator—genomic instability. Genomic E358K, M371K and R386K) overexpressed in human instability of WS is the result of the absence of cells were observed to have decreased nuclear rim nuclear enzymes that participate in DNA repair staining in conjunction with the formation of processes, while that of HGPS is secondary to intranuclear fo ci (Ostlund et al. 2001). A n defective nuclear structure. Other segmental independent study also demonstrated that three out progeroid syndromes are caused by mutations in of four LMNA mutant proteins (L85R, N195K and genes involved in DNA repair . For example, the L530) overexpressed in Hela cells form nuclear , whose manifestation includes aggregates and cause the relocation of endogenous skeletal and neurological abnormalities, is caused wildtype lamin (Raharjo et al. 2001). Abnormal by a mutation in CSB that encodes a DNA helicase localization of mutant lamins was also shown to be involved in transcription-coupled DNA r epair. associated with the loss of nuclear envelope- Ataxia , a disorder causing skin associated emerin. The authors of both studies noted atrophy and , is caused by that LMNA mutations associated with Emery- mutations in AT M, which encodes a protein kinase Dreifuss muscular dystrophy and dilated involved in DNA damage signaling. These cardiomyopathy cause abnormal lamin localization observations collectively suggest that the inability and loss of emerin. Primary fibroblasts isolated to maintain genomic stability could lead to from an individual with mandibuloacral dysplasia progeroid phenotypes in humans. The phenotypic showed a honeycomb pattern of lamin A distribution variability of these syndromes may reflect the and lobulation of nuclei (Novelli et al. 2002). By differences in the regulation and specific functions contrast, lamin A/C carrying mutations associated of the affected genes. The fact that WRN mutations with familial partial lipodystrophy behave in a and LMNA mutations can give rise to very similar manner indistinguishable from that of wildtype phenotypes suggests that there are common lamin (Ostlund et al. 2001, Raharjo et al. 2001). downstream event. A task for the futur e is to W e can tentatively conclude that ther e ar e at carefully delineate these downstream events—for least three non-exclusive models for the example, via microarray analysis of gene pathogenesis of laminopathies: expression and pr oteomics. Ideally, this r esear ch 1. Increased nuclear fragility: general will require robust animal models. While such alterations in nuclear structure resulting models may exist for the case of HGPS (Mounkes from LMNA mutation leading to cell et al. 2003), attempts to reproduce WS in transgenic instability and ultimately tissue atrophy mice have been disappointing (Lebel et al. 1998, (Burke et al. 2002). Lombard et al. 2000, Wang et al. 2000). 636 Oshima et al.

The accumulation of somatic DNA damage is a Both WRN and HGPS mutations may also potential unifying theory of aging (Martin et al. 1996). impact upon gene expression. Direct evidence that Currently, the leading causative pr ocess is oxidative this may be the case for WS cells has been published damage. According to this theory, natural by- (Balajee et al. 1999). For the case of HGPS, there are products of aerobic energy production, such as strong theoretical rationales for how alterations in (ROS), cause damage to the regulation of gene expression may occur with biological macromolecules, including both nuclear lamin A mutations, as discussed earlier in the essay and mitochondrial DNA (Martin et al. 1996). DNA (e.g., altered chromatin silencing, mislocalization of damage caused by ROS induces base modifications, nuclear proteins). single-strand breaks, or double-strand breaks. Base Which of these two broad aberrations in gene modifications can be repaired by base excision repair action is most significant for our understanding of and nucleotide excision r epair. Transcriptional- the pathogenesis of WS and HGPS? Which is most coupled repair may be particularly important. significant for our understanding of normative Double-strand DNA breaks can be repaired either by aging? This is a core challenge for future research homologous recombination or non-homologous end on the pathobiology of aging. joining. Despite the observation that many disease Acknowledgements genes responsible for the progeroid syndromes W e thank Mr. Joel Janes for his excellent participate in DNA repair systems, which in itself is editorial assistance. This work is supported by surprising, a direct connection has not been grants from the National Institutes of Health established between ROS-induced DNA damage and (CA78088 and HD044782) and the Progeria each of the progeroid syndrome genes. Research Foundation.

