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Title of Thesis

Institute/University/Department Details
Department of Biochemistry/ Quaid-i-Azam University, Islamabad
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
inherited ectodermal dysplasias, epidermal layer, placodes, mesenchyme, epidermal cell junctions, hypohidrotic ectodermal dysplasia, edar gene, edaradd gene

The blueprint for hair follicles, sweat glands, nails and teeth is laid down during embryonic development. This process starts with the formation of thickenings of the epidermal layer, called placodes. These are brought about by reciprocal signaling between the epithelial cells and the underlying mesenchyme. In humans, the inability to properly produce or pattern these placodes leads to diseases referred to as the ectodermal dysplasias (EDs).

Approximately, 200 distinct ectodermal dysplasias have been described in the literature. These disorders encompass a spectrum of developmental abnormalities of skin, hair, teeth, nails and sweat glands. Recent molecular studies have now begun to provide new insight into the pathogenesis of several ectodermal dysplasias. Indeed, over 30 EDs have been characterized through molecular analysis.

In the present study, five consanguineous, multigeneration Pakistani families with various types of EDs have been ascertained. Three of these families (A, B and C) were segregating autosomal recessive ED of hair and nail type while the other two families (D and E) were segregating autosomal recessive hypohidrotic ED. Pure hair and nail ED is a rare congenital disorder characterized by trichodysplasia (hypotrichosis, partial, or total alopecia) and dystrophy of finger and toe nails with no other associated abnormality. Various clinical forms of the disorder have been reported, the molecular bases of which are entirely unknown. To identify the gene underlying the hair and nail ED phenotype in the three Pakistani families (A, B and C), microsatellite markers from candidate gene regions were genotyped and two-point and multipoint parametric linkage analysis was performed.

In family A, the disease locus was mapped to a 16.6-centimorgan region on chromosome 12q12-q14.1 (Zmax = 8.2). The locus harbors six type II hair keratin genes. DNA sequence analysis revealed a homozygous missense mutation in the hair matrix and cuticle keratin KRTHB5, leading to substitution of a conserved arginine residue with histidine (R78H), located in the head domain. This mutation provides the first direct evidence in the molecular pathogenesis of pure hair-nail ectodermal dysplasias.

In family B, significant evidence of linkage was obtained for markers linked to type II keratin gene cluster on chromosome 12q13.13 (Zmax = 3.1). The linkage interval in this family maps to a 29.5 cM region according to the Marshfield genetic map. Sequence

analysis of the coding exons and splice junction sites of the hair keratin genes including KRTHBl, KRTHB3, KRTHB5 and KRTHB6, present in the disease interval, failed to identify a pathogenic mutation. These data suggest that the mutation may lie in a regulatory region of one of these hair keratin genes or in another gene within the linkage interval of family B on chromosome 12pll.l-q2l.1. In family C, the gene for pure hair and nail ED has been assigned to chromosome l7p 12q21.2. Significant evidence of linkage to this chromosomal region was found with multipoint Zmax > 4 at several markers. Haplotype analysis located the ED locus in a 24.2 cM genetic interval between markers D17S839 and D17S1299. The disease interval contains six epithelial keratin genes (KRT25A, KRT25B, KRT25C, KRT25D, KRT24 and KRTIO), and a homeobox gene (WHN). DNA sequence analysis of the coding exons and the splice junction sites of five epithelial keratin genes (KRT25A-KRT25D and KRT24) failed to reveal any sequence aberration in the affected individuals. However, the mutation may lie in a regulatory region of one of these epithelial keratin candidate genes.

Hypohidrotic ectodermal dysplasia (HED) is a genetic disease characterized by abnormal hair, teeth, and sweat gland development that may lead to life-threatening hyperthermia. Although most cases of HED display X-linked recessive inheritance, autosomal dominant and autosomal recessive forms also exist. X-linked HED is caused by mutations in the EDA gene, and the autosomal forms result from mutations in either the EDAR or EDARADD gene. In the present study, the molecular defect underlying autosomal recessive HED in eight patients belonging to two Pakistani families (D and E) has been investigated. Genotyping results showed linkage in both the families to the EDAR locus. DNA sequence analysis of EDAR gene in the two families led to the identification of two novel mutations. In family D, a missense mutation, replacing a non-polar glycine with polar serine (G382S) occurs in the death domain (DD), encoded by exon 12, of EDAR. In family E, sequence analysis revealed a four base pair deletion (718delAAAO) in exon 8 of the EDAR gene.

Download Full Thesis
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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
221.85 KB
2 1 Introduction 1
592.81 KB
  1.1 An Overview of Epidermis and its Associated Appendages 1
  1.2 Hair 4
  1.3 Nail 8
  1.4 Tooth 9
  1.5 Sweat Glands 10
  1.6 Epidermal Cell Junctions 11
  1.7 Keratin Intermediate Filaments 14
  1.8 Ectodermal Dysplasias 20
  1.9 Molecular Basis of Ectodermal Dysplasias 22
3 2 Materials and Methods 35
292.25 KB
  2.1 Families Studied 35
  2.2 Pedigree Analysis 35
  2.3 Clinical Examination 35
  2.4 Blood Sampling 35
  2.5 Genomic DNA Extraction 36
  2.6 Polymerase Chain Reaction (PCR) 36
  2.7 Agarose Gel Electrophoresis 37
  2.8 Polyacrylamide Gel Electrophoresis (PAGE) 37
  2.9 Genotyping 37
  2.10 Linkage Analysis 38
  2.11 Mutation Detection 38
  2.12 Sequence Alignments 38
4 3 Results 52
1600.94 KB
  3.1 Description of the Families Studied 52
  3.2 Clinical Findings 53
  3.3 Linkage Studies 54
  3.4 Mutation Analysis 58
5 4 Discussion 125
143.14 KB
  4.1 Ectodermal Dysplasia of Hair and Nail Type 125
  4.2 Hypohidrotic Ectodermal Dysplasia 132
6 5 References 135
250.54 KB
  5.1 Electronic data-base information 154