POLD1 Germline Mutations in Patients Initially Diagnosed with Werner Syndrome

Segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ. A prototypic example is the Werner syndrome (WS), caused by biallelic germline mutations in the Werner helicase gene (WRN). While heterozygous lamin A/C (LMNA) mutations are found in a few nonclassical cases of WS, another 10%–15% of patients initially diagnosed with WS do not have mutations in WRN or LMNA. Germline POLD1 mutations were recently reported in five patients with another segmental progeroid disorder: mandibular hypoplasia, deafness, progeroid features syndrome. Here, we describe eight additional patients with heterozygous POLD1 mutations, thereby substantially expanding the characterization of this new example of segmental progeroid disorders. First, we identified POLD1 mutations in patients initially diagnosed with WS. Second, we describe POLD1 mutation carriers without clinically relevant hearing impairment or mandibular underdevelopment, both previously thought to represent obligate diagnostic features. These patients also exhibit a lower incidence of metabolic abnormalities and joint contractures. Third, we document postnatal short stature and premature greying/loss of hair in POLD1 mutation carriers. We conclude that POLD1 germline mutations can result in a variably expressed and probably underdiagnosed segmental progeroid syndrome.     

Homozygous and compound heterozygous mutations at the Werner syndrome locus

The Werner syndrome (WS) is a rare autosomal recessive progeroid disorder. The Werner syndrome gene (WRN) has recently been identified as a member of the helicase family. Four distinct mutations were previously reported in three Japanese and one Syrian WS pedigrees. The latter mutation was originally described as a 4 bp deletion spanning a spliced junction. It is now shown that this mutation results in a 4 bp deletion at the beginning of an exon. Nine new WRN mutations in 10 additional WS patients, both Japanese and Caucasian, are described. These include three compound heterozygotes (one Japanese and two Caucasian). The new mutations are located all across the coding region.      

The mutational spectrum in Waardenburg syndrome

One hundred and thirty-four families or individuals with auditory-pigmentarysyndromes such as Waardenburg syndrome (WS) or probable neurocristopathieswere screened for mutations in the PAX3 and MITF genes. PAX3mutations were found in 20/25 families with definite Type 1WS and 1/2 with Type 3 WS, but in none of 23 with definite Type2 WS or 36 with other neurocristopathies. The PAX3 mutationsincluded substitutions of conserved amino acids in the paireddomain or the homeodomain, splice-site mutations, nonsense mutationsand frame-shifting insertions or deletions. No phenotype-geno-typecorrelations were noted within WS1 families. With MITF, mutationslikely to affect protein function were found in seven families,five of which had definite Type 2 WS. We conclude that Type1 and Type 3 WS are allelic and are normally caused by lossof function mutations in PAX3; that Type 2 WS is heterogeneous,with about 20% of cases caused by mutations in MITF, and thatindividuals with auditory, pigmentary or neural crest syndromeswhich do not fit stringent definitions of Waardenburg syndromeare unlikely to have mutations in either the PAX3 or MITF genes.The molecular pathology of MITF/microphthalmia mutations appearsto be different in humans and mice, with gene dosage havingmore significant effects in humans than in the mouse.     

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle

Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3′–5′ helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.     

The Werner syndrome protein at the crossroads of DNA repair and apoptosis

Werner syndrome (WS) is a premature aging disease characterized by genetic instability. WS is caused by mutations in a gene encoding for a 160 kDa nuclear protein, the Werner syndrome protein (WRN), which has exonuclease and helicase activities. The mechanism whereby WRN controls genome stability and life span is not known. Over the last few years, WRN has become the focus of intense investigation by a growing number of scientists. The studies carried out by many laboratories have provided a wealth of new information about the functional properties of WRN and its cellular partners. This review focuses on recent findings that demonstrate a functional interaction between WRN and two factors that bind to DNA breaks, Ku and poly(ADP-ribose) polymerase 1, and discuss how these interactions can influence fundamental cellular processes such as DNA repair, apoptosis and possibly regulate cell senescence and organismal aging.     

