Study On The Function Of A Case Of Werner Syndrome Caused By Novel Pathogenic Mutation Of WRN Gene

Objective Werner Syndrome(WS),also known as “adult progeria”,is a rare autosomal recessive genetic disorder.As a premature aging syndrome,WS is caused by mutations in the WRN gene,which is a multi-system systemic disease displayed with rapid aging after puberty with an estimated global incidence of 1:380,000-1:1,000,000.At present,only 20 cases of WS have been reported in our country.And a total of 157 different WRN pathogenic mutation sites have been recorded in the professional database of HGMD(The Human Gene Mutation Database).The present study was divided into two parts.we detected the pathogenic gene mutation sites of a patient with Werner syndrome in our hospital to identify the WRN gene mutation sites.Secondly,we verified the aging phenotype of normal human skin fibroblasts(HSF)caused by this mutation site through cytological experiments.Methods1.The clinical data of patients were collected.2.The DNA of patients’ peripheral blood was extracted,primers of 35 exons of WRN gene were designed,amplified by PCR,and the WRN gene sequence was detected by Sanger sequencing(the gold standard of gene sequence detection),and then compared with that of normal people to find mutation sites.3.The three-dimensional structure before and after the WRN gene mutation was simulated,and the spatial conformation before and after the WRN gene mutation was observed.4.The aging phenotype of human skin fibroblasts(HSF)at this mutation site was observed by cytological experiments:(1)Wild type WRN gene overexpression lentivirus,mutant WRN gene overexpression lentivirus and overexpression no-load lentivirus were constructed respectively,and the HSF cells were transfected into three groups,so that the target gene was overexpressed in the cells.Western blot(WB)was used to observe wild type group(WT group)and mutant group(MUT group).(2)Three groups of cells were stained respectively using β-galactosidase staining aging detection kit,and the positive rate of SA-β-gal staining of aging cells in each group was counted.Results1.According to the symptoms,clinical phenotype and auxiliary examination of patients,Werner syndrome could be diagnosed once the diagnostic criteria of Werner syndrome are met.2.Sanger sequencing found the mutation site of WRN gene in patients in this study,which was a new pathogenic mutation through database search.3.(1)Wild WRN lentivirus,mutant WRN lentivirus and empty negative control lentivirus were transfected into HSF cells respectively,and then the stable transformants of three groups of cells were screened out by puromycin,and the total proteins of the three groups of cells were extracted.The expression levels of p16 protein and LAMINB1 protein,which were indicators of cell aging among the groups,were detected by Western blot.With β-Actin protein as the internal reference control,the results showed that the expression level of aging index p16 was significantly increased and LAMINB1 was down regulated in WRN c.666-669 del TATT group,whether compared with WT group or NC group.(2)Three groups of cells were stained with SA-β-gal,and five visual fields were randomly observed.The positive rate of SA-β-gal staining of aging cells in each group was counted.Compared with WT group and NC group,the positive rate of SA-β-gal staining in mutational WRN c.666-669 del TATT group was significantly increased with statistical difference.Conclusions1.For a patient suspected of being diagnosed with Werner syndrome,the pathogenic mutation site NM?000553.6:exon7:c.666-669 del TATT:p.Ile222 fs of the WRN gene was detected by gene sequencing,and it was found that the patient was a new case of mutation.2.Confirmed by cytological experiments,the pathogenic mutation of WRN gene NM?000553.6:exon7:c.666-669 del TATT:p.Ile222 fs,could lead to the aging of HSF in human skin fibroblasts.