Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias
by Muhammad Tariq
Department of Biochemistry Faculty of Biological Sciences Quaid-I-Azam University Islamabad, Pakistan 2009
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias A thesis submitted in the partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry/ Molecular Biology
by Muhammad Tariq Department of Biochemistry Faculty of Biological Sciences Quaid-I-Azam University Islamabad, Pakistan 2009
In the name of Allah, the Most Gracious, the Most Merciful
He is the One who created the heavens and the earth, truthfully. Whenever He says, "Be," it is. His word is the absolute truth. All sovereignty belongs to Him the day the horn is blown. Knower of all secrets and declarations, He is the Most Wise, the Cognizant. (Al-Quran 6:73)
Dedicated to My sweet & beloved parents & brothers
Declaration
I hereby declared that the work presented in this thesis is my own effort and hard work and it is written and composed by me. No part of this thesis has been previously published or presented for any other degree or certificate.
Muhammad Tariq Contents
CONTENTS
Title Page No.
Preface
Acknowledgements I
List of Abbreviations III
List of Tables VII
List of Figures XI
Summary XIX
List of Publications XXIII
Chapter 1: Introduction 1
Human Skin 1
Epidermis 2
Dermis 2
Hypodermis 2
Ectodermal Appendages 2
Hair 2
Nail 3
Tooth 4
Sweat Glands 5
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias Contents
Eccrine Glands 6
Apocrine Glands 6
Ectodermal Dysplasias 6
Clinical Manifestations in Ectodermal Dysplasias 6
Classification of Ectodermal Dysplasias 7
Earlier Classification 7
1) Clinical Classification 7
2) Clinical-Genetic Classification 7
Modern Classification 8
1) Gene-based Classification (Molecular Classification) 8
2) Current and Online Genodermatoses Database 9
Ectodermal Dysplasias Involving Major Ectodermal 9 Appendages
Hypohidrotic Ectodermal Dysplasias (HED) 9
Ectodermal Dysplasias of Hair-Nail Type 10
Ectodermal Dysplasias of Hair-Teeth Type 11
Ectodermal Dysplasias of Nail-Teeth Type 11
Ectodermal Dysplasias of Hair-Nail-Teeth Type 11
Isolated Nail Disorders 12
Isolated Congenital Anonychia 12
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias Contents
Isolated Congenital Nail Dysplasia (ICND) 12
Isolated Congenital Nail Clubbing (ICNC) 13
Hereditary Nail Dysplasia 14
Syndromic Forms of Ectodermal Dysplasias 14
Trichorhinophalangeal Syndrome (TRPS) 14
Autosomal Recessive Ichthyosis with Hypotrichosis Syndrome 14 (ARIH)
Ellis-van Creveld Syndrome (EvC) 15
Cerebral Dysgenesis, Neuropathy, Ichthyosis, and 15 Keratoderma Syndrome (CEDNIK Syndrome)
Trichodentoosseous (TDO) Syndrome 16
Ectrodactyly, Ectodermal Dysplasia, and Cleft lip/palate 16 Syndrome (EEC)
Alopecias 17
Autosomal Recessive Alopecias 17
Congenital Atrichia 17
Recessive Hereditary Hypotrichosis 18
Human Nude Phenotype 19
Hypotrichosis with Juvenile Macular Dystrophy (HJMD) 20
Alopecia with Mental Retardation Syndrome (APMR) 20
Autosomal Dominant Alopecias 21
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias Contents
Monilethrix 21
Marie Unna Hereditary Hypotrichosis (MUHH) 21
Autosomal Dominant Localized Hereditary Alopecia 21
Hypotrichosis Simplex of the Scalp (HSS) 22
Chapter 2: Materials and Methods 23
Families Studied and Pedigree Analysis 23
DNA Analysis 23
Blood Sampling 23
DNA Extraction and Purification 24
Genotyping 25
Exclusion Mapping 25
Genome-wide Search 25
Polymerase Chain Reaction (PCR) 25
RT-PCR Analysis 26
Agarose Gel Electrophoresis 26
Polyacrylamide Gel Electrophoresis (PAGE) 26
DNA Sequencing 27
DNA Sequence Alignment 28
Statistical Analysis 29
Tables of Chapter 2 30
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias Contents
Chapter 3: Ectodermal Dysplasias 61
Results 62
Human Subjects and Clinical Findings 62
Family A 62
Family B 62
Family C 63
Family D 64
Family E 65
Family F 66
Linkage and Mutational Analysis 67
Family A 67
Family B 69
Family C 71
Family D 73
Family E 74
Families F 75
Discussion 75
Figures and Tables of Chapter 3 87
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias Contents
Chapter 4: Alopecias 139
Results 140
Human Subjects and Clinical Findings 140
Family G 140
Family H 140
Family I 141
Family J 141
Linkage and Mutational Analysis 142
Family G 142
Family H 143
Family I 143
Family J 144
Discussion 145
Figures and Tables of Chapter 4 149
Chapter 5: General Conclusion 169
Chapter 6: References 171
Literature References 171
Electronic Database Information 199
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias Preface Acknowledgements
ACKNOWLEDGEMENTS
All praises to be Almighty ‘Allah’ The most Merciful, without Allah’s divine help, man would not have been able to achieve anything in life. All regards, respects and blessings be upon the Holy Prophet Hazrat Muhammad (Sallallahu aliahi wa sallam), whose blessings and teachings flourished my thoughts and thrived my ambitions.
I’m greatly honored to pay my deep gratitude to my most learned, perfectionist and considerate supervisor Prof. Dr. Wasim Ahmad, Chairman Department of Biochemistry, Faculty of Biological Sciences, Quaid-I-Azam University Islamabad, under whose inspiring guidance, valuable suggestions, sympathetic attitude, immense patience and encouragements this research work was carried out. His energy, optimism, intelligence and continuous encouragement at every step during the course of this whole project enabled me to achieve my goals. I must say that without his support and kind efforts this task would have been impossible.
I contribute my special thanks and prayers to the family members on whom this research was conducted for their participation and cooperation.
I wish to express sincere thanks to my worthy seniors and colleagues Muhammad Jawad Hassan, Dr. Peter John, Muhammad Salman Chishti, Dr. Abdul Wali, Dr. Asma Gul, Ghazanfar Ali, Musharraf Jelani, Zahid Azeem, Naveed Wasif, Muhammad Ayub, Kamran Naqvi, Sulman Basit, Saadullah, Rizwana, Kulsoom, Bushra, Rabia and Gul Naz for their cooperation, suggestions and nice company during my research work, which is precious to me in all regards..
I present my the most heartiest thanks to my friends Sher Zaman Safi, Muhammad Tahir, Ajmal Khan, Fida Hussain, Arshad Islam, Akram Rahi, Sanaullah Gul, Saqib, Umar Zaman, Waheed, Ghayas Khattak, Muhammad Rafiq, Dr. Muhammad Khalid, Mohsin Shah, Fayyaz Shah, Azizullah, Sabir Zada, Javed Iqbal, Muhammad Sohail, Capt. Akhtar Nawaz, Mubarik Shah, Sabir Hussain, and Dr. Rizwan for their nice company and advice through out my research work and my stay at QAU. The good time spent with them can never be erased from my memories.
At this stage, I should pay the most heartiest thanks to all my M.Sc classmates at QAU Waqas Ahmad, Naveed Wasif, Irfanullah BTN, Kamran Naqvi, Hamdah
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias I Preface Acknowledgements
Shafqat, Bushra Ijaz, Gul Naz, Aneela Ijaz, Bushra, Shama, Gul Rahim, Aiesha, Saeed Khattak, Asad Razzaq, Mehwish, Salma, Saiqa, Saliha, Umara, Jehanzeb (Janzy), Naila, Abdul Hameed, Tahir Abbas, Rohee, Fakhar Islam, Munzir, Sadia Munawar, Sadia Naeem, Omais, Asim, Usman, Ashraf, and Shoukat for their cooperation, company and prayers. I convey my heartiest and sincerest acknowledgements to my all teachers since class prep, for their kind supervision without which I cannot attain this position. All of my teachers contributed equally in my knowledge and character-building.
No words can express my deepest gratitude and feelings to my dearest parents whose firm dedication, inbuilt confidence and untiring efforts led me to achievement of this goal. I owe deep gratitude to my brothers Shahid, Basharat, Abid and cousins Kamran, Fawad, Naazma, Imran, Saad, Mehran, Rameez, Shahroom, Shahzad, Saba Gul, Maria, Shandana and Moiz for their unmatched support, love and prayers during this period and console me to complete this research work.
Finally, I wish to thank the Higher Education Commission (HEC), Islamabad Pakistan, for awarding indigenous PhD fellowship and funding this project through research grants to my supervisor.
Muhammad Tariq
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias II Preface List of Abbreviations
LIST OF ABBREVIATIONS aa Amino Acid AD Autosomal Dominant ADHED Autosomal Dominant Hypohidrotic Ectodermal Dysplasia AH Autosomal Recessive Hypotrichosis APL Atrichia and Papular Lesions APMR Alopecia with Mental Retardation AR Autosomal Recessive ARHED Autosomal Recessive Hypohidrotic Ectodermal Dysplasia ARHS Autosomal Recessive Hypotrichosis Simplex ARIH Autosomal Recessive Ichthyosis with Hypotrichosis ARWH Autosomal Recessive Woolly Hair BMP Bone Morphogenetic Protein bp Basepair °C Centigrade CDH Cadherin cDNA Complementary Deoxyribonucleic Acid CDSN Corneodesmosin CEDNIK Cerebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma CLP Cleft Lip/Palate cm Centimeter cM Centimorgan cr Crinkled DL Downless dl downless DNA Deoxyribonucleic Acid dNTP Deoxynucleoside Triphosphate Del Deletion DLX Distal-less homeobox DSC Desmocollin DSG Desmoglein DSP Desmoplakin
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias III Preface List of Abbreviations
ED Ectodermal Dysplasia EDA1 Ectodysplasin A1 Isoform EDAR Ectodysplasin A1 Isoform Receptor EDARADD Ectodysplasin A1 Isoform Receptor Associated Death Domain EDTA Ethylenediamine Tetra Acetic Acid EEC Ectrodactyly, Ectodermal dysplasia, Cleft lip/Palate EEM Ectodermal Dysplasia, Ectrodyctyly and Macular Dystrophy ESTs Expressed Sequence Tags EVC Ellis-van Creveld EVPL Envoplakin EXT1 Exostosin 1 FGF Fibroblast Growth Factor fs Frameshift GJB6 Gap Junction Beta-6 GPCR G Protein Coupled Receptor HDF Hi Di Formamide HF Hair Follicle hHb Human Hair Basic HJMD Hypotrichosis with Juvenile Macular Dystrophy HME Hereditary Multiple Exostoses HR Hairless HSS Hypotrichosis Simplex of the Scalp HTS Hypotrichosis Simplex ICNC Isolated Congenital Nail Clubbing ICND Isolated Congenital Nail Dysplasia IQ Intelligence Quotient IRB Institutional Reveiw Board IRS Inner Root Sheath ORS Outer Root Sheath ins Insertion Kb Kilo basepair KRTHB Keratin Hair Basic LAH Localized Autosomal Recessive Hypotrichosis
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias IV Preface List of Abbreviations
LIPH Lipase-H LMB Limb-Mammary Syndrome LOD Logarithm of Odds LOR Loricrin LPA 2-Acyl Lysophosphatidic Acid Mg Milligram MIM Mendelian Inheritance in Man ml Milli Litre mM Milli Molar Mb Mega Basepair MR Mental Retardation MUHH Marie Unna Hereditary Hypotrichosis MSA Multiple Sequence Alignment MSX1 Muscle Segment Homeobox 1 NCBI National Center for Biotechnology Information NEMO Nuclear factor-kappa-B Essential Modulator ng Nanogram OD Optical Density OODD Odonto-Onycho-Dermal Dysplasia OPG Orthopantomogram p53 (Tumor) protein 53 p63 (Tumor) protein 63 PAGE Polyacrylamide Gel Electrophoresis PAX Paired Box PCR Polymerase Chain Reaction PG Prostaglandin
PGE2 Prostaglandin E 2 PGT Prostaglandin Transporter PHO Primary Hypertrophic Osteoarthropathy PIMS Pakistan Institute of Medical Sciences PKP1 Plakophillin 1 PTC Premature Termination Codon PVRL1 Polio Virus Receptor-Like 1
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias V Preface List of Abbreviations rpm Revolution Per Minute RSPO R-Spondin SDR Short chain Dehygrogenases/Reductases SDS Sodium Dodecyl Sulfate SHH Sonic Hedgehog SNAP29 Synaptosomal -Associated Protein 29 SNPs Single Nucleotide Polymorphism STR Short Tandem Repeat Ta Tabby Taq Thermophillus aquaticus TDO Tricho-Dento-Osseous TE Tris-EDTA TEMED N, N, N’, N’-Tetramethylethylene-diamine TGF Transforming Growth Factor TGM Transglutaminase TM Transmembrane TNF Tumor Necrosis Factor TNFR Tumor Necrosis Factor Receptor TP73L Tumor Protein 73-Like TRPS Trichorhinophalangeal Syndrome UCSC University of California Santa Cruz uM Micro Molar UV Ultra Violet μl Micro Liter VDR Vitamin D Receptor WHN Winged Helix Nude Wnt Wingless XEDAR X-Linked Ectodysplasin A1 Isoform Receptor XLHED X-Linked Hypohidrotic Ectodermal Dysplasia XPD Xeroderma Pigmentosum D ZNF Zinc Finger
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias VI Preface List of Tables
LIST OF TABLES
Table Title Page No. No.
2.1 Microsatellite markers used to test linkage to known genes/loci 30
involved in ectodermal dysplasias and alopecias
2.2 Primer sequences used for screening CDH7 gene in family A 33
2.3 Primer sequences used for screening CDH19 gene in family A 34
2.4 Primer sequences used for screening ZNF407 gene in family A 35
2.5 Primer sequences used for screening MFAP3L gene in family B 36
2.6 Primer sequences used for screening HMGB2 gene in family B 36
2.7 Primer sequences used for screening ASB5 gene in family B 37
2.8 Primer sequences used for screening ADAM29 gene in family B 37
2.9 Primer sequences used for screening VEGFC gene in family B 38
2.10 Primer sequences used for screening HAND2 gene in family B 38
2.11 Primer sequences used for screening ANXA10 gene in family B 39
2.12 Primer sequences used for screening MORF4 gene in family B 40
2.13 Primer sequences used for screening SAP30 gene in family B 40
2.14 Primer sequences used for screening HPGD gene in family B 41
2.15 Primer sequences used for screening HOXA1 gene in family C 42
2.16 Primer sequences used for screening HOXA2 gene in family C 42
2.17 Primer sequences used for screening HOXA3 gene in family C 43
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias VII Preface List of Tables
2.18 Primer sequences used for screening HOXA4 gene in family C 43
2.19 Primer sequences used for screening HOXA5 gene in family C 44
2.20 Primer sequences used for screening HOXA6 gene in family C 44
2.21 Primer sequences used for screening HOXA7 gene in family C 44
2.22 Primer sequences used for screening HOXA9 gene in family C 45
2.23 Primer sequences used for screening HOXA10 gene in family C 45
2.24 Primer sequences used for screening HOXA11 gene in family C 46
2.25 Primer sequences used for screening HOXA13 gene in family C 46
2.26 Primer sequences used for screening NEUROD6 gene in family C 47
2.27 Primer sequences used for screening FERD3L gene in family C 47
2.28 Primer sequences used for screening PRR15 gene in family C 47
2.29 Primer sequences used for screening EVX1 gene in family C 48
2.30 Primer sequences used for screening TWIST1 gene in family C 48
2.31 Primer sequences used for screening SP8 gene in family C 48
2.32 Primer sequences used for screening SP4 gene in family C 49
2.33 Primer sequences used for screening CCDC126 gene in family C 49
2.34 Primer sequences used for screening JAZF1 gene in family C 50
2.35 Primer sequences used for screening SNX10 gene in family C 50
2.36 Primer sequences used for screening TRA2A gene in family C 51
2.37 Primer sequences used for screening ITGB8 gene in family C 52
2.38 Primer sequences used for screening CBX3 gene in family C 53
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias VIII Preface List of Tables
2.39 Primer sequences used for screening TWISTNB gene in family C 53
2.40 Primer sequences used for screening AQP1 gene in family C 54
2.41 Primer sequences used for screening HNRNPA2B1 gene in family C 54
2.42 Primer sequences used for screening EDA1 gene in family D 55
2.43 Primer sequences used for screening EDAR gene in family E 56
2.44 Primer sequences used for screening TRPS1 gene in family F 57
2.45 Primer sequences used for screening P2RY5 gene in family G and H 58
2.46 Primer sequences used for screening DSG4 gene in family I 59
2.47 Primer sequences used for screening HR gene in family J 60
3.1 Clinical features of the affected individuals in family C 93
3.2 Two point and multipoint LOD score results between the 111 ectodermal dysplasia locus and chromosome 18 markers in family A
3.3 Two point and multipoint LOD score results between ICNC locus 118 and chromosome 4 markers in family B
3.4 Two point and multipoint LOD score results between ectodermal 129 dysplasia locus and chromosome 7 markers in family C
3.5 Neutral polymorphisms detected in genes sequenced in family C 131
3.6 Insertion and deletion mutations reported in the EDA1 gene 133
3.7 Mutations in the EDAR gene implicated in the pathogenesis of HED 136
3.8 Mutations in exon 6 of the TRPS1 gene associated with TRPS III 138
4.1 Mutations reported in the P2RY5 gene 162
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias IX Preface List of Tables
4.2 Mutations reported in the DSG4 gene causing autosomal recessive 166 hereditary hypotrichosis (LAH1) and autosomal recessive monilethrix
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias X Preface List of Figures
LIST OF FIGURES
Figure Title Page No. No.
3.1 Pedigree of family A segregating ectodermal dysplasia of hair, nail 87 and teeth type.
3.2 Clinical features of the affected individuals in family A 88
3.3 Pedigree of family B segregating autosomal recessive Isolated 89 Congenital Nail Clubbing (ICNC)
3.4 Clinical findings of the affected individuals in family B with 90 Isolated Congenital Nail Clubbing (ICNC)
3.5 Radiological findings of the affected individuals in family B with 91 Isolated Congenital Nail Clubbing (ICNC)
3.6 Pedigree of family C with a novel form of ectodermal dysplasia 92
3.7 Clinical features of an affected individual (V-4) in family C with a 94 novel form of ED: hypotrichosis
3.8 Clinical features of an affected individual (V-4) in family C with a 95 novel form of ED: nail and syndactyly findings
3.9 Clinical findings of an affected individual (V-4) in family C: oral 96 view
3.10 Pedigree of family D with X- linked hypohidrotic ectodermal 97 dysplasia (XLHED)
3.11 Clinical findings of the affected individuals in family D with X- 98 linked hypohidrotic ectodermal dysplasia (XLHED)
3.12 Pedigree of family E segregating autosomal recessive hypohidrotic 99
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XI Preface List of Figures
ectodermal dysplasia (ARHED)
3.13 Clinical findings in family E with autosomal recessive hypohidrotic 100 ectodermal dysplasia (ARHED)
3.14 Pedigree of family F segregating trichorhinophalangeal syndrome 101 type III (TRPS III)
3.15 Clinical findings of the affected individuals in family F with TRPS 102 III: facial deformity and hypotrichosis
3.16 Clinical findings of the affected individuals in family F with TRPS 103 III: brachydactyly
3.17 Radiological findings of an affected individual (IV-1) in family F 104 with TRPS III
3.18 Allele pattern obtained with marker D18S857 at 98.35-cM on 105 chromosome 18q22.1
3.19 Allele pattern obtained with marker D18S386, distal to D18S857 on 105 chromosome 18q22.1
3.20 Allele pattern obtained with marker D18S1373 at 102.35-cM on 106 chromosome 18q22.1-q22.2
3.21 Allele pattern obtained with marker D18S1131 at 102.51-cM on 106 chromosome 18q22.1-q22.2
3.22 Allele pattern obtained with marker D18S817 at 103.72-cM on 106 chromosome 18q22.2
3.23 Allele pattern obtained with marker ATA82B02 at 105.40-cM on 107 chromosome 18q22.3
3.24 Allele pattern obtained with marker D18S848 at 105.87-cM on 107 chromosome 18q22.3
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XII Preface List of Figures
3.25 Allele pattern obtained with marker D18S1106 at 106.63-cM on 107 chromosome 18q22.3
3.26 Allele pattern obtained with marker D18S541 at 107.11-cM on 108 chromosome 18q22.3
3.27 Allele pattern obtained with marker D18S1269 at 107.60-cM on 108 chromosome 18q22.3
3.28 Allele pattern obtained with marker D18S469 at 109.65-cM on 108 chromosome 18q22.3
3.29 Allele pattern obtained with marker GATA8C07 at 112.94-cM on 109 chromosome 18q22.3
3.30 Allele pattern obtained with marker D18S58 at 113.22-cM on 109 chromosome 18q22.3
3.31 Allele pattern obtained with marker D18S1121 at 115.67-cM on 109 chromosome 18q22.3
3.32 Pedigree and Haplotype Drawing of Family A Segregating 110 Ectodermal Dysplasia of Hair, Nail and Teeth Type
3.33 Ideogramatic representation of ED of hair, nail and teeth disease- 112 associated interval of 8.63-Mb flanked by markers D18S857 and D18S815 on chromosome 18q22.1-q22.3
3.34 Allele pattern obtained with marker D4S2952 at 169.13-cM on 113 chromosome 4q32.3
3.35 Allele pattern obtained with marker D4S3326 at 170.54-cM on 113 chromosome 4q32.3
3.36 Allele pattern obtained with marker D4S1502 at 171.12-cM on 114 chromosome 4q32.3
3.37 Allele pattern obtained with marker D4S2368 at 171.12-cM on 114
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XIII Preface List of Figures
chromosome 4q32.3
3.38 Allele pattern obtained with marker D4S2414 at 171.18-cM on 114 chromosome 4q32.3
3.39 Allele pattern obtained with marker D4S2979 at 172.31-cM on 115 chromosome 4q32.3
3.40 Allele pattern obtained with marker D4S2426 at 174.15-cM on 115 chromosome 4q33
3.41 Allele pattern obtained with marker D4S2373 at 174.15-cM on 115 chromosome 4q33
3.42 Allele pattern obtained with marker D4S621 at 175.84-cM on 116 chromosome 4q34.1
3.43 Allele pattern obtained with marker D4S2431 at 176.78-cM on 116 chromosome 4q34.1
3.44 Allele pattern obtained with marker D4S1501 at 184.46-cM on 116 chromosome 4q34.3
3.45 Pedigree of Family B Segregating Autosomal-Recessive Isolated 117 Congenital Nail Clubbing (ICNC)
3.46 Ideogramatic representation of ICNC disease-associated interval of 119 12.27-Mb flanked by markers D4S2952 and D4S415 on chromosome 4q32.3-q34.3
3.47 Sequence analysis of the HPGD gene mutation (c.577T>C) in 120 family B with Isolated Congenital Nail Clubbing (ICNC)
3.48 Tissue transcriptional analysis of the HPGD gene 121
3.49 Partial amino acid sequence comparison of human 15-PGDH with 122 other orthologs
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XIV Preface List of Figures
3.50 Allele pattern obtained with marker D7S488 at 31.04-cM on 123 chromosome 7p21.1
3.51 Allele pattern obtained with marker D7S815 at 32.52-cM on 123 chromosome 7p21.1-p15.3
3.52 Allele pattern obtained with marker D7S1802 at 34.63-cM on 124 chromosome 7p15.3
3.53 Allele pattern obtained with marker D7S2562 at 35.75-cM on 124 chromosome 7p15.3
3.54 Allele pattern obtained with marker D7S493 at 36.08-cM on 124 chromosome 7p15.3
3.55 Allele pattern obtained with marker D7S2190 at 39.92-cM on 125 chromosome 7p15.3
3.56 Allele pattern obtained with marker D7S2525 at 41.05-cM on 125 chromosome 7p15.2
3.57 Allele pattern obtained with marker D7S2848 at 46.61-cM on 125 chromosome 7p15.1
3.58 Allele pattern obtained with marker D7S2496 at 47.44-cM on 126 chromosome 7p15.1
3.59 Allele pattern obtained with marker D7S2492 at 47.44-cM on 126 chromosome 7p15.1
3.60 Allele pattern obtained with marker D7S632 at 48.65-cM on 126 chromosome 7p15.1
3.61 Allele pattern obtained with marker D7S817 at 50.85-cM on 127 chromosome 7p14.3
3.62 Allele pattern obtained with marker D7S484 at 53.72-cM with 127 similar allele pattern as of D7S817 on chromosome 7p14.3
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XV Preface List of Figures
3.63 Pedigree and Haplotype Drawing of Family C Segregating A Novel 128 Form of Ectodermal Dysplasia
3.64 Ideogramatic representation of a novel ED disease-associated 130 interval of 12.43-Mb flanked by markers D7S488 and D7S2491 on chromosome 7p21.1-p15.1
3.65 Sequence analysis of the EDA1 gene mutation in family D with 132 XLHED
3.66 Sequence analysis of the EDAR gene mutation 134 (c.399_404delGGTCTG) in family E with ARHED
3.67 Partial transcript representation of the EDAR gene mutation 135 (p.M133_C135delinsI) in family E with ARHED
3.68 Sequence analysis of the TRPS1 gene mutation in family F with 137 TRPS III
4.1 Pedigree of family G with hereditary autosomal recessive 149 hypotrichosis
4.2 Clinical features of family G with hereditary hypotrichosis 150
4.3 Pedigree of family H with hereditary autosomal recessive 151 hypotrichosis
4.4 Clinical features of family H with hereditary hypotrichosis 152
4.5 Pedigree of family I with hereditary autosomal recessive 153 hypotrichosis
4.6 Clinical features of family I with hereditary hypotrichosis 154
4.7 Pedigree of family J segregating congenital atrichia with papular 155 lesions (APL)
4.8 Clinical features of the family J with congenital atrichia and papular 156
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XVI Preface List of Figures
lesions (APL)
4.9 Allele pattern obtained with marker D13S328 at 50.18-cM on 157 chromosome 13q14.13-q14.2
4.10 Allele pattern obtained with marker D13S284 at 54.17-cM on 157 chromosome 13q14.3
4.11 Allele pattern obtained with marker D13S1245 at 54.74-cM on 158 chromosome 13q21.1
4.12 Allele pattern obtained with marker D13S169 at 55.14-cM on 158 chromosome 13q21.1
4.13 Allele pattern obtained with marker D13S1807 at 55.5-cM on 158 chromosome 13q21.1
4.14 Sequence analysis of the P2RY5 gene mutation (c.742A>T) in 159 family G with LAH3
4.15 Partial amino acid sequence comparison of human P2Y5 with other 160 orthologs
4.16 Sequence analysis of the P2RY5 gene mutation (c.69insCATG) in 161 family H with LAH3
4.17 Allele pattern obtained with marker D18S1108 at 50-cM on 163 chromosome 18q11.2
4.18 Allele pattern obtained with marker D18S478 at 54.85-cM on 163 chromosome 18q12.1
4.19 Allele pattern obtained with marker D18S847 at 57.41-cM on 164 chromosome 18q12.1
4.20 Allele pattern obtained with marker D18S36 at 59.09-cM on 164 chromosome 18q12.1
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XVII Preface List of Figures
4.21 Allele pattern obtained with marker D18S536 at 60.9-cM on 164 chromosome 18q12.1
4.22 Sequence analysis of the DSG4 gene in family I with autosomal 165 recessive hereditary hypotrichosis (LAH1)
4.23 Allele pattern obtained with marker D8S258 at 37.51-cM on 167 chromosome 8p21.3
4.24 Allele pattern obtained with marker D8S298 at 40.11-cM on 167 chromosome 8p21.3
4.25 Allele pattern obtained with marker D8S1786 at 41.41-cM on 168 chromosome 8p21.3
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XVIII Preface Summary
SUMMARY
Human skin is a very fascinating organ bearing important appendages (hair, nail, teeth, sweat glands), which play many important roles. A defect in a single gene may result in disruption in the development of these appendages. In this regard monogenic human skin disorders have provided a source for identification of genes and their functions.
