The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960

Review Genetic alterations in accelerated ageing syndromes Do they play a role in natural ageing?

Monika Puzianowska-Kuznickaa,b,∗, Jacek Kuznickic,d

a Department of Endocrinology, Medical Research Center, Polish Academy of Sciences, 1a Banacha Street, 02-097 Warsaw, Poland b Department of Biochemistry, Medical Center for Postgraduate Education, 99 Marymoncka Street, 01-813 Warsaw, Poland c Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, 4 Trojdena Street, 02-109Warsaw, Poland d Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warsaw, Poland Received 12 July 2004; received in revised form 25 October 2004; accepted 26 October 2004

Abstract

The molecular mechanisms leading to human senescence are still not known mostly because of the complexity of the pro- cess. Different research approaches are used to study ageing including studies of monogenic segmental . None of the known progerias represents true precocious ageing. Some of them, including Werner (WS), Bloom (BS), and Rothmund–Thomson syndromes (RTS) as well as combined xeroderma pigmentosa– (XP–CS) are charac- terised by features resembling precocious ageing and the increased risk of malignant disease. Such phenotypes result from the of the encoding involved in the maintenance of genomic integrity, in most cases DNA . Defec- tive functioning of these proteins affects DNA repair, recombination, replication and . Other segmental progeroid syndromes, such as Hutchinson–Gilford progeria (HGPS) and Cockayne syndrome are not associated with an increased risk of cancer. In this paper we present the clinical and molecular features of selected progeroid syndromes and describe the potential implications of these data for studies of ageing and cancer development. © 2004 Elsevier Ltd. All rights reserved.

Keywords: Ageing; Progeroid syndromes; ; Lamin A; ; Genomic integrity

Contents

1. Introduction ...... 948 2. Hutchinson–Gilford progeria ...... 948 3. Werner syndrome ...... 950

∗ Corresponding author. Tel.: +48 22 5991755; fax: +48 22 5991975. E-mail address: [email protected] (M. Puzianowska-Kuznicka).

1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.10.011 948 M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960

4. ...... 952 5. Rothmund–Thomson syndrome...... 953 6. Cockayne syndrome ...... 954

7. Summary...... 956 Acknowledgements ...... 956 References...... 956

1. Introduction drome, the responsible is still not known. In this review we describe the clinical and molecular features Little is known about the pathophysiology of hu- of the best known and most well studied progeroid man senescence. It is thought to be a complex process syndromes, with special emphasis on the function of involving genetic and environmental factors, affecting the mutated syndrome-responsible proteins, and po- several physiological pathways. Identification of the tential implications for the development of malignancy genes for age-related disorders, such as circulatory sys- (Tables 1 and 2). tem disorders, diabetes mellitus, cancer, dementia, os- teoporosis, etc., is difficult because most of these disor- ders are polygenic and their occurrence is strongly re- 2. Hutchinson–Gilford progeria lated to modifying environmental factors. The other ap- proach to the study of ageing is to identify the genes re- It is estimated that this autosomal dominant dis- sponsible for age-related monogenic hereditary disor- ease occurs in 1:4–8 million births. Only approxi- ders – so-called progeroid syndromes. In 1978, Martin mately 100 cases have been described in medical jour- (1978) defined 21 criteria of human ageing: 3 pheno- nals. The first description of this disease was given typic alterations in cells and 18 phenotypic alterations by Hutchinson and independently by Gilford, in 1886. in tissue or the total organism. He selected 83 autosomal At birth, affected infants appear normal (only a few dominant, 70 autosomal recessive, 9 sex-linked heredi- cases of intrauterine disease presentation are known tary disorders, and 3 chromosomal syndromes as ‘seg- (Rodriguez, Perez-Alonso, Funes, & Perez-Rodriguez, mental progeroid syndromes’. To date, no progeroid 1999)). Typical manifestations develop gradually be- syndrome is known that exactly resembles ‘physiolog- ginning from sixth to twelfth month of life, and are ical’ ageing, therefore the name ‘segmental syndromes’ evident by the first or second year of life. There is lit- is fully justified. Not all of them shorten patients’ tle phenotypic variation: all affected children are short, lifespan. Most features of ordinary ageing are present have a similar facial appearance with midface hypopla- in Werner syndrome (WS) and Hutchinson–Gilford sia, micrognathia, prominent eyes, protruding ears with progeria (HGPS) patients. Most extensively studied the absence of earlobes, delayed closure of fontanelles are the progeroid syndromes caused by defective heli- and sutures, alopecia, prominent scalp veins, atrophic case proteins, including Werner (WS), Cockayne (CS), skin, delayed dentition, and decreased subcutaneous Rothmund–Thomson (RTS), and Bloom (BS) syn- fat, thin limbs with prominent stiff joints, coxa valga, dromes, and xeroderma pigmentosa (XP) and trichoth- generalised osteodysplasia with osteolysis and patho- iodystrophy (TTD). In contrast, the gene which mu- logic fractures, dystrophic nails and high-pitched voice. tation is responsible for Hutchinson–Gilford progeria, Metabolic, endocrine, serum lipid and immunologic LMNA, has just recently been associated with this dis- examinations show no uniform abnormalities. There ease, and therefore little is known about the molec- are no signs of precocious brain ageing. HGPS chil- ular mechanisms leading to this progeria phenotype. dren’s intelligence and emotional development are nor- In other cases, e.g. Wiedemann–Rautenstrauch syn- mal. The median age at death is 13.4 years, and death M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 949