ReferencesReferencesReferences Bonne G, Di Barletta M R, Varnous S, Becane H M, Allsopp R C, Vaziri H, Patterson C, Goldstein S, Y ounglai Hammouda E H, Merlini L, Muntoni F, Greenberg E V, Futcher A B, Gr eider C W and Harley C B C R, Gary F, Urtizber ea J A, Duboc D, Fardeau 1992 Telomere length pr edicts r eplicative capacity M, Toniolo D and Schwartz K 1999 Mutations in of human fibroblasts; Proc. Natl. Acad. Sci. U S A the gene encoding lamin A/C cause autosomal 898989 10114-10118 dominant Emery- Dreifuss muscular dystrophy; Arlett C F and Harcourt S A 1980 Survey of Nat. Genet. 212121 285-288 radiosensitivity in a variety of human cell strains; Briata P, Bellini C, V ignolo M and Gherzi R 1991 Insulin Cancer Res. 404040 926-932 receptor gene expression is reduced in cells from a Badame A J 1989 Progeria; Arch. Dermatol. 125125125 progeric patient; Mol. Cell Endocrinol. 757575 9-14 540- 54 4 Brodsky G L, Muntoni F, Miocic S, Sinagra G, Sewry C Balajee A S, Machwe A, May A, Gray M D, Oshima and Mestroni L 2000 Lamin A/C gene mutation J, Martin G M, Nehlin J O, Brosh R, Orren D K associated with dilated cardiomyopathy with variable and Bohr V A 1999 The Werner syndro m e skeletal muscle involvement; Circulation 101101101 473-476 protein is involved in RNA polymerase II Br oers J L, Machiels B M, Kuijpers H J, Smedts F, van den transcription; Mol. Biol. Cell 101010 2655-2668 Kieboom R, Raymond Y and Ramaekers F C 1997 A- Beavan L A, Quentin-Hoffmann E, Schonherr E, and B-type lamins are differentially expressed in normal Snigula F, Leroy J G and Kr esse H 1993 Deficient human tissues; Histochem. Cell Biol. 107107107 505-517 expression of decorin in infantile progeroid Brown C A, Lanning R W, McKinney K Q, Salvino A R, patients; J. Biol. Chem. 268268268 9856-9862 Cherniske E, Crowe C A, Darras B T, Gominak S, Bertazzoni U, Scovassi A I, Stefanini M, Giulotto E, Greenberg C R, Grosmann C, Heydemann P, Mendell J Spadari S and Pedrini A M 1978 DNA R, Pober B R, Sasaki T, Shapir o F, Simpson D A, polymerases alpha beta and gamma in inherited Suchowersky O and Spence J E 2001 Novel and recurrent diseases affecting DNA repair; Nucleic. Acids Res. mutations in lamin A/C in patients with Emery- Dreifuss 5 5 2189-2196 muscular dystrophy; Am. J. Med. Genet. 102102102 359-367 Molecular Basis of Progeroid Syndromes 637

Brown W T 1979 Human mutation affecting aging - a Cool B L and Sirover M A 1990 Proliferation-dependent review; Mech. Dev. 999 325-336 regulation of DNA glycosylases in progeroid cells; ______2003 Hutchinson-Gilford progeria syndrome; in Mutat. Res. 237237237 211-220 Chromosomal Instability and Aging. Basinc Cciecne Cooper M P, Machwe A, Orren D K, Brosh R M, Ramsden and Clinical Implications pp. 245-262 eds F Hisama, D and Bohr V A 2000 complex interacts with and S M Weissman and G M Martin (New York: Marcel stimulates the Werner pr otein; Genes. Dev. 141414 907-912 Dekker, Inc.) Darlington G J, Dutkowski R and Brown W T 1981 Sister Burke B and Stewart C L 2002 Life at the edge: the chromatid exchange frequencies in Pr ogeria and Werner nuclear envelope and human disease; Nat. Rev. syndrome patients; Am. J. Hum. Genet. 