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Epidemiology and clinical aspects of Werner’s syndrome in North Sardinia: description of a cluster

European Journal of Dermatology. Volume 17, Number 3, 213-6, May-June 2007, Genes and skin

DOI : 10.1684/ejd.2007.0155

Summary

Author(s) : Maria Vittoria Masala, Carla Olivieri, Cesare Pirodda, Maria Antonietta Montesu, Maria Antonietta Cuccuru, Sara Pruneddu, Cesare Danesino, Decio Cerimele , Department of Dermatology, University of Sassari, Viale S Pietro N° 43, 09100 Sassari, Italy, Department of Human and Hereditary Pathology. Section of General Biology and Medical Genetics, University of Pavia. Pavia.

Summary : Werner syndrome (WS, MIM#277700) is a very rare autosomal recessive disorder. WS clinical signs include altered distribution of subcutaneous fat, juvenile bilateral cataracts, a mask-like face and bird-like nose, trophic ulcers of the feet, diabetes mellitus, and premature atherosclerosis. The habitus is characteristic, with short stature, stocky trunk and slender extremities. WS frequency has been roughly estimated to be 1: 100,000 in Japan and 1: 1,000,000-1: 10,000,000 outside of Japan. The only exception to the latter data can be seen in the clustering of WS in Sardinia. Since 2001, 5 new cases have been observed: 4 members of the same family and 1 sporadic case. Therefore, since 1982 the total number of cases described in North Sardinia amounts to 18: 15 are familial (11 members of the same family group) and 3 sporadic. A short clinical description of the 5 new cases is reported.

Keywords : Werner’s syndrome, Werner gene, Werner protein, Rec Q DNA helicase

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ARTICLE

Auteur(s) : Maria Vittoria Masala¹, Carla Olivieri², Cesare Pirodda¹, Maria Antonietta Montesu¹, Maria Antonietta Cuccuru¹, Sara Pruneddu¹, Cesare Danesino¹, Decio Cerimele¹

¹Department of Dermatology, University of Sassari, Viale S Pietro N° 43, 09100 Sassari, Italy
²Department of Human and Hereditary Pathology. Section of General Biology and Medical Genetics, University of Pavia. Pavia

accepté le 10 Janvier 2007

Werner syndrome (WS, MIM#277700) is a very rare autosomal recessive disorder. The Werner gene (WRN, MIM#604611) was cloned in 1996 on the short arm of chromosome 8 (8p12): it encodes a homolog of the E. coli Rec Q DNA helicase (RECQL2). The gene functionally interacts with a DNA polymerase, required for DNA replication and DNA repair, and therefore is related to the maintenance of DNA integrity.WS clinical signs include altered distribution of subcutaneous fat, juvenile bilateral cataracts, a mask-like face and bird-like nose, trophic ulcers of the feet, diabetes mellitus, and premature atherosclerosis. The habitus is characteristic, with short stature, stocky trunk and slender extremities [1]. About 1,270 cases were described worldwide up until 2002: approximately 80% of them were of Japanese ancestry and 70% were born of consanguineous marriages. WS frequency has been roughly estimated to be 1: 100,000 in Japan and 1: 1,000,000-1: 10,000,000 outside of Japan [2]. The only exception to the latter data can be seen in the clustering of WS in Sardinia [3, 4]. Indeed, the minimum prevalence of WS in North Sardinia has recently been estimated to be 1: 59,000 [4].13 patients affected by W.S. have been reported in North Sardinia over the last 25 years comprising: 7 members of one large family group, in 1979 [3]; three more cases, 1 sporadic case and two sisters, all belonging to different families, in 1982 [5]; 2 siblings, in 1994 [6]; finally, in 2003, 1 sporadic case [7]. Since 2003, 5 new cases have been observed: 4 members of the same large family group and 1 sporadic case. Therefore, the total number of cases described in North Sardinia amounts to 18 with 15 familial (11 members being of the same family group) and 3 sporadic.A short clinical description of the 5 new cases is reported.