Table 1 Selected progeroid syndromes – the summary of the genetic background Syndrome Gene Function Hutchinson–Gilford LMNA Lamin A Nucleus structure, mechanotransduction Werner WRN WRN DNA helicase DNA repair, recombination Bloom BLM BLM DNA helicase DNA repair, recombination Rothmund–Thomson RECQL4 RECQL4 DNA helicase Unknown, possibly as other helicases Cockayne CSA, CSB, XPB, XPD, CSA WD-repeat protein, Transcription-coupled DNA repair, transcription XPG CSB DNA helicase, XPB DNA helicase, XPD DNA helicase, XPG exonuclease

is usually the result of myocardial infarction or stroke. the observation of a case with 1q23 6 Mb deletion and Remarkably, no increase in cancer frequency is asso- two other cases of 1q20–24 inverted insertion in twins ciated with this syndrome. with progeria. In 2003 two reports describing muta- The first clues regarding the location of the gene tions within the lamin A/C gene (LMNA) in HGPS responsible for HGPS on 1q arose from patients were published simultaneously. Eriksson et

Table 2 Selected progeroid syndromes – the summary of the clinical signs Syndrome Age at presentation Clinical signs Length of life, typical cause of death Hutchinson–Gilford 6–12 months Short stature, midface hypoplasia, microg- 10–20 years, myocardial infarction, nathia, prominent eyes, alopecia, prominent stroke, no malignant disease scalp veins, atrophic skin, decreased subcuta- neous fat, osteoporosis with pathologic frac- tures, cardiovascular disease Werner Puberty Short stature, alopecia, atrophic skin, fat de- 40–50 years, myocardial infarction, posits on the trunk, trophic ulcerations of cancer the legs, diabetes mellitus type II, osteoporo- sis, juvenile bilateral cataract, hypogonadism, atherosclerosis, neoplastic disease Bloom At birth Short stature, narrow face, small mandible, Almost normal lifespan, cancer prominent nose and big ears, sensitivity to sunlight, teleangiectatic skin lesions, hyper- and hypopigmentation, variable degree of im- munodeficiency, diabetes mellitus, defective fertility, neoplastic disease Rothmund–Thomson 3–6 months Short stature, photosensitivity, polikiloderma, Normal lifespan, cancer hyperkeratosis, alopecia, cataract, reduced fertility, neoplastic disease (usually sarcoma) Cockayne CSA: 1–3 years, CSB: at Severe growth retardation with lack of sub- CSA: 20–40 years, CSB: 6–7 years, birth, XP–CS: first year cutaneous fat (so-called cachectic dwarfism), central nervous system deteriora- atrophic skin, sparse hair, progressive neu- tion, infections, no malignant disease, rodevelopmental abnormalities with micro- XP–CS: as CS, central nervous sys- cephaly, diffuse dysmyelination and calcium tem deterioration, cancer deposits in the cortex and basal ganglia, pro- gressive retinal atrophy and cataracts, sen- sorineural hearing loss, acute sun sensitivity. XP–CS – as above, and neoplastic disease 950 M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 al. (2003) revealed that 18 out of 20 classic HGPS ical sensitivity of the cell could be altered. Indeed, cases harboured an identical mutation, a GGC > GGT the experiments with lamin A/C-deficient mouse em- single-base substitution at position 1824 within exon bryo fibroblasts subjected to mechanical strain showed 11. Such substitution located to codon 608 did not alter defective nuclear mechanics and impaired mechani- the amino acid sequence of the encoded protein. Iden- cally activated gene transcription (Lammerding et al., tical mutation has been found by DSG et al. (2003) 2004; Worman & Courvalin, 2004). Other experiments in two patients who had a heterozygous C to T tran- show that cellular ageing of Hutchinson–Gilford proge- sition at nucleotide 1824. This change was not found ria syndrome fibroblasts is characterised by a period of in 300 control samples from healthy subjects or in the hyperproliferation and terminates with a large increase parents of the affected children. Further analysis re- in the rate of apoptosis (Bridger & Kill, 2004), and vealed that an additional cryptic donor splicing site has that fibroblast clones derived from HGPS donors fre- been introduced by this mutation in exon 11 at posi- quently fail to immortalise with telomerase despite the tion 1819–1820. As a result, lamin A encoded by the restoration of telomerase activity and the stabilisation mutated gene lacks 50 amino acids near the carboxy of telomere length (Wallis et al., 2004). Clearly, the cel- terminus, while lamin C also encoded by this gene is lular and molecular mechanisms that play a role in the normal. de Sandre-Giovannoli et al. also showed that pathophysiology of Hutchinson–Gilford progeria still both normal and truncated transcripts were produced await their explanation. by the same allele (T at position 1824). The mutation would then inhibit transcriptional processing of the nor- mal allele, acting as a dominant negative mutation. It is 3. Werner syndrome important to note that other LMNA mutations in HGPS patients have been described, namely R471C, R527C, The first written description of the clinical symp- G608S and 2036C>T (Cao & Hegele, 2003). toms of the syndrome was delivered by Werner in Lamin A/C proteins encoded by the LMNA gene 1904. It is estimated that, worldwide, there are cur- are ubiquitous components of the nuclear lamina, rently approximately 1300 people suffering from this a structure near the inner nuclear membrane and disease, which makes Werner syndrome the most com- the peripheral