The present study aimed at to investigate the genetic bases of two types of human hereditary skin disorders in Pakistani population: Ectodermal Dysplasias and Alopecias. Ten consanguineous Pakistani families with hereditary skin disorders including six (A-F) with various forms of ectodermal dysplasias and four (G-J) with alopecias were investigated.
Ectodermal dysplasias (EDs) are a large heterogeneous group of rare genetic disorders with structural or functional defects in one or more than one classical ectodermal appendages (hair, nail, teeth, and sweat glands). In three families (A, B, C) after exclusion of linkage to known genes, involved in human skin disorders, genome-wide homozygosity mapping was performed to identify the disease loci. Three other families (D, E, F) were directly screened for candidate genes on the basis of the phenotypes demonstrated by the affected individuals.
In family A, featuring autosomal recessive ectodermal dysplasia of hair-nail-teeth type, genome-wide search identified a novel locus on chromosome 18q22.1-q22.3 flanked by markers D18S857 and D18S815. Maximum two point LOD score of 2.73 was achieved at marker D18S541 while multipoint LOD score of 3.42 was obtained with several markers along the disease-interval. This locus spans a 17.32-cM region, which corresponds to 8.63-Mb on the sequence-based physical map (Build 36.1). Sequence analysis of the three candidate genes (CDH7, CDH9, ZNF407), located in the genetic interval, failed to reveal any pathogenic sequence variant causing the disease-phenotype in the family.
In family B, affected individuals demonstrated isolated, congenital, symmetrical, and bilateral clubbing of fingers- and toenails without any associated abnormality, inherited in autosomal recessive fashion. This condition has been named as Isolated Congenital Nail Clubbing (ICNC). Genome-wide homozygosity mapping in this
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XIX Preface Summary
family identified a novel locus for ICNC in a 13.25-cM genetic interval corresponding to 12.27-Mb on chromosome 4q32.3-q34.3 flanked by markers D4S2952 and D4S415. A maximum two-point LOD score of 2.98 (θ = 0.00) was obtained at marker D4S2368 while a maximum multipoint LOD score of 3.62 was obtained with several markers along the disease-interval. Ten candidate genes (ADAM29, MFAP3L, ASB5, MORF4, HMGB2, VEGFC, HAND2, ANXA10, SAP30, HPGD), located in the genetic interval of ICNC, were directly sequenced. Sequence analysis of the HPGD gene detected a homozygous missense mutation (c.577T>C; p.S193P) in the affected individuals of the family. This mutation probably results in the loss of enzyme function.
In family C, demonstrating a novel form of ectodermal dysplasia, genome-wide parametric linkage analysis localized a novel gene on chromosome 7p21.1-p15.1. The genetic interval spans 17.60-cM region, flanked by markers D7S488 and D7S2491, which corresponds to physical distance of 12.43-Mb (Build 36.1). Maximum two- point LOD score of 2.94 at zero recombination fraction (θ=0.00) was achieved for marker D7S2496, while maximum multi-point LOD score of 3.07 was achieved with several markers along the disease-interval. Sequence analysis of the 27 candidate genes (ITGB8, CCDC126, JAZF1, HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, SP4, SP8, TWIST1, FERD3L, NEUROD6, PRR15, EVX1, TWISTNB, CBX3, SNX10, HNRPA2B1, TRA2A, AQP1), located in the genetic interval, failed to detect any functional pathogenic sequence variant.
In family D, male and female affected individuals exhibited typical features of the X- linked hypohidrotic ectodermal dysplasia (XLHED). Sequence analysis of the EDA1 gene detected a novel 4-bp insertion mutation (c.913_914insTATA) in the affected individuals of the family. The mutation identified in the family leads to a frameshift and premature termination codon 2-bp downstream (p.305insIfsX306) in the same exon.
In family E, pedigree analysis and clinical features of the affected individuals were compatible with autosomal recessive hypohidrotic ectodermal dysplasia (ARHED). Sequence analysis of the EDAR gene identified a novel in-frame 6-bp deletion mutation (c.399_404delGGTCTG) in the affected individuals of the family. This in- frame deletion results in the skipping of three amino acids (Methionine at 133, Valine
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XX Preface Summary
at 134, Cystiene at 135) and insertion of a new amino acid (Isoleucine) (p.M133_C135delinsI) in one of the “cystiene-rich-regions” of the extracellular domain of the EDAR protein.
Affected individuals in family F demonstrated autosomal dominant trichorhinophalangeal syndrome type III. Sequence analysis of exon 6 of the TRPS1 gene from affected individuals detected a novel heterozygous missense mutation (c.2762G>T). This transversion results in the exchange of glycine residue with valine at position 921 (p.G921V) in the TRPS1 protein, which is the first residue after the C- terminal of the GATA zinc finger domain of the protein.
In the present study, four families (G, H, I, J) with hereditary alopecias were investigated. Alopecias are rare genetic disorders of hair. These may be isolated involving only hair as a defective entity or associated with other organs anomalies along hair defects.
In families (G, H, I, J), candidate gene approach was used to test linkage to four genes (DSG4, P2RY5, LIPH, HR) underlying autosomal recessive hereditary alopecias. Genotyping using highly polymorphic microsatellite markers, flanking these genes, showed linkage of the families to at least one of the candidate genes.
Two families G and H, featuring autosomal recessive hereditary hypotrichosis, showed linkage to LAH3 locus containing the P2RY5 gene on chromosome 13q14.11- q21.32. The affected individuals of family G revealed a novel missense mutation (c.742A>T; p.N248Y) in the P2RY5 gene located in the sixth (TM6: 228-253 amino acids) transmembrane domain of the P2Y5 protein. In family H, a previously described 4-bp insertion mutation (c.69insCATG) was detected in the affected individuals. This results in the 52 amino acids truncated P2Y5 protein (p.24insHfsX52).
Family I, demonstrating autosomal recessive hereditary hypotrichosis, was linked to LAH1 locus containing the DSG4 gene on chromosome 18q12.1. Sequence analysis of the DSG4 gene in the family identified a recurrent intragenic deletion mutation (c.Ex5-8del) in the affected individuals. This mutation was previously described in seven Pakistani families suggesting for a common ancestral mutation.
Family J with atrichia and papular lesion (APL) showed linkage to human hairless (HR) gene on chromosome 8p21.3. Sequence analysis of the HR gene in the affected
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XXI Preface Summary
and normal individuals of the family failed to identify any functional pathogenic sequence variant.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XXII Preface List of Publications
LIST OF PUBLICATIONS
The work presented in the thesis has been published in the following articles.
1) Peter John, Muhammad Tariq, Muhammad Arshad Rafiq, Muhammad Amin-Ud-Din, Dost Muhammad, Ishrat Waheed, Muhammad Ansar, Wasim Ahmad (2006). Recurrent intragenic deletion mutation in desmoglein 4 gene underlies autosomal recessive hypotrichosis in two Pakistani families of Balochi and Sindhi origins. Archives of Dermatological Research 298: 135- 137
2) Muhammad Tariq, Naveed Wasif, Muhammad Ayub, Wasim Ahmad (2007). A novel 4-bp insertion mutation in EDA1 gene in a Pakistani family with X-linked hypohidrotic ectodermal dysplasia. European Journal of Dermatology 17: 209-212
3) Muhammad Tariq, Naveed Wasif, Wasim Ahmad (2007). A novel deletion mutation in the EDAR gene in a Pakistani family with autosomal recessive hypohidrotic ectodermal dysplasia. British Journal of Dermatology 157: 207-209
4) Muhammad Tariq, Muhammad Salman Chishti, Ghazanfar Ali, Wasim Ahmad (2008). A novel locus for ectodermal dysplasia of hairs, nails and teeth type maps to chromosome 18q22.1-22.3. Annals of Human Genetics 72: 19- 25
5) Zahid Azeem, Musharraf Jelani, Gul Naz, Muhammad Tariq, Naveed Wasif, Syed Kamran-ul-Hassan Naqvi, Muhammad Ayub, Masoom Yasinzai, Muhammad Amin-ud-din, Abdul Wali, Ghazanfar Ali, Muhammad Salman Chishti, Wasim Ahmad (2008). Novel mutations in G protein-coupled receptor gene (P2RY5) in families with autosomal recessive hypotrichosis (LAH3). Human Genetics 123: 515-519
6) Muhammad Tariq, Saeed Ahmad, Wasim Ahmad (2008). A novel missense mutation in the TRPS1 gene underlies trichorhinophalangeal syndrome type III. British Journal of Dermatology 159: 476-478
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XXIII Preface List of Publications
7) Muhammad Tariq, Zahid Azeem, Ghazanfar Ali, Muhammad Salman Chishti, Wasim Ahmad (2009). Mutation in the HPGD gene encoding NAD+- dependent 15-hydroxyprostaglandin dehydrogenase underlies isolated congenital nail clubbing (ICNC). Journal of Medical Genetics 46: 14-20
8) Muhammad Tariq, Muhammad Ayub, Musharraf Jelani, Sulman Basit, Gul Naz, Naveed Wasif, Syed Irfan Raza, Abdul Khaliq Naveed, Saad ullah Khan, Zahid Azeem, Masoom Yasinzai, Abdul Wali, Ghazanfar Ali, Muhammad Salman Chishti, Wasim Ahmad (2009). Mutations in the P2RY5 gene underlie autosomal recessive hypotrichosis in 13 Pakistani families. British Journal of Dermatology 160: 1006-1010
9) Muhammad Tariq, Muhammad Nasim Khan, Wasim Ahmad (2009). Ectodermal dysplasia-cutaneous syndactyly syndrome maps to chromosome 7p21.1-p14.3. Human Genetics 125: 421-429
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias XXIV Chapter 1 Introduction
INTRODUCTION
The field of genetics in dermatology has progressed at great pace in last 15 years through an improved knowledge of the human genome, modern and advanced molecular biology techniques and comprehensively elaborated DNA databases. Many single gene skin disorders have been investigated and diagnosed at the molecular level by identifying their causative genes. However, a number of phenotypes related to skin is still to be elucidated awaiting their molecular genetics. Genetic research in dermatology is providing practical benefits for patients with rare genetic skin disorders in terms of accurate diagnosis, better genetic counseling and availability of DNA-based prenatal diagnosis. However, successful gene therapy for these disorders is still to be known or/and in developmental stages.
Genetic research on skin disorders in Pakistani population has a peculiarity due to high rate of consanguinity and large family size with different manifestations of skin. However, less attention has been given to this area of research due to lack of knowledge among affected families about their condition, rarity of skin disorders and lack of research expertise in this field. In the last 3 years, some advancement has been made in identifying novel genes for monogenic autosomal recessive skin disorders including ectodermal dysplasias and alopecias in Pakistani families.
Human Skin
Human skin is the interface between the internal organs and the external world. Skin and its associated appendages (hair, nail, teeth and many glands) play diverse functions as epidermal barrier and defense, immune surveillance, UV protection, thermoregulation, sweating, lubrication, pigmentation, sensation of pain and touch, and the protection of various stem cell niches in the skin. Despite having important appendages, skin due to its pliability, flexibility, and responsiveness, is a master in the art of self-defense- proving that a tissue need not to be hard in order to be tough (Ross and Christiano, 2006).
There are two major structural layers of the skin separated by a basement membrane; the epidermis and the dermis. In addition to these, there are also a number of other cell types and structures that assist skin functions (Chuong et al., 2002).
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 1 Chapter 1 Introduction
Epidermis
Epidermis is a stratified epithelium that is unique in the sense that it retains the ability to self-renew, proliferate and differentiate under both homeostatic and injury conditions by maintaining a population of mitotically active cells in the hair follicles and innermost basal layer (Segre, 2006). Normal adult skin comprises a thin surface epidermis nourished and maintained by a thicker dermis. Epidermis of the limb gives rise to a range of skin types (palmar, plantar and interfollicular epidermis) and varied appendages (including hair follicles, sebaceous glands, eccrine sweat glands and nails). Epidermis is further differentiated in five layers: stratum corneum, granular layer, upper spinous layer, lower spinous layer and basal layer. Epidermis’ primary role is protective and it provides protection largely through construction of an elaborate and highly organized outer surface, the stratum corneum. Stratum corneum is the primary interface and barrier between an organism and its outer environment and acts to prevent desiccation, toxin entry and microbial infection (Byrne et al., 2003).
Dermis
Dermis (Corium) is the immediate underlying layer to epidermis derived from the mesoderm linking epidermis and hypodermis. Dermis is composed of two layers: papillary layer and reticular layer. Sweat glands, sebaceous glands and hair follicles are present mostly in the dermis.
Hypodermis
Hypodermis (subcutaneous skin layer) is a deeper continuation of the dermis consisting of the loose connective tissue and adipose cells forming a deep layer of variable thickness. However, no adipose tissue is found in hypodermis of the eyelids, clitoris or penis (Clark, 1985).
Ectodermal Appendages
Hair
Hair is the filamentous keratinized skin appendage of the vertebrates. Besides protecting the body against coldness and wetness, hair is the sign of strength, power and beauty in human beings. Hairs are present all over the body surface, except on the
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 2 Chapter 1 Introduction thick skin of the soles and palms, the sides of the fingers and toes, the nipples, and the glans penis and the clitoris.
Hair shaft is composed of three different types of epithelial components: outermost layer or cuticle (protecting the cortex), middle layer or cortex (provides strength, colour and texture to the hair), and inner most layer or medulla (only present in large thick hair). The inner root sheath (IRS) surrounds the hair shaft and is composed of three cell types as well: the inner root sheath cuticle, Huxley’s layer, and Henle’s layer. IRS is surrounded by another layer outer root sheath (ORS), which composed of two cellular components: companion layer and ORS cells. All these cell types of the hair shaft except the ORS cells originate from the germinal epithelial cells at the base of the hair follicle (HF). These cells are apparently nourished by the dermal papilla by providing nutrients and growth factors (Van Steensel et al., 2000).
Responding to the Wnt and bone morphogenetic protein (BMP) signaling the ectodermal progenitors adopt an epidermal fate, which ultimately produce the hair placodes. Further dermal messages instruct the placodes to make the hair follicles. Mature hair follicles (HFs) a highly complex composite structure having seven concentric rings of terminally differentiating cells are derived from the matrix cells. HFs vary considerably in size and shape, depending on their location, but they all have the same basic structure. Hair follicle is one of the few organs of the body that undergoes cyclic bouts of degeneration and regeneration throughout life. Each hair follicle perpetually goes through three stages: growth phase (anagen), involution phase (catagen), and rest phase (telogen) (Fuchs, 2007).
Nail
Nail is the hard keratin structure growing over the tip of the finger or toe. The major function of the nail in human is the protection of the delicate terminal phalanx. Fingernails or toenails can be divided into several major sections. Each of these separate parts cooperates with the others to form a nail unit. A nail unit consists of nail folds, eponychium and cuticle, the matrix, nail plate, lunula, nail bed, and hyponychium. The area where the skin folds at the proximal end of nail plate is called the proximal nail fold. On each side of the nail plate, two more folds form tight seals along the sidewalls. These sidewall seals are called the lateral nail folds. The skin that lies directly on top of the newly developing nail plate is called the eponychium. The
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 3 Chapter 1 Introduction detached tissue that rides on and at the base of the nail plate, seeming to grow from under the edge of the nail fold. This thin layer of colorless tissue is the cuticle. Matrix is a small, glistening white patch of living tissue directly below eponychium and in close proximity to bony phalanx. It is an important component in entire nail unit, producing nail plate. The size and shape of the matrix determine the thickness, width, and curvature of the nail plate. Nail plate (also called natural nail) is mostly made of keratin, which is a special protein that creates the bulk of the nail plate. Nail plate is the horny end product of the matrix. Nail plate protects the nail bed and fingertip. The opaque, bluish white half-moon at the base of the nail plate is called the lunula. The lunula is the distal portion of the matrix (visible matrix). Not all fingers have a visible lunula. The nail bed is an area of pinkish tissue that supports the entire nail plate. The bed is enriched with many tiny blood vessels that feed and clean the tissue. The nail bed lies directly underneath the solid nail plate, starting at the matrix and ending at the free edge. The farthest or most distal edge of the nail unit is the hyponychium. The hyponychium is composed of living epidermis tissue. Its job is to protect the nail bed from pathogens (bacteria, fungi, viruses) under the free edge (Schoon, 2005).
Nail development starts around the ninth week of gestation and is completed during the fifth month of pregnancy, with development of the toenails lagging approximately 4 weeks behind the fingernails (Dawber et al., 2001). Proper epithelial-mesenchymal interactions both within the skin and with the underlying bone appear crucial for nail development, with bone morphogenic protein-4 (BMP4), fibroblast growth factor-4 (FGF4), Wnt7A and sonic hedgehog (Shh) all having important roles (Chuong et al., 1996). Transcription factors encoded by LMX1B and MSX1 are mutated in nail-patella syndrome and Witkop syndrome, respectively (Dreyer et al., 1998; Chen et al., 1998; Jumlongras et al., 2001). Ablation or ectopic expression of transcription factors such as Engrailed-1 in mouse models has also provided insights into nail development (Loomis et al., 1996).
Tooth
Teeth are specialized structural components of the craniofacial skeleton, found in the jaws of the many higher mammals and are comprised of the distinct mineralized tissues: enamel, dentin, and cementum. Developmental abnormalities in any of these tissues lead to isolated or associated dental conditions. Teeth are of different types and human dentition is composed of three basic tooth shapes: incisors, canines and
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 4 Chapter 1 Introduction multicuspids (molars, premolars). Tooth shape is determined very early in development by expression of different genes in different region of the mesenchyme of the jaw primordia (Tucker and Sharpe, 2004; Sartaj and Sharpe, 2006; Hu and Simmer, 2007). Structurally a tooth consists of three main parts: crown, root and the pulp cavity. The crown and the root consist of two layers of hard substance surrounding the dental pulp (soft tissue of the tooth).
In tooth morphogenesis, the dental epithelium and neural crest-derived mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth (Nakamura et al., 2008). Focal condensations of the migratory neural crest cells immediately beneath the oral epithelium are initial signs of tooth development. This initiation involves growth factors, transcription factors (MSX1, PAX9), signal receptors and other soluble morphogens. It is not surprising that such a complex process is prone to disturbances and may result in tooth agenesis (Hu and Simmer, 2007; Kapadia et al., 2007).
Recently, Liu et al. (2008) demonstrated that Wnt/beta-catenin signaling is active at multiple stages of tooth development. They indicated that tight regulation of this pathway is essential both for patterning tooth development in the dental lamina, and for controlling the shape of individual teeth. Transforming growth factor (TGF) beta 1, beta 2 and beta 3 are involved in the different stages of morphogenesis and differentiation of human developing dental organ and further it is indicated that TGF beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF beta 2 to cellular differentiation and TGF beta 3 to mineral maturation matrix (Sassá et al., 2008).
Sweat Glands
Sweat glands are highly active miniorgans of skin that fulfill a diversity of functions. They are simple coiled tubular glands and play some key roles in homeostasis maintenance and body temperature regulation. There are two types of sweat glands, eccrine sweat glands and apocrine sweat glands. Both types of glands are controlled by the sympathetic nervous system.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 5 Chapter 1 Introduction
Eccrine Glands
Eccrine glands are the most numerous types of sweat glands found all over the body with a density of 100-600/cm2, particularly on the palms of the hands, soles of the feet and forehead.
Apocrine Glands
Apocrine glands (scent glands) are mostly confined to the armpits (axilla), the areola of the breasts and the anal-genital area. Their secretory portion can be located in the dermis or in the hypodermis. They typically end in hair follicles rather than pores.
Ectodermal Dysplasias
Ectodermal dysplasias (EDs) are large, heterogeneous, and complex group of inherited disorders characterized by the structural and/or functional defects in at least one of the ectodermal appendages (hair, nails, teeth, sweat glands) with or without associated anomalies of other organs and systems during morphogenesis. EDs may be termed as pure EDs with only defects in classical ectodermal appendages like hair, nails, teeth, and sweat glands or ED/malformation syndromes or ED syndromes with defects in ectodermal appendages in association with other clinical manifestations. More than 200 EDs with different pathological and clinical conditions have been described in literature, however, very few of them are known at the molecular level with their causative genes. To date, all the EDs known exhibit all possible patterns of Mendelian inheritance (autosomal dominant or recessive, X-linked dominant or recessive) but sporadic cases have been also described.
Clinical Manifestations in Ectodermal Dysplasias
Clinically, EDs vary from mild to severe conditions depending on the type of ED. Skin manifestations in EDs include adermatoglyphia, reticular pigmentation, depigmentation, superficial dry scaling, atrophy, and hyperkeratosis. Hair abnormalities include hypotrichosis, partial or total alopecia. Nail changes include a wide range from slight, noticeable change to complete absence of the nails. Dental changes include hypodontia, oligodontia, conical and malformed teeth, and enamel defects leading to severe and early caries. Sweat glands may be absent or reduced with impaired function. Other anomalies associated with EDs include eye abnormalities, facial deformities, skeletal deformities, hearing impairment, mental
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 6 Chapter 1 Introduction retardation, cleft lips and palate, and other systemic conditions (Itin and Fistarol, 2004).
Classification of Ectodermal Dysplasias
Recent advancements in molecular genetics of the skin disorders urge a need for a comprehensive classification and categorization of these disorders helpful to dermatologists to diagnose and manage a particular skin disease easily.
Earlier Classification
1. Clinical Classification
Earlier classification was based solely on the clinical information described in different studies. In this regard, appreciable work was done by Freire-Maia, (1971, 1977), Freire-Maia and Pinheiro, (1988), and Pinheiro and Freire-Maia, (1987, 1994). They comprehensively reviewed all EDs from clinical point of view described so far. According to this earlier classification, ectodermal dysplasias were classified in two groups. Group A consists of ectodermal dysplasias of at least two classic ectodermal appendages without associated defect or abnormality. Group B consists of ectodermal dysplasias of at least one of the four ectodermal appendages (hair, nail, teeth, and sweat glands) plus one other associated ectodermal anomaly of the lips, ears, or dermatoglypics on palms and soles. In this classification, they used numbers for the basic affected ectodermal appendage like 1, 2, 3, 4, and 5 for hair, teeth, nails, sweat glands, and other associated ectodermal anomaly of the lips, ears, or dermatoglypics, respectively. So group A ectodermal dysplasias are donated by 1-2, 1-3, 1-4, 2-3, 2-4, 3-4, 1-2-3, 1-2-4, 1-3-4, 2-3-4 and 1-2-3-4 and group B ectodermal dysplasias are donated by 1-5, 2-5, 3-5 and 4-5. Further, Freire-Maia et al. (2001) classified 192 known EDs into 11 subgroups on the basis of the clinical information; each subgroup has EDs varying from 1 to 43. They also distinguished ‘pure EDs’, which are exclusively characterized by ectodermal signs, and ‘ED/malformation syndromes or ED syndromes’ in which ectodermal derivatives defects are associated with other malformations in different organs or tissues.
2. Clinical-Genetic Classification
In this classification molecular-genetic as well as clinical data were used to classify EDs in nine different groups (Priolo et al., 2000): Group I consists of ‘Pure EDs’
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 7 Chapter 1 Introduction having only ectodermal appendages defects with identified causative genes EDA1 and DL. Group II consists of diseases characterized by premature aging. Group III includes multiple congenital anomalies/EDs in association with skeletal abnormalities. Group IV includes ED syndromes characterized by premature aging and neoplasias. Group V diseases are characterized by skin associated disorders, such as keratodermas and skin fragility. Group VI includes diseases associated with deafness. Group VII includes ocular anomalies/retinal dystrophy. Group VIII consists of renal abnormalities. Group IX diseases are associated with the neuro-endocrine abnormalities.
Priolo and Lagana (2001) modified the clinical-genetic classification using biological mechanisms for the pathogenesis of the ectodermal dysplasias. They proposed two main groups for EDs. Group 1 consists of EDs with defects in the developmental regulation and epithelial-mesenchymal interactions, while group 2 consists of EDs with defects in cytoskeleton maintenance and cell stability. In group 1 major characteristic signs are involvement of major ectodermal derivatives, altered immune response or immunodeficiency, skeleton, and endocrines. In group 2 major characteristic signs are hyperkeratosis/keratodermas, deafness, cleft lip/palate (CLP), and retinal degeneration.
Modern Classification
1. Gene-based Classification (Molecular Classification)
Lamartine (2003) and Itin and Fistarol (2004) studied EDs with respect to their molecular and biochemical findings and proposed a new classification for EDs based on the function of the identified gene responsible for a disease. According to this classification causative genes for EDs are classified into four functional subgroups: cell-cell communication and signaling; cell adhesion; transcription regulation; and development. Cell-cell communication and signaling molecules include soluble ligand (EDA1 [MIM 300451]), receptors (EDAR [MIM 604095], XEDAR [MIM 300276]), adaptor (EDARADD [MIM 606603]), gap junction proteins (GJB6 [MIM 604418], GJB2 [MIM 121011]), and regulator of kinases (NEMO [MIM 300248]). Cell adhesion proteins consist of different adhesion and structural desmosomal molecules (PVRL1 [MIM 600644], PKP1 [MIM 601975], CDH3 [MIM 114021]). Genes in transcription regulation consist of different transcription factors (p63 [MIM 603273],
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 8 Chapter 1 Introduction
GATA3 [MIM 131320]) and DNA binding proteins (EVC [MIM 604831], TRPS1 [MIM 604386], XPD [MIM 126340]). Developmental genes for EDs include MSX1 [MIM 142983] and SHH [MIM 600725].
2. Current and Online Genodermatoses Database
Recently, Leech and Moss (2007) arranged alphabetically skin genetic disorders in tabulated form as current online database (online version: http://www.blackwell- synergy.com/loi/bjd). This table is designed in a fashion having genodermatosis names, their alternative name(s) if any, their abbreviated symbols if given, MIM number of genodermatosis, MIM number of identified gene if known, abbreviated gene symbols, chromosomal locus for condition and/or gene, gene product name and/or symbol and a brief function of the gene if known in a row for one particular genodermatosis.
Ectodermal Dysplasias Involving Major Ectodermal Appendages
Hypohidrotic Ectodermal Dysplasias (HED)
Hypohidrotic (anhidrotic) ectodermal dysplasias (HED) are the most common form of ectodermal dysplasias involving abnormalities of hair, teeth and eccrine sweat glands. HED exhibits all possible patterns of Mendelian inheritance (autosomal dominant or recessive, X-linked). However, X-linked HED (XLHED) is the most common variant of HED, with partial manifestations in females. XLHED is caused by mutations in the ectodysplasin (EDA1: ectodysplasin A1 isoform (EDA-A1) [MIM 300451]) gene, located on chromosome Xq12-q13.1. EDA1 has a murine homologue tabby (Ta), found mutated in 2 independent tabby mouse strains (Clarke et al., 1987; Zonana 1993; Kere et al., 1996; Srivastava et al., 1997; Wisniewski et al., 2002). More than 90 different mutations in the EDA1 gene have been implicated in the pathogenesis of the XLHED.
Autosomal recessive and dominant HED (ARHED and ADHED) are rare disorders characterized by similar clinical features as in XLHED and they are caused by mutations in ectodysplasin A1 isoform receptor (EDAR [MIM 604095]) gene (downless dl in mice), located on chromosome 2q11-q13 and EDAR-associated death domain (EDARADD [MIM 606603]) gene (crinkled cr in mouse), located on chromosome 1q42.2-q43. To date, 23 different mutations are known in the EDAR gene while only two mutations are known in the EDARADD gene associated with
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 9 Chapter 1 Introduction
ARHED and ADHED in different populations of the world (Monreal et al., 1999; Headon and Overbeek, 1999; Headon et al., 2001; Naeem et al., 2005; Chassaing et al., 2006; Tariq et al., 2007-Present study; Bal et al., 2007; RamaDevi et al., 2008; van der Hout et al., 2008; Valcuende-Cavero et al., 2008).
