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Genetic analyses of two cases of Werner’s Syndrome

European Journal of Dermatology. Volume 14, Number 6, 379-82, November-December 2004, Genes and skin

Summary

Author(s) : Yoko Sogabe, Masahito Yasuda, Yoko Yokoyama, Atsushi Tamura, Izumi Negishi, Kazunori Ohnishi, Tetsuya Shinozaki, Osamu Ishikawa , Department of Dermatology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan, Department of Orthopaedic Surgery, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan.

Summary : We report two cases of Werner’s syndrome (WS). First, a 42-year-old Japanese man was referred on suspicion of systemic sclerosis (SSc) because of scleroderma-like skin atrophy and foot ulcers. Second, a 51-year-old woman with malignant fibrous histiocytoma was referred on suspicion of premature aging syndrome. Because both patients had many typical manifestations compatible with WS, we made a clinical diagnosis of WS. Genetic analyses revealed a homozygous mutation, an A deletion at nucleotide 3677 of WS gene (WRN) in the first case and a homozygous mutation, a G to C substitution at one base upstream of exon 26 of WRN in the second case. Both mutations were consistent with those previously reported in Japanese WS patients.

Keywords : genetic diagnosis, malignant fibrous hystiocytoma (MFH), mutant allele specific amplification (MASA), Werner’s syndrome

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ARTICLE

Auteur(s) :, Yoko Sogabe^1,*, Masahito Yasuda¹, Yoko Yokoyama¹, Atsushi Tamura¹, Izumi Negishi¹, Kazunori Ohnishi¹, Tetsuya Shinozaki², Osamu Ishikawa¹

¹Department of Dermatology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan
²Department of Orthopaedic Surgery, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan

accepté le 9 Août 2004

Werner’s syndrome (WS) is a rare autosomal recessive disorder characterized by various clinical signs of premature aging: cataracts, scleroderma-like skin appearance, short stature, graying hair, and hair loss [1]. More variable features include increased risk of neoplasms, diabetes mellitus, hypogonadism, osteoporosis, soft tissue calcification, hyperkeratosis, and foot ulcers [1, 2]. The causative gene known as WRN was identified in 1996 [3]. It is composed of 35 exons that encode a DNA helicase [3]. To date, 22 different mutations of WRN have been identified in the world, 9 of which have been reported in Japan [4]. Three of the 9 mutations are major mutations in Japan [5]: mutation at nucleotide 1336, 1-base upstream of nucleotide 3370, and nucleotide 4144. A splice mutation occurring at one base upstream of nucleotide residue 3370 in exon 26 was the most frequently found mutation, accounting for 61% of total WRN mutations in Japan. Mutation at nucleotide 1336 in exon 9 accounted for 21% and mutation at nucleotide 4144 in exon 33 accounted for 9%. Because about 90% of WRN mutations occurring in Japanese patients are explained by one of these 3 mutations, genetic diagnosis is relatively simple [4, 5]. Matsumoto et al. have established 2 diagnostic systems for detecting 7 of the 9 mutations in Japanese patients: mutant allele specific amplification (MASA) and oligonucleotide ligation assay (OLA) [5]. We genotyped our patients using the MASA method.

Case reports

Patient 1

A 42-year-old man complaining of skin sclerosis and ulcers on bilateral feet was referred to our department with suspected systemic sclerosis (SSc). He was born in 1967 as the second of 3 offspring of a first cousin marriage. At the age of 15 years, he noticed his hair began to turn gray. In his early thirties, he noticed sclerotic skin changes of his fingers and toes without cyanotic change or Raynaud’s phenomenon. At the age of 35 years, he was treated surgically for bilateral cataracts.

The patient was 163.9 cm in height and 37.5 kg in weight. He spoke with a high-pitched voice. He had a bird-like appearance with a beak-shaped nose, multiple milliary-sized pigmentations, and teleangiectasias on the face (( Figure 1a )). The helices of the ears were atrophic and scar-like. The hair was dyed dark brown, but the lower parts of hair shafts were completely white (( Figure 1b )). The skin was atrophic and thin over his entire body. Subcutaneous fat tissue and muscles seemed to decrease slightly on palpation. The atrophic and sclerodermatous skin changes were more evident on distal sites of the limbs. There was blotchy, brown pigmentation over his limbs, with intermingled areas of depigmentation over the feet and the lower legs. He had flat feet and localized hyperkeratosis on the soles, especially on bone projection areas. Some skin ulcers covered with necrotic tissues or crusts were scattered on the soles, the dorsal aspects of the feet, and the right Achilles tendon (( Figure 1c )).

