Investigations On Gene Mutations Of Dyschromatosis Symmetrica Hereditaria And Epidermolytic Hyperkeratosis

Background1. Dyschromatosis symmetrica hereditaria （DSH, MIM 127400）, is also called reticulate acropigmentation of Dohi or symmetric dyschromatosis of the extremities. It is a pigmentary genodermatosis characterized by a mixture of hyperpigmented and hypopigmented macules and is localized on the back of the hands and feet. Some patients with DSH also have small freckle-like pigmented macules on their faces. The disease begins usually during infancy or childhood, commonly stops spreading before adolescence, and lasts for life. Apart from the skin lesions, there are no common associated disorders in DSH. DSH has been reported mainly in Japanese and Chinese. The DSH locus has been mapped to chromosome 1q21 and subsequently causative mutations have been identified within the double-stranded RNA-specific adenosine deaminase （DSRAD） gene. DSRAD （also called ADAR1） gene encodes RNA-specific adenosine deaminase. The enzyme converts adenosine to inosine in pre-mRNA.2. Epidermolytic hyperkeratosis （EHK; OMIM:113800）) is also called bullous congenital ichthyosiform erythroderma（BCIE）. It is a rare disorder of keratinization, approximately 1 of every 100,000-300,000 live births will be affected by this disorder. It is characterized by blistering and erythema at birth, and development of hyperkeratosis with increasing age, predominantly over large flexural joint areas and on palms and soles. Skin biopsies from patients showed marked epidermal acanthosis and hyperkeratosis, coarse keratohyaline granules, and mild vacuolization in the upper stratum spinosum. There is significant clinical heterogeneity in disease severity among affected individuals. Several studies have linked the disease to the typeⅡand typeⅡkeratin loci on chromosome 12q and 17q, respectively, and mutations have been described in highly conserved regions of keratrins 1 and 10.Objectives1. To investigate the gene mutation in four pedigrees with DSH, to explore the relationship between the mutation and clinical manifestations and further to establish basic for the genetic diagnosis and the genetic treatment.2. To investigate the gene mutation in a pedigree with epidermolytic hyperkeratosis, to explore the relationship between the mutation and clinical manifestations and further to establish basic for the genetic diagnosis and the genetic treatment.Methods1. Skin biopsies of patients with DSH were processed for haematoxylin and eosin staining. Genomic DNA of affected members, normal members of four pedigrees and 50 unrelated normal members was extracted with a whole blood genomic DNA extraction kit and was used as a template for the polymerase chain reaction （PCR）-mediated amplification of all exons of DSRAD gene. Direct sequencing of all PCR products of the whole coding regions of DSRAD was performed to identify the mutation.2. Skin biopsies of patients with EHK of the pedigree were processed for haematoxylin and eosin staining. Genomic DNA of affected members, normal members of the pedigree and 50 unrelated normal members was extracted with a whole blood genomic DNA extraction kit and was used as a template for the polymerase chain reaction （PCR）-mediated amplification of all exons of KRT1. Direct sequencing of all PCR products of the whole coding regions of KRT1 was performed to identify the mutation.Results1. Results of the investigations on gene mutations of dyschromatosis symmetrica hereditaria Combined with microscopic changes of affected lesions and clinical symptom, diagnosis of DSH was obtained in all pedigrees. Expected DNA fragments were amplified by PCR. The c.2433₂434delAG （p.T811fs） mutation was found in patients but not in the healthy members of family 1.The c.2565-2568delGACT（Lys 855fs） mutation was found in the affected members but not in the healthy individuals in family 2. We failed to find any mutation in PCR production of all exons of the DSRAD gene in family 3. A missense mutation 2747G→T in the DSRAD gene was found in the affected members in family 4 but not in the healthy individuals and in 50 unrelated controls.2. Result of the investigation on gene mutation of epidermolytic hyperkeratosis Combined with microscopic changes of affected lesions and clinical symptom, diagnosis of epidermolytic hyperkeratosis was obtained. Expected DNA fragments were amplified by PCR. Direct sequencing of the whole coding regions of KRT1 disclosed a heterozygous T to C transition at nucleotide position 623 of exon 2 of KRT1 in the patients. This nucleotide substitution resulted in the change of codon 208 for leucine to proline（L208P） in the 1A domain of keratin 1.Conclusions1. We detected two heterozygous mutation [c.2433₂434delAG （p.T811fs） and c.2565-2568delGACT（Lys 855fs）] in two Chinese pedigrees with DSH, which both lead to frameshift and premature translation termination within exon 8. The gene with these frameshift mutations would make the truncated protein with no double-strand RNA adenosine deaminase catatytic domain. The c.2565-2568delGACT（Lys 855fs） is the second mutation reported in exon 8 of DSRAD gene. We detected a new heterozygous missense mutation c.2747G>T（p.R916L） in an other Chinese pedigree with DSH, which alters one of the highly conserved amino acid residue in the putative deaminase catalytic domain. The mutation resulted in a replacement of the basic hydrophilic arginine （R） by the nonpolar hydrophobic amino acid Leucine （L）.We failed to find any mutation in DSRAD gene in family 3. We should do more work on it.2.we report a novel missense mutation in the 1A segment of theα-helical rod domain keratin 1 in a Chinese pedigree with EHK. This result expands the repertoire of KRT1 mutations underlying EHK and re-emphasizes the role of mutation in the highly conserved rod domain of keratin 1 in the pathogenesis of EHK.