333333 762-766 Mol. Cell Biol. 333 575-585 De Sandre-Giovannoli A, Bernard R, Cau P, Navarr o C, Cao H and Hegele R A 2000 Nuclear lamin A/C R482Q Amiel J, Boccaccio I, Lyonnet S, Stewart C L, Munnich mutation in canadian kindreds with Dunnigan- type A, Le Merrer M and Levy N 2003 Lamin a truncation familial partial lipodystrophy; Hum. Mol. Genet. 999 in Hutchinson-Gilford progeria; Science 300300300 2055 109-112 ______, Chaouch M, Kozlov S, Vallat J M, Tazir M, Kassouri ______and ______2003 LMNA is mutated in Hutchinson- N, Szepetowski P, Hammadouche T, Vandenberghe A, Gilford progeria (MIM 176670) but not in Stewart C L, Grid D and Levy N 2002 Homozygous W iedemann-Rautenstrauch progeroid syndro m e defects in LMNA, encoding lamin A/C nuclear- (MIM 264090); J. Hum. Genet. 484848 271-274 envelope proteins, cause autosomal recessive axonal Castro E, Edland S D, Lee L, Ogburn C E, Deeb S S, neuropathy in human (Charcot- Marie-Tooth disor der Brown G, Panduro A, Riestra R, T ilvis R, Louhija J, type 2) and mouse; Am. J. Hum. Genet. 707070 726-736 Penttinen R, Erkkola R, Wang L, Martin G M and DeBusk F L 1972 The Hutchinson-Gilford progeria Oshima J 2000 Polymorphisms at the Werner locus: syndrome. Report of 4 cases and review of the II. 1074Leu/Phe, 1367Cys/Arg, longevity, and literatur e; J. Pediatr . 808080 697-724 atherosclerosis; Am. J. Med. Genet. 959595 374-380 Epstein C J, Martin, G M, Schultz A L, Motulsky A G ______, Ogburn C E, Hunt K E, Tilvis R, Louhija J, Penttinen 1966 Werner ’s syndr ome: a r eview of its R, Erkkola R, Panduro A, Riestra R, Piussan C, Deeb symptomatology, natural history, pathologic featur es, S S, Wang L, Edland S D, Martin G M and Oshima J genetics and relationships to the natural aging 1999 Polymorphisms at the Werner locus: I. Newly process; Medicine 454545 172-221 identified polymorphisms, ethnic variability of 1367Cys/Arg, and its stability in a population of Eriksson M, Br own W T, Gordon L B, Glynn M W, Singer Finnish centenarians; Am. J. Med. Genet. 828282 399-403 J, Scott L, Erdos M R, Robbins C M, Moses T Y, Berglund P, Dutra A, Pak E, Durkin S, Csoka A B, Chen K Y, Chang Z F and Liu A Y 1986 Changes of Boehnke M, Glover T W and Collins F S 2003 Recurrent serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human de novo point mutations in lamin A cause Hutchinson- 423423 diploid fibroblasts; J. Cell Physiol. 129129129 142-146 Gilford progeria syndrome; Nature 423423423 293-298 Chen L, Lee L, Kudlow B A, Dos Santos H G, Sletvold Fatkin D, MacRae C, Sasaki T, Wolf f M R, Por cu M, O, Shafeghati Y, Botha E G, Gar g A, Hanson N B, Frenneaux M, Atherton J, Vidaillet H J, Jr ., Spudich Martin G M, Mian I S, Kennedy B K and Oshima J S, De Girolami U, Seidman J G, Seidman C, Muntoni 2003 LMNA mutations in atypical Werner ’s F, Muehle G, Johnson W and McDonough B 1999 syndrome; Lancet 362362362 (in press) Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy Clark M A and Weiss A S 1993 Elevated levels of and conduction-system disease; N. Engl. J. Med. glycoprotein gp200 in progeria fibroblasts; Mol. Cell 341341341 1715-1724 Biochem. 