chromatin. The nuclear lamina is in- mon known progeroid syndrome. The majority of pa- volved in many processes that occur inside the nu- tients are Japanese. The reason for this concentration cleus, including chromatin organisation, cell cycle within one ethnic group is not known. Most proba- and apoptosis regulation, DNA and RNA process- bly it is a result of inbreeding, although other rea- ing, etc. Microscopic analysis of HGPS fibroblasts sons are also possible. WS is an autosomal recessive probed with antibodies directed against lamin A re- progeroid syndrome most closely resembling ‘physio- vealed abnormalities of the nuclear membrane. Sim- logical’ ageing. Still, the typical WS phenotype con- ilarly, HGPS lymphocytes exhibit nuclear size and sists not only of typical ageing signs but also of other shape alterations with envelope interruptions and chro- features not commonly present in normal ageing (such matin extrusions. It is important to note that a wide as skin ulcers around the ankles and elbows, atheroscle- spectrum of human disorders – Emery–Dreifuss and rotic changes mostly in arterioles, osteoporosis affect- limb girdle muscular dystrophies, dilated cardiomy- ing mostly long bones of the lower extremities, and opathy with conduction disease, autosomal recessive calcification of cardiac valves). WS patients develop axonal neuropathy, mandibuloacral dysplasia, famil- normally until they reach puberty. The first sign of the ial partial lipodystrophy, Greenberg skeletal dysplasia, disease is absence of the pubertal growth spurt, which Pelger–Huet anomaly, and finally Hutchinson–Gilford results in short stature in the affected adult. By the progeria – are all ascribed to LMNA mutations. It is third decade of life, premature greying, loss of hair not known why the abnormalities within this gene re- and skin atrophy become apparent. Subcutaneous fat is sult in diseases with such diverse phenotypes. This deposited mostly on the trunk. In contrast, extremities is partly because the function of lamins is still not look thin. Trophic ulceration of the legs, diabetes melli- completely determined. Muscle manifestation of some tus type II, osteoporosis, juvenile bilateral cataract, hy- LMNA-mutated phenotypes suggests that the mechan- pogonadism and atherosclerosis develop. Werner syn- M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 951 drome is a cancer-prone disease. Compared with the Shimamoto, Goto, & Furuichi, 1997). This uniformity general population, an excess of soft tissue sarcomas, of molecular events possibly accounts for the low phe- osteosarcomas, myeloid disorders and benign menin- notypic variation observed in WS patients. It is impor- giomas have been observed in WS patients. In Japanese tant to note that missense changes in the WRN gene are subjects an excess of thyroid cancers and melanomas also known, and are typically considered polymorphic was also observed. The ratio between cancers of epithe- changes. It is possible, though, that mutations of this lial origin and sarcomas of mesenchymal origin in WS type might also result in WS phenotype. patients is 1:1, while in the general population it is 10:1. Wild type WRN protein binds to the single-stranded In addition, the primary sites of the cancers are also not part of the longer strand of DNA duplex and moves in typical in WS patients. For example, melanomas fre- a3 → 5 direction. It unwinds DNA-DNA and DNA- quently develop in regions not exposed to the sun, and RNA duplexes, DNA triple helix, G2 tetraplexes, and osteosarcomas frequently localise to the lower extrem- G4 tetraplexes made by two hairpin loops of d(CGG)n ities, whereas in the general population they localise to (interestingly, the sequences that can form G4 struc- upper extremities (Goto, Miller, Ishikawa, & Sugano, tures are present in telomeres, among others), and pro- 1996; Ishikawa, Miller, Machinami, Sugano, & Goto, motes branch migration of a . With re- 2000). There is a suggestion that the site of mutation gard to its exonuclease activity, double-stranded DNA in the WRN gene might be linked to the type of cancer: with multiple base-pair mismatches and structures sim- papillary thyroid carcinomas seem to be more com- ilar to Holliday junctions are more susceptible to di- mon in patients with mutation in the N-terminal end of gestion by WRN than DNA without mismatches (for a the WRN protein, while follicular carcinomas tend to review of WRN see Chen & Oshima, 2002). develop in WS patients bearing mutations within the WRN interacts with several protein components C-terminal end of this protein (Ishikawa et al., 1999). of the DNA replication complex, such as proliferat- WS patients typically die in the fifth decade of life (at ing cell nuclear antigen (PCNA) and topoisomerase I an average age of 47 years), owing to cardiovascular (Lebel, Spillare, Harris, & Leder, 1999). It also inter- disease or cancer. acts with DNA polymerase ␦, so one of its functions The genetic hallmark of WS is genomic instability. might be the recruitment of this polymerase to the com- The defect responsible for this syndrome was tracked plex secondary structures of DNA and alleviation of down in 1996, when WRN (RECQL2) gene was cloned stalled DNA synthesis (Kamath-Loeb, Loeb, Johans- and its mutations in WS patients found (Yu et al., son, Burgers, & Fry, 2001). A direct interaction be- 1996). The gene is localised on 8p11–12 and en- tween WRN and Bloom syndrome helicase (BLM) has codes a 1432 amino acid protein (WRN) homologous been shown. Both proteins colocalised to nuclear foci, to RecQ subfamily helicases (for a review of RecQ he- and BLM inhibited the exonuclease activity of WRN licases in cancer and ageing see Bachrati & Hickson, (von Kobbe et al., 2002). The absence of both WRN and 2003; Furuichi, 2001; Karow, Wu, & Hickson, 2000; BLM proteins synergistically increased hypersensitiv- Mohaghegh & Hickson, 2002). Indeed, WRN func- ity of the cell to genotoxic agents and UV light, com- tions as a 3 → 5 DNA helicase (Gray et al., 1997), pared with cells with deletions of either WRN or BLM but additionally acts as a 3 → 5 exonuclease (Huang proteins alone. This suggests that the two proteins may et al., 1998). The presence of exonuclease domain both be involved in DNA repair in a complementary makes WRN protein different from other RecQ heli- fashion (Inamura et al., 2002). In WS cells decreased cases. Helicase and exonuclease domains do not over- telomeric DNA repair efficiency was observed (Kruk, lap. Approximately 40 different mutations have been Rampino, & Bohr, 1995). WRN protein has been found described so far in the WRN gene. Mutations are either on telomeres (Shiratori et al., 1999), where it is re- homozygous or compound. They are frameshift muta- cruited by TRF2, a telomeric repeat binding factor es- tions, nonsense mutations or large genomic deletions, sential for correct telomeric structure (Machwe, Xiao, and result in the production of truncated WRN protein & Orren, 2004). WRN was shown to unwind up to 23 kb lacking a nuclear localisation signal. Therefore, trun- of a PCR-generated telomere repeat sequence (Ohsugi cated WRN fails to translocate into the nucleus, and et al., 2000). Telomeres found in lymphoblastoid cell cannot perform its physiological function (Matsumoto, lines originating from WS patients are unstable and 952 M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 of varying length. In WS fibroblasts telomeres shorten ing. It is estimated that 1 in 100 Ashkenazi Jews is a quickly, and cells stop dividing while telomeres are still carrier of the defective gene. Clinically, the syndrome quite long. Overexpression of telomerase in these cells is characterised by growth deficiency of prenatal on- decreases the rate of their senescence (Wyllie et al., set and other features that develop later, such as sen- 2000). These findings suggest that WRN protein plays sitivity to sunlight, teleangiectatic skin lesions, hyper- an important role in telomere maintenance. WRN has and hypopigmentation, variable degree of immunode- been shown to suppress increased homologous and il- ficiency (decreased levels of immunoglobulin A and M legitimate recombination (Yamagata et al., 1998). with recurrent respiratory and gastrointestinal tract in- Although global repair ability of WS cells is nor- fections), diabetes mellitus and defective fertility (men mal (Fujiwara, Higashikawa, & Tatsumi, 1977), the are infertile, women have reduced fertility). Sufferers absence of the above described functions of WRN re- have a narrow, birdlike face, small mandible, prominent sults in defective replication, inefficient transcription- nose and big ears. Despite patients’ short stature, the coupled DNA repair, deficient mismatch repair, and extremities (especially upper ones) are disproportion- chromosome rearrangements such as deletions and ately long with large hands and feet. Compared with multiple translocations, as well as increased spon- the general population BS patients are at 150–300-fold taneous mutation and deletions generated by non- increased risk of a wide variety of malignancies, in- homologous hyper-recombination (Fukuchi, Martin, cluding blood cell-derived and epithelial cancers. The & Monnat, 1989; Fukuchi et al., 1985). Altogether, cancers arise unusually early, with leukaemia develop- these alterations result in major genomic instability ing at an average age of 22 years and solid tumours in and cancer predisposition (Bachrati & Hickson, 2003; the fourth decade of life. Interestingly, a clinical dif- Furuichi, 2001; Mohaghegh & Hickson, 2002; Moser ference between leukaemia patients with and without et al., 2000). Interestingly, a direct interaction between BS is that leukaemia in BS usually presents itself with WRN and has been shown. In the absence of WRN, leukopenia rather than leukocytosis. p53-directed apoptosis was attenuated (Spillare et al., The genetic hallmark of Bloom syndrome is an 1999), while overexpression of WRN potentiated p53- inability to suppress hyper-recombination. The fre- dependent apoptosis (Blander et al., 1999). p53-null, quency of events is in- WRN-defective double mutant transgenic mice devel- creased 10-fold in BS cells in comparison to nor- oped a variety of tumours not detected in either type mal cells. The gene responsible for this condition was of single mutant (Lebel, Cardiff, & Leder, 2001). The cloned in 1995 (Ellis et al., 1995). BLM is located overexpression of MYC oncoprotein in WS fibroblasts on (locus 15q26.1) and encodes 1417 or after WRN depletion led to cellular senescence. So amino acid BLM (RECQL3) protein homologous to direct up-regulation of wild type WRN by MYC in nor- RecQ helicases. BLM is a 3 → 5 DNA helicase that, mal cells may promote MYC-driven tumourigenesis by in addition to unwinding duplex DNA, is able to unwind preventing cellular senescence (Grandori et al., 2003). G4 tetraplexes, triple