Ectodermal Dysplasias of Hair-Nail Type
Ectodermal dysplasia of hair and nails is a rare congenital disorder characterized by trichodystrophy (hypotrichosis, partial, or total alopecia) and dystrophy of the finger- and toenails with no other associated abnormality. ED of hair and nails may be pure or may be associated with other abnormality. Various clinical forms of the pure ED of hair and nails have been reported, with various degrees of severity in hypotrichosis and nail dystrophy. Barbareschi et al. (1997) reported an autosomal dominant pure hair-nail dysplasia in a mother and son. Calzavara-Pinton et al. (1991) described an autosomal recessive form of congenital onychodystrophy and severe hypotrichosis, nearly resulting in atrichia universalis. Eyebrows, eyelashes and body hair were completely absent in affected individuals in this family. In a Brazilian family, pure hair-nail dysplasia was inherited in an autosomal dominant pattern characterized by hypotrichosis of the whole body and short, fragile and spoon-shaped nails (Pinheiro and Freire-Maia, 1992). Harrison and Sinclair (2004) reported a hair-nail ED in a 3- year-old girl with generalized scalp hypotrichosis, absent eyebrows, short eyelashes and nail dystrophy in all digits.
An autosomal recessive form of ED of pure hair-nail type was described in a Pakistani family with 13 affected individuals. The patients had thin scalp and body hair and fine eyebrows and eyelashes. Nail dystrophy was present in all digits. Through homozygosity mapping, the disease locus was mapped to a 3.92-cM region on chromosome 10q24.32-q25.1 (Rafiq et al., 2005). Naeem et al. (2006a) in a large consanguineous Pakistani family mapped a gene for ED of hair-nail type segregating in autosomal recessive pattern on chromosome 12q13.13. In this family 4 males and 4 females had ectodermal dysplasia of the hair and nails with total alopecia and nail dystrophy present at birth. Upon sequencing six candidate type II hair keratin genes present in disease interval, a missense mutation (p.R78H) in the KRTHB5 [MIM 602767] gene was identified in all the affected individuals. Naeem et al. (2006b) mapped another gene for ED of hair-nail type in a Pakistani family on chromosome 17q12-q21.2 in a 24.2-cM region flanked by markers D17S839 and D17S1299.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 10 Chapter 1 Introduction
However, DNA sequencing of five epithelial keratin candidate genes, present in the disease locus, did not reveal any pathogenic mutation in the affected individuals. In continuation of studies on pure EDs of hair-nail, Naeem et al. (2007) mapped a locus for ED of hair-nail type to chromosome 12p11.1-q21.1 in a 29.5-cM region flanked by markers D12S2080 and D12S1040 in a Pakistani family with five affected individuals. However, DNA sequencing of six keratin candidate genes (KRTHB1- KRTHB6), present in the disease locus, did not reveal any pathogenic mutation in the affected individuals.
Ectodermal Dysplasias of Hair-Teeth Type
Trichodental dysplasia is an autosomal dominantly inherited disorder involving hair and teeth. Missing teeth, peg-shaped incisors, and shell teeth are the most common dental abnormalities. Hairs in most patients are fine, sparse, dull, and slow growing (Salinas and Spector, 1980; Kersey, 1987; Eteson and Clark, 1988).
Ectodermal Dysplasias of Nail-Teeth Type
Tooth and nail syndrome (Witkop syndrome) is a type of ED characterized by missing teeth and poorly formed nails, especially of toenails early in life. This condition is inherited in autosomal dominant pattern (Witkop, 1965; Giansanti et al., 1974; Hudson and Witkop, 1975; Wicomb et al., 2004). Later, a heterozygous stop mutation (p.S202X) was revealed in the homoedomain of the MSX1 [MIM 142983] gene, located at chromosome 4p16.1-p16.3, cosegregated with the phenotype in a 3- generation family with tooth and nail syndrome (Stimson et al., 1997; Jumlongras et al., 2001).
Ectodermal Dysplasias of Hair-Nail-Teeth Type
Ectodermal dysplasia of hair, nails and teeth type is a rare congenital disorder characterized by sparse and thin hairs, dystrophic finger- and toe nails, and missing and abnormal teeth. Ectodermal dysplasia involving hair, nails and teeth includes a condition called odontoonychodermal dysplasia (OODD), which is a rare congenital disorder characterized by hypotrichosis or dry hair with no or sparse eyebrows and eyelashes, dystrophic nails and misshapen teeth of variable forms with associated abnormalities like erythemathous lesions of facial region and hyperhidrosis with thickened soles and palms. Recently, it is reported that mutation in the WNT10A [MIM 606268] gene at chromosome 2q35-q36.2 is responsible for
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 11 Chapter 1 Introduction odontoonychodermal dysplasia phenotype (Zirbel et al., 1995; Megarbane et al., 2004; Adaimy et al., 2007). van Steensel et al. (2001) reported a 4-generation Dutch family with 14 individuals affected with autosomal dominant curly hair-acral keratoderma-caries syndrome. All the affected individuals showed curly brittle hair with premature hair loss, sparse eyebrows and eyelashes, premature loss of teeth due to caries, nail dystrophy, and acral keratoderma. Tariq et al. (2008, Present study) mapped a novel locus for pure ED of hair-nail-teeth type in a family of Pakistani origin having autosomal recessive pattern of inheritance to chromosome 18q22.1- q22.3.
Isolated Nail Disorders
Isolated Congenital Anonychia
Anonychia is a rare nail disorder characterized by the congenital absence or severe hypoplasia of the all finger- and toenails. Usually anonychia and its less severe phenotype hyponychia are associated with other genetic syndromes and limb anomalies. Recently in two parallel reports, Blaydon et al. (2006) and Bergmann et al. (2006) reported mapping of a locus for the autosomal recessive anonychia/hyponychia to chromosome 20p13 in families belonging to different ethnic groups of the world using homozygosity mapping and linkage analysis. They further identified a gene named R-spondin 4 (RSPO4 [MIM 610573]), mutations in which cause anonychia/hyponychia. RSPO4 gene covers ~44 kb of region in genome on chromosome 20p13 and consists of five exons encoding a secreted protein of 234 amino acids (Kim et al., 2006). R-spondin protein family consists of four members (Rspo1, Rspo2, Rspo3, Rspo4) encoded in the human and mouse genomes. Each member of the R-spondin family consists of five exons predicting to encode an N- terminal signal peptide (exon 1), two furin-type cystiene rich domain (exon 2 and 3), thrombospondin-type domain (exon 4) and a C-terminal putative nuclear localization signal (exon 5). The furin-like repeats encoded by exons 2 and 3 are believed to be required for activation and stabilization of β-catenin (Kazanskaya et al., 2004).
Isolated Congenital Nail Dysplasia (ICND)
Isolated congenital nail dysplasia (ICND) is a rare autosomal dominant disorder characterized by longitudinal streaks, thinning, and impaired formation of the nail plates, involving mostly all the finger- and toenails. In an effort to investigate a large
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 12 Chapter 1 Introduction
Southern German family over five generation, a novel locus was mapped for ICND to a 6-cM region between markers D17S926 and D17S1528 on chromosome 17p13. In this ICND family about 22 affected individuals (13 females and 9 males, aged 5 to 74 years) were examined, with nail dysplasia in all finger- and toenails with no associated anomaly. Further concentrating on the nail findings revealed longitudinal streaks and thinning of nail plates, mostly of all finger- and toenails, with some accentuation of the thumbnails and big toenails, poorly developed lunulae, longitudinal angular ridges of individual nail plates, platonychia and koilonychias, and notches and fissures of the free margins. Histological examination of the nail biopsies revealed an abnormal keratinization of the matrix epithelium with the broad granular layer and epithelial strands and buds extending from the nail bed, suggesting for ICND a disorder of epithelial origin (Krebsova et al., 2000; Hamm et al., 2000). No gene has been cloned for ICND so far.
Isolated Congenital Nail Clubbing (ICNC)
Hereditary nail clubbing (or digital clubbing) is a distinct rare genodermatosis entity characterized by the enlargement of the nail plate and terminal segments of the fingers and toes congenitally resulting from the proliferation of the connective tissues between the nail matrix and the distal phalanx (Myers and Farquhar, 2001). Nail clubbing may be an isolated abnormality or generally associated with a systemic disease. Nail clubbing is occasionally present from birth (congenital) without any underlying disease (Samman and Fenton, 1995). Hereditary nail clubbing is usually symmetrical and bilateral (in some cases unilateral). There may be different fingers and toes involved in varying degrees. Some fingers or toes are spared, but the thumbs are almost involved. Recently, Tariq et al. (2008, Present study) investigated a large Pakistani consanguineous family with multiple affected individuals with total congenital nail clubbing with no evidence of any other abnormality, segregating in autosomal recessive fashion, named Isolated Congenital Nail Clubbing (ICNC). They mapped a gene responsible for the ICNC through genetic parametric linkage analysis using microsatellite markers to a 13.25-cM disease interval between markers D4S2952 and D4S415 on chromosome 4q32.3-q34.3. This is the first report of the genetic linkage study and mapping of a gene for the ICNC.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 13 Chapter 1 Introduction
Hereditary Nail Dysplasia
Rafiq et al. (2004) reported a large six generation Pakistani consanguineous family having 14 affected individuals with a novel form of isolated hereditary nail dysplasia, segregating in autosomal recessive fashion. Although in affected individuals at birth, both finger- and toenails were normal, onychodystrophy starts at the age of 7-8 years differentially affecting finger- and toenails in the family. Dystrophy of the fingernails leads to onycholysis, and of toenails leads to anonychia, starting at the same time. After excluding linkage to type I and type II keratins genes and some of the ectodermal dysplasia genes and performing a genome wide scan with microsatellite markers, a novel locus for hereditary nail dysplasia was mapped to a 5-cM region on chromosome 17q25.1-q25.3 between markers D17S1807 and D17S937.
Syndromic Forms of Ectodermal Dysplasias
Trichorhinophalangeal Syndrome (TRPS)
Trichorhinophalangeal syndrome (TRPS) is a dominantly inherited condition characterized by the developmental defects of hair, face and selected bones. Three different clinical forms of TRPS are known as TRPS I, TRPS II and TRPS III sharing some major clinical features like slow growing hair, bulbous tip of the nose, long flat philtrum, thin upper vermilion border, and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations, and short stature (Momeni et al., 2000; Ludecke et al., 2001).
TRPS III (Sugio-Kajii syndrome) is associated with severe brachydactyly and short stature resulting from the progressing shortening of all the phalanges and metacarpals and some long bones (Ludecke et al., 2001). TRPS II (Langer-Giedion Syndrome, LGS) is a contiguous gene syndrome combining the clinical features of the TRPS I and mental retardation (MR), and hereditary multiple exostoses (HME) due to a de novo deletion of the two contiguous EXT1 and TRPS1 genes at chromosome 8q24.1 (Buhler et al., 1987).
Autosomal Recessive Ichthyosis with Hypotrichosis Syndrome (ARIH)
Recently, a novel autosomal recessive ichthyosis with hypotrichosis syndrome (ARIH) in a consanguineous Israeli-Arab family with three affected individuals was described. Clinical findings included thickened, grayish, scaling skin and curly,
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 14 Chapter 1 Introduction sparse, fragile, brittle and slow growing hair. ARIH was mapped to chromosome 11q24.3 by homozygosity mapping for a missense mutation in membrane-type serine protease 1 (ST14 [MIM 606797]) gene resulting in glycine to arginine substitution (p.G827R) in the highly conserved peptidase S1-S6 domain of matriptase protein. This study suggested that matriptase plays a significant role in epidermal desquamation based on histological study of the affected individuals with ARIH (Basel-Vanagaite et al., 2007). Matriptase protein is a type II transmembrane serine protease of the S1 trypsinlike family (Hooper et al., 2001).
Ellis-van Creveld Syndrome (EvC)
Ellis-van Creveld syndrome (EvC) (also known as Chondroectodermal dysplasia/Mesoectodermal dysplasia) is a rare autosomal recessive disorder, was first described by Ellis and Van Creveld (1940). This syndrome comprises a tetrad of clinical manifestations characterized by disproportionate dwarfism, bilateral postaxial polydactyly, ectodermal dysplasia, and congenital heart malformations. Mutations in the EVC1 [MIM 604831] and EVC2 [MIM 607261] genes are associated with Ellis- van Creveld syndrome; these two genes lie in a head-to-head configuration on chromosome 4p16.1 that is conserved from fish to man and may be functionally related (Ruiz-Perez et al., 2000, 2003; Galdzicka et al., 2002).
Cerebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma Syndrome (CEDNIK Syndrome)
Sprecher et al. (2005) studied two unrelated, consanguineous Arab Muslim families from northern Israel segregating in autosomal recessive pattern, comprising seven affected individuals (four males and three females). The affected individuals displayed a novel neurocutaneous syndrome termed as Cerebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma syndrome (CEDNIK syndrome) and characterized by severe developmental abnormalities of the nervous system as well as aberrant differentiation of the epidermis. All patients had progressive microcephaly and facial dysmorphism including elongated faces, antimongolian eye slant, slight hypertelorism, and flat broad nasal root. Palmoplantar keratoderma and ichthyosis appeared between 5 and 11 months of age, with progressive worsening during the second year of life. By the age of 8–15 months, psychomotor retardation became apparent as major developmental milestones, like unaided sitting and walking, were
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 15 Chapter 1 Introduction not attained. By homozygosity mapping, a gene named synaptosomal -associated protein 29 (SNAP29 [MIM 604202]), coding for a SNARE protein involved in intracellular vesicle fusion, was identified. Mutation in this gene is responsible for CEDNIK phenotype.
Trichodentoosseous Syndrome (TDO)
Trichodentoosseous syndrome (TDO) is an autosomal dominant condition characterized by dysplastic nails, curly hair, bone sclerosis of the long bones and calvarium, taurodontism, and enamel hypoplasia that occurs with hypomaturation/hypocalcification defects. There are two distinct types of the TDO, namely TDO-I and TDO-II. Hart et al. (1997) mapped the TDO syndrome locus to a 12-cM region flanked by markers D17S932 and D17S809 on chromosome 17q21 through genetic linkage analysis, investigating 4 families with a total of 39 TDO- affected members. They found that 2 clinical features, taurodontism and enamel hypoplasia, were fully penetrant in all the affected individuals, while bone and hair features were variably expressed. By genomic cloning and sequencing, 2 human distal-less homeobox gene family members, DLX3 [MIM 600525] and DLX7 [MIM 601911] were identified on TDO locus on chromosome 17q21. Further, it was revealed that 4-bp deletion (c.571_574delGGGG) in the human DLX3 gene resulting in frameshift and premature stop codon and a functionally altered protein, was responsible for TDO phenotype in several families (Price et al., 1998a,b; Islam et al., 2005).
Ectrodactyly, Ectodermal Dysplasia, and Cleft lip/palate Syndrome (EEC)
The ectrodactyly, ectodermal dysplasia, cleft lip/palate syndrome (EEC syndrome) is an autosomal dominant dysplasia syndrome, whose pleiotropic effects involve mainly ectodermal structures. The most common clinical manifestations are ectodermal dysplasia, ectrodactyly of hands and feet, cleft lip/palate, and tear-duct anomalies (Sankhyan et al., 2006).
There are various clinical forms of EEC syndrome with one or more phenotypic differences. Qumsiyeh (1992) mapped locus for the classic EEC syndrome 1 (ECC1) on chromosome 7q11.2-q21.3. Maas et al. (1996) found great variability in the clinical manifestations of EEC in the affected members of large Dutch kindred with specific genitourinary anomalies, termed as EEC3. EEC3 was mapped to a 3-cM
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 16 Chapter 1 Introduction region on chromosome 3q27 by critical recombination event between markers D3S1580 and D3S1314 overlapping with limb-mammary syndrome (LMS). This colocalization and the overlapping clinical features of these disorders strongly suggested that the same gene is involved in this form of EEC syndrome (EEC3) and LMS. Further analysis and fine mapping identified mutations in tumor protein p73- like (TP73L [MIM 603273]) gene, causing EEC3 (Celli et al., 1999; van Bokhoven et al., 1999; 2001; Akahoshi et al., 2003; Reisler et al., 2006). Alopecias
Collectively structural, growth or developmental abnormalities of the hair are known as alopecias. There are many forms of genetic alopecias or hair loss having abnormalities in hair shaft structure and hair follicle morphogenesis, maintenance and cycling, many of them have been diagnosed at the molecular level by identifying their causative genes. These various forms of hair loss show extensive variation in age of onset, severity, and associated ectodermal abnormalities. Alopecias may be isolated (pure alopecias) involving only hair as a single entity or part of a syndrome (syndromic alopecias) involving hair with other associated abnormalities like mental retardation, erythroderma, keratodermas, immunodeficiencies, blindness and deafness. Different alopecias exhibit different modes of inheritance i.e. autosomal recessive, autosomal dominant and complex inheritance.
Autosomal Recessive Alopecias
Congenital Atrichia
Congenital atrichia or atrichia with papular lesions (APL) is a rare autosomal recessive disorder characterized by complete irreversible hair loss including scalp, axilla and body shortly after birth in combination with the development of papular lesions of keratin-filled cysts on various regions of the body. Patients are born with normal hairs but these are shed almost completely during the first weeks or months of life and never regrow. Histologically, the affected scalp skin lacks the mature hair follicles (Ahmad et al., 1998a, b; Zlotogorski et al., 2002). APL results from failure to initiate the first adult catagen. The gene responsible for the pathogenesis of APL was mapped to chromosome 8p21.3 (Ahmad et al., 1998a; Nothen et al., 1998) and further a homozygous pathogenic mutation (p.T1022A) was identified in the human hairless (HR [MIM 602302]) gene causing this condition (Ahmad et al., 1998a).
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 17 Chapter 1 Introduction
To date, 38 different mutations in the HR gene have been implicated in the pathogenesis of APL from different populations around the world, thereby establishing the molecular basis of this condition. These include 8 missense, 11 nonsense, 12 deletion, 2 insertion and 5 splice site mutations (Ahmad et al., 1998a,b; 1999a,b; Cichon et al., 1998; Zlotogorski et al., 1998; 2002; Kruse et al., 1999; Sprecher et al., 1999a,b; Aita et al., 2000; Paradisi et al., 2003; Ahsoor et al., 2005; John et al., 2005; Wali et al., 2006a; Betz et al., 2007; Michailidis et al., 2007; Kim et al., 2007; Kraemer et al., 2008; Roelandt et al., 2008).
Recessive Hereditary Hypotrichosis
Hereditary hypotrichosis with autosomal recessive pattern is a rare hair disorder characterized by sparse hair on scalp, sparse to absent eyebrows and eyelashes and less dense axillary, pubic and body hair. To date, three different forms of recessive hereditary hypotrichosis have been known with similar clinical features and different genetic loci. Kljuic et al. (2003) and Rafique et al. (2003) reported five unrelated Pakistani families with hereditary hypotrichosis named as localized autosomal recessive hypotrichosis (LAH1). Upon genome wide linkage analysis, LAH1 has been mapped to chromosome 18q12.1 containing four desmoglein genes (DSG1, DSG2, DSG3, DSG4) and three desmocollin genes (DSC1, DSC2, DSC3). Further, a homozygous intragenic deletion mutation (c.Ex5-8del) was detected encompassing exon 5–8 in the desmoglein 4 (DSG4 [MIM 607892]) gene at chromosome 18q12.1 underlying LAH1 in two families of Pakistani origin (Kljuic et al., 2003), which was later reported in six additional Pakistani families with different ethnic background, suggesting for dispersion for a common ancestral chromosome harboring the same deletion (Rafiq et al., 2004; Moss et al., 2004; John et al., 2006b-Present study). This mutation generates an in-frame deletion creating a predicted protein with missing amino acids from 125 to 335. The amino acid sequence in this region is believed to be critical in cadherin–cadherin interaction and dimerization, necessary for proper cell adhesion (Boggon et al., 2002; He et al., 2003). A missense mutation (p.A129S) and a single nucleotide deletion (c.87delG) in the DSG4 gene have also been reported in an Iraqi and Pakistani family with LAH1, respectively (Messenger et al., 2005; Wajid et al., 2007).
A large Pakistani consanguineous family with hereditary hypotrichosis was investigated segregating in an autosomal recessive pattern and a novel genetic locus
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 18 Chapter 1 Introduction was mapped to a 7.1-cM on chromosome 3q26.33-q27.3 flanked by markers D3S2314 and D3S1602, designated as ‘AH’ (LAH2) (Aslam et al., 2004). The LAH2 disease interval was narrowed down to a 350-kb region on chromosome 3q27 containing only 4 genes (MAP3K13, TMEM41A, SENP2, and LIPH) through linkage analysis on Mari families with hereditary hypotrichosis (Kazantseva et al., 2006). Upon sequence analysis of the lipase H (LIPH [MIM 607365]) gene present in the LAH2 candidate interval in these families revealed a homozygous deletion mutation (985-bp) underlying LAH2 phenotype and this deletion spans exon 4, and the flanking intronic sequences. Deletion of exon 4 does not alter the reading frame of the LIPH gene; however, it does delete a highly conserved domain containing evolutionarily invariant amino acid residues, including a catalytic triad conserved in lipases. LIPH is a phosphatidic acid-selective phospholipase A1 (PLA1) that produces 2-acyl lysophosphatidic acid (LPA), which is a lipid mediator with diverse biologic properties (Kazantseva et al., 2006). Further, two deletion mutations c.346– 350delATATA and c.659-660delTA in exon 2 and exon 5, respectively, of the LIPH gene was revealed in large Pakistani families with autosomal recessive hereditary hypotrichosis. These mutations lead to frameshift and downstream premature termination codons (Ali et al., 2007; Jelani et al., 2008).
Wali et al. (2007a) mapped the third genetic locus for autosomal recessive hereditary hypotrichosis (LAH3) in a 17.35-cM region on chromosome 13q14.11-q21.32 in two Pakistani consanguineous families demonstrating clinical features similar to other forms of hereditary hypotrichosis: LAH1 and LAH2. Most recently, Pasternack et al. (2008) and Shimumora et al. (2008) in parallel reports identified mutations in a G protein-coupled receptor (P2RY5 [MIM 609239]) gene on chromosome 13 responsible for autosomal recessive hypotrichosis simplex and autosomal recessive woolly hair in three Saudi Arabian and six Pakistani families, respectively. The region containing the P2RY5 gene overlaps with that of LAH3 region on chromosome 13q14.11-q21.32.
Human Nude Phenotype
Congenital alopecia, severe T-cell immunodeficiency, and nail dystrophy syndrome (also known as Human nude phenotype) was described in a Italian family segregating in autosomal recessive pattern with two affected sisters (Pignata et al., 1996). Frank et al. (1999) identified a homozygous nonsense mutation (p.R255X) in the winged helix
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 19 Chapter 1 Introduction nude (WHN [MIM 600838]) gene, also known as FOXN1 on chromosome 17q11.q12 causing congenital alopecia, severe T-cell immunodeficiency, and nail dystrophy syndrome in two sisters described earlier, and confirming that this syndrome is human homologue of the ‘nude’ mouse model (Nehls et al., 1994; Segre et al., 1995). Recently, Amorosi et al. (2008) identified a human fetus homozygous for a mutation in the FOXN1 gene that lacked the thymus and also had abnormal skin, anencephaly and spina bifida.
Hypotrichosis with Juvenile Macular Dystrophy (HJMD)
Hypotrichosis with juvenile macular dystrophy (HJMD) is a rare autosomal recessive disorder characterized by congenital sparse and short hair, heralding progressive degeneration of the retinal pigment epithelium, which leads to blindness by the second decade of life. A classical cadherin family member, P-cadherin encoded by cadherin 3 gene (CDH3 [MIM 114021]) has been shown to be involved in the hair follicle (HF) morphogenesis (Jamora et al., 2003) and mutations in the CDH3 gene cause HJMD. To date, 13 different mutations in the CDH3 gene including three missense, three nonsense, four deletions, one insertion and two splice site, have been reported responsible for HJMD in different ethnic groups of the world, proving clinical heterogeneity for this condition (Sprecher et al., 2001; Indelman et al., 2002, 2003, 2005, 2007; Shimomura et al., 2008; Jelani et al., 2008). Recently, Shimomura et al. (2008) hypothesized that CDH3 could be a direct transcriptional target gene of p63 and further revealed experimentally that p63 directly interact with two distinct regions of the CDH3 promoter. The CDH3 gene is located at chromosome 16q22.1 comprising of 16 exons.
Alopecia with Mental Retardation Syndrome (APMR)
Alopecia with mental retardation syndrome (APMR) is a rare autosomal recessive form of alopecia characterized by hair loss on the scalp, absence of the eyebrows, eyelashes, axillary and pubic hair, and mild to severe mental retardation (Benke and Hajianpour, 1985; Vogt et al., 1988). Through molecular linkage studies, three different genetic loci have been mapped on three different human chromosomal regions, suggesting genetic heterogeneity for APMR syndrome. John et al. (2006a) mapped the first APMR locus named alopecia with mental retardation syndrome 1 (APMR1) in an 11.49-cM region on chromosome 3q26.33-q27.3 in a family with
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 20 Chapter 1 Introduction complete hair loss and severe mental retardation (IQ 25-30). Alopecia with mental retardation syndrome 2 (APMR2) locus was identified in a 9.6-cM region on chromosome 3q26.2-q26.31 in a large family with total alopecia and mild to moderate mental retardation (IQ 53-61) (Wali et al., 2006b). In continuation with the study on alopecias, Wali et al. (2007b) mapped the APMR3 locus in a 10.9-cM region on chromosome 18q11.2-q12.2 in a family with total alopecia by birth and severe mental retardation (IQ 25-30). To date, no causative gene has been cloned for APMR.
Autosomal Dominant Alopecias
Monilethrix
Monilethrix is a rare congenital hereditary structural defect of the hair shaft. Classically, monilethrix is inherited in autosomal dominant pattern; however, autosomal recessive inheritance is also followed in some sporadic cases. Affected individuals have normal hair at birth, but within the first few months of life develop fragile, brittle hair that tends to fracture and produce varying degrees of dystrophic alopecia. Monilethrix is caused by mutations in three hair keratin genes located at chromosome 12q13: hHb1, hHb3, and hHb6 (Healy et al., 1995; Winter et al., 1997a, b; Korge et al., 1999; Horev et al., 2003; van Steensel et al., 2005). It is now proposed that LAH and moniletrix is clinically overlapped with moniletrix-like hair abnormalities in LAH caused by mutations in the DSG4 gene and proved the existence of autosomal recessive form of monilethrix (Schaffer et al., 2006; Shimomura et al., 2006; Zlotogorski et al., 2006).
Marie Unna Hereditary Hypotrichosis (MUHH)
Marie Unna hereditary hypotrichosis (MUHH) is a rare autosomal dominant hair disorder that is characterized by coarse, wiry, twisted hair developed in early childhood and is followed by the development of alopecia. To date, two genetic loci have been mapped for MUHH on human chromosome 8p21.3 and 1p21.1-1q21.3, suggesting genetic heterogeneity for MUHH (van Steensel et al., 1999; Yang et al., 2005). No causative gene has been identified for MUHH yet.
Autosomal Dominant Localized Hereditary Alopecia
Recently, a Chinese family was reported with a novel form of autosomal dominant localized alopecia clinically and genetically distinct from previously reported conditions. The hair loss in this family involved only scalp hair in the frontal and
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 21 Chapter 1 Introduction occipital areas, and complete absence of eyebrows and eyelashes. However, body, axillary, and pubic hairs were normal. In search for a gene through linkage analysis, a novel locus for this form of autosomal dominant alopecia was mapped on chromosome 2p25.1–2p23.2 in a 28.30-cM region covering about 19-Mb physical distance (Wang et al., 2007). Gene responsible for this form of alopecia is still to be known.