120120120 51-60 341341 Fidzianska A, Toniolo D and Hausmanowa-Petrusewicz Colige A, Roujeau J C, De la Rocque F, Nusgens B and Lapiere C M 1991 Abnormal gene expression in I 1998 Ultrastructural abnormality of sarcolemmal skin fibroblasts from a Hutchinson-Gilford patient; nuclei in Emery-Dreifuss muscular dystrophy Lab. Invest. 646464 799-806 (EDMD); J. Neurol. Sci. 159159159 88-93 Conover C A, Dollar L A, Rosenfeld R G and Hintz R Fisher D Z, Chaudhary N and Blobel G 1986 cDNA L 1985 Somatomedin C-binding and action in sequencing of nuclear lamins A and C reveals fibroblasts from aged and progeric subjects; J. Clin. primary and secondary structural homology to Endocrinol. Metab. 606060 685-691 intermediate filament proteins; Proc. Natl. Acad. Constantinou A, Tarsounas M, Karow J K, Brosh R M, Sci. U S A 838383 6450-6454 Bohr V A, Hickson I D and West S C 2000 Werner ’s Fry M and Loeb L A 1999 Human werner syndrome syndrome protein (WRN) migrates Holliday DNA helicase unwinds tetrahelical structures of the junctions and co- localizes with RPA upon r eplication fragile X syndrome repeat sequence d(CGG)n; J. arrest; EMBO Rep. 111 80-84 Biol. Chem. 274274274 12797-12802 638 Oshima et al.

Fukuchi K, Martin G M and Monnat R J, Jr. 1989 Mutator Huang S, Li B, Gray M D, Oshima J, Mian I S and Campisi phenotype of Werner syndr ome is characterized J 1998 The premature ageing syndrome protein, WRN, by extensive deletions; Proc. Natl. Acad. Sci. U S A is a 3'—>5' exonuclease; Nat. Genet. 202020 114-116 868686 5893-5897 Ishikawa Y, Sugano H, Matsumoto T, Furuichi Y, Miller Gahan P B and Middleton J 1984 Euploidization of human R W and Goto M 1999 Unusual features of thyroid hepatocytes from donors of different ages and both carcinomas in Japanese patients with Werner sexes compared with those fr om cases of Werner ’s syndrome and possible genotype-phenotype syndrome and progeria; Exp. Gerontol. 191919 355-358 relations to cell type and race; Cancer 858585 1345-1352 Garg A, Speckman R A and Bowcock A M 2002 Jones K L, Smith D W, Harvey M A, Hall B D and Multisystem dystrophy syndrome due to novel Quan L 1975 Older paternal age and fresh gene missense mutations in the amino-terminal head and mutation: data on additional disor ders; J. Pediatr . alpha-helical rod domains of the lamin A/C gene; 868686 84-88 Am. J. Med. 112112112 549-555 Khalifa M M 1989 Hutchinson-Gilford progeria syndrome: Genschel J and Schmidt H H 2000 Mutations in the report of a Libyan family and evidence of autosomal LMNA gene encoding lamin A/C; Hum. Mutat. 161616 recessive inheritance; Clin. Genet. 353535 125- 1 32 451-459 Kieras F J, Br own W T, Houck G E, Jr. and Zebr ower Giro M and Davidson J M 1993 Familial co-segregation M 1986 Elevation of urinary hyaluronic acid in of the elastin phenotype in skin fibroblasts from W erner ’s syndr ome and progeria; Biochem. Med. Hutchinson-Gilford progeria; Mech. Ageing Dev. Metab. Biol. 363636 276-282 707070 163-236 Kodadek T 1998 Mechanistic parallels between DNA Goldstein S, Wojtyk R I, Harley C B, Pollar d J W, replication, r ecombination and transcription; Trends Chamberlain J W and Stanners C P 1985 Protein Biochem. Sci. 232323 79-83 synthetic fidelity in aging human fibr oblasts; Adv. Lebel M and Leder P 1998 A deletion within the Exp. Med. Biol. 190190190 495-508 murine induces Gordon L B, Harten I A, Calabro A, Sugumaran G, sensitivity to inhibitors of topoisomerase and loss Csoka A B, Brown W T, Hascall V and Toole B P of cellular proliferative capacity; Proc . Natl . Acad . 