helix, Holliday junctions, bubbles Physiologically, such interactions may be of great im- and forked DNA. BLM is necessary for normal double portance for the prevention of the accumulation of ge- stranded break repair (Langland et al., 2002). netic aberrations that, if not removed (such as in Werner The alterations affecting BLM gene are missense, syndrome), can lead to premature senescence or neo- nonsense or frameshift mutations, and large genomic plastic transformation. deletions. All but missense mutations result in prema- ture translation termination and production of the trun- cated BLM protein. Like truncated WRN, shortened 4. Bloom syndrome BLM protein also fails to translocate to the nucleus since the nuclear localisation signal normally present in The first description of the syndrome, by Bloom, its C-terminus is deleted. Although BLM protein bear- was published in 1954. BS is an autosomal recessive ing a missense mutation is present in the nucleus, its disorder that may occur in different ethnic groups but function is abolished due to the lack of ATPase and/or is most common in Ashkenazi Jews (descendants of DNA helicase activity. The absence of physiological Eastern European Jews), owing to intensive inbreed- function of BLM results in a markedly elevated rate of M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 953 mutations, and a high frequency of chromosomal aber- p53-mediated apoptosis is defective in BS fibrob- rations and sister chromatid exchange (SCE), owing lasts and can be rescued by expression of the normal to excessive recombination. Expression of wild type BLM gene (Wang et al., 2001). Further inactivation of BLM in BS cells can decrease their high SCEs to nor- p53 prevented the death of damaged BS cells and de- mal levels. layed recruitment of BRCA1 to nuclear foci (Davalos BLM physically interacts with topoisomerase III␣ & Campisi, 2003). Expression of BLM in p53 wild (Johnson et al., 2000). Expression of a BLM deletion type cells causes an anti-proliferative effect that is not mutant defective in topoisomerase III␣ binding results present in p53-deficient cells. p53-mediated transacti- in intermediate SCE levels. This suggests the involve- vation is attenuated in BS fibroblasts (Garkavtsev, Kley, ment of both proteins in the regulation of recombina- Grigorian, & Gudkov, 2001). After hydroxyurea treat- tion in somatic cells (Hu et al., 2001). It is proposed that ment, p53 and BLM co-localised with each other and interaction of RecQ helicase with topoisomerase III␣ with RAD51 at sites of stalled DNA replication forks. might be a well conserved mechanism that disrupts re- The absence of p53 enhanced the rate of spontaneous combination intermediates between erroneously paired sister chromatid exchange in BS cells. These results nucleic acid molecules (Harmon, DiGate, & Kowal- indicate that p53 and BLM functionally interact during czykowski, 1999). Indeed, it has been shown that BLM resolution of stalled DNA replication forks (Sengupta and topoisomerase III␣ catalyse the resolution of a re- et al., 2003). Interactions between p53 and BLM pro- combination intermediate containing a double Holliday tein suggest that Bloom syndrome cancer-prone phe- junction. This mechanism prevents exchange of flank- notype may in part be a result of the deregulation of the ing sequences (Wu & Hickson, 2003). p53 tumour suppressor pathway. BLM interacts with single-stranded DNA-binding Interestingly, it has been shown that human BLM protein – . Such binding stim- can prevent premature ageing in yeast (Heo et al., ulates BLM protein helicase activity (Brosh et al., 1999). However, the role of this protein in ordinary 2000). BLM also interacts with RAD51, the protein human ageing remains to be elucidated. accumulating in nuclear foci that are thought to corre- spond to sites of recombinational repair (Wu, Davies, Levitt, & Hickson, 2001). This and the ability of BLM 5. Rothmund–Thomson syndrome to unwind G4 structures suggest the involvement of this protein in DNA replication and repair. BLM also Rothmund first described this syndrome in 1868. interacts with 5-flap endonuclease/5–3 exonuclease Only approximately 300 cases have been described (FEN-1), a genome stability factor involved in Okazaki since then in English language scientific journals. Some fragments processing and DNA repair. BLM protein sufferers are born with skeletal abnormalities such stimulates both the endonucleolytic and exonucleolytic as underdeveloped or absent thumbs and/or forearm cleavage activity of FEN-1 (Sharma et al., 2004). bones. Prominent forehead, prognathism and saddle BLM interacts with so-called BRCA1-associated nose make patients’ faces characteristic. Erythema- genome surveillance complexes (BASCs) which con- tous plaques accompanied by oedema appear on the tain BRCA1, among others. Many BASC-forming pro- cheeks at around age 3–6 months. Subsequently, polik- teins play a role in the recognition of damaged DNA ilodermatous changes with telangiectasia, skin atrophy or unusual DNA structures (Wang et al., 2000). This and regions of hypo- and hyperpigmentation develop. and the ability of BLM to unwind difficult DNA struc- Growth retardation results in dwarfism. Greying and tures further support the involvement of this protein in loss of hair, juvenile cataract, and hypogonadism with DNA repair. Thus, the absence of normal function of delayed puberty are further clinical signs of the dis- BLM