The clinical features of the patient led us to suspect WS rather than SSc. Routine laboratory studies revealed slight anemia (RBC 3.96 × 10⁶/mm³, Hb 12.3 g/dl), and type IIa hyperlipidemia (total cholesterol 284 mg/dl, triglyceride 106 mg/dl). Urinary and serum hyaluronic acid (HA) levels were slightly elevated, 525 ng/mg cre (204-460 ng/mg cre) and 88.2 ng/ml (≦50 ng/ml), respectively. There were subcutaneous calcifications on the bilateral Achilles tendons. Multiple brain embolization and mild brain atrophy were detected by magnetic resonance imaging (MRI). Glucose intolerance, endocrinological abnormalities, and antinuclear antibodies were not detected. Chest radiography, Ga-scintigraphy, and computed tomography (CT) of the whole body showed no abnormalities. Evidence of internal malignancies was not found. After informed consent was given, a biopsy of the forearm skin was performed. The biopsy specimen showed atrophic epidermis and hyalinized collagen fibers in the lower dermis.

Patient 2

A 51-year-old woman with a tumor on the right upper arm was referred on suspicion of premature aging syndrome. She was born in 1951 as the third of 4 offspring of a non-consanguineous marriage. While in her twenties, her hair began to turn gray. At the ages of 28 and 29 years, she was surgically treated for bilateral cataracts. In her late forties, she noticed a small nodule near the right elbow joint. The nodule had grown over the years, and she visited our orthopaedic surgery department.

The patient was 145 cm in height and 45 kg in weight. Her hair, eyebrows, and eyelashes were almost white, and lower eyelashes were almost lost. Multiple milliary-sized pigmentations and teleangiectasias were seen on her face (( Figure 2a )). Her skin was atrophic and thin over the extremities because of muscle atrophy and loss of subcutaneous fat tissue. The atrophic and sclerodermatous changes were more evident in the distal sites of the limbs. There was blotchy, brown pigmentation over her limbs, with intermingled areas of depigmentation over the feet and the lower legs. She had flat feet and hyperkeratosis on the left sole. There was a hard tumor (10 × 8 cm) near the right elbow. The surface was irregular with poor mobility to the underlying tissue (( Figure 2b )). The tumor, which was surgically removed by orthopaedists, was pathologically diagnosed as a storiform-pleomorphic type of malignant fibrous histiocytoma (MFH).

Results of routine laboratory tests were within normal limits. Urinary HA level was slightly elevated (474 ng/mg cre) and serum HA level was in the normal range. There were subcutaneous calcifications on the bilateral Achilles tendons (( Figure 2c )). A left renal cyst was detected by MRI. Glucose intolerance, endocrinological abnormalities, and antinuclear antibodies were not detected. Chest radiography and brain CT showed no abnormalities. Evidence of internal malignancies was not obtained. Skin biopsy of the forearm showed atrophic epidermis, epidermal hyperkeratosis, and normal dermal collagen fibers.

Both cases met the diagnostic criteria of WS proposed by the Ministry of Health, Labor and Welfare as “definite” [6].

Genetic analysis

After obtaining informed consent, genomic DNA was extracted from the expanded fibroblasts (patient 1) or the peripheral mononuclear cells (patient 2). We screened the 8 types of WRN mutation frequently found in Japanese patients using the MASA method [5]. Homozygous mutations in exon 29 (patient 1) and at one base upstream of exon 26 (patient 2) were detected. The fragment spanning exon 29 was amplified with an upper primer specific to a wild-type allele (5′-CTGAGTCTCCTGCT-3′) or specific to a mutated allele (5′-CTGAGTCTCCTGCC-3′) and a common lower primer (5′-TTGAAAGACAATCTCATTTAGGGG-3′). The fragment containing exon 26 was amplified using an upper primer specific to a wild-type allele (5′-GCCAATTTCTACCC-3′) or specific to a mutated allele (5′-GCCAATTTCTACCG-3′) and a common lower primer (5′-GGTGGAAGGCTTTTTCCCGTCAGC-3′). Amplification was carried out under the following conditions: 40 cycles of 30 sec at 94 °C, 30 sec at 49 °C (exon 29) or 38 °C (exon 26), and 30 sec at 72 °C. The amplified DNA fragments were electrophoresed in a 1.5% agarose gel. To establish DNA sequencing, DNA fragments containing an expected mutation point were amplified using the PCR method. The fragment containing exon 29 was amplified with an upper primer (5′-TGGAAAGACATTCTCATTTAGGGG-3′) and a lower primer (5′-CTAGTCCTTTATTTTTAGAGCTTC-3′). The fragment spanning exon 26 was amplified with an upper primer (5′-GGTGGAAGGCTTTTTCCCGTCAGC) and a lower primer (5′-GACTTATCCTTTCCTCACAGATCC-3′). Amplification was carried out under the following conditions: 30 cycles of 30 sec at 94 °C, 30 sec at 56 °C (exon29) or 60 °C (exon26), and 30 sec at 72 °C. The PCR products were subcloned into pBluescript SKII⁺ (STRATAGENE; CA, USA). Two or 3 clones of each fragment were sequenced using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems; CA, USA), an ABI PRISM^®310 Genetic Analyzer (Applied Biosystems; CA, USA), and Gene Scan^® Analysis software (Applied Biosystems; CA, USA).

