Detection Of Interaction Between Wild Type ADAR1 Protein And Its R916W Mutant Using Yeast And Mammalian Two Hybrid Systems And Gene Locus And Mutation Screening In Marie Unna Hereditary Hypotrichosis

Dyschromatosis Symmetrica Hereditaria(DSH, MIM 127400) is a skin disorder caused by mutation of ADAR1. By now, more than fourty mutations of ADAR1 in DSH have been revealed. Previously, we have reported two novel mutations of ADAR1, which are a nonsence mutation p.513x and a missence mutation p.R916W in two Chinese DSH families. Although many mutaions have been found in DSH, the pathogenic mechanism is still obscure.Double-stranded RNA-specific adenosine deaminase (ADAR) was identified as a developmentally regulated dsRNA unwinding activity in early antisense experiments with Xenopus oocytes. The enzyme converts adenosine to inosine in dsRNA, which destabilizes the dsRNA helix. The RNA modifying activity of ADAR1 is important for various functions. Among these are site-specific RNA editing of transcripts of the glutamate receptors which are channels for the neurotransmitter L-glutamate in the brain. ADAR1 also functions to modify viral RNA genomes and may be responsible for hypermutation of certain negative-stranded viruses, such as measles, which may result in lethal measles inclusion body encephalitisAs a highly conserved housekeeping gene, ADAR1 plays an important role in many kinds of biological processes. ADAR1 functions as a RNA editase which catalyses adenosine to inosine in pre-mRNA. Editing in coding sequence by ADAR1 can lead to alteration of codons and functions of the affected genes, whereas editing of the noncoding sequence, mainly involving the intron and 3'UTR, can affect mRNA splicing, translation, and stability. ObjectiveRecently, it is clarified that dimerization is essential for the RNA editing activity of ADAR1. Furthermore, we previously revealed that the development of DSH was attributed to the haploinsufficiency of ADAR1 protein due to its gene mutations. Therefore, the affection of these mutations on the formation and activity of the homodimer may be a key to obtain insight into the correlation between the genotype and the phenotype of DSH.MethodThe present study was designed to detect protein-protein interaction between the wild type ADAR1 (ADAR1^WT) and its R916W mutant (ADAR1^R916W) using yeast and mammalian two-hybrid systems, and to gain some insight into the molecular mechanism by which the missense mutations in the ADAR1 gene cause DSH.ResultThe result showed that yeast cells co-transformed with plasmids pGADT7-ADAR1^WT and pGBKT7-ADAR1^WT or plasmids pGADT7-ADAR1^WT and pGBKT7-ADAR1^R916W failed to form colonies on SD/-Trp/-Leu/-His/-Ade plates, indicating a lack of protein-protein interaction. HeLa cells co-transfected with plasmids pACT-ADAR1^WT, pBIND-ADAR1^WT and pG5luc showed significant increase in luciferase activity (P＜0.05). Whereas cells co-transfected with pACT-ADAR1^WT, pBIND-ADAR1^R916W and pG5luc exhibited considerable decrease in luciferase activity (P＜0.05), retaining 66% of the activity compared with the wild-type counterpart.ConclusionThis indicates that the RNA editing activity of ADAR1 may decrease in patients with DSH, which serves as possible mechanism of DSH. Marie Unna hereditary hypotrichosis (MUHH) is a rare autosomal dominant disorder. In most MUHH pedigrees, the affected members usually have little or no body hair, eyelashes, eyebrows, and secondary sexual hair. Apart from hair loss, there are no common associated disorders in MUHH. Light microscopic examination from hair of the scalp could show thick, irregular, and twisted hair.There are some studies on the molecular basis of MUHH. In a large Dutch family with MUHH, linkage with marker D8S258 on 8p was detected by a genome-wide search and informative recombinants placed the MUHH locus in a 2.4 cM interval between D8S258 and D8S298 on 8p22-p21. This locus was conformed by other several genetic linkage studies and the genetic interval was further narrowed.ObjectiveBecause the MUHH locus mapped to the same region of chromosome 8 as the human homolog of the mouse 'hairless' gene, which had been shown to carry recessive mutations responsible for congenital atrichia, our work then focused on mutation screening of HR.MethodIn this study, in order to refine the MUHH locus to a narrow chromosome region and screen the candidate genes causing the disease, we performed genotyping and two-points linkage analysis in a Chinese family with MUHH, using 4 high-density microsatellite markers mapped at 8p21, meanwhile, we amplified and sequenced all the exons of two candidate genes, HR and RAI16, by PCR. The mutation found by automatic sequencing was identified using restriction enzyme Smal in both all the families and 80 normal persons.ResultThe result showed that in the pedigree, significant evidence for linkage was observed in 8p21, with a maximum two-point LOD score of 3.14 (θ= 0). Through all the exons of RAI16 and HR have been sequenced, no mutation was found, until a base substitutaion G2104T was observed in the 7th exon.ConclusionThis study provides a refined map location for isolation of the gene causing MUHH in the region of 8p21 and excludes the possibility of all the exons in both HR and RAI16 as candidates for the disorder.