in BS cells leads to increased recombination and ease. RTS sufferers are at increased risk of cancer. In defective DNA replication and repair. An important al- the majority of cases they suffer from osteosarcoma or teration observed in BS cells is decreased DNA ligase I skin cancer. The cancers develop at a younger age than activity (Runger & Kraemer, 1989), which can further would normally be expected. Even though some clini- contribute to the defective processing of DNA in these cal symptoms suggest precocious ageing, the patients’ cells. lifespan is not altered (providing that the neoplastic 954 M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 disease is diagnosed early and is adequately treated). tion groups: milder CSA and severe CSB. Some pa- Inheritance is autosomal recessive. tients with xeroderma pigmentosa phenotypes caused The disease is caused by abnormalities within the by mutations in the XPB, XPD, and XPG genes also RECQL4 gene localised on the long arm of chromo- present clinical features of CS (XPB-CS, XPD-CS, some 8 (position 8q24.3) (Kitao et al., 1999). As in the XPG-CS). Only approximately 25% of ‘pure’ CS pa- above-described WS and BS, this gene encodes another tients are diagnosed with CSA, the rest being diagnosed protein homologous to RecQ helicases. Its function has with CSB. There is marked variability in the clinical not been extensively studied, but based on its structural manifestation of this disease. In CSA the child’s devel- similarity to WRN and BLM helicases it is predicted to opment is normal during the first years of life, although be an ATP-dependent enzyme unwinding DNA struc- so-called ‘early CSA’ has also been described. CSB tures. manifests itself intrauterinally. The cardinal features Mutations of RECQL4 gene found in RTS patients of CS are: senile-like appearance (atrophic skin, sparse affect either the coding sequence or the splice junctions hair), severe growth retardation with a lack of sub- and frequently result in the production of truncated pro- cutaneous fat (cachectic dwarfism), progressive neu- tein. The correlation of mutation type with cancer de- rodevelopmental abnormalities with microcephaly, dif- velopment has been studied. Truncating RECQL4 mu- fuse dysmyelination and calcium deposits in the cor- tations were found in two-thirds of 33 RTS patients tex and basal ganglia, progressive ocular abnormali- examined. However, all RTS patients suffering from ties including retinal atrophy and cataract, sensorineu- osteosarcoma had such mutations. In other words, there ral hearing loss, and acute sun sensitivity. The appear- was a significant correlation between truncating muta- ance of cataract during the first 3 years of life indicates tions and the risk of osteosarcoma (Wang et al., 2003). the severe phenotype and predicts early death. Despite As in other syndromes associated with helicase dys- the sensitivity to ultraviolet radiation, only xeroderma function, RTS cells are characterised by marked ge- pigmentosa–Cockayne syndrome (XP–CS) sufferers nomic instability associated with chromosomal rear- have an increased frequency of skin cancers. CSA pa- rangements resulting in acquired somatic mosaicism. tients usually die in the second or third decade of life RECQL4 transcripts are down-regulated in cells from (the mean age of death is 12.5 years), while death in RTS patients (Kitao, Lindor, Shiratori, Furuichi, & Shi- CSB patients occurs much earlier, usually in the sixth to mamoto, 1999). Embryonic fibroblasts from transgenic seventh year of life. The most common cause of death mice with deletions within the RecQ helicase domain is deterioration of the central nervous system or a vari- of RECQL4 genes show a defect in cell proliferation ety of respiratory tract infections. However, XP–CS pa- (Hoki et al., 2003). tients (as all XP patients) are at 1000-fold increased risk The interactions of RECQL4 with other proteins are of developing skin cancer (basal cell carcinomas, squa- still unknown. Interestingly, RECQL4 mutations have mous cell carcinomas and melanomas), mostly in sun also been found in another inborn genetic defect – RA- exposed areas. Tumours occur by the fourth to eighth PADILINO syndrome characterised by growth retar- year of life and most patients die in the second or third dation, forearm, thumb and patella defects, infantile decade of life, owing to malignancy. diarrhoea and hypogonadism, but not by a significant The disease is caused by CSA or CSB mutation. CSA cancer risk (Siitonen et al., 2003). (ERCC8) defective in Cockayne syndrome group A was cloned in 1995. It is located on the long arm of (5q12.3) and encodes CSA protein be- 6. Cockayne syndrome longing to the family of WD-repeat proteins (Henning et al., 1995). CSB (ERCC6), mutations in which re- CS is another autosomal recessive segmental sult in Cockayne syndrome group B, was cloned in progeroid syndrome, first described by Cockayne in 1992 (Troelstra et al., 1992). It is localised to lo- 1936. It is estimated that a child suffering from CS cus 10q11, and encodes another member of the he- is born once in 250,000 live births. Cell fusion stud- licase family. Mutations found in CS genes are ho- ies have identified five CS complementation groups. mozygous or compound, missense, nonsense mutations Most sufferers belong to the ‘pure’ CS complementa- or deletions (Cao, Williams, Carter, & Hegele, 2004; M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 955