Hypotrichosis Simplex of the Scalp (HSS)
Hypotrichosis simplex of the scalp (HSS) is a rare autosomal dominant form of isolated alopecia limited to scalp. HSS is clinically characterized by progressive loss of scalp hair beginning at the middle of first decade leading to almost complete loss of scalp hair by the third decade, although normal hair at birth and in the first years of life. In HSS eyebrows, eyelashes, beard and other body hair are normal. Gene responsible for the HSS was mapped to chromosome 6p21.3 in two Danish families (Betz et al., 2000) and nonsense mutations (p.Q215X, p.Q200X, p.Y239X) were identified in the corneodesmosin (CDSN [MIM 602593]) gene associated with HSS (Levy-Nissenbaum et al., 2003; Davalos et al., 2005).
In the present study, ten consanguineous Pakistani families with human skin disorders including six with pure and syndromic form of ectodermal dysplasias and four with hereditary alopecias have been investigated. Genetic linkage mapping and DNA sequencing of candidate genes have been performed to identify the disease genes.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 22 Chapter 2 Materials and Methods
MATERIALS AND METHODS
Families Studied and Pedigree Analysis
In the present study, ten consanguineous Pakistani families including six (A, B, C, D, E, F) with various forms of inherited ectodermal dysplasias and four (G, H, I, J) with hereditary alopecias were investigated. These families were recruited from different regions of Pakistan. Approval of the study was obtained from the Institutional Review Board (IRB), Quaid-I-Azam University, Islamabad, Pakistan. All families were visited at their residential places, informed about the purpose of the study, and written consents were obtained to publish photographs and X-rays of the affected individuals. Family information was collected from elders of the families. Affected members of the families were examined at the Department of Dermatology, Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan.
Pedigrees of the families were constructed as described by Bennett et al. (1995). The pattern of inheritance of the disease in each pedigree was deduced by observing the mode of transmission of the disease phenotype within the family. Males and females were symbolized by squares and circles, respectively. Unfilled and blackened squares and circles represent normal and affected individuals, respectively. Crossed symbols indicate deceased individuals while circles with a black dot indicate carrier females. Each generation was denoted by Roman numerals (I, II, III, IV, V, VI) while individuals within a generation were denoted by Arabic numerals (1, 2, 3, 4). Double marriage lines indicate consanguineous unions.
DNA Analysis
Blood Sampling
Peripheral blood samples, 6-10 ml, were collected from all affected and normal individuals, available at the time of the study, in 10 ml vacutainer tubes (BD vacutainer K2 EDTA 18 mg) with the help of the 5 ml (BD 0.60 mm X 25 mm, 23 G X 1 TW) and 10 ml (BD 0.8 mm X 38 mm 21 G X 1 ½ TW) syringes. Blood samples were stored at 4°C.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 23 Chapter 2 Materials and Methods
DNA Extraction and Purification
Genomic DNA was extracted from the venous blood samples using the standard Phenol-Chloroform Method (Sambrook and Russel, 2001). Approximately, 0.75 ml of blood was taken in a 1.5 ml microcentrifuge tube (Axygen, USA); mixed with equal volume of solution A (0.32 M Sucrose (BDH, England), 10 mM Tris (BDH, England) pH 7.5, 5 mM MgCl2 (Sigma, Germany), 1% Triton X-100 (Sigma, Germany)) and was kept at room temperature for 10-15 minutes after mixing the contents. The tube was then centrifuged for 1 minute at 13,000 rpm in microcentrifuge (Centrifuge 5415R, Eppendorf, Germany) and after discarding the supernatant, the pellet was resuspended in 400 μl of solution A. Centrifugation was repeated and after discarding the supernatant, the nuclear pellet was resuspended in 400 μl of solution B (10 mM Tris pH 7.5, 400 mM NaCl (BDH, England), 2 mM EDTA (BDH, England) pH 8.0), 12 μl of SDS (BDH, England) (20%) and 6 μl of proteinase K (Sigma, Germany) (20 mg/ml) and incubated at 37C overnight. On the following day, 0.5 ml of a fresh mixture of equal volume of solution C (Phenol, BDH, England) and D (24 volumes of Chloroform (BDH, England) and 1 volume of Isoamyl alcohol (BDH, England)) was added in the tube, mixed thoroughly and centrifuged for 10 minutes at 13,000 rpm. The aqueous phase (upper layer) was transferred to a new microcentrifuge tube and equal volume of solution D was added. Centrifugation was then carried out again at 13,000 rpm for 10 minutes. The aqueous phase was placed in a new tube and 55 μl sodium acetate (BDH, England) (3 M, pH 6) and equal volume of chilled isopropanol (BDH, England) was added. Tubes were then inverted several times to precipitate the DNA. Centrifugation was then carried out again at 13,000 rpm for 10 minutes to pellet the DNA. The DNA pellet was washed with chilled 70% ethanol (BDH, England) and dried (Concentrator 5301, Eppendorf, Germany) at 37C. After evaporation of residual ethanol, DNA was dissolved in appropriate amount of DNA dissolving buffer e.g. Tris-EDTA (1XTE) buffer (Qbiogene, North America) and stored at 4°C. Genomic DNA was quantified by calculating its optical density (OD) at 260 nm and diluted to 40-50 ng/µl for amplification by polymerase chain reaction (PCR).
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 24 Chapter 2 Materials and Methods
Genotyping
Exclusion Mapping:
The families, presented here, were initially tested for linkage to known genes/loci involved in human hereditary skin disorders (ectodermal dysplasias and alopecias) using highly polymorphic microsatellite markers tightly linked to these loci. Table 2.1 lists the known genes/loci involved in human hereditary skin disorders, their chromosomal location and microsatellite markers (average heterozygosity > 70%) with their genetic location (cM). Information about the genetic location of the markers and their product sizes amplified through PCR was obtained from Rutgers combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004) and genome database (http://www.gdb.org). Upon observing the positive and convincing linkage to a known gene in a family, corresponding gene was sequenced to identify a functional variant causing the disease phenotype in that family.
Genome-wide Search:
After excluding the linkage to known genes/loci involved in human hereditary skin disorders, genome-wide homozygosity linkage mapping was performed using DNA samples of maximum four affected individuals from the family excluded from the linkage (Lander and Botstein, 1987). For genome-wide linkage scan, 396 short tandem repeat (STR) microsatellite markers from Linkage Mapping Set (Invitrogen Co, San Diego, California, USA) were used. These markers are spaced approximately at 10-cM apart and are located on the 22 autosomes and the X and Y chromosomes.
Polymerase Chain Reaction (PCR)
PCR amplification of the microsatellite markers was performed with 25 µl reaction mixture in 0.2 ml microtube (Axygen, USA). The reaction mixture contained 1 µl genomic DNA as template (40 ng), 0.3 µl (20 µM) of each forward and reverse primer, 2.5 µl 10X PCR buffer (500 mM Tris-HCl and 100 mM KCl) (MBI,
Fermentas, Life Sciences, UK), 1.5 µl MgCl2 (25 mM) (MBI, Fermentas, Life
Sciences, UK), 0.5 µl dNTPs (0.25 mM of each dNTP), 0.2 µl Taq DNA polymerase (0.5 unit) (MBI, Fermentas, Life Sciences, UK) and 18.7 µl PCR water. The thermocycling conditions were as follows: initial 5 min denaturation of template DNA at 95°C as first step followed by 33 cycles of 95°C for 1 min (denaturation), 57°C for
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 25 Chapter 2 Materials and Methods
1 min (annealing), and 72°C for 1 min (polymerization), followed by a final extension of 10 min at 72°C in thermo cyclers (Biometra, Germany).
RT-PCR Analysis
RT-PCR analysis of the HPGD gene in normal skin and brain cDNA libraries (Invitrogen, San Deigo, CA) was performed according to the manufacturer’s protocol using a primer set designed from HPGD mRNA (NM_000860.3). The forward and reverse primers, amplifying 355-bp fragment, were designed from cDNA sequence corresponding to exon 4 (5’-GTGAAGGCGGCATCATTATC-3’) and exon 7 (5’- TGGTGCTATTATGAAGATCAC-3’), respectively.
Agarose Gel Electrophoresis
Genomic DNA was analyzed on agarose gel (1%), which was prepared by mixing 0.5 g agarose (BDH, VWR International Ltd. England) in 50 ml 1XTBE (Tris Borate EDTA) buffer (89 mM Tris, 89 mM Borate (Sigma, Germany), 2 mM EDTA pH 8.3). PCR amplified products were checked for amplification on agarose gel (2%). 5 µl ethidium bromide (10 mg/ml) (ICN Biomedical Inc., Ohio) was added to the gel to facilitate visualization of DNA after electrophoresis. Before loading to wells of the gel, genomic DNA and PCR amplified products were mixed with equal volume of bromophenol blue dye (0.25% Bromophenol Blue (Sigma, Germany) in 40% Sucrose solution). DNA ladder of 100-bp (MBI, Fermentas, Life Sciences, UK) was used to determine the size of amplified products. Electrophoresis was performed at 120 volts for half an hour in horizontal gel electrophoresis apparatus (Whatman, Biometra, Germany) having 1XTBE buffer. Genomic DNA and PCR amplified products were visualized by placing the gel on UV Transilluminator (Biometra, Germany).
Polyacrylamide Gel Electrophoresis (PAGE)
The amplified PCR products of the microsatellite markers used in exclusion and genome-wide genome homozygosity mapping were resolved on 8% non-denaturing polyacrylamide gel. Composition of 50 ml 8% polyacrylamide gel included: 13.5 ml 30% acrylamide solution (29 g/1 g ratio of Acrylamide (MERCK, Germany) and N, N Methylene-bis-acrylamide (BDH, England)), 5 ml 10XTBE (0.89 M Tris, 0.89 M Borate, 0.02 M EDTA), 0.35 ml 10% Ammonium persulphate (Sigma, Germany), 17.5 l TEMED (N, N, N’, N’-Tetramethylethylene-diamine) (Sigma, Germany) and 31.13 ml distilled water. This mixture was poured between two glass plates held apart
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by a spacer of 1.5 mm thickness. After inserting the comb, gel was allowed to polymerize for 20-30 minutes at room temperature. Amplified products were mixed with bromophenol blue dye (0.25% Bromophenol Blue in 40% Sucrose solution). DNA ladders of 50-bp, 20-bp, and 10-bp (MBI, Fermentas, Life Sciences, UK) were also used to determine the allele sizes of microsatellite markers. Electrophoresis was performed at 120 volts for 90-120 minutes in vertical gel electrophoresis apparatus (Whatman, Biometra, Germany) having 1XTBE buffer and the gel was stained with ethidium bromide (10 µg/ml) solution for visualization on UV Transilluminator (Biometra, Germany). Gels were photographed by using electrophoresis documentation and analysis system DC 290 (Kodak, Digital Sciences, USA).
DNA Sequencing
To search for pathogenic mutations biallelic (bi-directional) sequencing was performed of the 46 genes including CDH7, CDH19, ZNF407, MFAP3L, HMGB2, ASB5, ADAM29, VEGFC, HAND2, ANX10, MORF4, SAP30, HPGD, HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, NEUROD6, FERDL3, PRR15, EVX1, TWIST1, SP8, SP4, CCDC126, JAZF1, SNX10, TRA2A, ITGB8, CBX3, TWISTNB, AQP1, HNRNPA1B1, EDA1, EDAR, TRPS1, P2RY5, DSG4, and HR. All exons and intron-exon boundaries of these genes were PCR amplified from genomic DNA using primers designed from intronic and exonic sequences. Primers were designed using Primer3 software (Rozen and Skaletsky, 2000). The primer sequences, their amplified products (amplicons) and annealing temperatures are described in Tables 2.2-2.47. To get a complete chromatogram for a targeted DNA sequence, two thermocycling reactions (1st and 2nd PCR) and two purifications protocols (1st purification and 2nd purification) were performed. One thermocycling reaction is followed by one purification protocol consecutively.
Amplification thermocycling reaction (1st PCR or amplification PCR) was performed for 35 cycles to amplify a targeted exon of a gene using a particular primer set (forward and reverse) in 50 µl reaction mixture. The reaction mixture contained 3 µl genomic DNA as template (40 ng), 2.5 µl of each forward and reverse primer (0.02 µg/µl), 5 µl 10X PCR buffer (500 mM Tris-HCl and 100 mM KCl) (MBI, Fermentas,
Life Sciences, UK), 3 µl MgCl2 (25 mM) (MBI, Fermentas, Life Sciences, UK), 1 µl dNTPs (0.25 mM of each dNTP), 0.4 µl Taq DNA polymerase (1 unit) (MBI,
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Fermentas, Life Sciences, UK) and 32.6 µl PCR water. Thermocycling conditions were same as described earlier under the polymerase chain reaction. The amplified products were purified (1st purification) using Rapid PCR Purification System 9700 (Marligen Biosciences, Ijamsville, MD, USA).
The purified PCR products were then subjected to sequencing thermocycling reaction (2nd PCR or sequencing PCR) for 30 cycles using a particular primer (forward or reverse) in 10 µl reaction mixture. The reaction mixture contained 1 µl DNA template (200 ng), 1 µl primer (forward or reverse), 1.5 µl Big Dye Terminator version 3.1 ready reaction mixture and 1 µl 5X sequencing buffer (PE Applied Biosystems, Foster City, CA, USA) and 5.5 µl PCR water. Thermocycling conditions were as follows: initial 1 min denaturation of template DNA at 95°C as first step followed by 28 cycles of 95°C for 30 seconds (denaturation), 48-62°C for 30 seconds (annealing), and 72°C for 4 min (polymerization), followed by a final extension of 10 min at 72°C. Then 2nd PCR products were subjected to ethanol purification (2nd purification). The purified products were resuspended in 20 µl HDF (Hi-Di Formamide) (PE Applied Biosystems, Foster City, CA, USA) and were placed in 0.5 ml septa tubes to be sequenced directly in an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
DNA Sequence Alignment
Chromatograms of exons from the affected and normal individuals were compared with the corresponding control gene sequences obtained from Ensembl database (http://www.Ensembl.org) to identify any functional sequence variant. Sequence variants were identified via Bioedit sequence alignment editor, version 6.0.7 (Tom Hall, Isis Pharmaceuticals Inc.). Whenever a functional sequence variant was revealed in an exon of a gene, all available samples from the same family were sequenced for that exon of the gene. For further confirmation of the functional mutation and exclusion of that mutation to be a non-pathogenic polymorphism, 100 chromosomes were sequenced for that exon in a population to whom the family belonged. Identified sequence variants and mutations were named according to the Human Genome Variation Society recommendations (http://www.hgvs.org/mutnomen/).
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 28 Chapter 2 Materials and Methods
Statistical Analysis
The National Center for Biotechnology Information (NCBI) Build 36 sequence-based physical map was used to determine order of the genome scan and fine mapping markers (International Human Genome Sequence Consortium, 2001). The Rutgers combined linkage-physical map of the human genome (Kong et al., 2004) was utilized for genetic map distances in linkage analysis for the genome scan and fine mapping markers. PEDCHECK (O'Connell and Weeks, 1998) was used to identify Mendelian inconsistencies while the MERLIN (Abecasis et al., 2002) program was utilized to detect potential genotyping errors that did not produce Mendelian inconsistencies. Haplotypes were constructed using SIMWALK2 (Weeks et al., 1995; Sobel and Lange, 1996). Two-point linkage analysis was carried out using the MLINK program of the FASTLINK computer package for the genome scan and fine mapping markers (Cottingham et al., 1993). Multipoint linkage analysis was performed using ALLEGRO version 2 (Gudbjartsson et al., 2005). An autosomal recessive mode of inheritance with complete penetrance and a disease allele frequency of 0.001 were used. Equal allele frequencies were assumed in two-point and multipoint analysis for the fine mapping markers because it was not possible to estimate allele frequencies from the founders, since these markers were only genotyped in these families.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 29 Chapter 2 Materials and Methods
Tables of Chapter 2
Table 2.1: Microsatellite markers used to test linkage to known genes/loci involved in ectodermal dysplasias and alopecias
Genetic Chromosomal Microsatellite S. No. Candidate Genes / Loci Location Location Markers * (cM) * DXS8380 56.66 1 Ectodysplasin A1 (EDA1) Xq13.1 DXS8040 57.49 DXS111 58.30
D1S2346 163.01 D1S305 163.48 2 Loricrin (LOR) gene 1q21.3 D1S1153 164.80 D1S2624 166.65
D1S1723 216.60 D1S2615 217.55 3 Plakophilin (PKP1) gene 1q32.1 D1S2655 218.81 D1S2686 218.94
D1S235 261.73 EDAR-associated Death D1S2850 264.43 4 1q43 Domain (EDARADD) gene D1S517 271.85 D1S1149 273.11
D2S340 127.83 Ectodysplasin A1 Isoform D2S1889 127.83 5 2q13 Receptor (EDAR) gene D2S1893 128.62 D2S1891 128.62
D3S1564 185.79 D3S2328 188.02 D3S3520 190.47 Alopecia with Mental D3S2427 195.85 Retardation Syndrome 1, 2 D3S1754 198.14 6 3q26.2-q27.3 (APMR1, APMR2) & Lipase D3S2314 201.92 H (LIPH) gene D3S1571 204.89 D3S3609 205.94 D3S3592 206.82 D3S1617 208.48
D6S265 53.32 D6S273 53.70 7 Corneodesmosin (CDSN) gene 6p21.33 D6S1615 53.70 D6S2414 54.89
Continued
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 30 Chapter 2 Materials and Methods
Continued
D8S298 40.11 D8S1786 41.41 8 Hairless (HR) gene 8p21.3 D8S1733 41.59 D8S1752 42.55 D10S1710 121.11 D10S1267 122.40 Ectodermal Dysplasia of Hair- 9 10q24.32-q25.1 D10S1264 123.53 Nail locus D10S254 124.14 D10S1741 125.50 D11S4104 127.01 Poliovirus receptor-Like 1 D11S4171 128.16 10 11q23.3 (PVRL1) gene D11S924 128.16 D11S1299 128.16
D11S4123 145.13 Ichthyosis with Hypotrichosis D11S4150 146.09 11 11q24.3 Syndrome (ST14) gene D11S4463 150.29 D11S4131 152.89 D12S1698 55.91 D12S59 58.19 D12S1589 60.06 Ectodermal Dysplasia of Hair- D12S85 63.46 13 Nail Locus, Plakophilin 2 12p11.1-q21.1 D12S1661 65.12 (PKP2) & Type II Keratin genes D12S347 67.53 D12S398 69.75 D12S1644 75.14 D12S1610 80.27 D13S175 D13S633 0.55 Gap Junction Proteins (GJB6 & 3.10 14 13q12.11 GJB2) genes D13S127 6.97 5 8.75 D13S787
D13S1312 48.03 Localized Autosomal Recessive 13q14.11- D13S168 51.33 15 Hypotrichosis (LAH3) and q21.32 D13S284 54.17 P2RY5 gene D13S1807 55.50
12.33 D14S1430 16 Transglutaminase 1 (TGM1) gene 14q12 15.00 D14S581 15.36 D14S64
Continued
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 31 Chapter 2 Materials and Methods
Continued
D16S3107 86.57 17 Cadherin 3 (CHD3) gene 16q22.1 D16S3025 86.57 D16S3095 86.61
D17S926 4.52 Isolated congenital nail dysplasia D17S1840 5.64 18 17p13 (ICND) D17S1529 6.58 D17S1528 9.44 D17S1824 55.21 D17S1880 58.69 Ectodermal Dysplasia of Hair-Nail D17S1293 62.44 19 Locus, Junction Plakoglobin (JUP) 17p12-q21.1 D17S907 65.07 & Type I Keratin genes D17S1788 67.87 D17S1814 70.10 D17S1860 73.63 D17S1301 115.63 Hereditary nail dysplasia locus & D17S1839 116.93 20 17q25.1-q25.3 Envoplakin (EVPL) gene D17S1817 117.94 D17S937 120.70 D18S877 56.26 Desmogleins (DSGs), D18S847 57.41 Desmocollins (DSCs) genes & 21 18q11.2-q12.1 D18S36 59.09 Alopecia with Mental Retardation D18S456 60.41 Syndrome 3 (APMR3) D18S57 62.36 D20S103 2.17 Transglutaminase 3 (TGM3) & R- D20S199 4.99 22 20p13 Spondin (RSPO4) genes D20S906 5.65 D20S113 6.98 D20S847 54.80 23 Transglutaminase 2 (TGM2) gene 20q11.23 D20S834 55.60 D20S478 57.96
*STS microsatellite markers and average-sex distance in cM (Centimorgan) according to the Rutgers combined linkage-physical human genome map (Build 36.1) (Kong et al., 2004)
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Table 2.2: Primer sequences used for screening CDH7 gene in family A
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
2 GAAGGCTATTCAGCAGTGGTG GTGTTTACCATCCCCATAGC 496 52
3 GTACTCCTGCCTGACATAAG GTTCCCCTCCCTAACTGAC 533 52
4 CTCTTTGGGGCTGGTTTAC TGCTTACCAAAGTAGACATATTG 431 53
5 TTCAGTATTGAGAAAACCACG AATGCAAATATATGATGGCTTC 483 50
6 GAAGTTGTGCTACAATGATGCC GTTCCCTAAGAGTCTGACAC 450 55
7 CATGACAGAGTCCTTCCTAAG GGAACAAAGGAACATCAGAGCG 451 55
8 GACAGATCAGAGAGTCAGTAGG CCTGTGAAGGTAATATAGGCCC 496 55
9 CAACAATTTTCTCATCAGGG TGGGTAAGGTAGGAAACAGC 808 57
10 CTCCCTAGCTCATTCAGGGAG CCATAATCAGCATTCCTGGGG 324 52
11 CTGCAACTCCAGAGATTTCC ACAAAACAAACACCATCCAG 497 54
12 GAAAACCTGTAACTCAGCGTCC TCTCCAGCAAGGGGAGTTCTTG 707 55
† bp = Base pair ‡ oC = Centigrade
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Table 2.3: Primer sequences used for screening CDH19 gene in family A
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
2 GTCAGATGCAGAAGTTGTAACC GTGGATGTTTTAGGTGCTTGC 568 57
3 ACTCTTCCTTCTCCCTGGTG CTTCTTTTATTCCTGTGAAGGC 710 52
4 GAACAAGGAGGTGAATTGAGC CAGTCTCCTAGAAGTATCAGC 427 53
5 GTCACATTGCAGTGGTTC AGCCATGAAGTTAGTGGC 520 56
6 GGCATGAACCCAGGAGG TCATCTCTAAAATCCTTGTCTCC 512 54
7 AGGTAAGTGACAATACACCCAG AAGCAGAGGTGGCAATGAG 603 57
8 GGCTATGTAGAGCATGCATTG GTAGCCTTGTCCCCATGTATAG 317 57
9 TTACAGCCCATGGCAAATAC TGTCTCATTTTACCTTTACCTGC 915 57
10 TTTGACAGTTATTTTCTGCAATG TCTTTTGCTTGATTTCCAAAC 379 57
11 TGTCCCTAGCACATGTGAC GTTCAGTTCAGCACCACC 403 55
12 CTTGAGCTTGAAATGAGAGC TCCATAGACTAGGGCTTTCC 687 55
† bp = Base pair ‡ oC = Centigrade
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 34 Chapter 2 Materials and Methods
Table 2.4: Primer sequences used for screening ZNF407 gene in family A
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1-1* TTTGAAGAATGTATTGAAAGGG ACATGTCTGCAGAACCAGC 960 53
1-2* ATGGCATGCGATTACTACG AACTGTGCTATCAGAAGCTGG 849 51
1-3* CAAGAAGATCCCGTTCTGG ACGGCAGTGAAATGTGAAC 917 55
2 GGAAGACTCACTGAAACAAAGTC TGGGAGAAGAATGTCATAACTG 492 57
3 TATGCAAGCTTTGGTGTAGC TCCAAATGCTTTCAACTACTTC 442 58
4 CGCAAAGCAGAGAATAAATG TTCACACACAAGCACAAATG 589 57
5 CATCTGGAAGACATCTACCAG GACCTTCAAGTGCACAGATG 483 57
6 GTCCTAAGTGTGTGGAGTC GGTAATTAGCTCCCAACCG 394 55
7 TTGGGATTTGGTATTTCTGG GTTGAGTGGGATCAAACTGG 593 55
8 GTGCGGTCACTGAATTAGG GGTGGCCAGTGTGACTCTC 927 55
† bp = Base pair ‡ oC = Centigrade * Exon 1 amplified by 3 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 35 Chapter 2 Materials and Methods
Table 2.5: Primer sequences used for screening MFAP3L gene in family B
Exon Amplicon Annealing No. Forward Primer (5′→3′) Reverse Primer (5′→3′) (bp)† Temp. (°C)‡
