| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 91 === Genetic factor plays an important role in determining the development of human skin disorders. Except for anomalies associated with chromosomal aberrations, single-gene and multiple gene disorders are known to cause skin phenotypes, easily detected clinically. In this thesis research, molecular genetic and biochemical approaches were taken to investigate the pathogenesis of three diseases: neurofibromatosis type 1 (NF1), epidermolytic palmoplantar keratoderma (EPPK), and primary cutaneous amyloidosis (PCA). The former two are inherited by autosomal dominant mode, while the latter is mostly sporadic in nature.
Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome affecting the nervous system. Mutations of the NF1 gene are different among patients, making NF1 molecular diagnosis difficult. Molecular genetic analysis of NF1 patients in Taiwan has not been reported before; therefore, a mutation screen protocol needs to be established to analyze NF1 mutations in our population. A RT-PCR based DNA diagnosis procedure was established to investigate the NF1 gene mutation in neurofibromatosis type I. Five overlapping segments covering approximately 8.6 Kb of the NF1 gene were amplified and sequenced to identify genetic alteration(s) in the coding region. Four new mutations in three patients were uncovered by this protocol. We further investigated what caused the cDNA deletion by PCR, using genomic DNA as a template. We found that a recombination between homologous intronic sequences caused the 7260-8167 deletion of the NF1 gene in the first patient. In the other two patients, we identified a single-base substitution in intron 13, designated IVS13+1G>A, in the second case, and an intron 3 mutation, IVS3+1G >T, in the third case. Both mutations affected the splicing donor signal and caused frame-shift and truncation of the NF1 protein.
Epidermolytic palmoplantar keratoderma (EPPK) is a rare autosomal disorder of the skin. So far, only thirty-two kindreds of the keratin 9 gene have been documented in the English literature. We identified a four-generation family from Taiwan with typical clinical and histopathological features of EPPK. To identify the mutation of the new EPPK family and to determine whether the mutation in this Chinese family fall to the 1A region in the keratin 9 rod domain, we have analyzed the coding sequence of the keratin 9 gene in the family members and reviewed the mutation spectrum of familial EPPK in the literature. Whole blood sample was collected from affected and normal individuals of the family as well as 50 controls. Polymerase chain reaction was carried out to amplify the keratin 9 gene sequence. The PCR products were subjected to direct DNA sequencing for coding sequence analysis. A novel point mutation, designated 542T>G, (numbering from the first nucleotide of GenBank accession no. S69510) was identified. The mutation converts a leucine codon (CTC) to an arginine codon (CGC) at amino acid position 159 of keratin 9 protein in a conserved hydrophobic residue of the keratin heptad repeats. By literature review, we found that all familial EPPK mutations cluster within a 16-amino-acid region in the 1A rod domain of keratin 9 protein, and that the Taiwanese family adds a new base substitution type to the list of rare inherited mutations causing EPPK.
Primary cutaneous amyloidosis (PCA) is a late-onset, slowly progressing skin disease prevalent in Southeast Asia and South America. The nature of cutaneous amyloid in PCA remains unknown. To understand the molecular basis of PCA pathogenesis, we investigated the nature of amyloid deposit by immunofluorescence and RNA in situ hybridization. RNA in situ hybridization revealed that melanocytes produced amyloid precursor protein in the epidermis, while immunofluorescence microscopy showed specific antibodies against A4 amyloid peptide reacted with the amorphous amyloid material in the papillary dermis. Together, the results indicate that amyloid deposit in PCA is probably originating from melanocytes. Additionally, we established a method for extracting amyloid proteins for biochemical characterization. We concluded that PCA can serve as a model for studying amyloid formation in neural crest-derived cells in the skin.
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