Clinical And Basic Studies Of Werner Syndrome With Newly Identified Phenotypes

Werner syndrome （WS, OMIM catalog#277700） is regarded as an autosomal recessive human genetic instability syndrome whose phenotype mimics premature aging-patients appear to age rapidly after puberty. WS was first reported by Otto Werner in1904. It was regarded as a potential model of accelerated human aging.WS has a wide clinical spectrum. Young patients with relatively normal physical and mental development who appears to be growing stagnation from10to18years old. These clinical manifestations mainly involved in four systems:connective tissue involvement rate was100%at age of27±10years old with the clinical manifestation of short stature, recalcitrant leg ulcers, skin sclerosis, premature greying, alopecia, skin atrophy, osteoporosis, cataract and hyaluronic acid urine; endocrine and metabolic system involvement rate was80%at age of36±9years old with the clinical manifestation of diabetes mellitus, hypogonadism, thyroid dysfunction, hyperlipidemia; Immune system involvement rate was80%at age of40±10years old with the clinical manifestation of autoimmune disease; nervous system involvement rate was50%at age of40±10years old with the clinical manifestation of brain atrophy, alzheimer’s disease and schizophrenia etc. Skin ulcers appear at age of 34.7±9.6years old. The typical cause of about50%death is either cancer or cardiovascular disease and occurs at a median age of54years. The disease is rare, there are10domestic cases reported before2010using CNKI database search and the first case was reported in1983. From1916to2002, there are1300cases reported all around the world, including more than1000cases reported in Japan. The prevalence of WS is1/100,000in Japan and the frequency ranges from1/1,000,000to1/10,000,000in other area, this maybe caused by the relatively high level of consanguinity in Japanese populations. There are7cases with complete data in China and one case is children and6cases are adults. In these7cases,2patients were caused by parents’consanguineous marriage,6adult patients had cataract,5patients had leg or foot ulcers, one patient had meningioma leading to left facial numbness. Our case manifested as prominent progeroid disorders, multiple system （skin, skeleton, skeletal muscle and fat） degeneration. The case could be classified as a novel Werner syndrome which manifested with sensorineural hearing loss, and no diabetes mellitus and cataract. This is the first case of Werner syndrome with sensorineural hearing loss in Chinese. Refer to foreign literature, only Uhrhammer, etc. reported one WS patient with hearing loss and no cataract in France.http://www.pathology.washington.edu/research/werner/registry/diagnostic.html shows the Diagnostic Criteria of Werner Syndrome:Cardinal signs and symptoms （onset over10years old）:①Cataracts （bilateral）;②Characteristic dermatological pathology （tight skin, atrophic skin, pigmentary alterations, ulceration, hyperkeratosis, regional subcutaneous atrophy） and characteristic facies （’bird1facies）;③Short stature;④Parental consanguinity （3rd cousin or greater） or affected sibling;⑤Premature greying and/or thinning of scalp hair. Further signs and symptoms:①Diabetes mellitus;②Hypogonadism （secondary sexual underdevelopment, diminished fertility, testicular or ovarian atrophy）;③Osteoporosis;④Osteosclerosis of distal phalanges of fingers and/or toes （x-ray diagnosis）;⑤Soft tissue calcification;⑥Evidence of premature atherosclerosis （e.g. history of myocardial infarction）;⑦Mesenchymal neoplasms, rare neoplasms or multiple neoplasms;⑧Voice changes （high-pitched, squeaky, or hoarse voice）;⑨Flat feet. Definite:All the cardinal signs and two further signs; Probable:The first three cardinal signs and any two others; Possible:Either cataracts or dermatological alterations and any four others; Exclusion:Onset of signs and symptoms before adolescence （except stature, since current data on pre-adolescent growth patterns are inadequate）.The main differential diagnosis which have several skin aging and/or deafness and pigmentation include: systemic scleroderma, acrogenia, laminopathy （including progenia, mandible acra dysplasia, atypical WS, etc.）, Rothmund-Thomson syndrome, KID syndrome, Cockayne syndrome, congenital dyskeratosis, etc.Werner syndrome is a rare disease which manifested as a wide clinical spectrum. Based on the the patient’s presentation, this is the first case of Werner