2 CATGACCTGCTGTGAACAGTG GTGCTGGCTCCAAAGCTCCTG 447 52
3-1* GCATGTGAAGTCGACGTTC GGATTGTGAGACCTCATCC 599 60
3-2* CTCTAGAGCTTGCCAAAGTC ATCGGTAGAAGGTTCTGCAG 440 55
3-3* GAACTGCAGGACTATCATGG TCCAGTCCCATTCACTGCAG 654 57
Table 2.6: Primer sequences used for screening HMGB2 gene in family B
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
2 GGACATTTCTGTCAGGAAAGG GTCTGCCTGGAACTCTTAAG 646 57
3/4¶ GATCTTGTCGTCTTTGGATG CTACAAGTCTGTCTGCAGG 684 56
5 GCCTAGTGGGATAAGTGCTC GAAGCTAGTATTGAGCTGCAC 411 58
† bp = Base pair ‡ oC = Centigrade ¶ Two consecutive exons amplified by a single pair of primer set * Exons amplified by 2-3 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 36 Chapter 2 Materials and Methods
Table 2.7: Primer sequences used for screening ASB5 gene in family B
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 CTTTCCTCAGGACCAGCTG GCCACGAAGTGAGTTACAAG 439 57
2 CACCCTGTTGAACACTGAG GCTGCACATATTGTGCAGAG 524 55
3 CTGGAAACTCGTTTGCCTG CTACCGAGAATGAAGGAAAG 328 57
4/5¶ GCCCATTCCAATGAATTATCTGC GTGAGCTAGGAAGCCGACTTATC 875 57
6 CCTTCCTCTATTGCCTTTCC GCCATTGTTACTTTCCATGCC 388 55
7 GTGGTCATGCTATCCTAGG CTGGGTGATCTCACACTCAC 369 58
Table 2.8: Primer sequences used for screening ADAM29 gene in family B
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. ζ (bp)† Temp. (°C)‡
5-1* ACCACCTGTGACTCCAGC TCCTCCAGCAACTTTGAGTC 760 57
5-2* CCACTCAGAAGCAAAGTTC CTCTCCTTACAGGGAATTCC 922 52
5-3* GACTGCAAGTTCCTACCATC GGAGTTTGAACATCTTGCTGC 806 56
5-4* GGCTATGGAGGTAGTGTTGAC GTATGCCACACATATGCCATG 794 55
† bp = Base pair ‡ oC = Centigrade * Exon 5 of the ADAM29 gene amplified by 4 sets of overlapping primers ζ Only exon 5 represents the coding sequence of the ADAM29 gene ¶ Two consecutive exons amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 37 Chapter 2 Materials and Methods
Table 2.9: Primer sequences used for screening VEGFC gene in family B
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1 CTGTGAGGCTTTTACCTGAC GAGCACTGAGCTCAGTAAC 525 57
2 GTGTTAGGGAACGGAGCATAG CCCTAAAGGTGTATTCGGTG 432 55
3 GGATCCCATCAGCTGTCTC GCATGAGGCTTTGACTCCAC 451 55
4 CATAGCGTCCTGCGTACACTAG GCCTCTAAAATACGCTTCCCACTG 502 54
5 GTTGTGGGTAGAGAGAGTTTGG GGGTACAAACATGAGTATGTTCC 462 59
6 GGAACATACTCATGTTTGTACCC TCCAGTCTCTCATGCAAGTTTAGC 636 60
7 GCTTACAATACCTCATCCTTCC CATGGTTCAGGAAAGACAGAC 498 56
Table 2.10: Primer sequences used for screening HAND2 gene in family B
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1-1* GTATGAGCACGAGAGGATTC TGTTGATGCTCTGAGTCCTG 661 57
1-2* GACTACAGCATGGCCCTGTC CATCCTTACACAACCTGGTGC 536 57
2 CTAGGGTAGCGCTAACCTTG GAGAGGGGAAGGAAATTGCAC 433 55
† bp = Base pair ‡ oC = Centigrade * Exon 1 of the HAND2 gene amplified by 2 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 38 Chapter 2 Materials and Methods
Table 2.11: Primer sequences used for screening ANXA10 gene in family B
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1 CTCAAGTCATGCTGTATCC GGATTCTTAGGTATGCTC 506 57
2 AGGTTGGTCTTCAACTCCTGAC GAGGCACTTATGGATTTAAGGC 487 55
3 AGGAGATAGGAAGGATCCAGG GGTCACTGTGTGAGTGTGAAC 503 55
4 CAGGGGTGAGATCAAATCAAC CCCTTGACCACAACATTTCC 468 57
5 CTTCAGGGTCAAATAGGTAG GGTCCTCAGTGAATCCTATG 412 57
6 AGTCCTGTGCTTATGTTACC GAACATGGCTTCAGTAAATG 424 60
7/8¶ CAGGAAATGAACACTTACTCCAG CTTTGAGATAACTGACTTTCCGC 507 52
9 AGCAGTGAGTTAGTGGGTC GTACTCTTCCCCTATGGAG 521 56
10 CCTGTGGCTTATAGTGTTACC CCAGTTTTTCTCACTTGGGTG 357 56
11 TCACTGTCAGCATGCGGTAAG CTAAGTTAGCACCTGCCGATG 504 60
12 TCATTGGGAGCAGTACTTAGG GGCAAATTCAGGATAGTAGGC 443 57
† bp = Base pair ‡ oC = Centigrade ¶ Two consecutive exons amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 39 Chapter 2 Materials and Methods
Table 2.12: Primer sequences used for screening MORF4 gene in family B
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* GGTTCCGGAGAGCAGAGTACTC GTGCCCAACATTAGGTTGAAG 525 55
1-2* AACCATGGCTTGTTGATGACTG CACTAACATCTTCAAAGCACATCG 575 57
Table 2.13: Primer sequences used for screening SAP30 gene in family B
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 CTCAGCACTGTCCACTGTTTC CCAAACACAGACTCCACGAG 632 55
2 AGATGGAACCAACCCCTCCTTC GTGTAAATGCATGTCAACTGGC 507 56
3 TTGCAGCTCTGTTGTCAC CCTAAGTGGCAGACAAAC 516 60
4 TCACACTATCTCTTGTTCG CAGAGAAAATCCTCTGTAG 423 55
†bp = Base pair ‡ oC = Centigrade * Exon 1 of the MORF4 gene amplified by 2 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 40 Chapter 2 Materials and Methods
Table 2.14: Primer sequences used for screening HPGD gene in family B
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 GCAAAGATCGCGAAGCTTG ACTTCTGAGGTGTGCTCAC 472 52
2 GTGAGCACACCTCAGAAGTG GCTATTGGGCTGTCAGAAGG 446 55
3 CCAAGCTGCCAGATTGATG GCCAATCCCTGAGTTAAGC 555 55
4 GGCAAACCCAAAGAATCCAGG GGAGTCTCACCACAACCTTTG 570 55
5 GGCTACTGAGTTTCACAAAGC GGCCTATTGCATCTTGCATTTC 570 55
6 CATTGTTACATAGCTGGGAGG CTCCCAGAGAGTTTGCCAAAC 439 55
7 CCTGCCAAAATGATGGAAGG TACAACCTAGCCTTTGGTCC 625 55
†bp = Base pair ‡ oC = Centigrade
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 41 Chapter 2 Materials and Methods
Table 2.15: Primer sequences used for screening HOXA1 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* GTGATCCATCACTGCGGAAG GCACTGAAGTTCTGTGAGCC 531 57
1-2* CGACGACCGCTTCCTAGTG CTCTCCATGCACTACAACAC 640 57
2 CTCCACAGCCCGATTTGTG GCACCAAGTCTCTGGTCCAG 669 55
Table 2.16: Primer sequences used for screening HOXA2 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 CACCCTAGCAGCGATATTCTATG GACAAGGCAACCAAGCATCTCTC 653 56
2-1* CAATAGCTCTACTGCATCCAGGG GTTGACAAGCAGTTGGGAACAG 649 55
2-2* GGAAGGCTACACTTTTCAGC CCTCAACACTTAAAGGAGGG 525 57
†bp = Base pair ‡ oC = Centigrade * Exons amplified by 2 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 42 Chapter 2 Materials and Methods
Table 2.17: Primer sequences used for screening HOXA3 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* GAGAGTAAGATGGATTTGCGCGG TGTTGCTGGCATTCTGAGGAGGG 576 56
1-2* CAAGGCACACGAACTGAGTGAGG CAGACATGCTCCATCGCTCCTAG 444 55
2-1* TGGGACCTGACGGATGCAGGAAC CCATGAGCGTGCGGGTCATAGTC 637 57
2-2* TCTGAACTCTATGCATTCGCTGG GCGGAGAAGAGAAAAGGAAGG 632 60
Table 2.18: Primer sequences used for screening HOXA4 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* AGGAGCGGCTCGAACTTTGGTG TGCAGGACGTGGCTCGCATG 596 56
1-2* CCACTGCCTCCTACTACG GCACATACCCACATCTCAC 561 54
2 GTATCTTGCGTGAACTTGGTG CAACCAGCACAGACTCTTAAC 669 55
† bp = Base pair ‡ oC = Centigrade *Exons amplified by 2 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 43 Chapter 2 Materials and Methods
Table 2.19: Primer sequences used for screening HOXA5 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 ACCAAGTACATGTCCCAGTC CTGGGAGAAATGAGACCAAG 781 52
2 GAGAAGGAAATTCGGGAGGG CGAGAACAGGGCTTCTTCAC 514 55
Table 2.20: Primer sequences used for screening HOXA6 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 GATTTGCTGCTGTCGCTTTTGG GAAGCCAAAGAAACTTTGCTGG 698 58
2 CATTCAGTATGCTTTGGGCC GTATTGGCTGTGTGTGTGAGG 643 55
Table 2.21: Primer sequences used for screening HOXA7 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 CCGTCCAAAAGAAAATGGGG CATACAATGCTATGGGCTCC 701 55
2 GACTAGGCCAGGAGGAAGGTG TGGGAGCTGGAGTAGGTGATG 598 57
† bp = Base pair ‡ oC = Centigrade
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 44 Chapter 2 Materials and Methods
Table 2.22: Primer sequences used for screening HOXA9 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* CGTTATTGTTCTGCTGGACGGGC CAGGAGCGCATGTACCTGCCGTC 505 55
1-2* TTGGCGCCTCGTGGAACCCAGTG TCCCAAACACCAACTTCTGGCTC 539 57
2 CTGACTGCCTTTCCTAACCAG CCTGAACAGGGTTTGCCTTGG 538 55
Table 2.23: Primer sequences used for screening HOXA10 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* CTACGAAACCAAACTGGGAG GAGACTTTGGGGCATTTGTC 659 60
1-2* CATAGACCTGTGGCTAGACG AAGTTCACAAGGTCAGCCTG 755 52
2 TGTGACTTGGGACATCTCTC CAGCCCTGCACAGATGTAAC 536 55
† bp = Base pair ‡ oC = Centigrade * Exons amplified by 2 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 45 Chapter 2 Materials and Methods
Table 2.24: Primer sequences used for screening HOXA11 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* GGATGGGGATAGATTTCCACGTC TAGAAATTGGACGAGACTGCGGG 510 57
1-2* GGCAATCTGGCCCACTGCTACTC TTTCAGCACTCGCCACGTGATCC 657 57
2 ATCTGTGGCTGAGCCTCCAACTG GGCTATCTCCATGCATCCCTCTC 561 52
Table 2.25: Primer sequences used for screening HOXA13 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* GTGCAGAGCTGGAGTCGGAG GCTGCCGAAGTAGCCATAGG 733 52
1-2* CAACCAGTGCCGCAACCTGATG GGGCAGACCAGGAAGAGAACAG 778 52
2 CTAGGCTTTAGCAGAAGACAGG TGCCAGTCTCTGTCTCTTTCTC 517 55
† bp = Base pair ‡ oC = Centigrade * Exons amplified by 2 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 46 Chapter 2 Materials and Methods
Table 2.26: Primer sequences used for screening NEUROD6 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* ATCAGGCCCAGAATCAGTC GGACGAATGTGAGCAGATC 633 55
1-2* GACGCTCTGGACAACTTAAG GGAGGACTTAAGGGACCTTC 455 57
1-3* CATGGGACTCTTGATAATTCC GTAGAAACAGAATCACAGTGC 578 57
Table 2.27: Primer sequences used for screening FERD3L gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 GTGATGGTGCCCCATCAGATTC GAAGACCAGACGTTCTGTCTTG 718 55
Table 2.28: Primer sequences used for screening PRR15 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 ACGTGACTTCAACCAACCCAG TGGAATGAGACAACGCGTAAG 688 55
† bp = Base pair ‡ oC = Centigrade * Exons amplified by 2 sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 47 Chapter 2 Materials and Methods
Table 2.29: Primer sequences used for screening EVX1 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1 CTCTTGCAACCAAGATCCGTC CGCAGATAGCAGAGAGAGTTG 666 55
2 CAGTACCCTAGCAGAAACAG CTGTCACGAGGGTCAATCTG 545 55
3 GGTGTCTGCTTTGGCTGTTCC TTTCCCCCTTGGCGTCCTAG 776 55
Table 2.30: Primer sequences used for screening TWIST1 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1-1* CTTTTTGGACCTCGGGGCCATC CCTCGTAAGACTGCGGACTCCC 542 57
1-2* CAAGCGCGGCAAGAAGTCTGC GAGGAAATCGAGGTGGACTG 526 55
Table 2.31: Primer sequences used for screening SP8 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1/2¶ GGCTTTGTGTGCAAGAGAGTG GCCTGGAAAACAGAGTAGTCG 729 53
3-1* CTGGTGTCCGACTCGTTCAG CTCTGCCTCCTGGCAGTTG 780 52
3-2* ACCTCATGGACGGCTTCAAG CACAGAAGGAAGAAGGAAGAG 794 55
† bp = Base pair ‡ oC = Centigrade * Exons amplified by 2 sets of overlapping primers ¶ Two consecutive exons, 1 and 2, amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 48 Chapter 2 Materials and Methods
Table 2.32: Primer sequences used for screening SP4 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
1/2¶ ATTCGCGGAAAAAGAGGCAGAG CTCAGAAAGATGCTTTGAGTGG 814 52
3-1* GTGTTCCCAAGGGCATTTTG GAAGCTGTCCTGTTAGCAGC 796 52
3-2* CAACTTCAGACAGTGGAAGG CAACTGATTTGCCCTGAAGGTG 899 53
3-3* ATCCAACAGCCTCAGCAACAG CTAATCAGAAAGCTCTGTCCC 716 54
4 GATACATAGGCAACCTGAGCC TAGTGACCATCATTCCCAGCC 517 57
5 GAACTACAGTTATGGCATC GTAAATGCCTTCTCCTTC 452 57
6 GCAACTAGTGAAACTGTAC CAACACTTCACGTGTTGAC 523 57
Table 2.33: Primer sequences used for screening CCDC126 gene in family C
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
3 GTCGTCTGTTCTGTTACTG GCAAAGACCTATGCATGAAG 539 55
4 CAGATGACCCAGTAGTTGTTC CATGAATCCAGCATACTCCTTG 426 57
† bp = Base pair ‡ oC = Centigrade * Exon amplified by 3 sets of overlapping primers ¶ Two consecutive exons, 1 and 2, amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 49 Chapter 2 Materials and Methods
Table 2.34: Primer sequences used for screening JAZF1 gene in family C
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1 CTCCTCTCCTCACCCCAC TCAGCGCCTCGGGCTACTC 648 52
2 CACTGTTGATGTTTGGAATTCCC GAGCATTCCATATACAGGTTGC 441 57
3 GTCGTGCATGTCTGAAACTC GTCTGTGTCGTCATCTGTC 393 57
4 CTGGTCTCTTAGGTGGGAAC CGCACTCTAATGCAGGAGAG 514 55
5 GACTACACTCTAAACAGCTGC GGGTATGATGATTACATGTGC 497 57
Table 2.35: Primer sequences used for screening SNX10 gene in family C
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
2 TCACATGTCAGGGACTGTGG CTATGCAGGCACCAATGGTG 342 52
3 GTGTTCTAGATTTGCTCGTG GCACATCTTCACATGTGAAG 523 57
4 GTTCCTGGGTTATGTGCAAG CTTTCATGGCCTATACCCATC 476 57
5 GATGGGTATAGGCCATGAAAG CAGAAACAGGAACACTCTTCC 421 55
6 AGCTTCATGTTTTTACCCACC ACTATGATGAGCTGTCACAGC 597 57
7 CCTGTCTCTCTTTGTGACTGG CCTAACATCATGACAGTGAGC 308 55
† bp = Base pair ‡ oC = Centigrade
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 50 Chapter 2 Materials and Methods
Table 2.36: Primer sequences used for screening TRA2A gene in family C
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1 TGGTTGCCGACTCTTTCCTC GGAACCCAGAAGCCATCTTG 306 52
2 CTGGAGGGTGTTGTCTAACC TGGAACTCCTGGGCTCAAGC 450 57
3 CTCAGAAGCAAGGAGGAAGG TGCTTACGAAAGATGGTGGC 460 57
4 GCTCATCTGCTCTAGTTCTC CTGGACGCAAGATCCTATC 486 55
5 CAATGCCCTGTACAAGCTAC GCAGGGTTTTGCCATGTTGC 388 57
6 CAGAATACCATGGGAGTCAG CTCTTTCTGCCAATGAGACC 489 55
7-8 GGTCTCATTGGCAGAAAGAG GAGTAGGAAGAATCCACAGC 496 56
† bp = Base pair ‡ oC = Centigrade
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 51 Chapter 2 Materials and Methods
Table 2.37: Primer sequences used for screening ITGB8 gene in family C
Exon Amplico Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. n (bp)† Temp. (°C)‡
1 GGAAAACGTCCTAGCGACACTC CTTGAAGTTCTCTGATGACCG 358 57
2 GAAGGTCCTTGTCCTCTAC CTGATGCACCTTCAAGGCAC 494 57
3 GATGCAGAAAGTTGGAGTAGG CTTGATTCTGAGTCCACTGTG 488 55
4 GTGATGATTGTGCTAGGTGC GCCCAATCTACAGTTCATCTC 533 58
5 CGTCGTATAAAGTTTCCCTGG CACCTTGGTGAGGAGTAAGAG 440 60
6 GCTATTTACGCAGCAGCGTTG CTCTCTCATGGCTTATCCTTG 442 58
7 CATAGTCCTTCCCAGGTAC CAGGCTGGACTCAAGTGTATC 466 57
8 GTTCTGGCTGCTCTACTAGG ACTCCTCCTCTCGAGATGAC 355 55
CAAGTAGGAACTTTGAGGAGA 9 GGGTCATTATGTGGAAACACTG 462 55 G
10 GCTTCAGATAGGCTTGCACTC CTGTTGCCATGCCCTAATTTC 837 52
11 GATGCTGTTCCCATTCATTGG GTCACAATGTATCTGGCAACG 452 57
12 CCTTCGATGTAATGATGGAG CTGGGATTACAGGTGTGAG 395 57
13-14¶ GCTTGGTTATGGGACATGGAC GCAATTTGTCTCCTCCTGTGAC 797 53
† bp = Base pair ‡ oC = Centigrade ¶ Two consecutive exons, 13 and 14, amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 52 Chapter 2 Materials and Methods
Table 2.38: Primer sequences used for screening CBX3 gene in family C
Exon Amplico Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. n (bp)† Temp. (°C)‡
2 CACATGGTTAGGACACGTAC GAGCTGGGAAATATGTGGATG 353 57
3 GTGTCTGCCCTGGGATATAG AGTCTGCTCACCTTCAACTG 468 57
4 TAGTATCTGCAGTACAGAGG CCAAGATCATGCCATTGCAC 420 55
5 GCTCTAGTCATTTCCATCTC GGTATTGAAGTGCTCCAGGA 434 58
6 TCCTGGAGCACTTCAATACC GTAACCAGTGCTATGGATGC 418 60
Table 2.39: Primer sequences used for screening TWISTNB gene in family C
Exon Amplico Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. n (bp)† Temp. (°C)‡
1 TGCGTAATGCAGCGAAAAGAG CGGATCGCGACTCTAGAAATC 529 57
2 GCAGAAGGATCAATTGAGCC GGATGGGACATATATCGTGTG 519 54
3 ATGTTTTGGCATGACATGTGG GGAAGGTAATTGTGCTTTCAG 726 55
4 TGCCATGGTTATGTGTCTTGTAC TTGGCTAGTCTTGACATTGTAG 86 52
† bp = Base pair ‡ oC = Centigrade
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 53 Chapter 2 Materials and Methods
Table 2.40: Primer sequences used for screening AQP1 gene in family C
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1 CATCCATCCAGAGGAGGTCTGTG TTCCTCAGTGTCCCCATCTGCAC 659 57
¶ 2-3 GATGGGCTCTGAAGGCCCATCTG CCTGATTTCCTGTCCTCTGGCTG 836 50
4 GATTTGGCTCTCCTACCTGCC GGCAGATCTTGGGGAAGTGAC 442 55
Table 2.41: Primer sequences used for screening HNRNPA2B1 gene in family C
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
1 CAGTTCTCACTACAGCGCCAG GGGGAAGCTGTTTGTACGCTC 296 57
¶ 2-4 CTTGTGTAGATCAGCTGAGGC GGTCCAGAAACCCAACACACC 782 57
¶ 5-7 GTGGATACAAGCGAAGTATGG AGAGCTTGCCAGGATACAATC 853 55
8 AACTGGTAGCACTGGTGTGTC ACTGGACCACTATCACCCAAG 333 58
¶ 9-10 AGTAACCCCAGCCACCAAATG TACATGGAGCACTGCCCACAG 738 60
11 CTGCTAACTGGCTGCAAAGG TACTCCTGCAGCTATGTCTC 280 55
† bp = Base pair ‡ oC = Centigrade ¶ Exons amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 54 Chapter 2 Materials and Methods
Table 2.42: Primer sequences used for screening EDA1 gene in family D
Exon Amplicon Annealing No. Forward Primer (5′→3′) Reverse Primer (5′→3′) (bp)† Temp. (°C)‡
2 AGCCGATGGCAGGACAGTAG AACCTGCCCTGGGCAACTTC 657 55
3 GGTACAGGTAGACTGTCTATG CTCCCAAAGTGCTAGGATTAC 322 55
4 TGGATCCTTGCCAAAAGC AGAGTGAAACTCCGTCTC 448 55
5 TGAGGCAGGAGAATCGCTTG CTCTCAGGATCACCCACTC 526 57
6 TGCACTCTGACTCTTCCTCC TCAGAATCTCCGGGGTGTTC 313 58
7 CCAGGATGGAAACATGGGAC TCGTATGCCAACGGTACCTC 252 55
8 TTCTAGGCTACCCTGGTTGC CCATTGGATGGACTTGGCTG 350 59
8 GTCAATTCACCACAGGGAGG AGTCAAGCAGGCCTTGTCAC 500 60
† bp = Base pair ‡ oC = Centigrade
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 55 Chapter 2 Materials and Methods
Table 2.43: Primer sequences used for screening EDAR gene in family E
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
2 AGCTTCAGCTTCATGTCTGC GGGACTATGATCAGCATTCC 329 55
3 GACTTAGCAGCAGTGAGCAC TGCCATCAATCTCAACGTCC 441 55
4 CAAGAGTAGCTTCTGGAGAC CACTTAGGAGACACAGAGGC 520 57
5 GGAAACTGAGTGGACAGAGC ACTCAAGGCTCAGATGTGGC 408 58
6 CCTCAGGCCCATTCATGATC GGAAGCGCACCATAATCATC 294 54
7/8¶ TCAGTCTAAGCAAGCACCTC ATGGTTCAGCATGTGAGAGC 547 55
9 CTGCTCTCACATGCTGAACC GCTAGCCTGTCAGTTCACTC 306 57
10 ACCGGTGTATGTCTCGTCTC GCTGTGAACTTGTCACTGTC 495 55
11 GGAAAACCTGAAGAAGGCCC TCTGACCAAAGTGCGGCAAC 367 57
12 CTGGCAGCGTTCTAGGTGTC GTCTGGCTCCTTGAACATCC 651 59
† bp = Base pair ‡ oC = Centigrade ¶ Two consecutive exons, 7 and 8, amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 56 Chapter 2 Materials and Methods
Table 2.44: Primer sequences used for screening TRPS1 gene in family F
Exon Amplicon Annealing Forward Primer (5′→3′) Reverse Primer (5′→3′) No. (bp)† Temp. (°C)‡
2 TGCAGATGTACAGATCAGCTC CCATAAGACTATAGCCACCCTC 286 59
3-1* AGGAAGCATTTGATTATGCAC ACCTTGACAATTGGCTTGAC 647 57
3-2* AAAGAGCAGAGGCAGATGAC GCTACCTGTCTGGTACTGGG 646 57
4-1* ATACTTGGCAGTACCCTCCC TGTTCTAGCAGTTTAAGTGAGC 523 57
4-2* TACTCAGTGCCCATAAAGCC CTGGGCTGCAAAGTCCTC 487 57
4-3* GCCACTTCTCCGTCATTATC ATCACTCTCAACAATTCCCG 522 55
5 AAATGCCTGTTAATTACATTCC CATGAGTTACTTGCTGCCAC 936 58
6 ACCTCATTATGGGCAGTGTG ACTGCAAGCCAGGGAATG 347 60
7-1* AGCATGGTTTATATTTGTGAGG AGGACTGCCTCTCTCAGAAC 506 56
7-2* ATGCAACCTTTGCACATTC TACATTTGGTGGTGCCTTC 480 57
7-3* GCCTTATCCCACCTTCAATC GGTCTTCATAAGACATTACAAG 500 57
† bp = Base pair ‡ oC = Centigrade * Exons amplified by multiple sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 57 Chapter 2 Materials and Methods
Table 2.45: Primer sequences used for screening P2RY5 gene in family G and H
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡
2-1* CTGTTGAAGAACCCAGCGG CCCTGAGAGTGGGTAGACTG 690 53
2-2* CTTCACAACACGGAATTGGC TGGGTACATTGTCCTTACTGC 595 55
2-3* GGGTAACAATGCCTCAGAAG GGCACCACATAATTTAAGTCAG 847 52
† bp = Base pair ‡ oC = Centigrade * Exon amplified by multiple sets of overlapping primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 58 Chapter 2 Materials and Methods
Table 2.46: Primer sequences used for screening DSG4 gene in family I
Exon Amplicon Annealing No. Forward Primer (5′→3′) Reverse Primer (5′→3′) (bp)† Temp. (°C)‡
2 GTATCCCAACCTGCTGTAGA AGAGATAGAGGACAGCAGCT 708 55
3 CTCACACTGTAAGACACCTG GAGCAGTGAAGCCTGCAAAT 236 50
4 TGGTAAAGAAACCCACTCCC TTTGGGTTCAGTCTGCCATG 362 55
5 CTACAGTCTGAATTCACTGG CAAGTGTGGCTTTTTCTGTC 353 55
6/7¶ GAATTCAGGAGAAGGCCAAC GTCCACACATAGGACAGAAC 751 55
8 TCTCCTGATTGGACTATGGG GGCAGATTCTGTCTCTAAGG 378 50
9 AACAGCGTATCTCCTGGACC GGGTAGAACAAACTGGCCAC 650 55
10 AGTTTCGCACATTGTAGCTG TAAGGTGTTTAGGGCTTTCC 344 55
11 CCTACAAGTTCCATGGCATC GGCAAGAACTGTGGAAACAG 406 55
12 GCCCACCAAGGAATTTCCAT CCATGAACCTAACCATCCCA 411 55
13/14¶ GTGACTTCCTAAACCGAGCA CCCAAAGAGACTGACAGACT 521 55
15 CCAGCGCTGTTAAACCAACA AGGCCTACTACCATTGTGAG 314 55
† bp = Base pair ‡ oC = Centigrade ¶ Two consecutive exons, 13 and 14, amplified by a single pair of primer set
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 59 Chapter 2 Materials and Methods
Table 2.47: Primer sequences used for screening HR gene in family J
Annealing Exon Amplicon Forward Primer (5′→3′) Reverse Primer (5′→3′) Temp. No. (bp)† (°C)‡ 2-1 * GCCTTACTGGTTTGAGCTGC TGAGATGGCCACCACTATGC 548 58
2-2 * TCCTGAGCACCCCAGACTCC CTTGGGGTTGACTGTGGGGC 614 61
3-1 * GAGGGCTTCAGTATTCTCCC AGTGGGTGGGTAGGATGAAC 494 56
3-2 * GAATCCTTGCCCGCTCTTCC CTGAGGAACTCCCAGAGAGC 757 58
4 CATCCTCAGACTCCCTGCTC TGGCTGTGTCTTCCTCCTGC 474 59
5 CTGCCACTCTCAGCAAGTGC CCTTAGGTCTAGGAGCTGGC 393 58
6 CTCTCCATGGAAGCTGCTCC GCCAACGAATGACCACAGGC 360 59
7 GCTGTGTCTCTATGTGACCC GGTGGTGAGTGTAGACCAAC 393 56
8 AGCTTCCCGTCTGATTGTCC GGGAATTAGCCTGATCCCAC 370 57
9 AAGGTGTTTGGAGGCATGTC 421 55 GGTAGAAGTCCATGAGCAAC
10 ATGTTGGTGATGCGGTCATC 462 56 TGCAGGAAAAGCAGTAGAGC 11 AGCGAATACACATGGCCTTC TAAGGGCAGTAGAACAGCTC 529 55
12 TCCCCGAGCTGTTCTACTGC 430 60 ACAGGAGGAGACAGAACGGC
13 AGCGTAAGTGTCCCCAACAC ACATGAGAGTACCAGGGACC 358 57
14 CCTGGTACTCTCATGTTTGC TGGAATCAGAGAAGCGCTTC 358 55
15 ACTCCTGACCTCAGGTGATC TCCAGGCCTGAAAGGAAGTC 357 56
16 TCAGCATCCTGGTGCATGCC TTGGGTCTGTGCAGCTCACC 400 61
17 CTGCCCTTCAAGACTTGACC CTCAGTGACTTCAAGGCCTC 465 56
18 GAATCTGCTCTCTGAGAGCC AGGGTGGGATCTGCTATGTC 347 56
19 CTGGGATTACAGGTGTGAGC AGATCTTTTGGCAGGAGGGC 677 57
† bp = Base pair ‡ oC = Centigrade * Exons amplified by overlapping sets of primers
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 60 Chapter 3 Ectodermal Dysplasias
ECTODERMAL DYSPLASIAS
Ectodermal dysplasias (EDs) are rare heterogeneous group of genetic disorders manifesting anomalies of classical ectodermal appendages like hair, nails, teeth, and sweat glands. These ectodermal organs originate from two adjacent tissue layers: the epithelium and the mesenchyme. Sequential and reciprocal interactions between the epithelium and the mesenchyme regulate the early steps of development in all the ectodermal organs (Pispa and Thesleff, 2003). Specific interactions among the germ layers may lead to a wide range of ectodermal dysplasias when genes important for development are mutated or otherwise altered in expression (Itin and Fistarol, 2004). Identification of genes through molecular genetics involved in the pathogenesis of different EDs greatly enhance the knowledge and understanding about the germ layers interactions during morphogenesis and differentiation of the ectodermal appendages.
Functionally EDs were classified into four main subclasses on the basis of genes involved: (1) cell-cell communication and signaling (2) cell adhesion (3) transcription regulation and (4) development (Lamartine, 2003). Cell-cell communication and signaling class include genes encoding extracellular ligands, receptors, adaptors and gap junction proteins (EDA1, EDAR, EDARADD, XEDAR, NEMO, GJB2, GJB6). ED genes involved in cell adhesion encode different kinds of structural and desmosomal proteins (PVRL1, PKP1, CDH3) mediating interactions between cell membranes and cytoskeleton. Genes in transcription regulation consist of different transcription factors and DNA binding proteins (p63, GATA3, EVC, TRPS1, XPD). EDs are also caused due to mutations in genes involved in development of several tissues like MSX1 and SHH.
