Study On The Mutations Of ADAR1 Gene In The Patients With Dyschromatosis Symmetrica Hereditaria

Background: Dyschromatosis symmetrica hereditaria (DSH)(also called"reticulate acropigmentation of Dohi") is an autosomal dominant disease characterized by a mixture of hyperpigmented and hypopigmented macules distributed on the dorsal aspects of the extremities and freckle-like macules on the face.DSH has been reported mainly in Japan and China, although a few cases were described among Koreans, Indians, Europeans and South Americans. No racial difference in the condition has been observed, but the disorder might be distributed mainly in East Asia. The lesions usually appear in infancy or early childhood, commonly stop spreading before adolescence, and last for life. The DSH locus has been mapped to chromosome 1q21 and then, in 2003, pathogenic mutations were identified in the double-strand RNA-specific adenosine deaminase(ADAR1) gene. ADAR1 spans 30 kb and contains 15 exons. It encodes RNA-specific adenosine deaminase composed of 1226 amino acid residues, with a calculated molecular mass of 139 kDa. ADAR1 protein catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates, which results in the creation of alternative splicing sites or alternations of the codon and thus leads to functional changes in the protein. The ADAR1 gene is expressed ubiquitously, but the target gene(s) in the skin still remain unknown, and also the molecular pathogenesis of DSH has not been clarified yet. So far, there has been reported more than 70 mutations, including mis-sense mutations, splice-site mutations, frame-shift mutations, nonsense mutations. In order to present precise information for gene diagnosis, prenatal diagnosis and gene therapy in future, we want to detect new mutations in patients with DSH of Shandong province, China in this study.Objective:(1) To detect the mutations in ADAR1 gene of patients with DSH. (2) To analyze the characteristic of the clinical feature and mutations in patients, and to investigate the genotype-phenotype correlation. (3) To detect mutation of one patient with atypical clinical features in a family of DSH. Materials and Methods: After informed consent, 6ml peripheral blood were gained from patients. DNA was extracted from samples using Takaba kit. Polymerase chain reaction (PCR) amplification of the ADAR1 gene was performed using 13 pairs of primers spanning all 15 exons and flanking sequence of ADAR1 gene. Direct sequencing was performed to screen the mutations in ADAR1 gene. And then RNA was extracted from peripheral blood of two families. Reverse transcription-polymerase chain reaction (RT-PCR) was used for investigating the expression of mRNA. Control samples obtained from DNA source of State Key Laboratory of Genetic Engineering, Fudan University. Results: Nine mutations were identified in ten families and one sporadic case, including four missense mutations(c.K1105N,c.G1047A,c.F1095L,c.G1067R), two frameshift mutations (p.Q779fs-792X, p.P441fs-463X), two splice-site (c.2886-5T>C, c.3021-2G>A), and one nonsense mutation(p.R1096fs), of which eight mutations were first reported. But we failed to detect any mutation in two families. In addition, for one patient, whose clinical phenotypes did not accord with typical clinical manifestation of DSH, we made the diagnose of DSH by direct DNA sequencing.Conclusion: Eight new mutations were reported including four missense mutations(c.K1105N,c.G1047A,c.F1095L,c.G1067R), two frameshift mutations (p.Q779fs-792X, p.P441fs-463X), two splice-site (c.2886-5T>C, c.3021-2G>A). We diagnosed the first case by DNA sequencing.