Colella, Nardo, Botta, Lehman, & Stefanini, 2000; Ren Based on the fact that xeroderma pigmentosa com- et al., 2003). Both proteins encoded by CS genes are plementation group A patients who are completely de- components of the transcription-coupled DNA repair ficient in nucleotide excision repair do not develop CS (TCR) subpathway of the nucleotide excision repair symptoms, it is suggested that, in addition to NER de- (NER) system. Briefly, TCR involves damage recog- fects, transcription deficiency contributes to the devel- nition by the stalled RNA polymerase II complex (Mu opment of CS clinical symptoms. This is supported by & San-car, 1997), local DNA unwinding, incision on the fact that TFIIH with which CS proteins interact dur- both sides and removal of damaged oligonucleotide, ing transcription-coupled repair, is not only a factor in- gap-filling DNA synthesis with the intact complemen- volved in DNA repair, but is also a transcription factor, tary strand as a template, ligation of the newly syn- and that RNA polymerase II-dependent transcription is thesised oligonucleotide and the resumption of RNA decreased in CS cell extracts (Balajee, May, Dianov, synthesis. More than 20 proteins are involved in this Friedberg, & Bohr, 1997). Abnormal transcription- process, including all XP proteins, mutations in which coupled repair of oxidative DNA damage was detected result in xeroderma pigmentosa types A through G, or in CS cells, but not, as mentioned before, in XPA cells a combined syndromes XPB-CS, XPD-CS and XPG- that are completely NER deficient (Leadon & Cooper, CS, and both CSA and CSB proteins, damage of which 1993). Defective TCR of oxidative damage was also results in Cockayne syndrome A or B, respectively (for found in XPG–CS cells but not in XPG cells (Cooper, review see de Boer & Hoeijmakers, 2000). The unwind- Nouspikel, Clarkson, & Leadon, 1997), and in XPB-CS ing step of TCR requires the presence of TFIIH com- and XPD-CS cells but not in XPB or XPD cells. In ad- plex. TFIIH was originally identified as a factor crucial dition, it has been shown that unrepaired 8-oxoguanine for transcription initiation. It consists of nine subunits blocked transcription by RNA polymerase II (Le Page including XPB and XPD, the two helicases that unwind et al., 2000). These data further support the notion that double stranded DNA in 3 → 5 and 5 → 3 direction, CS phenotype also results from the defective TCR of respectively. Subsequently, the damaged strand is ex- oxidative lesions. cised by the two exonucleases. The one restricting at An interesting feature of CS is the unchanged risk of the 3 side of the damage site is XPG. cancer, despite UV hypersensitivity. In fact, it has been After UV irradiation or oxidative DNA damage, demonstrated that cancer-predisposed Ink4a/ARF−/− CSA protein rapidly translocates to the nuclear matrix mice develop fewer neoplastic lesions if their CSB gene in a CSB-dependent manner. CSA interacts with CSB had been disrupted (Lu et al., 2001). Based on the data and p44 subunit of TFIIH (Henning et al., 1995). In regarding Cockayne syndrome and xeroderma pigmen- UV-irradiatedcells CSA co-localises with the phospho- tosa molecular defects, this can be explained by normal rylated form of RNA polymerase II engaged in RNA global genome nucleotide excision repair (GG-NER) in transcript elongation (Kamiuchi et al., 2002). It is pro- CS cells and abnormal GG-NER in XP cells. In fact, posed that CSA is required for efficient DNA repair GG-NER defect seems to be of crucial importance for only during the elongation stages of RNA polymerase neogenesis, since XPC patients develop cancers while II transcription (Tu, Bates, & Pfeifer, 1998). CSB inter- only their GG-NER but not their TCR function is af- acts with the stalled transcription complex (van Gool et fected. In other cases of XP both GG-NER and TCR are al., 1997) and plays a role in the recruitment of TFIIH defective, so XP patients, including XPB-CS, XPD-CS to the damaged DNA. CSB also induces chromatin re- and XPG-CS patients, suffer from neoplastic disease. modelling (Citterio et al., 2000). The conformational What kind of XPB, XPD or XPG mutations result changes in the stalled complex or its translocation from either in xeroderma pigmentosa or XP–CS is a matter the lesion site as well as chromatin remodeling may al- of interest. It has been reported that XPG-CS patients low CSA to attach and the repair complex to access had truncating mutations, while XPG patients had mis- the damaged DNA strand. Thus, both CSA and CSB sense mutations that resulted in the production of full- are components of RNA polymerase II-associated com- length XPG protein defective in its exonuclease