In the present study, six Pakistani families (A-F) with various forms of ectodermal dysplasias have been located and investigated. Genetic linkage mapping strategy and automated DNA sequencing were performed to identify the causative genes.
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RESULTS
Human Subjects and Clinical Findings
Family A
Family A, manifesting ectodermal dysplasia (ED) of hair, nails and teeth, resides in district Lodhran of the Punjab province of Pakistan. Five-generation pedigree consists of four affected individuals including three males (V-1, V-2, V-3) and one female (V- 5) (Figure 3.1). The affected individuals were born of the normal parents (IV-1 and IV-2), being first cousins, suggesting autosomal recessive pattern of inheritance of ED phenotype in the family. The ages of the affected individuals ranged from 10 to 19 years at the time of the study.
All the affected individuals of family A have fine, thin, weak, and in some cases sparse hairs on the scalp that could be painlessly plucked without force. Eyebrows and eyelashes were either absent or sparse, and body hairs were thin. Affected individuals have misshaped, large and irregular maxillary and mandibular teeth. Some of the teeth were missing. The finger-and toenails were dystrophic in appearance and showed a variable degree of severity with age. Nail plates of the fingernails of affected individuals were extremely thin and flat (Figure 3.2). The patients have normal sweating. Eye abnormalities, skeletal abnormalities, ichthyosis, oral leukokeratosis, palmoplantar keratosis and flexure pigmentation were absent. Results of routine laboratory tests, including white blood cell counts and granulocyte function, were normal. Heterozygous carrier individuals have normal hair, nails and teeth, and were clinically indistinguishable from genotypically normal individuals.
Venous blood samples were collected from seven individuals, including four affected (V-1, V-2, V-3, V-5) and three normal members (IV-1, IV-2, V-4) of the family.
Family B
Family B with isolated congenital nail clubbing (ICNC) originates from district Bhimbar, Azad Jammu and Kashmir, Pakistan. This is a large family comprising of six generations with eleven affected individuals including four males (III-2, IV-7, IV- 9, V-9) and seven females (IV-5, V-2, V-4, V-6, V-7, VI-1, VI-2) (Figure 3.3). An affected individual (III-2) was deceased while four members (IV-9, V-4, V-6, V-9) were not present at the time of the study. All affected individuals were born of
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consanguineous marriages, suggesting autosomal recessive pattern of inheritance of ED in the family.
Affected individuals in family B were born with total bilateral symmetrical nail clubbing in all finger- and toenails with no other associated abnormalities. Neurological problems, congenital malformations, facial dysmorphisms and systemic conditions (lung, heart, and gastrointestinal) were not observed in any of the affected individuals. Affected individuals have normal skin and sweating, and never complain of bone and joint pains. The affected individuals ranged from 2 to 50 years at the time of the study however, they were fully aware of the abnormality since childhood. They have normal height and proportionate stature and were not different in any respect, besides congenital clubbing, from their healthy relatives. Clubbing was clearly and equally pronounced in all the affected individuals, appeared to remain fixed in degree with respect to age. The nails were shiny, hypoplastic, thickened, long, broad, more curved from cuticle to tip and convex in every diameter (resulting in widening of the fingertips) (Figure 3.4). ECG performed on two affected individuals (IV-7, V-2) showed normal QRS complex. Heterozygous carrier individuals had normal finger- and toenails and were clinically indistinguishable from genotypically normal individuals.
Radiographs of hands and feet of the two affected individuals (IV-7, V-2) of the family showed appearance of stubby soft tissue accumulation at terminal phalanges with normal bones and their angles (Figure 3.5). Radiographs of chest, shoulders, elbows and thorax disclosed no abnormality in the affected individuals.
Venous blood samples were collected from twelve individuals, including six affected (IV-5, IV-7, V-2, V-7, VI-1, VI-2) and six normal members (IV-4, IV-6, IV-8, V-1, V-3, VI-3) of the family.
Family C
Family C with a novel form of ectodermal dysplasia belongs to district Rawlakot, Azad Jammu and Kashmir, Pakistan. The five-generation pedigree contains four affected individuals including one male (V-4) and three females (V-2, V-5, V-6) (Figure 3.6). All affected individuals were born of a consanguineous phenotypically normal couple (IV-1 and IV-2), suggesting autosomal recessive pattern of inheritance of this form of ED in the family.
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The ages of the affected individuals (V-2, V-4, V-5, V-6) ranged from 15-32 years at the time of the study. The clinical features of the affected individuals included hair loss, tooth enamel hypoplasia, nail hypoplasticity, keratoderma, epidermal and follicular hyperkeratosis, partial syndactyly, hyperhidrosis, and facial features with prominent pinnae, pointed nose and thin upper lips (Table 3.1). Hairs on scalp were sparse, hard, and slow-growing with follicular hyperkeratosis. Eyebrows and eyelashes were sparse to absent (Figure 3.7). Teeth were cone or conical and cylindrical shaped having roughness due to lack of enamel formation. Orthopantomogram (OPG) of an affected individual (V-4) revealed opaque roots and crown showing absence of enamel. There was generalized spacing (interdental spaces) in both arches (enamel hypoplasia). Teeth roots were almost complete (showing little bulbous appearance) as observed in OPG (Figure 3.9). Both finger- and toenails were flat and hypoplastic (Figure 3.8). Bilateral cutaneous partial syndactyly of fingers (double), sparing thumbs and little fingers, and toes (triple), sparing thumbs only, were observed in the affected individuals (Figure 3.8). They were unable to close their hands completely, having apparently long palms and short fingers. In addition, skin of the affected individuals was stiff, hard, and stretched showing mild epidermolytic hyperkeratosis. Affected individuals have normal hearing, mental condition, eyesight, height, and growth. The parents of the affected individuals were healthy and clinically indistinguishable from genotypically normal individuals of the family. X-rays of hands, feet, ankle, knee, and elbow joints from affected individual (V-4) showed no radiological evidence of any bony lesion or injury and joint spaces remained intact. X-ray of chest of the same individual revealed that lungs appear normal in translucency and vascular pattern with no evidence of acute consolidation/pneumothorax. However, transverse cardiac diameter was increased with CTR of 14:26 (cardiomegaly). Pulmonary hili were normal in size and shape.
Venous blood samples were collected from nine individuals, including four affected (V-2, V-4, V-5, V-6) and five normal members (IV-1, IV-2, V-1, V-3, V-7) of the family.
Family D
Family D with clinical manifestations of hypohidrotic ectodermal dysplasia (HED) resides in a remote area of district Jhang, Punjab province, Pakistan. This five-
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generation pedigree has five affected males (IV-2, IV-5, IV-6, V-1, V-3) and three partially affected females (IV-3, V-5, V-6) (Figure 3.10). The partially affected status of females and pedigree analysis showed that the disease phenotype segregates in X- linked pattern in this family.
All the five affected male individuals (IV-2, IV-5, IV-6, V-1, V-3) of the family D showed the characteristic features of HED, including fine and curly sparse hair, absent eyebrows and eyelashes, conical teeth, diminished sweating, absence of axillary and pubic hair, dry and thin skin, periorbital wrinkling and hyperpigmentation, protruding prominent lips, pointed chin, frontal bossing and saddle-shaped nose (Figure 3.11). Three female carriers (IV-3, V-5, V-6) exhibited significant clinical features of HED. Female carriers V-5 (Figure 3.11) and V-6 have sparse hair on scalp and conical teeth. The carrier V-5 showed reduced secretion of sweat. Saddle nose and thin skin were observed in the carrier IV-3. Female carriers III-4, IV-1, and V-4 had no observable features of HED and their status was determined by gene analysis. All affected males and female carriers have normal finger- and toenails.
Venous blood samples were collected from fifteen individuals, including five affected (IV-2, IV-5, IV-6, V-1, V-3), and three normal males (III-3, IV-4, V-2) and seven females (III-4, IV-1, IV-3, IV-7, V-4, V-5, V-6) of the family.
Family E
Family E with clinical manifestations of hypohidrotic ectodermal dysplasia (HED) resides in a remote area of district Jhang, Punjab, Pakistan. This four-generation pedigree has three affected individuals including two males (IV-1, IV-3) and one female (IV-2) (Figure 3.12). All the three affected individuals are present in the fourth generation (IV) of the pedigree. An affected male (IV-1) died at the age of six years. Affected individuals were born of phenotypically normal parents, suggesting autosomal recessive pattern of inheritance of HED phenotype in the family.
Clinical findings in all the affected individuals were compatible with classical HED features. These included fine and curly sparse hair, absent eyebrows and eyelashes, absence of axillary and pubic hair, conical teeth, diminished sweating, dry and thin skin, protruding prominent lips, hyperpigmentation of the skin around the eyes and mouth and saddle-shaped nose (Figure 3.13). Affected individuals have normal
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finger- and toenails. The parents of the affected individuals were healthy and clinically indistinguishable from genotypically normal individuals of the family.
Venous blood samples were collected from six individuals, including two affected (IV-2, IV-3) and four normal individuals (III-3, III-4, IV-4, IV-5) of the family.
Family F
Family F, demonstrating typical features of autosomal dominant trichorhinophalangeal syndrome (TRPS), belongs to district Lower Dir, North West Frontier Province, Pakistan. This family consists of ten affected individuals including nine males (I-2, II-2, II-3, III-2, III-4, III-7, III-8, III-9, IV-1) and one female (III-6) (Figure 3.14). Two affected males (I-2, II-2) were deceased at the time of the study. Pedigree analysis strongly supported the autosomal dominant pattern. The ages of the affected individuals ranged from 6 to 55 years at the time of the study.
Affected individuals showed typical features of TRPS III including fine, dry, weak, slow-growing and in some cases sparse and brittle hypopigmented hair on scalp that could be painlessly plucked without force, absent or thin body hair, few eyelashes, sparse axillary and pubic hair, speech impairment, thin upper lip, large nasal bridge, bulbous pear-shaped nose, long and flat philtrum, high bossed forehead (Figure 3.15), severe short stature and hip malformation. All the affected individuals have short and broad finger- and toenails (racket nails), dental malocclusion, and severe brachydactyly with distorted, swollen and broadened fingers (Figure 3.16). The affected individuals showed normal intelligence having good social dealings.
Radiological study of a 17 years old affected individual (IV-1) revealed short and broad metacarpal bones with radiolucent distal ends and proximal margin sclerosis. Phalanges were also short and broad with less clear cone-shaped epiphyses of the proximal phalanges due to short bone age (Figure 3.17). In the pelvis region both femora necks were short and broad having increased angles with abnormal and broad heads (Figure 3.17). Radiographs of the shoulders, elbows and thorax disclosed no abnormalities.
Venous blood samples were collected from fourteen individuals, including eight affected (II-3, III-2, III-4, III-6, III-7, III-8, III-9, IV-1) and six normal members (II-1, II-4, III-1, III-10, III-11, IV-2) of the family.
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Linkage and Mutational Analysis
Family A
Family A was initially tested for linkage to known genes, involved in ectodermal dysplasias (EDs) and alopecias phenotypes, using highly polymorphic microsatellite markers tightly linked to these regions (Chapter 2: Materials and Methods: Table 2.1). Examination of the haplotypes did not reveal any region of homozygosity in the affected individuals, thus excluding the family from linkage to known genes. Human genome-scan was thus carried out using the DNA samples of four affected individuals (V-1, V-2, V-3, V-5) (Figure 3.1). In the course of screening 396 markers, four markers (D2S1360 at 2p24.2, D7S1799 at 7q22.1, D16S2644 at 16q24.1, ATA82B02 at 18q22.3) from the decode genetic map (Kong et al., 2002) were found to be homozygous in all the four affected individuals. Upon testing the rest of the family members, linkage to three of these regions were excluded. Evidence suggestive of genetic linkage was obtained with marker ATA82B02, located at 105.40-cM on chromosome 18q22. All affected individuals were homozygous and normal individuals were heterozygous at this marker (Figure 3.23). In order to fine map the locus, additional markers were selected from the Rutgers combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004). Twenty markers were proximal to ATA82B02 (D18S68, D18S465, D18S483, D18S382, D18S875, D18S857, D18S1367, D18S477, D18S386, D18S1365, D18S979, D18S19, D18S466, D18S1373, D18S1131, D18S5, D18S1092, D18S817, D18S61, D18S1125) and 23 markers were distal to ATA82B02 (D18S848, D18S390, D18S1361, D18S488, D18S1091, D18S485, D18S1106, D18S541, D18S1269, D18S1358, D18S43, D18S850, D18S469, D18S874, GATA8C07, D18S486, D18S58, D18S1161, D18S1112, D18S815, D18S1121, D18S823, D18S1371). Eleven of these markers (D18S382, D18S979, D18S19, D18S5, D18S61, D18S390, D18S1361, D18S1358, D18S43, D18S486, D18S1112) were not informative in this family. Electropherograms of the ethidium bromide stained 8% non-denaturing polyacrylamide gel for markers, which established linkage to chromosome 18q22.1- q22.3 and segregated with the disease phenotype are shown in Figures 3.18-3.31. Analysis of the marker genotypes within this region with PEDCHECK (O'Connell and Weeks, 1998) and MERLIN (Abecasis et al., 2002) did not elucidate any genotyping error. Genotypes for the genome and fine mapping markers were analyzed
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using two-point and multipoint linkage analysis (Chapter 2: Statistical Analysis). Table 3.2 summarizes the two point LOD score obtained for total 33 markers. The maximum two-point LOD score of 2.73 at zero recombination was achieved with marker D18S541 at 107.11-cM. Multipoint linkage analysis gave a maximum LOD score of 3.42 at several markers (D18S1365, D18S466, D18S1373, D18S1131, D18S1092, D18S817, D18S1125, ATA82B02, D18S848, D18S488, D18S1091, D18S485, D18S1106, D18S541, D18S1269, D18S850, D18S469 D18S874, GATA8C07, D18S58). When the marker allele frequencies were varied for the fine mapping markers from 0.2 to 0.8, the maximum multipoint LOD score varied from 3.42 to 3.23, respectively, and remained at several markers including D18S1125, ATA82B02, D18S848, D18S488, D18S1091 and D18S485.
Haplotypes using SIMWALK2 were constructed to determine the critical recombination events (Figure 3.32). Two historic recombination events, observed in all the four affected subjects (V-1, V-2, V-3, V-5), defined the centromeric and telomeric boundaries of the linkage interval. The first recombination event occurred between markers D18S1367 and D18S477, which defined the centromeric boundary of the linkage interval. A second recombination event occurred between markers D18S1161 and D18S815 defining the telomeric boundary of the linkage interval. The disease interval (region of homozygosity) is flanked by markers D18S857 (98.35-cM) and D18S815 (115.67-cM) on chromosome 18q22.1-q22.3 and spans 17.32-cM region according to the Rutger combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004). This region corresponds to 8.63-Mb on the sequence-based physical map (Build 36.1) (International Human Genome Sequence Consortium, 2001) (Figure 3.33).
To search for a causative gene responsible for a novel ED of hair, nail, and teeth type in family A, three candidate genes CDH7 [MIM 605806], CDH19 [MIM 603016] and ZNF407 [GenBank accession # NM_017757.2], were sequenced in two affected (V-1, V-3) and one normal (V-4) individuals of the family. Sequence analysis with the standard sequence of the exons and splice junctions of these genes (www.ensembl.org/Homo_sapiens) failed to identify any functional sequence variant, which could cause ED of hair, nail, and teeth type in family A.
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Family B
The family B was first tested for linkage to known candidate genes, involved in hereditary isolated nail disorders and nail disorders associated with other clinical features, by using highly polymorphic microsatellite markers (Chapter 2: Materials and Methods: Table 2.1). These included: RSPO4 gene at 20p13 (D20S103, D20S105, D20S117, D20S199, D20S906), ICND locus at 17p13 (D17S926, D17S849, D17S1840, D17S1529, D17S1528), hereditary nail dysplasia locus at 17q25.1-q25.3 (D17S1807, D17S1301, D17S1839, D17S1817, D17S937), ectodermal dysplasias of hair and nail type loci at 10q24.32-q25.1 (D10S1710, D10S1267, D10S1264, D10S254, D10S1741), at 17p12-q21.1 encompassing type I keratins (D17S1824, D17S1540, D17S1788, D17S1814, D17S800, D17S1860), and at 12p11.1-q21.1 encompassing type II keratins (D12S291, D12S85, D12S339, D12S297, D12S270, D12S96, D12S329, D12S1686).
After excluding the linkage to known genes involved in hereditary nail disorders, genome-wide homozygosity mapping was carried out using DNA samples from four affected individuals (V-2, V-7, VI-1, VI-2) of the family (Figure 3.3). The affected individuals showed homozygosity with 8 microsatellite markers including D2S1776 (2q24.3), D4S2368 (4q32.3), D9S934 (9q33.1), D11S2006 (11q12.1), D13S1493 (13q13.1-q13.2), D16S539 (16q24.1), D20S481 (20q13.12) and D22S1045 (22q12.3- q13.1). Upon testing DNA from rest of the family members, linkage to seven of these regions was excluded. All affected individuals of the family were found to be homozygous at marker D4S2368, located at 171.12-cM on chromosome 4q32.3. Unaffected individuals of the family were heterozygous at this marker (Figure 3.37). In order to fine map the region, 44 additional markers (D4S413, D4S3351, D4S2982, D4S2993, D4S3046, D4S1603, D4S1528, D4S2398, D4S3339, D4S3337, D4S2306, D4S2952, D4S1566, D4S2388, D4S620, D4S3326, D4S1502, D4S2414, D4S2979, D4S1597, D4S243, D4S2426, D4S2373, D4S1545, D4S621, D4S1595, D4S2992, D4S2991, D4S2290, D4S1539, D4S3246, D4S3035, D4S3028, D4S3030, D4S3338, D4S415, D4S1552, D4S2967, D4S1501, D4S1529, D4S1530, D4S1584, D4S3041, D4S1554) were selected from the Rutgers combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004) and genotyped in all the affected and unaffected individuals of the family. Nineteen markers (D4S413, D4S1528, D4S3339, D4S2388, D4S620, D4S243, D4S1545, D4S2992, D4S2991, D4S2290, D4S1539,
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D4S3246, D4S3035, D4S3028, D4S3030, D4S1552, D4S2967, D4S1530, D4S1584) were uninformative and were not considered for further analyses. Electropherograms of the ethidium bromide stained 8% non-denaturing polyacrylamide gels for markers that showed genetic linkage to chromosome 4q32.3-q34.3 and segregated with the disease phenotype are shown in Figures 3.34-3.44. After genotyping all the fine mapping markers, the data were analyzed using two-point and multipoint linkage analysis (Chapter 2: Statistical Analysis). Analysis of the genome scan and fine mapping markers genotypes within this region with PEDCHECK and MERLIN did not elucidate any genotyping errors. The results of the two-point and multipoint linkage analyses are presented in Table 3.3. The highest two-point LOD score at zero recombination fraction (θ=0.00) was 2.98 at marker D4S2368 (171.12-cM). Maximum multi-point LOD score of 3.62 was achieved with several markers including D4S3326, D4S1502, D4S2368, D4S2414, D4S1597, D4S2373 along the disease-interval.
To determine the most likely critical candidate linkage interval containing the gene for the ICNC, haplotypes were constructed using SIMWALK2 for fully informative markers mapped on chromosome 4q32.3-q34.3. Examination of the haplotypes showed that disease-associated alleles cosegregated with the phenotype of the ICNC in this family (Figure 3.45). A recombination event between markers D4S2952 (169.13-cM) and D4S3326 (170.54-cM) in two individuals, IV-1 and IV-2, defined the centromeric boundary of the disease interval. The telomeric boundary of the interval corresponds to a recombination event between markers D4S2431 (176.78- cM) and D4S415 (182.38-cM), which occurred in an individual V-2. The linkage interval flanked by markers D4S2952 (169.13-cM) and D4S415 (182.38 cM) contains 12.27-Mb physical distance and is 13.25-cM long according to Rutgers combined linkage-physical map of the human genome (Build 36.1) (Figure 3.46).
Ten candidate genes (MFAP3L [GenBank accession # NM_021647], HMGB2 [MIM 163906], ASB5 [GenBank accession # NM_080874.2], ADAM29 [MIM 604778], VEGFC [MIM 601528], HAND2 [MIM 602407], ANXA10 [MIM 608008], MORF4 [MIM 116960], SAP30 [MIM 603378], HPGD [MIM 601688]), located in the ICNC disease-interval on chromosome 4q32.3-q34.1 (Figure 3.46), were sequenced in two affected (VI-1, VI-2) and one normal (V-1) individuals of the family B. Sequence analysis was performed with the standard sequence of the exons and splice junctions
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of the above genes (www.ensembl.org/Homo_sapiens). A single homozygous T to C transition was identified at cDNA position 577 (c.577T>C) in exon 6 of the NAD+- dependent 15-hydroxyprostaglandin dehydrogenase (HPGD) gene in both the affected individuals. This transition exchange a hydroxylic uncharged polar serine residue to aliphatic nonpolar cyclic proline at amino acid position 193 (p.S193P) of the 15- PGDH protein. This mutation (p.S193P) was present in homozygous state in all the affected individuals and in heterozygous state in the obligate heterozygous carriers of the family (Figure 3.47), confirming cosegregation of the disease phenotype in the family. This particular genetic change in the HPGD gene was not detected in 300 chromosomes screened by direct sequencing, taken from the same population to which the present ICNC family belongs. RT-PCR analysis of the normal human skin and brain cDNA libraries (Invitrogen, San Deigo, CA) showed high expression of the HPGD gene in these tissues (Figure 3.48) The HPGD gene expression in several other tissues (lung, placenta, hair follicle, colon, eye, urothelial tissues, liver, pancreas) has been reported previously (Anggard et al., 1971; Tai, 1976; Pichaud et al., 1995; Basu and Stjernschantz, 1997). Multiple sequence alignment (MSA) of PGDH protein sequences from different vertebrate species revealed that serine at position 193 is a conserved residue (http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene) (Figure 3.49).
Family C
The family C was initially tested for linkage to known genes, involved in human hereditary ectodermal dysplasias, by genotyping highly polymorphic microsatellite markers (Chapter 2: Materials and Methods: Table 2.1). This included three ectodermal dysplasia of hair and nail types loci at 10q24.32-q25.1 (D10S1710, D10S1267, D10S1264, D10S254, D10S1741), at 17p12-q21.1 encompassing type I keratins (D17S1824, D17S1540, D17S1788, D17S1814, D17S800, D17S1860), and at 12p11.1-q21.1 encompassing type II keratins (D12S291, D12S85, D12S339, D12S297, D12S270, D12S96, D12S329, D12S1686); Desmogleins (DSGs) and desmocollins (DSCs) gene cluster at 18p21.1 (D18S877, D18S847, D18S36, D18S456), SNAP29 gene at 22q11.2 (D22S944, D22S873, D22S1026, D22S446, D22S308), ST14 gene at 11q24.3 (D11S4123, D11S4150, D11S4463, D11S4131) and autosomal recessive hypotrichosis locus (LAH2) at 3q26.2-q27.3 (D3S1571,
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D3S3578, D3S3592, D3S1617). Genotyping results showed that the family was not linked to any of the known loci tested.
After exclusion of the family from linkage to known genes, human genome scan was performed to identify a causative gene. DNA samples from four affected individuals (V-2, V-4, V-5, V-6) of the family (Figure 3.6) were used in genome-wide linkage search. During this search nine regions including D1S551 (1p31.1), D4S2397 (4p15.2); D5S1456 (5q35.1); D7S1802 (7p15.3); D7S1808 (7p15.2-p15.1); D10S1230 (10q26.12); D13S895 (13q33.3); D19S714 (19p13.12); D21S1437 (21q21.1) were found homozygous in the affected individuals. Seven of these regions were excluded by genotyping all DNA samples available for the study. Affected individuals of the family were homozygous at two markers D7S1802 at 34.63-cM and D7S1808 at 42.92-cM on chromosome 7p21.1-p15.1. Normal individuals of the family were heterozygous at these two markers (Figure 3.52). This region was further refined and saturated by genotyping 28 additional microsatellite markers (D7S2557, D7S2508, D7S507, D7S3051, D7S488, D7S2532, D7S815, D7S1802, D7S2562, D7S493, D7S2458, D7S1487, D7S2463, D7S2190, D7S1791, D7S2525, D7S2449, D7S2564, D7S1808, D7S2848, D7S2496, D7S2492, D7S2491, D7S632, D7S526, D7S817, D7S484, D7S2250) selected from Rutger combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004). All these markers were fully informative in the family. Electropherograms of the ethidium bromide stained 8% non-denaturing polyacrylamide gel for markers that showed genetic linkage to chromosome 7p21.1-p15.1 and segregated with the disease phenotype are shown in Figures 3.50-3.62. After genotyping fine mapping and genome-scan markers, the data were analyzed using two-point and multipoint linkage analysis (Chapter 2: Statistical Analysis). Maximum two-point LOD score of 2.94 at zero recombination fraction (θ=0.00) was achieved for marker D7S2496 at 47.44-cM. However, maximum multi- point LOD score of 3.07 was achieved with several markers including D7S2562, D7S493, D7S2463, D7S2190, D7S2525, D7S2449, D7S1808, D7S2848, D7S2496, D7S2492 along the disease-interval (Table 3.4).
Haplotypes were constructed using SIMWALK2 for fully informative markers in the chromosomal region 7p21.1-p14.3 to delineate the most likely disease-interval containing the gene for the ED in this family (Figure 3.63). A historic recombination event was detected between markers D7S488 and D7S2532 in all the affected
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individuals defined the telomeric boundary of the linkage interval. The centromeric boundary of the linkage interval was defined by a recombination between markers D7S2492 and D7S2491, which has occurred in two normal individuals (V-1 and V-7) Therefore, the most likely candidate region for the novel ED in family C is between markers D7S488 at 31.04-cM (telomeric) and D7S2491 at 48.65-cM (centromeric) spanning 17.60-cM corresponding to 12.43-Mb region on chromosome 7p21.1-p15.1 according to the sequence-based physical map (Build 36.1) (International Human Genome Sequence Consortium, 2001).
The candidate region of the novel ectodermal dysplasia mapped on chromosome 7p21.1-p15.1 in family C, contains many functionally characterized genes (Figure 3.64). Twenty seven of these genes including (HOXA1 ([MIM 142955]), HOXA2 [MIM 604685], HOXA3 [MIM 142954], HOXA4 [MIM 142953], HOXA5 [MIM 142952], HOXA6 [MIM 142951], HOXA7 [MIM 142950], HOXA9 [MIM 142956], HOXA10 [MIM 142957], HOXA11 [MIM 142958], HOXA13 [MIM 142959], NEUROD6 [MIM 611513], FERD3L [GenBank accession # NM_152898.2], PRR15 [GenBank accession # NM_175887.2], EVX1 [MIM 142966]), TWIST1 [MIM 601622], SP8 [MIM 608306], SP4 [MIM 600540], CCDC126 [GenBank accession # NM_138771.3], JAZF1 [MIM 606246], SNX10 [GenBank accession # NM_013322.2], TRA2A [GenBank accession # NM_013293.3], ITGB8 [MIM 604160], CBX3 [MIM 604477], TWISTNB [MIM 608312], AQP1 [MIM 107776], HNRPA2B1 [MIM 600124]) were selected for mutational screening. The basis of selection of these genes for sequence analysis was their function and expression data, available at the University of California-Santa Cruz (UCSC) human genome database. These genes were directly sequenced in two affected (V-4, V-5) and one normal (IV- 2) individuals of the family. Sequence analysis with standard sequence of the exons and splice junctions of these genes (www.ensembl.org/Homo_sapiens) failed to identify any functional sequence variant, which could be responsible for the ED in family C. However, sequence analysis detected several known and novel polymorphisms in these genes (Table 3.5).