syndrome with sensorineural hearing loss in China. The relationship between genotype and phenotype of Werner syndrome is very complicated as reported. Most of WS are caused by mutations in a gene, WRN. The molecular basis of WS is mutations within the WRN gene located on chromosome8p11-12. The gene spans35exons and encodes a protein of1432aminoacids belonging to the RecQ-type family of DNA helicases. More than85mutations within this gene have been characterized in WS patients of different ethnic origins from the database （http://www.pathology. washington.edu/research/werner/database/）. Sequence analysis of the WRN coding region detects mutations in both alleles for approximately90%of affected individuals. The most common pathologic variant is c.1105C>T, which accounts for20%-25%of mutations in the European and Japanese populations. These mutations include base substitution, deletion, insertion and non-synonymous SNP and are found in coding regions and splicing sites of30exons of WRN gene. Domestic scholars Zhao et al. reported one31-year-old male patient with cataract and no diabetes. Automated DNA sequencing identified the mutation in the patient as c.3250de1G in exon25of WRN. It shortened the synthetic WRN protein, thus reduced activity of the halicase. Hao et al. reported one case of WS patient with meningioma and found a new heterozygous mutation as c.2806insA in exon18of WRN. Taiwan scholars Yeong et al. reported two siblings suffered from WS with cataract, chronic leg ulcers and no diabetes. DNA sequencing revealed the mutation in the patient as3264-5delAG in exon25of WRN. French scholars Uhrhammer et al. reported one WS patient with hearing loss and no cataract. The variant of WRN gene is c.3496A>T leading to p.Lys1166>X. It is the first time that we detect the mutations of WRN gene in a Chinese patient with novel Werner syndrome.Werner syndrome protein （WRN） is one of a family of five human RecQ helicases implicated in the maintenance of genome stability. The conserved RecQ family also includes RecQl, BLM （Bloom syndrome protein）, RecQ4（Rothmund-Thomson syndrome protein） and RecQ5in humans. Defects in three of the RecQ helicases, RecQ4, BLM, and WRN, cause human pathologies linked with cancer predisposition and premature aging. Mutations in the WRN gene are the causative factor of Werner syndrome （WS）. WRN is one of the best characterized of the RecQ helicases and the roles are known in DNA replication, repair, transcription and telomere maintenance. Studies both in vitro and in vivo indicate that the roles of WRN in a variety of DNA processes are mediated by post-translational modifications, as well as several important protein-protein interactions.The relationship between genotype and phenotype of Werner syndrome is very complicated as reported. The disease shows genetic heterogeneity. Patients of WS are advised to keep good living habits and regular physical examination, preventing possible complications such as atherosclerosis and all kinds of malignant tumors etc. At present, most of the genetic disease cannot be cured. We hope that future genetic diagnosis and treatment of WS can benefit from our research.Objective1. Based on the clinical features of the case and overall review on the related literatures, we aim to identify the clinical characteristics including involved tissue origin, clinical manifestation, differentiation diagnosis, treatment and prognosis of Werner syndrome. To identify the clinical spectrum and the clinical diagnostic criteria of Werner syndrome and to improve the understanding of the novel Werner syndrome in China.2. To develop a rapid and simple reverse transcription-polymerase chain reaction （RT-PCR） detecting the mutations of WRN gene in a Chinese patient with novel Werner syndrome and to discuss initially molecular mechanism and pathogenesis in this case of Werner syndrome.MethodsThe patient and his family signed our informed consent and all of the clinical and experimental research meet the Helsinki declaration.1. Collect the patients detailed clinical materials, perfect the screening examination of the system and laboratory inspection, review the literatures, affirm clinical diagnosis and differential diagnosis of the case.Case reportThe patient is a38-year-old Chinese male patient with gradual lipodystrophy, muscular dystrophy, premature aging, sensorineural hearing loss and deep ulcers of feet. Spontaneous subcutaneous adipose tissue progressively