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Description:
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Natural diseases caused by keratin mutations are rare and have only been reported in humans . We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10 . Affected dogs have a tendency to form shallow erosions or blisters following mild trauma , which is first noted after the birthing process . As the dogs age , they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions . The main histologic and ultrastructural features include : marked hyperkeratosis , epidermal hyperplasia , prominent vacuolation of the upper suprabasal layers , eosinophilic intracytoplasmic aggregates (keratin bundles ) , numerous and frequently enlarged keratohyaline granules , and epidermal hyperplasia . Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance . The keratin 10 mutation was defined as a G -T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5 . The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site . Real -time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35 -fold decrease in KRT10 message .
Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers . After 21 days of culture , normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum . Affected epidermis largely reproduced the expected morphologic alterations . Immunoblotting and immunohistochemistry for keratin 10 protein and real -time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations .
A diagnostic PCR assay was established for detection of the carrier state . Global analysis of gene expression between normal , carrier and affected dogs was performed with DermArray cDNA microarrays . Affected and carrier dogs showed differential regulation of 320 and 298 genes , respectively , between normal dogs . In affected dogs , 217 were upregulated and 103 were downregulated . In carrier dogs , 222 were upregulated and 76 were downregulated . 72 genes (65 upregulated , 7 downregulated ) were altered in both affected and heterozygous dogs . |