Case reports

Patient 1

V. P., a 38-year-old female, came to our observation in March 2001 complaining of progressive scleroderma-like skin changes which had started 25 years before. This was associated with bilateral cataracts, and pains in her feet and legs while standing or walking which had started 8 years previously (figure 1), family A, case III).

Family history revealed that her parents were first cousins and her brother had died of Cooley’s anaemia.

On physical examination, we observed: low stature, spread and exaggerated wrinkles in the face and the neck (figure 2), and thin grey hair. Her feet were slender, subcutaneous fat had disappeared, and hyperkeratotic calluses were present. The bony vault was exaggerated because of subcutaneous fat disappearance (figure 3). The voice was high-pitched.

X-rays revealed widespread osteoporosis, and lamellar calcinosis of the heel tuberositas. Laboratory investigation revealed hypercholesterolemia, mild increase in hepatic enzymes (sGOT 57 U/L, sGPT 63). Endocrinology evaluation revealed precocious menopause.

Patient 2

In June 2002 M. G., female (figure 1), family A, case IV) and V.P.’s first cousin came under our observation. Family history showed that her parents were consanguineous, she was the third affected in a family of nine siblings; indeed, she was a sister of the two patients described in 1979 (3).

M.G.’s past history revealed that menarche had occurred at the age of 13. She was affected by Cooley’s anaemia and so subject to frequent blood transfusions. In addition, she had developed a serious heart failure and diabetes. Bilateral cataracts had developed in the third decade. Physical examination showed small stature (128 cm), and thin grey hair. The voice was hoarse. All extremities showed marked atrophy of the subcutaneous tissue and muscle. Patient presented bilateral trophic ulcers at the perimalleolar and heel regions. The patient died, aged 36, of cachexia.

Patient 3

V. M., a 32-year-old female, born of consanguineous parents (figure 1), family A, case I) and cousin on the maternal side of patient n°1.

She came under our observation in May 2003 complaining that from the age of 18 she had suffered from loss of hair and precocious greying and fine wrinkles in the periocular areas. In that same period she had observed the spontaneous loss of some molars and tapering in the feet due to progressive loss of subcutaneous fat. At 29 years of age, visual disturbances had developed and a diagnosis of bilateral posterior cataract had been made; polyarthralgia and ostealgia had also appeared. At the age of 30, the patient had become pregnant and was delivered of a normal child after a normal pregnancy. At the age of 31, arthralgia and ostealgia had worsened with the appearance of calcinosis on the soles of the feet.

Our physical examination revealed high pitched voice, low stature (145 cm tall), peribuccal and periocular wrinkles, and thin grey hair. The extremities were very slender with a serious loss of subcutaneous fat. The patient presented flat feet, and muscle wasting in the legs.

Laboratory findings were normal, X-ray tests revealed widespread osteoporosis, and soft tissue calcification.

Patient 4

V.A.D., aged 25, female (figure 1), family A, case II) and V.M.’s younger sister. She came to our department in June 2003, after her sister’s diagnosis of WS. She was 137 cm tall and her clinical history revealed that her growth had ceased at the age of 12 (figure 4). Menarche had occurred at the age of 13. She referred recent widespread arthralgias in the foot, knee and lumbar regions. At clinical observation she showed: short stature, bird-like facies, reduction of subcutaneous tissues in the legs which appeared thin, and plantar hyperkeratosis. Slight opacity of the lenses was observed, but no cataract had yet developed. Laboratory findings were normal.

Patient 5

We observed M.D., aged 33, male (figure 1), family B, case V) in 2004. His parents were related: M.D.’s mother was her mother-in-law’s second cousin. Past history revealed that growth had stopped at 15, at 165 cm, and at the same age the first grey hairs had appeared. Since childhood the patient had had problems with his sight. During the second decade bilateral cataracts developed and were extracted at the age of 30.

At physical examination we noted: characteristic bird-like facies, slender extremities, and a prominent abdomen. The skin was taut and slightly hyperpigmented, fine wrinkles were present in the periocular and peribuccal regions and the hair was completely grey, with sparse pubic and axillary hair. In addition, we observed slight atrophy of the testes. Laboratory findings showed hypercholesterolemia, and an increase in hepatic enzymes (sGOT 61 U/I, sGPT67 U/I).