disappeared and muscular dystrophy occurred after the age of10years. At the same time, progeroid features including bird-like face, gray hair and skin changes occurred, older-like pigmentation in her sclerodermatous skin over the neck and distal lower limbs were found too. He spoke with mild hoarse voice. He was born at full term after an uneventful pregnancy and delivery. Development was considered unremarkable during the first10years of life except infantile penis and scrotum. After the age of20years, there was no beard, pubic hair, prominentia laryngea and genitalia growth be found yet. No consanguinity was reported. Systematic clinical and biological investigations of his family members showed no lipodystrophy, skin alterations, progeroid features and diabetes. Tests for fasting glycemia, postprandial glycemia and lipid profile of them showed normal. His height was160cm, weight35Kg （BMI13.67Kg/m2）, and blood pressure is normal. Physical examination revealed bird-like face, exophthalmos, gray hair. Loss of subcutaneous fat involving all of the body including the upper and lower extremities, the trunk, the gluteal region as well as the face was noticed. All of the skin was dry and older-like pigmentation in his sclerodermatous skin over the neck and distal lower limbs. His skin of the extremities was atrophic and thin with a pair of flat feet. Markedly muscular dystrophy from the upper and lower extremities was found, but muscular strength was relatively normal. Examination of eyes showed right eye pterygium, both iris atrophy, crystalline lens and fundus oculi were normal. Examination of oral cavity revealed tooth column not neat and outstanding. He got infantile penis and scrotum, without pubic hair. The abdominal examination showed no abnormality. Cardiac examinations showed no abnormality. Abdominal ultrasound revealed a small prostate with the size of25mm×18mm×20mm and showed inhomogenous echo. X-ray showed the bone density of target area of both hands and feet was decreased. The lipid profile showed low level of high density lipoprotein （HDL）（1.05mmol/L）, low level of ApoAl0.90g/L and high level of ApoB1.02g/L. Hormone tests showed serum total testosterone was at a low.level of0.382ng/mL.Markers of immunolgical diseases were negative. Tumor markers were within normal range. Liver function test showed aspartate aminotransferase （AST）, aminoleucine transferase （ALT） were normal, hepatitis virus markers test showed HBsAb and HbcAb were positive. Skin biopsy revealed no pathological changes. Brainstem electric response audiometry （BERA） revealed bilateral moderately severe sensorineural hearing loss. Electromyogram revealed normal. Thyroid function test showed TSH, FT3, FT4, T3and T4were normal. Muscle enzyme test showed creatine kinase23.0u/L （normal level:34.0～270.0u/L）, lactate dehydrogenase, a-hydroxyl butyrate dehydrogenase, creatine kinase isozyme were normal. Bone mineral density measurement revealed osteoporosis （lumbar1,2,3,4and L1-L4, L2-L4, femoral neck, upper femoral neck, Wards triangle, greater trochanter etc, T value<-2.5）. Electrocardiogram showed normal sinus rhythm and incomplete right bundle branch block. Result of Schirmer test was left1mm/min and right3mm/min which revealed dry eye syndrome.2. Blood samples were collected from a sporadic patient with WS, his unaffected brother and30unrelated population-matched controls. Skin tissues were collected from the patient and3unrelated age-matched healthy controls.3. WRN gene mutation analysisTo develop a rapid and simple reverse transcription-polymerase chain reaction （RT-PCR） strategy formulation analysis of the WRN gene, to identify pathogenic mutations in a Chinese patient with novel type WS. The principle is:the total RNA of blood cells are extracted, the mRNA is used as template, using Oligo （dT） and reverse transcriptase reverse transcription into cDNA. Then using cDNA as template for PCR amplification, then using ABI. PRISM3700automatic sequencing instrument direct sequencing. Using Chromas software （version2.23） and Clustalx software analyse sequencing results and compare with cDNA sequences of WRN gene （GenBank registration number:NM₀00553.4） to find probable mutations. DNA of blood cells are extracted. Design and synthesis the primers of the exon which have the suspicious mutations. After Polymerase Chain Reaction （PCR） amplification, then using the3730XL type DNA sequencing instrument direct sequencing. Using Chromas