2003 Hyaluronan is not elevated in urine or serum Sci U S A 959595 13097-13102 in Hutchinson-Gilford Progeria Syndrome; Hum. Leverenz J B, Y u C E and Schellenberg G D 1998 Genet. 113113113 178-187 Aging-associated neuropathology in Werner Goto M 1997 Hierarchical deterioration of body systems syndrome; Acta. Neuropathol. (Berl) 969696 421-424 in Werner ’s syndr ome: implications for normal Li B and Comai L 2000 Functional interaction between ageing; Mech. Ageing Dev. 989898 239-254 Ku and the werner syndrome protein in DNA ______, Miller R W, Ishikawa Y and Sugano H 1996 Excess end processing; J. Biol. Chem. 275275275 28349-28352 of rare cancers in Werner syndrome (adult progeria); ______and ______2001 Requirements for the Cancer Epidemiol. Biomarkers Prev. 555 239-246 nucleolytic processing of dna ends by the werner Gray M D, Shen J C, Kamath-Loeb A S, Blank A, syndrome protein-ku70/80 complex; J. Biol. Chem. Sopher B L, Martin G M, Oshima J and Loeb L A 276276276 9896-9902 1997 The Werner syndrome protein is a DNA Lipman J M, Applegate-Stevens A, Soyka L A and Hart helicase; Nat. Genet. 171717 100-103 R W 1989 Cell-cycle defect of DNA repair in Guernsey D L, Koebbe M, Thomas J E, Myerly T K progeria skin fibroblasts; Mutat. Res. 219219219 273-281 and Zmolek D 1986 An altered response in the Little J B, Epstein J and Williams J R 1975 Repair of induction of cell membrane (Na + K)ATPase by DNA strand breaks in progeric fibroblasts and aging thyroid hormone is characteristic of senescence human diploid cells; Basic Life Sci. 793-800 in cultur ed human fibroblasts; Mech. Ageing Dev. Lloyd D J, Trembath R C and Shackleton S 2002 A 333333 283-293 novel interaction between lamin A and SREBP1: Harmon F G and Kowalczykowski S C 1998 RecQ implications for partial lipodystrophy and other helicase, in concert with RecA and SSB proteins, laminopathies; Hum. Mol. Genet. 111111 769-777 initiates and disrupts DNA recombination; Genes Lombard D B, Beard C, Johnson B, Marciniak R A, Dev. 121212 1134-1344 Dausman J, Bronson R, Buhlmann J E, Lipman R, Hoehn H, Bryant E M, Au K, Norwood T H, Boman H Curry R, Sharpe A, Jaenisch R and Guarente L 2000 and Martin G M 1975 Variegated translocation Mutations in the WRN gene in mice accelerate mosaicism in human skin fibroblast cultures; mortality in a -null background; Mol. Cell Biol. Cytogenet. Cell Genet. 151515 282-298 202020 3286-3291 Molecular Basis of Progeroid Syndromes 639

Maciel A T 1988 Evidence for autosomal recessive Nakamura K D, Turturro A and Hart R W 1988 Elevated inheritance of progeria (Hutchinson Gilford); Am. c-myc expression in progeria fibroblasts; Biochem. J. Med. Genet. 313131 483-487 Biophys. Res. Commun. 155155155 996-1000 Maquart F X, Bellon G, Gillery P, Borel J P, Labeille B, Nakura J, Wijsman E M, Miki T, Kamino K, Yu C E, Risbourg B and Denoeux J P 1988 Increased secretion Oshima J, Fukuchi K, Weber J L, Piussan C, Melaragno of fibronectin and collagen by progeria (Hutchinson- M I and et al. 1994 Homozygosity mapping of the Gilfor d) fibr oblasts; Eur. J. Pediatr. 