Follow-up on the surviving patients is still underway at three-monthly intervals.

In all patients the clinical diagnosis was made on the basis of major and minor criteria proposed by the International Registry of Werner syndrome group [2], and was confirmed by genetic analyses. After informed consent, we obtained peripheral blood from all patients, with exception of patient n°4, for cytogenetic and genomic molecular analyses.

Chromosomes were studied with standard procedures and at least 20 metaphases for each subject were analysed. Chromosomal aberrations found by cytogenetic analysis in affected subjects were as expected for WS [8].

In the molecular analyses, the DNA was amplified by PCR using primers used for exons 18 and 19 and primers designed by C.O. for IVS 18. With this test we were able to characterise the disease causing mutation at genomic DNA level in all five cases, identifying a homozygous g. nt 77177 a>g mutation (Genbank acc# AY442327) [9].

Discussion

The WRN protein is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3’-5’ helicase, a 3’-5’ exonuclease and an ATPase. In vitro WRN binds and is catalytically active toward non-canonical DNA structures such as recombination intermediates, replication forks, repair intermediates and telomeric ends. Deficiency in WRN results in a cellular phenotype of genomic instability [10]. More than 40 different mutations were detected up until 2004, including deletions, nucleotide additions, point mutations inactivating splice acceptor sites of some of the exons and point mutations in the coding sequence of the gene [2]. The clinical features of patients affected by WS are strikingly similar in spite of the fact that different mutations may be at work in different patients. The common final alteration of the genetic defect and the pleiotropic effects of WRN could go some way to explaining this conundrum. A further point of interest is that despite the numerous mutations found, phenotypical aspects remain the same worldwide.

Most of mutations result in a truncated WRN protein that lacks the C-terminal and the nuclear localization signal (WLS). A splice mutation occurring at one base upstream of nucleotide 3370 in exon 26 was the most frequent mutation found in Japan, accounting for 61% of total WRN mutations: mutation at nucleotide 1336 in exon 9 accounted for 21% and mutation at nucleotide 4144 in exon 33 accounted for 9% [11]. Thus, the inability of WRN to be transported into the nucleus would seem to be critical for the pathogenesis of WS.

In our four patients, the genetic molecular studies showed a homozygous g. nt 77177 a> g mutation (Genbank acc AY442327). This mutation inserts a new 5’ splice site (↑ At/Gt). At 3’ cryptic splice site (↓ag) is present 106 bp upstream and explains the 106 bp insertion [9]. This mutation had been seen in another family member A by Yu et al. [12], who had found a cDNA 105 bp insertion between exons 18 and 19 of WRN gene, but effected no further characterizations. The presence of this homozygous mutation in two unrelated families would suggest a founder effect. Microsatellite analysis in order to verify this hypothesis is currently in progress.

The description of five new WS cases in North Sardinia confirms the high prevalence of the disease reported in our previous papers [3, 5]. Four of these new patients are offspring of the family grouping reported in 1982; the subjects affected at that time were seven, so the total number of patients in this family now amounts to eleven.

The large number of WS patients in Sardinia may have at least two explanations. First, the poor connections between Sardinia and the Italian mainland which, until 50 years ago, amounted to one daily boat service. In addition, even internal communications on the island itself were hampered, due to the scarcity of road and railway networks. Second, the frequency of consanguineous marriage, partly due to this geographic isolation and partly to avoid the excessive fragmentation of small land and sheep holdings which were the main property assets and nutritional sources for country families.

The appearance of four new cases in the large family group previously described (family A) during the last five years has induced us to start a program of genetic counselling in collaboration with the Department of Medical Genetics of the University of Pavia. In addition, a proposal to Health Service authorities is underway for the implementation of an educational campaign aimed at heightening awareness among the population, so as to prevent the emergence of new WS cases.

Acknowledgements

Financial support: none. Conflict of interest: none.

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