Family D
The pedigree structure of family D and clinical features of the affected individuals were compatible with X-linked hypohidrotic ectodermal dysplasia (XLHED). Therefore, the entire coding portion and intron-exon boundaries of the EDA1 gene,
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located on chromosome Xq12-q13.1, were sequenced in all the 15 individuals of the family. Sequence analysis of exon 8 of the EDA1 gene [GenBank accession # NM_001005609.1] from affected males (IV-2, IV-5, IV-6, V-1, V-3) in the family revealed a novel 4-bp insertion at cDNA position 913 (c.913_914insTATA) (Figure 3.65) resulting in frameshift and a premature stop codon 2-bp downstream in the same exon (p.305insIfsX306). For nucleotide numbering, A of the first ATG was used as position +1. This insertion was present in the heterozygous state in female carriers (III-4, IV-1, IV-3, V-4, V-5, V-6) within the family (Figure 3.65). This mutation was not found in healthy males (III-3, IV-4, V-2) and a healthy female (IV-7) of the family (Figure 3.65). A list of insertion and deletion mutations implicated in the EDA1 gene to date is presented in Table 3.6.
Family E
Autosomal recessive pattern of inheritance and hypohidrotic ectodermal dysplasia (HED)-related clinical features of the affected individuals of family E led to screen the candidate genes reported earlier to be responsible for HED. The entire coding portion (12 exons) and intron–exon boundaries of the EDAR gene [GenBank accession # NM_022336.2], located on chromosome 2q11-q13, were sequenced in two affected (IV-2, IV-3) and four normal individuals (III-3, III-4, IV-4, IV-5) of the family. The sequence analysis of exon 5 of the EDAR gene from affected individuals revealed a novel 6-bp in-frame deletion mutation starting at nucleotide position 399 (c.399_404delGGTCTG) (Figure 3.66). For nucleotide numbering, A of the ATG translation initiation site was used as position +1. For amino acid numbering the initiation methionine of the EDAR protein was used as position 1. This deletion was present in the heterozygous state in obligate carriers III-3 and III-4 (Figure 3.66). The mutation (c.399_404delGGTCTG) results in skipping of three amino acids (Methionine at 133, Valine at 134, Cysteine at 135) and insertion of a new amino acid (Isoleucine) (p.M133_C135delinsI), in one of the “cystiene-rich-regions” of extracellular domain of EDAR protein (Figure 3.67). The mutation was not identified in two normal individuals (IV-4, IV-5) of the family (Figure 3.66). This in-frame deletion mutation was not identified in a panel of 200 chromosomes of Punjabi speaking Pakistani population to whom this family belongs and the mutation was not identified outside the family.
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To date, 23 different mutations, responsible for hypohidrotic ectodermal dysplasia (HED), have been reported in the EDAR gene (Table 3.7).
Family F
The autosomal dominant mode of inheritance and clinical features of the affected individuals of the family F, compatible with trichorhinophalangeal syndrome type III, led to directly screen the human the TRPS1 gene [GenBank accession # NM_014112] for a causative mutation. The entire coding (exon 3-7), non-coding (exon 2) and intron-exon boundaries of the TRPS1 gene, located on chromosome 8q24.1, were sequenced in fourteen individuals including eight affected and six normal members of the family (Figure 3.14). Sequence analysis of exon 6 of the TRPS1 gene from affected individuals revealed a heterozygous missense mutation at cDNA position 2762 (c.2762G>T) (Figure 3.68). This transversion results in the exchange of glycine residue with valine at position 921 (p.G921V) in the TRPS1 protein, which is the first residue after the C-terminal of the GATA zinc finger domain of the protein (http://au.expasy.org/uniprot/Q9UHF7). The normal individuals of the family were homozygous for the wild type sequence c.2762G (Figure 3.68). To ensure that this particular genetic variation does not represent a non-pathogenic polymorphism, a panel of 100 unaffected unrelated ethnically control individuals (200 chromosomes) was screened and this mutation was not identified outside the family. Mutations in the TRPS1 gene, associated with the pathogenesis of TRPS III, reported to date are presented in Table 3.8.
DISCUSSION
In this study, six Pakistani families with various forms of ectodermal dysplasias were investigated. In three families (A-C), linkage studies led to the identification of three novel ectodermal dysplasia loci on different chromosomes. Three other families (D-F) showed linkage to known genes involved in human hereditary skin disorders.
In family A, clinical features of the affected individuals included thin and fine hair on scalp, dystrophic and flat nails, absent or sparse eyebrows and eyelashes, missing and abnormal teeth and thin body hair. These features are different from affected individuals with EDs of hair, nail, and teeth types reported earlier (Zirbel et al., 1995; van Steensel et al., 2001; Megarbane et al., 2004; Naeem et al., 2006a,b).
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In family A, genome-wide scan mapped the disease locus on chromosome 18q22.1- q22.3. The candidate linkage interval for this novel form of ED spans 17.32-cM region flanked by markers D18S857 and D18S815 according to the Rutgers combined linkage-physical map (Build 36.1) of the human genome (Kong et al., 2004), which corresponds to 8.63-Mb on the sequence-based physical map (International Human Genome Sequence Consortium, 2001). This is the first locus on chromosome 18 harboring a causative gene for ectodermal dysplasia.
According to The National Center for Biotechnology Information (NCBI) (Build 36.1) sequence-based physical map, there are 26 known and several predicted genes, and several expressed sequence tags (ESTs) in the linkage interval between markers D18S857 and D18S815. This includes Cadherin 7 (CDH7 [MIM 605806]) and Cadherin 19 (CDH19 [MIM 603016]), Thioredoxin domain containing 10 (TXNDC10 [GenBank accession # NM_019022]), Docking protein 5-like (DOK6 [GenBank accession # NM_152721]), CD226 [MIM 605397], Rotanin (RTTN [MIM 610436]), Suppressor of cytokine signaling 6 (SOCS6 [MIM 605118]), Cerebellin 2 precursor (CBLN2 [MIM 600433]), Neuropilin and tolloid-like protein 1 isoform 2 (NETO1 [MIM 607973]) gene, F-box protein 15 (FBXO15 [MIM 609093]) gene, Cytochrome b-5 isoform 1 (CYB5A [MIM 250790]) gene, CNDP dipeptidase 2 (CNDP2 [MIM 609064]), carnosinase 1 (CNDP1) and ZNF407.
Two of the cadherin genes, CDH7 and CDH19, are strong candidates for ED of hair, nail and teeth type. These are type II classical cadherin from the cadherin superfamily. These two cadherins encode membrane glycoproteins and comprised of five extracellular cadherin repeats, a transmembrane region and a highly conserved cytoplasmic tail (Kools et al., 2000). The cadherins are a family of cell surface molecules involved in the structural and functional organization of cells in various tissues. These proteins act as mediators of selective cell-cell adhesion. Some of the other cadherins have been shown to be responsible for various forms of hereditary diseases. For instance, mutations in the desmoglein 4 (DSG4 [MIM 607892]) gene result in localized autosomal recessive hypotrichosis (LAH1) (Kljuic et al., 2003; Rafiq et al., 2004; Messenger et al., 2005; John et al., 2006b; Wajid et al., 2007). Mutations in the cadherin-3 (CDH3 [MIM 114021]) gene on chromosome 16q22.1 is responsible for congenital hypotrichosis associated with juvenile macular dystrophy (HJMD), an autosomal recessive disorder characterized by hair loss, heralding
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progressive macular degeneration and early blindness (Sprecher et al., 2001; Indelman et al., 2007; Shimomura et al., 2008; Jelani et al., 2008). Levy-Nissenbaum et al. (2003) identified nonsense mutations in the gene corneodesmosin (CDSN [MIM 602593]), located on chromosome 6p21.3, in three families suffering from hypotrichosis simplex of the scalp (HSS). CDSN, a glycoprotein expressed in the epidermis and inner root sheath (IRS) of hair follicles, is a keratinocyte adhesion molecule. Another strong candidate gene in this region is a zinc-finger ZNF407, a transcription factor.
Although cadherins, CDH7 and CDH19, and a zinc-finger ZNF407 were strong candidate genes for ED of hair, nail and teeth type, these were screened and were found to be negative for sequence variants. Because only exons and splice junction sites of the CDH7, CDH9 and ZNF407 genes were sequenced, the possibility of a functional variant in the regulatory regions of these three genes cannot be ruled out. On the other hand, 23 other known genes and several expressed sequenced tags are present in the candidate interval. Further fine-mapping and sequencing work are required in order to identify the gene which causes ED of hair, nail and teeth type in family A.
In family B, clinical features of the affected individuals included bilateral symmetric nail clubbing in all finger- and toenails by birth without any associated abnormality, a condition named as Isolated Congenital Nail Clubbing (ICNC). A condition related to this was first described by Von Eiselsberg in 1911 (Horsfall, 1936).
In family B, genome-wide homozygosity mapping led to the identification of a novel locus for isolated congenital nail clubbing (ICNC) in a 13.25-cM genetic interval flanked by markers D4S2952 and D4S415 on chromosome 4q32.3-q34.3. This corresponds to a physical distance of 12.27-Mb (Build 36.1). Twenty seven functional candidate genes were located in the disease interval of the family and ten of them were sequenced. Sequence analysis of a candidate gene HPGD detected a homozygous missense mutation (p.S193P) in the affected individuals of the family.
The HPGD gene (NM_000860.3; EC 1.1.1.141) consists of 7 exons spanning 31-Kbp of genomic DNA on chromosome 4q34.1. According to UniProtKB database (UniProtKB/Swiss-Prot accession # P15428), HPGD encodes a 266 amino acids protein with helical structure. The 15-PGDH, a member of short chain non-
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metalloenzyme family of dehydrogenases, is a dimeric enzyme composed of two identical subunits approximately 28.9 kDa with a variety of substrates like prostaglandins, lipoxins, and hydroxyl fatty acids (Tai et al., 2002).
Prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2α) are the two most representative prostaglandins in human and mouse skin (Narumiya et al., 1999). The
PGE2, a major prostaglandin and ubiquitous lipid mediator is produced by cyclooxygenases including COX-1 and COX-2, and microsomal PGE synthase (mPGES) actions in epithelial cells from membrane stores of arachidonic acid
(Jakobsson et al., 1999; Smith et al., 2000). The PGE2 has been implicated as a mediator in a number of physiological systems including intracellular pressure, gastric acid secretion, renal salt and water transport (Breyer and Breyer, 2000). Signals from PGs are terminated by selective PG uptake across the plasma membrane through prostaglandin transporter (PGT) and oxidation in the cytosol (Nomura et al., 2004).
The 15-PGDH, residing in cytosol, catalyses the first step of prostaglandins (PGs) and related eicosanoids catabolism, the oxidation of the 15-hydroxyl group, which results in a reversible inactivation of these biologically active compounds into their inactive 15 keto-metabolites which exhibit greatly reduced biological activities (Anggard, 1966; Anggard et al., 1971; Ensor and Tai, 1995; Okita and Okita, 1996). This enzyme utilizes NAD (+) specifically as a coenzyme and ubiquitously present in mammalian tissues. Coggins et al. (2002) generated the HPGD-deficient mice (hpgd-/-
) demonstrating the involvement of 15-PGDH in regulating the PGE2 metabolism.
The primary structures of 15-PGDH from various species revealed that it is homologous except for two regions, the C-terminal domain and residues from 205 to 224 (Hohl et al., 1993). Sequence alignment categorized this enzyme into short chain dehydrogenases/reductases (SDR) family. Several amino acid residues of human 15- PGDH have been proved to be critical for enzyme function. Three glycine residues at amino acid positions 12, 16, and 18 are present in the putative coenzyme binding site having a “Rossmann fold” structure with a conserved glycine pattern (GlyXaaXaaXaaGlyXaaGly) found near the N-terminal. Two residues Tyr-151 and Lys-155, located in the motif of TyrXaaXaaXaaLys near to the central part of the SDRs, and serine 138 are essential for catalytic activity of 15-PGDH (Chavan et al., 1993; Krook et al., 1993; Ensor and Tai, 1994, 1996; Jörnvall et al., 1995). Zhou et al. (2001) suggested that the C-terminal region appears to be more important for the
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interaction of the enzyme with prostaglandin substrates than with the coenzyme. A + 3D-model for ternary 15-PGDH-NAD -PGE2 predicted the presence of a deep cleft between a core domain (having most of the polypeptides) and a small lobe (two α- helices J and K that protrude outside from the core). This cleft is presumed to be the binding site for PEG2. The core domain consists of seven-stranded parallel beta-sheets (A-G), flanked each one by α -helices (A-I), resembling the “Rossmann fold” structure of the SDR family (Tai et al., 2006).
The missense mutation (p.S193P) identified in the present study lies in the eleventh helix (amino acid 193-195) of 15-PGDH protein [http://www.expasy.org/uniprot/P15428]. Uppal et al. (2008) through protein modeling studies revealed that serine 193 is a part of network of hydrogen bonds lining the hydrophobic reaction cavity of 15-PGDH. As a general rule, the introduction of proline residue (helix breaker) within an α -helix reduces the stability of the helix by creating a slight bend due to lack of the hydrogen bond, therefore, it is predicted that the mutation p.S193P will probably disrupt the structure of the helix number eleven of 15-PGDH. This will result ultimately in loss of enzyme function. While a paper describing the mutation in HPGD gene in family B has being finalized, Uppal et al. (2008) reported mutations in HPGD gene, resulting in loss of enzymatic function, in four families with primary hypertrophic osteoarthropathy (PHO). The PHO is characterized by nail clubbing, periostosis, acro-osteolysis, painful joint enlargement and skin manifestations including thickening of the skin of face and scalp, coarsening of facial features, hyperhidrosis, and seborrhea (Jajic et al., 2001; Uppal et al., 2008). However, these clinical features were not observed in the affected individuals of the ICNC family, presented here. Additionally, the heterozygous carrier individuals of the ICNC family have no observable clubbing phenotype as has been reported in families with PHO. Since 15-PGDH can metabolize several substrates, suggesting altered metabolism of different molecule (s), which may be the cause of variations in clinical phenotypes in patients with PHO and ICNC. The involvement of 15-PGDH in the pathogenesis of the ICNC may open up interesting perspectives into the function of this enzyme.
In family C, the molecular basis of a novel form of ectodermal dysplasia was investigated and the gene was mapped on chromosome 7p21.1-p15.1. The disease- interval for this unique form of ED spans a 17.60-cM region, flanked by markers
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D7S488 and D7S2491, according to the Rutgers combined linkage-physical map (Build 36.1) of the human genome (Kong et al., 2004). This region corresponds to 12.43-Mb on the sequence-based physical map (International Human Genome Sequence Consortium, 2001). This is the third human ED gene mapped on chromosome 7. Previously, two other forms of ED pallister-hall syndrome (PHS) and ectrodactyly-ectodermal dysplasia-cleft lip/palate syndrome 1 (EEC1) were mapped on chromosome 7p13 and 7q11.2-q21.3, respectively. Clinical features of the affected individuals with PHS and EEC1 are different from the ED patients reported here.
According to The National Center for Biotechnology Information (NCBI) (Build 36.1) sequence-based physical map, there are more than 50 known and several predicted genes, and several expressed sequence tags (ESTs) in the linkage interval between markers D7S488 and D7S2491. Some of these genes are histone deacetylase 9 (HDAC9 [MIM 606543]), TWIST1 [MIM 601622], FERD3L [GenBank accession # NM_152898.2], TWIST neighbor (TWISTNB [MIM 608312]), integrin, beta 8 precursor (ITGB8 [MIM 604160]), ATP-binding cassette, sub-family B (MDR/TAP), member 5 (ABCB5 [MIM 611785]), SP8 [MIM 608306], SP4 [MIM 600540], dynein, axonemal, heavy chain 11 (DNAH11 [MIM 603339]), nucleoporin like 2 (NUPL2 [GenBank accession # NM_007342]), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3 [MIM 608259]), transformer-2 alpha (TRA2A [GenBank accession # NM_013293.3]), neuropeptide VF precursor (NPVF [GenBank accession # NM_022150]), heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1 [MIM 600124]), src kinase associated phosphoprotein 2 (SKAP2 [GenBank accession # NM_003930]), RAM2 [GenBank accession # NM_018719], Rap guanine nucleotide exchange factor 5 (RAPGEF5 [GenBank accession # NM_012294]), coiled-coil domain containing 126 (CCDC126 [GenBank accession # NM_138771.3]), DFNA5 gene [MIM 608798], MMP6, HOXA gene cluster (HOXA1 ([MIM 142955]), HOXA2 [MIM 604685], HOXA3 [MIM 142954], HOXA4 [MIM 142953], HOXA5 [MIM 142952], HOXA6 [MIM 142951], HOXA7 [MIM 142950], HOXA9 [MIM 142956], HOXA10 [MIM 142957], HOXA11 [MIM 142958], HOXA13 [MIM 142959]), JAZF zinc finger 1 (JAZF1 [MIM 606246]), CARD4 [MIM 605980], neurogenic differentiation 6 gene (NEUROD6 [MIM 611513]), PDE1C [MIM 602987], coiled- coil domain containing 129 (CCDC129 [GenBank accession # NM_194300]), growth hormone releasing hormone receptor (GHRHR [MIM 139191]), indolethylamine N-
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methyltransferase (INMT [MIM 604854]), corticotropin releasing hormone receptor 2 (CRHR2 [MIM 602034]), WIPF3 [GenBank accession # NM_001080529], cAMP responsive element binding protein 5 (CREB5 [GenBank accession # NM_001011666]), secernin 1 (SCRN1 [GenBank accession # NM_0014766]), PRR15 [GenBank accession # NM_175887.2], 3-hydroxyisobutyrate dehydrogenase (HIBADH [MIM 608475]), even-skipped homeobox 1 (EVX1 [MIM 142996]), chromobox homolog 3 (CBX3 [MIM 604477]), sorting nexin 10 (SNX10 [GenBank accession # NM_013322.2]), TAX1BP1 [MIM 605326], AQP1 [MIM 107776].
Interestingly, HOXA gene cluster is present within the genetic disease-interval of a novel form of ED in family C on chromosome 7p15.2. Therefore, HOXA genes were considered as strong candidates for ED in this family.
The HOX genes are an evolutionarily conserved class of transcription factors playing important roles during development and hematopoiesis. HOX play an essential role during embryogenesis by binding to DNA through a highly conserved 183-nucleotide sequence called the homeodomain. The homeodomain containing proteins are transcription factors that play a role in determination of segment identity of the developing embryo along various body axes (Gehring, 1993). During evolution, cluster duplication gave rise to four mammalian HOX clusters, located on different chromosomes, with a total of 39 HOX genes found in 13 different paralogous groups. In each cluster, the spatial and temporal pattern of expression of the HOX genes corresponds to their physical position within the cluster (Krumlauf, 1994). In human four HOX clusters HOXA, HOXB, HOXC, and HOXD are located on chromosomes 7, 17, 12, and 2, respectively.
Recently, mutations in several genes of HOXA cluster have been implicated in causing human disorders. Homozygous mutations were identified in the HOXA1 gene causing Bosley-Salih-Alorainy syndrome (BSAS) and/or Athabascan brainstem dysgenesis syndrome (ABDS) (Bosley et al., 2008). Similarly, a homozygous mutation in the HOXA2 gene was implicated in the pathogenesis of autosomal recessive microtia in an Iranian family (Alasti et al., 2008). Chen et al. (2007) reported heterozygous sequence variants in the HOXA4 gene in Chinese patients with hypospadias. Two other HOX genes, HOXB6 and HOXA4 play important roles in the epidermal development and differentiation of various tissues at week 10-17 of gestation (Stelnicki et al., 1998; Komuves et al., 2000). In addition, mice and chicken
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models also show that HOXB6 gene products are involved in controlling pattern formation in developing limbs (Wedden et al., 1989; Schughart et al., 1991). Ota et al. (2007) showed the relationship between the HOXA7 gene expression and epithelial ovarian cancer progression. Genetic alterations of the HOXA10 gene affect the severity of endometriosis in Taiwanese population (Wu et al., 2008).
Another strong candidate gene for ED in family C is integrin beta 8 (ITGB8). Integrins are transmembrane adhesion molecules mediating critical cytosolic signaling events. Integrins act to regulate both physiologic and pathologic events, including complex processes such as angiogenesis, tumor growth, and metastasis. Mutations in many integrin family members cause several genetic defects (Wehrle-Haller and Imhof, 2003).
On the basis of function and expression data, several genes from the candidate interval of family C were selected for mutational screening. These included HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, NEUROD6, FERDL3, PRR15, EVX1, TWIST1, SP8, SP4, CCDC126, JAZF1, SNX10, TRA2A, ITGB8, CBX3, TWISTNB, AQP1, and HNRNPA1B1. These genes were sequenced directly in two affected and one normal individual of the family and found to be negative for any functional sequence variant. Because only exons and splice junction sites of these genes were sequenced, the possibility of a functional variant in the regulatory regions of these genes cannot be ruled out. On the other hand, several other known genes and expressed sequenced tags are present in the candidate interval.
In the present study, two families D and E showed clinical features of hypohidrotic ectodermal dysplasias (HED). In family D, five males were affected with X-linked hypohidrotic ectodermal dysplasia (XLHED). Three females in this family showed mild phenotypes of XLHED. Generally, in XLHED males are fully affected with this disorder, however, one-third of female carriers have no obvious clinical features, another one-third have minimal findings (missing of few teeth), and a final one third have clinically significant involvement, but to lesser degree than that in affected males (Pinheiro and Freire-Maia, 1979; Freire-Maia and Pinheiro, 1982). This clinical variation among female carriers is due to random X inactivation (Zonana, 1993). However, HED phenotype inheriting in autosomal recessive and dominant mode is
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equally pronounced in affected males and females (Monreal et al., 1999; Headon et al., 2001; Naeem et al., 2005; Chassaing et al., 2006).
In family D, a novel 4-bp insertion mutation (c.913_914insTATA) has been detected in exon 8 of the EDA1 gene causing X-linked hypohidrotic ectodermal dysplasia (XLHED). This insertion mutation is a direct 4-bp tandem repeat. Such repeats, like classical microsatellite loci, are comparatively prone to mutation by slipped strand mispairing (Mazzarella and Schlessinger, 1998). As a result, the copy number of tandem repeats is liable to fluctuate, introducing a deletion or an insertion of one or more repeat units (Strachen and Read, 2004). Frameshifting deletions or insertions result in abolition of gene expression. The mutation identified in this family leads to a frameshift and premature termination codon 2-bp downstream in the same exon, predicting to cause nonsense mediated decay of the mRNA or premature protein truncation (Urlaub et al., 1989). The insertional mutation (c.913_914insTATA) identified in this family is located in the TNF homology domain, which spans 142 amino acid region (250-391) of EDA protein. Mutations in this domain can affect the overall structure of EDA, receptor binding site and interaction site (Hymowitz et al., 2003).
According to Human Gene Mutation Database, 85 different pathogenic mutations have been reported (http://www.hgmd.cf.ac.uk), mostly clustering in functionally important domains of the EDA protein: (1) TNF-homology domain, responsible for binding to receptor, mutations in which impair binding of both splice variants to their receptors; (2) collagen-like domain, indispensable for trimerization of the ligand, mutations in this domain inhibit trimerization of the TNF homology region; and (3) the consensus furin recognition site (the protease cleavage site), mutations in which prevent proteolytic cleavage of the EDA (Schneider et al., 2001; Wisniewski et al., 2002). About 80% of the EDA1 gene mutations are small intragenic changes, including point mutations, small deletions, and insertions. However, large deletions, including complete exon loss or even complete gene have also been reported (Vincent et al., 2001) (Table 3.6).
In family E, a novel 6-bp homozygous deletion mutation has been detected in the EDAR gene causing autosomal recessive hypohidrotic ectodermal dysplasia (ARHED). This is the third novel deletion mutation (c.399_404delGGTCTG) identified in exon 5 of the EDAR gene. The deletion mutation identified is located in
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the extracellular domain, spanning from amino acid residues 27-187 of the EDAR protein (http://ca.expasy.org/cgi-bin/get-sprot-entry/Q9UNE0). This deletion results in skipping of the three amino acids methionine, valine and cystiene and insertion of a new amino acid isolucine (p.M133_C135delinsI), in one of the “cystiene-rich- regions” of the extracellular domain. EDAR like other TNFR-like receptors have elongated structures by a scaffold of disulfide bridges. These disulfide bonds form “cysteine rich domains” (CRDs) that are the basic hallmark of the TNFR superfamily (Smith et al., 1994). These elongated chains of TNF receptors fit in the interacting “grooves” of their ligand trimers (Banner et al., 1993). Skipping of cys135 in the deletion mutation (c.399_404delGGTCTG), reported here, abolishes a disulfide bridge that exists between cys135 and cys148 (http://ca.expasy.org/cgi-bin/get-sprot- entry/Q9UNE0) (which is likely to produce unstable protein), changes the secondary structure of protein and/or alters the interaction site for ligand binding.
To date, 23 pathogenic mutations in the EDAR gene including 14 missense, 2 nonsense, 3 splice sites and 4 deletions have been reported (Table 3.7). Thirteen of the missense mutations were identified in the two functional domains of the EDAR protein: extracellular and death domains (Monreal et al., 1999; Shimomura et al., 2004 ; Naeem et al., 2005; Chassaing et al., 2006 ; Tariq et al., 2007-Present study ; RamaDevi et al., 2008; van der Hout et al., 2008; Valcuende-Cavero et al., 2008). The two nonsense mutations p.E354X and p.R358X identified in the EDAR gene lead to complete abolishment of the death domain (Monreal et al., 1999; van der Hout et al., 2008). The three splice sites mutations reported disturb the splicing of either exon 3 or exon 6 (Monreal et al., 1999; Shimomura et al., 2004; Chassaing et al., 2006). The four deletion mutations c.Ex4del, c.399_404delGGTCTG, c.718_721delAAAG and c.478delC result in the formation of either truncated or no EDAR protein (Monreal et al., 1999; Naeem et al., 2005; Tariq et al., 2007-Present study; RamaDevi et al., 2008).
The EDA, an epithelial morphogen, is a type II transmembrane protein with a C- terminal TNF domain, which binds to its receptor EDAR. EDAR is a NF-кB- activating member of the TNF receptor family (Yan et al., 2000; Kumar et al., 2001; Koppinen et al., 2001). EDAR protein contains an extracellular ligand binding N- terminal domain, single transmembrane region and intracellular region containing death domain (DD) (Headon and Overbeek, 1999). EDAR through its DD interacts
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with the DD of its adapter EDARADD which further interacts with tumor necrosis factor receptor-associated factors (TRAFs 1, 2, 3) leading to the downstream activation of NF-κB and JNK/c-fos/c-jun pathways (Headon et al., 2001; Doffinger et al., 2001; Yan et al., 2002; Cui et al., 2002). These signaling pathways lead to cell death, proliferation or differentiation and are important in the early epithelial- mesenchymal interaction that regulates ectodermal appendage formation (Schneider et al., 2001; Locksley et al., 2001).
Hypohidrotic ectodermal dysplasia (HED) results from mutation in EDA1, EDAR and EDARADD genes; in all cases these mutations disrupt the morphogenesis of ectodermal structures including hair, teeth, and eccrine sweat glands (Kere et al., 1996; Monreal et al., 1999; Headon et al., 2001). This is an unprecedented example of naturally occurring mutations in ligand (EDA1), receptor (EDAR) or adaptor (EDARADD) giving rise to the same phenotypic disease characterized by a defect in the proper development of epidermal appendages.
Family F, in the present investigation, showed features of trichorhinophalangeal syndrome (TRPS). This is a rare genetic disorder characterized by alopecia and skeletal deformities. Three different clinical forms of TRPS have been reported as TRPS I, TRPS II and TRPS III, phenotypically distinguishable from each other. Trichorhinophalangeal syndrome type III is a subtype of TRPS associated with severe brachydactyly and short stature resulting from the progressive shortening of all the phalanges and metacarpals and some long bones (Niikawa et al., 1986; Kajii et al., 1994; Momeni et al., 2000; Lüdecke et al., 2001).