147147147 442 W erner syndrome locus (WRN); Genomics 232323 600-608 Markiewicz E, Dechat T, Foisner R, Quinlan R A and Novelli G, Muchir A, Sangiuolo F, Helbling-Lecler c A, D’Apice Hutchison C J 2002 Lamin A/C binding protein M R, Massart C, Capon F, Sbraccia P, Federici M, LAP2alpha is required for nuclear anchorage of Lauro R, Tudisco C, Pallotta R, Scarano G, Dallapiccola retinoblastoma protein; Mol. Biol. Cell 131313 4401-4413 B, Merlini L and Bonne G 2002 Mandibuloacral dysplasia Martin G M, Austad S N and Johnson T E 1996 Genetic is caused by a mutation in LMNA-encoding lamin analysis of ageing: role of oxidative damage and A/C; Am. J. Hum. Genet. 717171 426-431 environmental stresses; Nat. Genet. 131313 25-34 O’Brien M E and Weiss A S 1995 Hutchinson-Gilford ______and Oshima J 2000 Lessons from human progeria fibroblasts exhibit metabolically normal progeroid syndromes; Nature 408408408 263-266 uridine uptake and RNA synthetic rates; Biochem. ______, ______, Gray M D and Poot M 1999 What Biophys. Res. Commun. 210210210 225-230 geriatricians should know about the Werner Ogihara T, Hata T, Tanaka K, Fukuchi K, Tabuchi Y syndrome; J. Am. Geriatr. Soc. 474747 1136-1144 and Kumahara Y 1986 Hutchinson-Gilford ______, Sprague C A and Epstein C J 1970 Replicative progeria syndrome in a 45-year-old man; Am. J. life-span of cultivated human cells. Ef fects of donor ’s Med. 818181 135-138 age, tissue, and genotype; Lab. Invest. 232323 86-92 Ohsugi I, Tokutake Y, Suzuki N, Ide T, Sugimoto M and Matsumoto T, Shimamoto A, Goto M and Furuichi Y 1997 Fur uichi Y 2000 Telomere repeat DNA forms a Impaired nuclear localization of defective DNA helicases large non-covalent complex with unique cohesive in Werner ’s syndr ome; Nat. Genet. 161616 335-336 pr operties which is dissociated by Werner syndr o m e DNA helicase in the presence of replication protein McKusick V A 1998 Mendelian Inheritance in Man: A A; Nucleic. Acids. Res. 282828 3642-3648 Catalog of Human Genes and Genetic Disorders (Baltimore: The Johns Hopkins University Press). Oshima J 2000 Comparative aspects of the Werner syndrome gene; In. V ivo 141414 165-172 Millis A J, Hoyle M, McCue H M and Martini H 1992 Differential expression of metalloproteinase and ______2000 The Werner syndr ome protein: an update; tissue inhibitor of metalloproteinase genes in aged Bioessays. 222222 894-901 human fibroblasts; Exp. Cell Res. 201201201 373-379 ______, Brown W T and Martin G M 1996 No detectable M o rozov V, Mushegian A R, Koonin E V and Bork P mutations at Werner helicase locus in pr ogeria; 1997 A putative nucleic acid-binding domain in Lancet. 348348348 1106 Bloom’s and Werner ’s syndr ome helicases; Trends ______, Huang S, Pae C, Campisi J and Schiestl R H 2002 Biochem. Sci. 222222 417-418 Lack of WRN results in extensive deletion at Moser M J, Oshima J and Monnat R J, Jr. 1999 WRN nonhomologous joining ends; Cancer Res. 626262 547-551 mutations in Werner syndrome; Hum. Mutat. 