| Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant inherited skin disorder characterized by diffuse yellow thickening of palms and soles sharply bordered with erythematous margins. Histologically and ultrastructurally, EPPK presents cytolysis of keratinocytes and abnormal aggregation of keratin filaments in the suprabasal layers of epidermis. EPPK patient shows the typical symptoms within several weeks or months after birth.Here, four EPPK families including 48 patients and 106 normal members from Ningbo, Zhejiang Province, China, were investigated. Denaturing high performance liquid chromatograph (DHPLC) and DNA sequencing were used to screen KRT9 gene for sequence variations. A novel insertion-deletion (indel) mutation, 497delAinsGGCT, was identified in exon 1 of KRT9, resulting in the change of Tyrosine166 to Tryptophan and Leucine (Y166delinsWL). Allele specific-PCR (AS-PCR) confirmed that (i) this indel was co-segregated with all affected individuals in four EPPK pedigrees, and (ii) all EPPK patients were heterozygous for the mutation. This is the first revealed indel in highly conserved helix initiation motif of rod domain of keratin 9, and has been accepted by the Human Intermediate Filament Mutation Database (http://www.interfil.org). The results suggested that this novel indel might be the causative mutation of these EPPK cases and provided the molecular basis of classification, molecular diagnosis, prenatal diagnosis and be helpful for the new drug and gene therapy of this disease.In addition, we confirmed that PCR products from heterozygous mutation contain not only wide-type and mutant homoduplexes, but also two heteroduplexes, by the indel (497delAinsGGCT) heterozygotes. Thus, in our opinion, it is not necessary to heat and cool PCR products obtained from heterozygous mutation in advance before using mutation screening methods such as DGGE, TGGE, CSGE, DHPLC and HA, etc.
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