Pathways and functions of the Werner syndrome protein

Mutations in human WRN (also known as RECQ3) gene give rise to a rare autosomal recessive genetic disorder, Werner syndrome (WS). WS is a premature aging disease characterized by predisposition to cancer and early onset of symptoms related to normal aging including osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and atherosclerosis. This review focuses on the functional role of Werner protein (WRN) in guarding the genetic stability of cells, particularly by playing an integral role in the base excision repair, and at the telomere ends. Furthermore, in-depth biochemical investigations have significantly advanced our understanding of WRN protein regarding its binding partners and the site of protein-protein interaction. The mapping analysis of protein interaction sites in WRN for most of its binding partners have revealed a common site of protein-protein interaction in the RecQ conserved (RQC) region of WRN.     

WRN participates in translesion synthesis pathway through interaction with NBS1

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of polη and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner.     

p53-Mediated apoptosis is attenuated in Werner syndrome cells

The WRN DNA helicase is a member of the DExH-containing DNA helicase superfamily that includes XPB, XPD, and BLM. Mutations in WRN are found in patients with the premature aging and cancer susceptibility syndrome known as Werner syndrome (WS). p53 binds to the WRN protein in vivo and in vitro through its carboxyl terminus. WS fibroblasts have an attenuated p53- mediated apoptotic response, and this deficiency can be rescued by expression of wild-type WRN. These data support the hypothesis that p53 can induce apoptosis through the modulation of specific DExH-containing DNA helicases and may have implications for the cancer predisposition observed in WS patients.     

A homozygous MITF mutation leads to familial Waardenburg syndrome type 4

Waardenburg syndrome (WS) is a genetic disorder characterized by hearing loss and pigmentary abnormalities with variable penetrance. Though heterozygous mutations in MITF are a major cause for Waardenburg syndrome type 2 (WS2), homozygous mutations in this gene and the associated phenotype have been rarely characterized. In this study, we identified a novel p.R223H mutation in MITF in a Chinese Han family with variable WS features. Both parents carried a heterozygous p.R223H mutation. They had normal hearing, and premature greying of the hair is their only pigmentary abnormality. In contrast, their two children both carried a homozygous p.R223H mutation and had classic WS features including profound hearing loss, heterochromia irides and marked pigmentary abnormalities in hair and skin. Interestingly, the two affected children also have persistent chronic constipation since the neonatal period, symptoms suggestive of Waardenburg syndrome type 4 (WS4). Our study revealed a likely association between homozygous mutations in MITF and WS4, which implies a dosage effect for the underlying pathogenesis mechanism.     

A frameshift mutation in the HuP2 paired domain of the probable human homolog of murine Pax-3 is responsible for Waardenburg syndrome type 1 in an Indonesian family.

Waardenburg syndrome type 1 (WS1) is an autosomal dominant disorder characterized by deafness, dystopia canthorum, heterochromia iridis, white forelock, and premature greying. A similar phenotype is caused in the mouse by mutations in the Pax-3 gene. This observation, together with comparisons of conserved syntenies in the murine and human genetic maps, suggested that at least some WS1 mutations should occur in HuP2, the probable human homolog of Pax-3. Two mutations in the HuP2 sequence of individuals with WS1 have been reported recently. Both of them occur in the highly conserved paired box region of the gene, which encodes a DNA binding domain. The functional consequences of these mutations are at present speculative. We report here a 14 bp deletion in the paired domain encoded by exon 2 of HuP2 in an Indonesian family segregating for WS1. This frameshift mutation results in a premature termination codon in exon 3. The HuP2 product is a truncated protein lacking most of the paired domain and all of the predicted homeo domain. We propose that the WS1 phenotype in this family is due to loss of function of HuP2 and discuss two mechanisms for the dominant effect of this mutation.     