131313 Ostlund C, Bonne G, Schwartz K and Worman H J 2001 271-279 Properties of lamin A mutants found in Emery-Dreifuss Mounkes L C, Kozlov S, Hernandez L, Sullivan T and muscular dystrophy, cardiomyopathy and Dunnigan- Stewart C L 2003 A progeroid syndrome in mice type partial lipodystrophy; J. Cell Sci. 114114114 4435-4445 is caused by defects in A-type lamins; Nature 423423423 Ozaki T, Saijo M, Murakami K, Enomoto H, Taya Y and 298-301 Sakiyama S 1994 Complex formation between lamin Muchir A, Bonne G, van der Kooi A J, van Meegen M, A and the retinoblastoma gene product: Baas F, Bolhuis P A, de Visser M and Schwartz K 2000 identification of the domain on lamin A required Identification of mutations in the gene encoding lamins for its interaction; 999 2649-2653 A/C in autosomal dominant limb girdle muscular Passarino G, Shen P, Van Kirk J B, Lin A A, De Benedictis dystrophy with atrioventricular conduction disturbances G, Cavalli Sforza L L, Oefner P J and Underhill P A (LGMD1B); Hum. Mol. Genet. 999 1453-1459 2001 The werner syndrome gene and global Myers R S, Kuzminov A and Stahl F W 1995 The sequence variation; Genomics 717171 118-122 recombination hot spot chi activates RecBCD Phillips G J, Prasher D C and Kushner S R 1988 Physical recombination by converting Escherichia coli to a and biochemical characterization of cloned sbcB and recD mutant phenocopy; Proc. Natl. Acad. Sci. xonA mutations from Escherichia coli K-12; J. USA 92 6244-6248 Bacteriol. 170170170 2089-2094 640 Oshima et al.

Raffaele Di Barletta M, Ricci E, Galluzzi G, Tonali P, Mora M, Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat Morandi L, Romorini A, Voit T, Orstavik K H, N, Nagashima K, Stewart C L and Burke B 1999 Merlini L, Trevisan C, Biancalana V, Housmanowa- Loss of A-type lamin expression compromises Petrusewicz I, Bione S, Ricotti R, Schwartz K, Bonne G nuclear envelope integrity leading to muscular and Toniolo D 2000 Different mutations in the dystrophy; J. Cell Biol. 147147147 913-920 LMNA gene cause autosomal dominant and Suzuki N, Shimamoto A, Imamura O, Kuromitsu J, Kitao autosomal recessive Emery-Dreifuss muscular S, Goto M and Furuichi Y 1997 DNA helicase activity dystrophy; Am. J. Hum. Genet. 666666 1407-1412 in Werner ’s syndr ome gene product synthesized in a Raharjo W H, Enarson P, Sullivan T, Stewart C L and Burke B baculovirus system; Nucleic Acids Res. 252525 2973-2978 2001 Nuclear envelope defects associated with LMNA Szamosi T, Szollar J, Meggyesi V, Wilhelm O, Bodanszky mutations cause dilated cardiomyopathy and Emery- H and Matyus J 1984 Serum cholesterol and Dreifuss muscular dystrophy; J. Cell Sci. 114114114 4447-4457 triglyceride levels in progeria as a model of ageing; Rainbow A J and Howes M 1977 Decreased repair of Mech. Ageing Dev. 282828 243-248 gamma ray damaged DNA in progeria; Biochem. Tokunaga M, Wakamatsu E, Sato K, Satake S, Aoyama Biophys. Res. Commun. 747474 714-719 K, Saito K, Sugawara M and Yosizawa Z 1978 Rober R A, Weber K and Osborn M 1989 Differ ential Hyaluronuria in a case of progeria. (Hutchinson- timing of nuclear lamin A/C expression in the various Gilfor d syndrome); J. Am. Geriatr. Soc. 262626 296-302 organs of the mouse embryo and the young animal: Tollefsbol T O and Cohen H J 1984 Werner ‘s syndr ome: a developmental study; Develop. 