func- plexes, and their possible role is to assist this poly- tion (Nouspikel, Lalle, Leadon, Cooper, & Clarkson, merase in dealing with transcription blocks and the re- 1997). The role of the C-terminal end of XPG protein sumption of transcription. in the development of CS phenotype is supported by 956 M. Puzianowska-Kuznicka, J. Kuznicki / The International Journal of Biochemistry & Cell Biology 37 (2005) 947–960 the fact that homozygous transgenic mice with XPG nomic damage accumulate with age, and genomic in- lacking the last 360 amino acids exhibit growth retar- stability characterises one of the age-related diseases dation and short lifespan, although this phenotype was – malignant disorder. Some clues to their potential in- milder than in XPG-null mice (Shiomi et al., 2004). volvement in normal ageing or in longevity promo- Individuals suffering from XPD-CS were shown to ex- tion come from the analysis of polymorphisms of their hibit unique XPD mutations. Even though the extent genes in different age groups. The analysis of a poly- of their NER defect was similar to XPD patients with morphism at amino acid 1367 of WRN protein (Cys other XPD mutations, their cells were extremely sensi- (TTG)/Arg(CTG) in WRN gene) in Finnish newborns, tive to UV light (van Hoffen et al., 1999). The reason centenarians and the general population were similar for such hypersensitivity was the introduction of DNA across age groups suggesting that there is no associa- breaks not in close proximity to the lesion site (as a tion of this WRN polymorphism with longevity. How- necessary NER step) but in other locations. Therefore ever, if there is no association with longevity, there in addition to their inability to remove oxidative dam- might be link between the gene and common age- age, XPD-CS cells break their own DNA after UV ir- related diseases. 1367 Cys/Arg seems to be associated radiation (Berneburg et al., 2000). Another mechanism with a variation in the risk of myocardial infarction responsible for the development of certain phenotypes in the Japanese population, with 1367 Arg allele being has been proposed namely the relative expression of less common but protective against this disease (Castro mutated XPB genes (Riou et al., 1999). Others have et al., 1999, 2000). Frameshift mutation in the cod- demonstrated that mutations found in two XPB-CS pa- ing region of the BLM gene has been found in geneti- tients decreased TFIIH transcriptional activity by pre- cally unstable sporadic gastrointestinal tumours (Calin, venting promoter opening (Coin, Bergmann, Tremeau- Herlea, Barbanti-Brodano, & Negrini, 1998). It might Bravard,& Egly, 1999). be of interest to check the sequence/function of the other proteins implicated in progeroid syndromes as- 7. Summary sociated with malignant disease in different human tu- mours characterised by marked genomic instability. A number of genes, including mitochondrial, Fas, Until now, there is many more data suggesting FasL, MHC class I, different lipid metabolism genes, the involvement of different lipid metabolism genes etc., have been studied in ordinary ageing. Some stud- than genome integrity genes in the control of human ies delivered negative results while others described lifespan. Although progress in resolving the molecular potential associations (Barzilai et al., 2003; Niemi et function of helicases and other progeroid syndrome- al., 2003). A genomewide linkage study performed on associated proteins is rapid, our understanding of 308 centenarians and near-centenarians found a statisti- their role in ordinary ageing (if any) remains to be cally significant linkage within chromosome 4. Further elucidated. refining of the search identified a microsomal transfer protein gene encoding protein involved in lipoprotein synthesis as a factor potentially modulating lifespan Acknowledgements (Geesaman et al., 2003). Another approach to the study of ageing is the func- This work was supported by State Committee for tional analysis of the genes involved in the development Scientific Research grant PBZ-KBN-022/PO5/1999 of progeroid syndromes. Unfortunately, none of the ‘Genetic and environmental factors of longevity’ co- known progerias, including those most closely resem- ordinated by the International Institute of Molecular bling ordinary ageing (Hutchinson–Gilford and Werner and Cell Biology in Warsaw. syndromes) represent true precocious ageing. Still, a disturbed function of such proteins in these disorders References implies their role in ‘physiological’ ageing. This is further supported by the fact that many of these pro- Bachrati, C. Z., & Hickson, I. D. (2003). 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