Role for the Werner syndrome protein in the promotion of tumor cell growth

Werner syndrome (WS) is a premature aging and cancer-prone disease caused by loss of the RecQ helicase WRN protein. Cultured WS fibroblasts display high genomic instability and senesce prematurely. Epigenetic inactivation of the WRN gene occurs in numerous tumor types, in which WRN demonstrates tumor suppressor-like activity (Agrelo et al., 2006). However, the role of WRN in tumors that express WRN protein is unknown. Here we report that the inhibition of WRN expression strongly impairs growth of 12 out of 15 cancer cell lines tested. For those cell lines in which WRN depletion induced high cell death, the majority of the surviving proliferative clones exhibited WRN expression. Growth arrest induced by WRN depletion was characterized by an accumulation of cells in the G2/M cell cycle phases and an increase in DNA damage. Importantly, WRN depletion inhibited tumor growth in vivo in SCID mouse xenograft models. Altogether, these findings support a dual role for WRN in tumorigenesis; tumor suppressor-like activity in tumors with WRN inactivation and the promotion of proliferation and survival in tumors that express WRN. These findings suggest a possible therapeutic role for WRN as an anti-cancer target, and highlight the importance of WRN protein status for tumorigenesis and clinical treatments of patients.     

JAG1 mutations are found in approximately one third of patients presenting with only one or two clinical features of Alagille syndrome

Alagille syndrome is a multisystem disorder characterized by highly variable expressivity, most frequently caused by heterozygous JAG1 gene mutations. Classic diagnostic criteria combine the presence of bile duct paucity on liver biopsy with three of five systems affected; liver, heart, skeleton, eye and dysmorphic facies. The aim of this study was to determine the prevalence and distribution of JAG1 mutations in patients referred for routine clinical diagnostic testing. Clinical data were available for 241 patients from 135 families. The index cases were grouped according to the number of systems affected (heart, liver, skeletal, eye and facies) and the mutation frequency calculated for each group. JAG1 mutations were identified in 59/135 (44%) probands. The highest mutation detection rates were observed in patients with the most frequent presenting features of Alagille syndrome; ranging from 20% (one system) to 86% (five systems). The overall mutation pick-up rate in a clinical diagnostic setting was lower than in previous research studies. Identification of a JAG1 gene mutation is particularly useful for those patients with atypical or mild Alagille syndrome who do not meet classic diagnostic criteria as it provides a definite molecular diagnosis and allows accurate genetic counselling for the family.     

Presence of a major WFS1 mutation in Spanish Wolfram syndrome pedigrees

Wolfram syndrome (WS) is an autosomal recessive neurodegenerative disease mainly characterized by familial diabetes mellitus and optic atrophy. WS patients frequently present with other clinical features such as diabetes insipidus, renal abnormalities, psychiatric disorders, and a variety of neurologic symptoms: deafness, ataxia, peripheral neuropathy. A gene responsible for Wolfram Syndrome (WFS1) has been recently identified on chromosome 4p16.1. Twenty-two Wolfram patients from 16 Spanish families were screened for mutations in the WFS1 coding region by SSCP analysis and direct sequencing. Since WS has been considered a mitochondrial disorder for some time, mitochondrial DNA (mtDNA) in these families was also examined. WFS1 mutations were detected in 75% of families (12 of 16). One of these mutations, an insertion of 16 base pairs in exon 4, turned out to be notably frequent in Spanish pedigrees. As many as 50% of pedigrees with WFS1 mutations harbored this insertion, either in one (33% of cases) or in two chromosomes (67%). Ten other mutations were identified: 7 missense changes, 2 deletions, and 1 nonsense mutation. Only 3 of these changes had been previously described in non-Spanish pedigrees. Large mtDNA rearrangements and LHON point mutations were detected in four and six families, respectively. No correlation could be established between WFS1 gene mutations and specific point mutations or rearrangements in mtDNA. We would suggest first screening for the 16-bp insertion in exon 4 when a new Spanish WS case is reported.     

The spectrum of WRN mutations in Werner syndrome patients

The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in the range of 46-48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are available to qualified investigators.     