105105105 365-378 An underdiagnosed disorder resembling premature Saintigny Y, Makienko K, Swanson C, Emond M J, aging; Age 777 75-88 Monnat R J Jr 2002 Homologous recombination Tsuchiya H, Tomita K, Ohno M, Inaoki M and Kawashima resolution defect in Werner syndr ome; Mol. Cell A 1991 Werner ’s syndr ome combined with quintuplicate Biol. 22 6971-6988 malignant tumors: a case report and review of literature Saito H M, R E 1991 Immortalization of Werner syndro m e data; Jpn. J. Clin. Oncol. 212121 135-142 and progeria fibroblasts; Exp. Cell Res. 192192192 373-379 W ang L, Ogburn C E, War e C B, Ladiges W C, Youssoufian Satoh M, Imai M, Sugimoto M, Goto M and Furuichi Y H, Martin G M and Oshima J 2000 Cellular Werner 1999 Pr evalence of Werner ’s syndr ome heterozygotes phenotypes in mice expressing a putative dominant- in Japan; Lancet. 353353353 1766 negative human WRN gene; Genetics 154154154 357-362 Sephel G C, Sturrock A, Giro M G and Davidson J W ang S M, Nishigori C K, Zhang J M and Takebe H M 1988 Increased elastin production by progeria 1990 Reduced DNA-repair capacity in cells originating skin fibroblasts is controlled by the steady-state from a progeria patient; Mutat. Res. 237237237 253-257 levels of elastin mRNA; J. Invest. Dermatol. 909090 Yamabe Y, Sugimoto M, Satoh M, Suzuki N, 643-647 Sugawara M, Goto M and Furuichi Y 1997 Down- Shen J C and Loeb L A 2000 The Werner syndro m e regulation of the defective transcripts of the gene: the molecular basis of RecQ helicase- W erner ’s syndr ome gene in the cells of patients; deficiency diseases; Trends Genet. 161616 213-220 Biochem. Biophys. Res. Commun. 236236236 151-154 Shumaker D K, Lee K K, Tanhehco Y C, Craigie R and Yannone S M, Roy S, Chan D W, Murphy M B, Huang W ilson K L 2001 LAP2 binds to BAF.DNA complexes: S, Campisi J and Chen D J 2001 Werner syndro m e requirement for the LEM domain and modulation protein is regulated and phosphorylatedby DNA- by variable regions; Embo. J. 202020 1754-1764 dependent protein kinase; J. Biol. Chem. 276276276 Speckman R A, Garg A, Du F, Bennett L, Veile R, 38242-38248 Arioglu E, Taylor S I, Lovett M and Bowcock A M Y e L, Miki T, Nakura J, Oshima J, Kamino K, Rakugi H, 2000 Mutational and haplotype analyses of families Ikegami H, Higaki J, Edland S D, Martin G M and with familial partial lipodystrophy (Dunnigan Ogihara T 1997 Association of a polymorphic variant variety) reveal recurrent missense mutations in the of the Werner helicase gene with myocar dial globular C-terminal domain of lamin A/C; Am. J. infarction in a Japanese population; Am. J. Med. Hum. Genet. 666666 1192-1198 Genet. 686868 494-498 Stefanini M, Orecchia G, Rabbiosi G and Nuzzo F Yu C E, Oshima J, Fu Y H, Wijsman E M, Hisama F, Alisch 1986 Altered cellular response to UV irradiation R, Matthews S, Nakura J, Miki T, Ouais S, Martin G M, in a patient affected by premature ageing; Hum. Mulligan J and Schellenberg G D 1996 Positional cloning Genet. 737373 189-192 of the Werner ’s syndr ome gene; Science 272272272 258-262 Stuurman N, Heins S and Aebi U 1998 Nuclear lamins: Zebrower M, Kieras F J and Brown W T 1986 Urinary their structure, assembly, and interactions; J. Struct. hyaluronic acid elevation in Hutchinson-Gilford Biol. 122122122 42-66 progeria syndrome; Mech. Ageing Dev. 353535 39-46