software （version2.23） and Clustalx software analyse sequencing results and compare with DNA sequences of WRN gene （GenBank registration number: NC₀00008.9） to verify the mutations. Specific steps are as follows:3.1Total RNA extraction from peripheral blood and cDNA sequencing:3.1.1Extract total RNA from2anticoagulant venous blood samples of the patient and his elder brother3.1.2Reverse transcription3.1.3Design and synthesis primers3.1.4PCR system optimization and amplification3.1.5PCR product purification3.1.6PCR purification product sequencing3.2DNA extraction from peripheral blood and DNA sequencing validation:3.2.1Extract DNA from anticoagulant venous blood samples of the patient and his elder brother and30normal healthy controls3.2.2Design and synthesis primers3.2.3PCR system optimization and amplification3.2.4PCR product sequencing4. Bioinformatics analysisThrough the Internet database （such as http://blast.ncbi.nlm.nih.gov/Blast.cgi; http://www.ncbi.nlm.nih.gov/genbank, etc.）, sequence alignment and analyze the results.5. Fluorescence quantitative PCR analysisRNA extraction from skin tissue samples of the patient and three cases of normal healthy control, reverse transcription, PCR system optimization, PCR amplification and sequencing, using GapDH as internal reference, detect mRNA expression quantity of WRN gene, repeat three times and get consistent results.5.1Determine the gene sequence and target species5.2Design and synthesis primers5.3RNA extraction from skin tissue samples, reverse transcription, PCR system optimization and amplification5.4Construct plasmid standard of two gene5.5Draw standard curve of two gene5.6SYBR quantitative PCR analysis for two loci of three samples5.7Data Analysis6. Immunohistochemical method to detect WRN protein expressionUsing Immunohistochemical method to detect WRN protein expression of skin tissue samples of patient and three cases of normal healthy controls, repeat three times and get consistent results..Results1. The case manifested as the prominent progeroid disorders with multiple system （skin, skeleton, skeletal muscle and fat） degeneration and it Conform to the clinical diagnosis standard of Werner syndrome. It’s presentation could be classified as a novel Werner syndrome which manifest with sensorineural hearing loss, right eye pterygium, both iris atrophy, and no diabetes mellitus and cataract. 2. We describe a novel and rapid RT-PCR-based method for WRN gene mutation analysis in a Chinese patient with novel type WS.2967+237A>G、3309+26C>T、 synonymous mutation c.2361G>T and c.3237G>A are found in the WRN gene of the patient and these are also observed in part of the30unrelated normal control individuals. There is no pathogenic mutation in exon regions of WRN gene. The variations belong to single nucleotide polymorphisms （SNPs） and the exon coding SNPs （cSNPs） had been reported abroad.3. Since all these sequencing revealed no pathogenic mutations, we subsequently further explored the sequencing of WRN gene at mRNA level. To determine the transcriptional quantities of mRNA of WRN gene, we performed real-time PCR. Similarly, it revealed no upregulation or downregulation compared with controls. Thus, the transcription process of WRN gene in patient’s skin was normal.4. By using immunohistochemical method, we demonstrate that the overall expression level of WRN on skin tissue from the patient and controls have no obviously differece.Conclusions1. Werner syndrome is a rare disease which manifested as a wide clinical spectrum. Based on the the patient’s presentation, it could be classified as a novel type Werner syndrome. This is the first case of Werner syndrome with sensorineural hearing loss in China.2. Reverse Transcription-Polymerase Chain Reaction （RT-PCR） can be used simply and rapidly in WRN gene mutation analysis and it can greatly save the cost. It is worthy to be popularized in gene mutation analysis of monogenic disease.3. Four SNPs are present in the WRN gene of the sporadic patient with the novel Werner syndrome, but there is no pathogenic mutation in exon regions of WRN gene. It further confirms that the relationship between genotype and phenotype of Werner syndrome is very complicated as reported. It shows genetic heterogeneity of this disease.4. Compared with normal healthy controls, there is no obvious abnormity in mRNA level and WRN protein expression in skin tissue of the patient. There may be other factors which cause the disease.