Polymorphisms at the Werner locus: II. 1074Leu/Phe, 1367Cys/Arg, longevity, and atherosclerosis

Werner syndrome (WS) is a progeroid syndrome caused by autosomal recessive null mutations at the WRN locus. The WRN gene encodes a nuclear protein of 180 kD that contains both exonuclease and helicase domains. WS patients develop various forms of arteriosclerosis, particularly atherosclerosis, and medial calcinosis. The most common cause of death in Caucasian subjects with WS is myocardial infarction. Previous studies have identified specific polymorphisms within WRN that may modulate the risk of atherosclerosis. Population studies of the 1074Leu/Phe and 1367Cys/Arg polymorphisms were undertaken to evaluate the role of WRN in atherogenesis. Frequencies of the 1074Leu/Phe polymorphisms in Finnish and Mexican populations revealed an age-dependent decline of 1074Phe/Phe genotype. In Mexican newborns, but not in Finnish newborns, the 1074Leu/Phe and 1367Cys/ Arg polymorphisms were in linkage disequilibrium. Among coronary artery disease subjects, there was a tendency for the 1074Phe allele to be associated with coronary stenosis in a gene dose-dependent manner. Furthermore, the 1367Arg/Arg genotype predicted a lower degree of coronary artery occlusion, as measured by NV50, when compared to the 1367Cys/Cys or 1367Cys/Arg genotypes. However, these tendencies did not achieve statistical significance. Samples from Mexican patients with ischemic stroke showed a trend of haplotype frequencies different from that in a control group of Mexican adults. These data support the hypothesis that WRN may mediate not only WS, but may also modulate more common age-related disorders and, perhaps, a basic aging process.     

Coronary artery disease in a Werner syndrome-like form of progeria characterized by low levels of progerin, a splice variant of lamin A

Classical Hutchinson-Gilford progeria syndrome (HGPS) is caused by LMNA mutations that generate an alternatively spliced form of lamin A, termed progerin. HGPS patients present in early childhood with atherosclerosis and striking features of accelerated aging. We report on two pedigrees of adult-onset coronary artery disease with progeroid features, who were referred to our International Registry of Werner Syndrome (WS) because of clinical features consistent with the diagnosis. No mutations were identified in the WRN gene that is responsible for WS, among these patients. Instead, we found two novel heterozygous mutations at the junction of exon 10 and intron 11 of the LMNA gene. These mutations resulted in the production of progerin at a level substantially lower than that of HGPS. Our findings indicate that LMNA mutations may result in coronary artery disease presenting in the fourth to sixth decades along with short stature and a progeroid appearance resembling WS. The absence of early-onset cataracts in this setting should suggest the diagnosis of progeroid laminopathy. This study illustrates the evolving genotype-phenotype relationship between the amount of progerin produced and the age of onset among the spectrum of restrictive dermopathy, HGPS, and atypical forms of WS.     

Telomere ResQue and preservation--roles for the Werner syndrome protein and other RecQ helicases

Werner syndrome is an autosomal recessive disorder resulting from loss of function of the RecQ helicase, WRN protein. WS patients prematurely develop numerous clinical symptoms and diseases associated with aging early in life and are predisposed to cancer. WRN protein and many other RecQ helicases in general, seem to function during DNA replication in the processing of stalled replication forks. Genetic, cellular and biochemical evidence support roles for WRN in proper replication and repair of telomeric DNA, and indicate that telomere dysfunction contributes to the WS disease pathology.     

Bloom's syndrome gene suppresses premature ageing caused by Sgs1 deficiency in yeast

BACKGROUND: Bloom's syndrome (BS) is an autosomal recessive disorder causing short stature, immunodeficiency, and an increased risk of cancer. Increased rates of sister chromatid exchange and chromosomal aberration have been observed in cells having defects in the BLM gene. Among five kinds of human RecQ helicases cloned, the mutations in WRN and RecQL4 have been known as the causes of premature ageing. Little is, however, known about the function of BLM helicase in ageing. RESULTS: We show that human BLM, but not WRN can prevent the premature ageing and the increased homologous recombination at the rDNA loci caused by sgs1 mutation. Unexpectedly, the levels of ERCs (extrachromosomal rDNA circles), the products of homologous recombination, formed in 7-generation cells of the wild-type or the sgs1:BLM strain were comparable with those of the sgs1 or the sgs1:WRN age-matched-old cells. CONCLUSION: These results imply that BLM helicase may have an important role in human ageing. In addition, these data suggest that the accumulated ERCs per se may be not the cause of premature ageing in yeast, inconsistent with the model proposed by Sinclair & Guarente. We discuss a new model, which explains how Sgs1 or BLM helicase suppresses premature ageing in yeast.