Patient 1 bore a homozygous mutation at nucleotide 3677 in exon 29 (( Figure 3a )), an A deletion (( Figure 4a )). Patient 2 carried a homozygous mutation at one base upstream of exon 26 of WRN (( Figure 3b )), a G to C substitution (( Figure 4b )). Both mutations cause a frameshift leading to premature termination [3, 4].

Discussion

In 1904, Otto Werner first described 4 siblings having the appearance of premature aging with cataracts and sclerodermatous skin changes in his doctoral thesis “Cataract in combination with scleroderma” [1, 7]. He thought it to be a variant form of Rothmund-Thomson syndrome, a kind of premature aging syndrome with photosensitivity. In 1934, Oppenheimer and Kugel established the eponym Werner’s syndrome as distinct from Rothmund-Thomson syndrome [8]. Since then, 1150 patients have been reported worldwide, of which 850 were from Japan [4].

Japan has the highest frequency of this disease in the world. One reason for this high incidence among Japanese is considered to be the custom of consanguineous marriages in local areas [9]. Several prefectures in Japan have an aggregation of WS patients, where the frequency of consanguineous (mostly first cousin) marriage of their parents has been relatively high. Another is its geographical isolation with a relatively low population exchange [4].

A variety of skin manifestations have been described in many case reports and reviews on WS. By the second decade of life, most WS patients show sclerodermatous skin changes such as atrophy, pigmentation, and depigmentation [1, 2, 8]. Hyperkeratosis also occurs, often associated with ulcers on the feet and ankles [8]. Ulceration often becomes severe and amputation is required in certain cases. From these skin manifestations and poor knowledge of SSc and WS, WS might have been misdiagnosed as SSc. The difficulty in lifting up the WS skin mainly results from a decrease of connective tissues, subcutaneous adipose tissues, and muscles [1]. In contrast, the difficulty in lifting up of SSc skin results from an accumulation of hyalinized collagen fibers. Diagnosis, however, cannot be made only from histological findings of the skin because hyalinization of collagen fibers is sometimes seen in a patient’s skin in WS [1].

There are several progeroid syndromes in the world, and all of them are segmental or unimodal progeria, not global progeria. WS is recognized as the most suitable model of human natural aging, because it shows a variety of clinical and biological manifestations similar to normal aging, sequentially from prepuberty. Therefore, WS has been studied intensively over the past 20 to 30 years in the fields of biochemistry, cell biology, and genetics. The genetic locus of WS was mapped onto the short arm of chromosome 8 using linkage analysis [10], and the responsible gene (known as WRN) has been identified in 8p11-12 region by positional cloning [3]. The WRN gene encodes a protein consisting of 1432 amino acids and is homologous to RecQ-type DNA helicase [3].

Twenty-two different mutations of WRN have been found, and 9 gene mutations have been identified in Japanese patients with WS [4, 10-12]. All mutations result in a predicted truncated protein due to either a nonsense mutation or mutations leading to a frameshift [13]. No missense WRN mutations have been found in WS patients [13]. The predicted truncated proteins lack nuclear localization signals in the C-proximal region. Therefore, mutant WRN products cannot be transported into the nucleus where the DNA helicase is supposed to function [14]. This finding also explains the enigma that most WS patients have similar clinical phenotypes regardless of different genotypes [15, 16]. However, the exact functions of the intact and mutated WRN protein are still not known. The relationship between genotype and phenotype has not been clarified, either.

Our patients had many typical manifestations suggesting WS, and met the diagnostic criteria of WS proposed by the Ministry of Health, Labor and Welfare as “definite” [6]. Not only clinical symptoms, but also genetic mutations were consistent with WS. Patient 1 had an A deletion in exon 29, and patient 2 had a G to C substitution at one base upstream of exon 26. The latter is the most common mutation in Japan, accounting for 61% of Japanese WS patients [5].

Glucose intolerance, and elevation of urinary and serum HA levels are seen with a high frequency in WS patients [16]. In our patients, however, glucose tolerance was normal. Patient 1 had a slight elevation of urinary and serum HA level and more severe sclerodermatous changes with ulceration, although he was younger than patient 2. It is conceivable that WS patients have some variations in their symptoms. The phenotype in WS may be dependent not only on the WRN gene mutation but also on other factors: patient’s age, life style, environment, and genetic background in different ethnic groups [15, 17]. We assume that many factors are involved and modify aging processes due to WRN per se.

References

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