In family F, a novel missense mutation (p.G921V) has been detected in the TRPS1 gene causing trichorhinophalangeal syndrome type III (TRPS III). This transversion results in the exchange of a hydrophilic, non-optically active and non-polar glycine residue with a hydrophobic bulky valine at position 921 (p.G921V) in the TRPS1 protein. The glycine at this position is the first residue after the C-terminal of the GATA zinc finger domain (896-920aa) of the protein (http://au.expasy.org/uniprot/Q9UHF7) and this is evolutionary conserved in human, mouse and Xenopus (Malik et al., 2001), suggesting its importance in the region. As discussed earlier by Lüdecke et al. (2001), TRPS1 protein with missense mutations have a decreased affinity to DNA, because of altered GATA zinc finger and exert a dominant negative effect as a component of multimeric protein complex.
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The TRPS1, located on chromosome 8q24.1, is a common gene responsible for the etiology of the both TRPS I and TRPS III consisting of five coding exons (3-7). TRPS1 protein is a vertebrate nuclear transcription factor of the GATA family encoding 1281 amino acids with nine putative zinc-finger domains, including a single carboxy-terminal GATA-type zinc finger (894-920 aa), and two nuclear localization signals (NLS1: 886-891 aa and NLS2: 946-952 aa) (Momeni et al., 2000; Lüdecke et al., 2001; Malik et al., 2001). Very little is known about the TRPS1 other than that it functions as GATA family sequence-specific transcriptional repressor (Malik et al., 2001).
Most recently, Itoh et al. (2008) revealed TRPS1 as a regulator of chondrogenesis and apoptosis in ATDC5 cells, and found TRPS1 as downstream target of GDF5 (MIM 601146) signaling pathway. The TRPS1 and GDF5 mutually regulate their expression in a feedback manner. Mutations in both of these molecules cause brachydactyly. Interestingly, Kunath et al. (2002) summarized the correlation of the expression pattern of the TRPS1 in the developing hair and skeleton with the phenotype observed in TRPS patients.
To date, more than 35 different mutations have been reported in the TRPS1 gene leading to TRPS I and TRPS III (Momeni et al., 2000; Lüdecke et al., 2001; Hilton et al., 2002; Gentile et al., 2003; Kaiser et al., 2004; Gonzalez-Huerta et al., 2007; Rossi et al., 2007; Tariq et al., 2008-Present study). However, only missense mutations in exon 6 of the TRPS1 gene, which encodes a presumptive GATA DNA-binding zinc finger domain of TRPS1 protein, are accounted for TRPS III (Lüdecke et al., 2001; Hilton et al., 2002; Kobayashi et al., 2002; Tariq et al., 2008-Present study) (Table 3.8). The mutation p.G921V, identified in family F is the sixth novel missense mutation in the TRPS1 gene as the underlying genetic defect for type III trichorhinophalangeal syndrome. This is the first report from the Pakistani population describing and diagnosing a large family with TRPS III.
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Figures and Tables of Chapter 3
1 2 I
1 2 3 4 II
1 2 III
1 2 IV
1 2 3 4 5 V
Figure 3.1: Pedigree of family A segregating ectodermal dysplasia of hair, nail and teeth type. Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate a consanguineous marriage.
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Figure 3.2: Clinical features of the affected individuals in family A. a: Phenotypic appearance of an affected individual (V-2) showing thin and sparse hair on scalp and missing eyebrows and eyelashes. b, c: Dystrophic finger- and toenails of an affected individual (V-1). d: Affected individual V-1 with missing and misshaped teeth.
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1 2 I
1 2 3 4 II
1 2 3 4 5 6 7 8 9 10 III
1 2 3 4 5 6 7 8 9 10 11 IV
1 2 3 4 5 6 7 8 9 V
1 2 3 VI
Figure 3.3: Pedigree of family B segregating autosomal recessive Isolated Congenital Nail Clubbing (ICNC). Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate consanguineous marriages.
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Figure 3.4: Clinical findings of the affected individuals in family B with Isolated Congenital Nail Clubbing (ICNC). Clubbing of fingernails (a) and right thumb (b) in an affected individual (IV-7), and clubbing of toenails in an affected individual (V-2) (c).
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Figure 3.5: Radiological findings of the affected individuals in family B with Isolated Congenital Nail Clubbing (ICNC). Radiographs of hands of individuals (V-2) (a), and (IV-7) (b) and feet of individuals (V-2) (c), and (IV-7) (d) showing normal bones and their angles with stubby soft tissue accumulation at the terminal phalanges.
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1 2 I
1 2 3 4 II
1 2 3 4 III
1 2 IV
1 2 3 4 5 6 7 V
Figure 3.6: Pedigree of family C with a novel form of ectodermal dysplasia. Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate consanguineous marriages.
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Table 3.1: Clinical features of the affected individuals in family C Appendage /organ Clinical features Sparse and slow growing hair on scalp, sparse eyebrows and Hair eyelashes, sparse to absent armpit and pubic hair, thin to absent body hair, follicular hyperkeratosis Thickened, flat, hypoplastic and yellowish or discolored in both Nails finger- and toenails Cone or conical and cylindrical shaped, generalized spacing Teeth (interdental spaces) in both arches (enamel hypoplasia), ill-defined teeth surface morphology
Sweating Hyperhidrosis at hands, face and scalp
Rough, stiff, hard, stretched, and more or less scaly appearance of Skin entire body, mild epidermolytic hyperkeratosis, mild to severe dystrophic palmoplantar keratoderma
Facial features Large prominent pinnae, pointed nose, thin upper lips
Bilateral cutaneous (soft tissue) partial syndactyly of fingers (double Syndactyly involving three fingers) sparing thumbs and little fingers, and toes (triple involving four fingers) sparing thumbs
Skeleton Increased transverse cardiac diameter (cardiomegaly)
Hearing, height, growth and mental Normal status
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a b
c
Figure 3.7: Clinical features of an affected individual (V-4) in family C with a novel form of ED. a, b: Phenotypic appearance of the affected individual showing thin, sparse scalp hair, and sparse eyebrows and eyelashes. c: A closer view of the same affected individual (IV-4) showing sparse eyebrows and eyelashes, and conical teeth.
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a b
c d
Figure 3.8: Clinical features of an affected individual (V-4) in family C with a novel form of ED: a) Hand (right) with thick, rough, and hard palms indicating palmo- keratoderma, also showing partial and double syndactyly sparing thumb and little finger. b) Hand (left) showing partial syndactyly and hypoplastic fingernails c) A closer view of hypoplastic fingernails. d) Hypoplastic toenails with partial and triple syndactyly of fingers sparing thumbs.
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a
b
Figure 3.9: Clinical findings of an affected individual (V-4) in family C: a) Oral view showing cylindrical or conical shaped teeth with enamel hypoplasia and generalized interdental spacing in both arches b) Orthopantomogram (OPG) of the same individual showing absence of enamel and ill-defined teeth surface morphology due to exposure of soft dentin.
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Figure 3.10: Pedigree of family D with X- linked hypohidrotic ectodermal dysplasia (XLHED). Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Symbols with black dots represent genotypically carrier females. Crossed symbols represent deceased individuals.
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Figure 3.11: Clinical findings of the affected individuals in family D with X-linked hypohidrotic ectodermal dysplasia (XLHED): (a) Phenotypic appearance of an affected male (IV-2) having sparse hair on scalp, hyperpigmentation, saddle nose, prominent lips and absent eyebrows and eyelashes. (b) The same individual with only four permanent, conical teeth. (c) Affected male (V-1) with sparse hair on scalp. (d) Female carrier (V-5) with thin and sparse hair on scalp and scanty eyebrows and eyelashes.
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Figure 3.12: Pedigree of family E segregating autosomal recessive hypohidrotic ectodermal dysplasia (ARHED). Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate consanguineous marriages.
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a b
c
Figure 3.13: Clinical findings in family E with autosomal recessive hypohidrotic ectodermal dysplasia (ARHED): (a) Phenotypic appearance of an affected individual (IV-3) having curly hair on scalp, saddle nose, prominent lips and absent eyebrows and eyelashes. (b) Affected individual (IV-3) with sparse and thin hair on scalp. (c) The same individual (IV-3) with absent teeth in lower jaw and two irregular or conical teeth in upper jaw.
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Figure 3.14: Pedigree of family F segregating trichorhinophalangeal syndrome type III (TRPS III). Circles and squares represent females and males, respectively. Crossed symbols represent deceased individuals. Filled symbols represent affected individuals.
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Figure 3.15: Clinical findings of the affected individuals in family F with TRPS III: (a) Phenotypic appearance of an affected individual (IV-1) having prominent ears, long nasal bridge with bulbous nose and high bossed forehead (b) Sparse hair on the scalp of an affected individual (III-4).
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Figure 3.16: Clinical findings of the affected individuals in family F with TRPS III: (a) Severe brachydactyly with swollen and broadened fingers, and racket nails in an affected individual (II-3) (b) brachydactyly of toes with dystrophic nails in an affected individual (III-8) of the family.
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b
Figure 3.17: Radiological findings of an affected individual (IV-1) in family F with TRPS III: (a) radiograph of the hands showing short metacarpals and less clear cone- shaped epiphyses of the phalanges (b) radiograph of the pelvic region showing abnormal necks and heads in the femora.
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Genotyping using Microsatellite Markers
Family A Figures 3.18-3.31 represent electropherograms of ethidium bromide stained 8% non- denaturing polyacrylamide gels (PAGEs) obtained by genotyping microsatellite markers mapped in the disease-interval of family A on chromosome 18q22.1-q22.3. Genetic positions (in centimorgan) for these marker loci were obtained from Rutgers combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004). Lane 1:- V-1 (Affected) Lane 4:- IV-1 (Normal) Lane 7:- V-5 (Affected) Lane 2:- V-2 (Affected) Lane 5:- IV-2 (Normal) Lane 3:- V-4 (Normal) Lane 6:- V-3 (Affected)
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.18: Allele pattern obtained with marker D18S857 at 98.35-cM on chromosome 18q22.1, showing heterozygous status of the affected individuals (V-1, V-3, V-5) of family A. This marker defined the centromeric boundary of the disease interval.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.19: Allele pattern obtained with marker D18S386, distal to D18S857 on chromosome 18q22.1, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
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V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.20: Allele pattern obtained with marker D18S1373 at 102.35-cM on chromosome 18q22.1-q22.2, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.21: Allele pattern obtained with marker D18S1131 at 102.51-cM on chromosome 18q22.1-q22.2, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.22: Allele pattern obtained with marker D18S817 at 103.72-cM on chromosome 18q22.2, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
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V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.23: Allele pattern obtained with marker ATA82B02 at 105.40-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.24: Allele pattern obtained with marker D18S848 at 105.87-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.25: Allele pattern obtained with marker D18S1106 at 106.63-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-3, V-5) of family A.
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V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.26: Allele pattern obtained with marker D18S541 at 107.11-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.27: Allele pattern obtained with marker D18S1269 at 107.60-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.28: Allele pattern obtained with marker D18S469 at 109.65-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
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V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.29: Allele pattern obtained with marker GATA8C07 at 112.94-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.30: Allele pattern obtained with marker D18S58 at 113.22-cM on chromosome 18q22.3, showing homozygosity among the affected individuals (V-1, V-2, V-3, V-5) of family A.
V-1 V-2 V-4 IV-1 IV-2 V-3 V-5
Figure 3.31: Allele pattern obtained with marker D18S1121 at 115.67-cM on chromosome 18q22.3, showing heterozygous status of the affected individuals (V-1, V-2, V-3, V-5) of family. This marker defined the telomeric boundary of the disease interval.
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1 2 I
1 2 3 4 II
1 2 III
1 2 IV Markers cM 10 11 10 11 D18S68 94.23 10 9 10 9 D18S465 96.46 7 6 7 6 D18S483 96.46 3 2 3 2 D18S875 98.16 6 5 7 5 D18S857 98.35 5 6 5 5 D18S1367 98.35 5 6 6 5 D18S477 98.93 1 2 2 1 D18S386 NA 4 5 4 4 D18S1365 NA 3 4 4 3 D18S466 101.53 6 7 7 6 D18S1373 102.35 4 5 5 4 D18S1131 102.51 5 6 5 5 D18S1092 102.83 1 2 3 1 D18S817 103.72 9 10 9 9 D18S1125 104.56 1 2 1 1 ATA82B02 105.4 2 2 3 2 D18S848 105.87 8 9 8 8 D18S488 105.9 1 1 2 1 D18S1091 105.9 7 7 8 7 D18S485 106.63 5 6 6 5 D18S1106 106.63 5 6 7 5 D18S541 107.11 3 4 5 3 D18S1269 107.6 1 2 2 1 D18S850 109.65 4 5 5 4 D18S469 109.65 4 5 4 4 D18S874 110.66 1 2 1 1 GATA8C07 112.94 10 10 11 10 D18S58 113.22 11 11 12 11 D18S1161 114.38 1 2 1 2 D18S815 115.67 1 2 1 2 D18S1121 115.67 1 2 1 2 D18S823 NA 6 5 6 5 D18S1371 117.29
Markers cM Band 5 1 2 3 4 V Centromere 11 10 11 10 11 10 11 10 11 10 D18S68 94.23 9 10 9 10 9 10 9 10 9 10 D18S465 96.46 6 7 6 7 6 7 6 7 6 7 D18S483 96.46 2 3 2 3 2 3 2 3 2 3 D18S875 98.16 5 6 5 6 5 6 5 6 5 6 D18S857 98.35 18q22.1 5 5 5 5 5 5 5 5 5 5 D18S1367 98.35 5 5 5 5 5 5 6 5 5 5 D18S477 98.93 1 1 1 1 1 1 2 1 1 1 D18S386 NA 4 4 4 4 4 4 4 4 4 4 D18S1365 NA 3 3 3 3 3 3 4 3 33 D18S466 101.53 6 6 6 6 6 6 7 6 6 6 D18S1373 102.35 4 4 4 4 4 4 5 4 4 4 D18S1131 102.51 5 5 5 5 5 5 5 5 5 5 D18S1092 102.83 1 1 1 1 1 1 3 1 11 D18S817 103.72 9 9 9 9 9 9 9 9 9 9 D18S1125 104.56 1 1 1 1 1 1 1 1 1 1 ATA82B02 105.4 2 2 2 2 2 2 3 2 2 2 D18S848 105.87 8 8 8 8 8 8 8 8 8 8 D18S488 105.9 1 1 1 1 1 1 2 1 1 1 D18S1091 105.9 7 7 7 7 7 7 8 7 7 7 D18S485 106.63 5 5 5 5 55 6 5 5 5 D18S1106 106.63 5 5 5 5 5 5 7 5 5 5 D18S541 107.11 3 3 3 3 3 3 5 3 3 3 D18S1269 107.6 1 1 1 1 1 1 2 1 1 1 D18S850 109.65 4 4 4 4 4 4 5 4 4 4 D18S469 109.65 4 4 4 4 4 4 4 4 4 4 D18S874 110.66 1 1 1 1 1 1 1 1 1 1 GATA8C07 112.94 10 10 10 10 10 10 11 10 10 10 D18S58 113.22 11 11 11 11 11 11 12 11 11 11 D18S1161 114.38 2 1 2 1 2 1 1 2 2 1 D18S815 115.67 18q22.3 2 1 2 1 2 1 1 2 2 1 D18S1121 115.67 2 1 2 1 2 1 1 2 2 1 D18S823 NA 5 6 5 6 5 6 6 5 5 6 D18S1371 117.29 Telomere Figure 3.32: Pedigree and Haplotype Drawing of Family A Segregating Ectodermal Dysplasia of Hair, Nail and Teeth Type For genotyped individuals, haplotypes of the closely linked microsatellite markers on chromosome 18q are shown beneath each symbol. Disease-interval is flanked by two markers with arrows (D18S857 and D18S815) containing the gene responsible for the disease phenotype in family A.
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Table 3.2: Two point and multipoint LOD score results between the ectodermal dysplasia locus and chromosome 18 markers in family A
Genetic Physical Multipoint Two point LOD score at recombination fraction θ= Marker Distance(cM)¶ Distance (bp)# LOD score 0.00 0.01 0.05 0.10 0.20 0.30 0.40 D18S68 94.23 59688619 -0.877 -1.09 -0.63 -0.21 -0.09 -0.04 -0.01 0.01 D18S465 96.46 61046802 -1.107 -1.07 -0.62 -0.19 -0.08 -0.04 -0.01 0.01 D18S483 96.46 61065854 -1.107 -1.01 -0.56 -0.14 -0.03 -0.01 -0.01 0.01 D18S875 98.16 61843509 -1.520 -0.92 -0.47 -0.05 0.05 0.03 -0.01 0.00 D18S857 98.35 62328759 -1.517 -0.46 0.00 0.42 0.51 0.43 0.25 0.08 D18S1367 98.35 62703136 1.000 1.04 1.01 0.90 0.76 0.49 0.27 0.10 D18S477 98.93 63254495 3.236 2.60 2.55 2.32 2.02 1.43 0.83 0.28 D18S386 NA 63945560 3.395 2.23 2.18 1.98 1.72 1.21 0.69 0.22 D18S1365 NA 64295077 3.420 1.34 1.31 1.19 1.04 0.73 0.43 0.17 D18S466 101.53 64574879 3.423 2.47 2.42 2.20 1.91 1.35 0.78 0.25 D18S1373 102.35 64874759 3.425 2.65 2.59 2.36 2.06 1.46 0.85 0.29 D18S1131 102.51 64966865 3.425 2.55 2.49 2.26 1.97 1.39 0.81 0.27 D18S1092 102.83 65405890 3.425 2.16 2.11 1.91 1.66 1.16 0.67 0.25 D18S817 103.72 65580098 3.425 2.41 2.36 2.15 1.88 1.33 0.78 0.27 D18S1125 104.56 66883860 3.425 1.54 1.51 1.38 1.21 0.87 0.53 0.23 ATA82B02 105.40 67064497 3.425 1.02 1.00 0.90 0.79 0.55 0.32 0.11 D18S848 105.87 67451773 3.425 1.47 1.44 1.30 1.13 0.80 0.48 0.18 D18S488 105.90 67555441 3.425 1.51 1.48 1.35 1.18 0.85 0.52 0.22 D18S1091 105.90 67607678 3.425 1.32 1.29 1.17 1.02 0.73 0.43 0.16 D18S485 105.90 67972247 3.425 1.78 1.74 1.59 1.39 1.00 0.61 0.26 D18S1106 106.63 67996318 3.425 2.60 2.55 2.32 2.02 1.43 0.83 0.28 D18S541 107.11 68325170 3.425 2.73 2.67 2.44 2.14 1.54 0.93 0.36 D18S1269 107.60 68363143 3.425 2.61 2.55 2.32 2.04 1.45 0.87 0.32 D18S850 109.65 69374296 3.425 2.23 2.18 1.98 1.72 1.21 0.69 0.22 D18S469 109.65 69455057 3.425 2.55 2.49 2.26 1.97 1.39 0.81 0.27 D18S874 110.66 69659473 3.424 1.34 1.31 1.19 1.04 0.73 0.43 0.17 GATA8C07 112.94 69792802 3.421 1.02 1.00 0.90 0.79 0.55 0.32 0.11 D18S58 113.22 70195435 3.416 1.87 1.83 1.67 1.47 1.06 0.66 0.28 D18S1161 114.38 70399261 3.373 1.89 1.85 1.69 1.48 1.08 0.67 0.29 D18S815 115.67 70962574 -1.496 -0.89 -0.44 -0.02 0.08 0.04 -0.01 -0.01 D18S1121 115.67 70977404 -1.496 -0.89 -0.44 -0.02 0.08 0.04 -0.01 -0.01 D18S823 NA 71179457 -1.495 -0.89 -0.44 -0.02 0.08 0.04 -0.01 -0.01 D18S1371 117.29 71239724 -1.026 -0.99 -0.54 -0.12 -0.01 0.00 -0.01 0.00 ¶Average-sex distance in cM is according to the Rutgers combined linkage-physical human genome map (Kong et al., 2004) #The physical position is based according to build 36.1 of the human genome (International Human Genome Sequence Consortium 2001) Markers D18S857 and D18S815 (in boldface) flank the disease-associated interval for ED of hair, nail and teeth type
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 111 Chapter 3 Ectodermal Dysplasias
p Chromosome 18 q
CDH7, CDH19, DSEL DOK5, CD226, RTTN, CBLN2, NETO1, CYB5A, TXNDC10, CCDC102B SOCS6, DKFZp434G145 FBX015,CNDP1, CNDP2,ZNF407 D18S857 D18S815 (98.35-cM) (115.67-cM) (ED of hair, nail and teeth region) 8.63-Mb
Figure 3.33: Ideogramatic representation of ED of hair, nail and teeth disease- associated interval of 8.63-Mb flanked by markers D18S857 and D18S815 on chromosome 18q22.1-q22.3. The candidate genes located in the region are also shown. Physical (Mb) and genetic (cM) distances are depicted according to the sequence-based physical map (Build 36.1) (International Human Genome Sequence Consortium, 2001) and Rutgers combined linkage-physical map (Build 36.1) (Kong et al., 2004), respectively.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 112 Chapter 3 Ectodermal Dysplasias
Family B Figures 3.34-3.44 represent electropherograms of ethidium bromide stained 8% non- denaturing polyacrylamide gels (PAGEs) obtained by genotyping microsatellite markers mapped in the disease-interval of family B on chromosome 4q32.3-q34.1. Genetic positions (in centimorgan) for these marker loci were obtained from Rutgers combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004). Lane 1:- IV-4 (Normal) Lane 5:- V-1 (Normal) Lane 9:- VI-3 (Normal) Lane 2:- IV-7 (Affected) Lane 6:- VI-2 (Affected) Lane 10:- IV-6 (Normal) Lane 3:- IV-5 (Affected) Lane 7:- V-7 (Affected) Lane 11:- VI-1 (Affected) Lane 4:- V-2 (Affected) Lane 8:- IV-8 (Normal) Lane 12:- V-3 (Normal)
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.34: Allele pattern obtained with marker D4S2952 at 169.13-cM on chromosome 4q32.3, showing heterozygous status of the affected individuals (V-2, VI-2, VI-1) of family B. This marker defined the centromeric boundary of the disease interval.
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.35: Allele pattern obtained with marker D4S3326 at 170.54-cM on chromosome 4q32.3, showing homozygosity among the affected individuals (IV-7, IV-5, V-2, VI-2, V-7, VI-1) of family B.
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IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.36: Allele pattern obtained with marker D4S1502 at 171.12-cM on chromosome 4q32.3, showing homozygosity among the affected individuals (IV-7, IV-5, V-2, VI-2, V-7, VI-1) of family B.
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.37: Allele pattern obtained with marker D4S2368 at 171.12-cM on chromosome 4q32.3, showing homozygosity among the affected individuals (IV-7, IV-5, V-2, VI-2, V-7, VI-1) of family B.
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.38: Allele pattern obtained with marker D4S2414 at 171.18-cM on chromosome 4q32.3, showing homozygosity among the affected individuals (V-2, VI- 2, VI-1) of family B.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 114 Chapter 3 Ectodermal Dysplasias
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.39: Allele pattern obtained with marker D4S2979 at 172.31-cM on chromosome 4q32.3, showing homozygosity among the affected individuals (IV-7, IV-5, V-2, VI-2, V-7, VI-1) of family B.
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.40: Allele pattern obtained with marker D4S2426 at 174.15-cM on chromosome 4q33, showing homozygosity among the affected individuals (IV-7, IV- 5, V-2, VI-2, V-7, VI-1) of family B.
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.41: Allele pattern obtained with marker D4S2373 at 174.15-cM on chromosome 4q33, showing homozygosity among the affected individuals (IV-7, IV- 5, V-2, VI-2, V-7, VI-1) of family B.
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IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.42: Allele pattern obtained with marker D4S621 at 175.84-cM on chromosome 4q34.1, showing homozygosity among the affected individuals (IV-7, IV-5, V-2, VI-2, V-7, VI-1) of family B.
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.43: Allele pattern obtained with marker D4S2431 at 176.78-cM on chromosome 4q34.1, showing homozygosity among the affected individuals (IV-7, IV-5, V-2, VI-2, V-7, VI-1) of family B.
IV-4 IV-7 IV-5 V-2 V-1 VI-2 V-7 IV-8 VI-3 IV-6 VI-1 V-3
Figure 3.44: Allele pattern obtained with marker D4S1501 at 184.46-cM on chromosome 4q34.3, showing heterozygous status of the affected individuals (IV-7, IV-5, V-2) of family B. This marker defined the telomeric boundary of the disease interval.
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1 2 I
1 2 3 4 II
1 2 3 4 5 6 7 8 9 10 III
1 2 3 4 5 6 7 8 9 10 11 IV
Markers cM
9 8 8 9 8 8 8 8 8 8 D4S1603 164.77 9 6 5 5 5 5 6 5 6 5 6 D4S2398 167.3 2 1 1 2 1 2 1 2 1 2 D4S3337 168.2 3 2 2 2 2 3 2 3 2 3 D4S2952 169.13 2 1 1 1 2 1 1 1 2 1 D4S3326 170.54 2 1 1 1 3 1 1 1 3 1 D4S1502 171.12 11 10 10 10 12 10 10 10 12 10 D4S2368 171.12 2 1 1 1 3 1 1 1 3 1 D4S2414 171.18 11 10 10 10 11 10 10 10 11 10 D4S1597 172.31 2 1 1 1 2 1 1 1 2 1 D4S2373 174.15 2 1 1 1 2 1 1 1 2 1 D4S1595 176.6 2 1 1 1 2 1 1 1 2 1 D4S2431 176.78 3 1 1 2 3 1 1 1 3 1 D4S415 182.38 2 1 1 3 2 1 2 1 2 1 D4S1529 186.02 1 2 2 1 2 1 2 1 2 1 D4S3041 193.64 5 4 4 6 4 5 4 5 4 6 D4S1554 195.79
1 2 3 4 5 6 7 8 V
Markers cM
8 8 8 9 9 8 8 8 D4S1603 164.77 6 5 5 5 6 5 5 5 D4S2398 167.3 2 1 1 2 2 1 1 1 D4S3337 168.2 3 2 2 2 3 2 2 2 D4S2952 169.13 1 2 1 1 2 1 1 1 D4S3326 170.54 1 2 1 1 2 1 1 1 D4S1502 171.12 10 11 10 10 11 10 10 10 D4S2368 171.12 1 3 1 1 2 1 1 1 D4S2414 171.18 10 11 10 10 11 10 10 10 D4S1597 172.31 1 2 1 1 2 1 1 1 D4S2373 174.15 1 2 1 1 2 1 1 1 D4S1595 176.6 1 2 1 1 2 1 1 1 D4S2431 176.78 1 1 1 2 3 1 1 1 D4S415 182.38 1 2 1 3 2 1 1 1 D4S1529 186.02 1 2 2 1 1 2 1 2 D4S3041 193.64 5 4 4 6 5 4 5 4 D4S1554 195.79
3 1 2 Markers cM cM Band VI Centromere 4q32.2 8 8 8 8 8 8 D4S1603 164.77 5 6 5 6 6 5 D4S2398 167.3 1 2 1 2 2 1 D4S3337 168.2 2 3 2 3 3 2 D4S2952 169.13 4q32.3 1 1 1 1 1 2 D4S3326 170.54 1 1 1 1 1 2 D4S1502 171.12 10 10 10 10 10 11 D4S2368 171.12 1 1 1 1 1 3 D4S2414 171.18 10 10 10 10 10 11 D4S1597 172.31 1 1 1 1 1 2 D4S2373 174.15 1 1 1 1 1 2 D4S1595 176.6 1 2 D4S2431 176.78 1 1 1 1 HPGD 1 1 1 1 1 1 D4S415 182.38 4q34.3 2 1 2 1 1 2 D4S1529 186.02 2 1 2 1 1 2 D4S3041 193.64 4q35.1 4 5 4 5 5 4 D4S1554 195.79 Telomere Figure 3.45: Pedigree of Family B Segregating Autosomal-Recessive Isolated Congenital Nail Clubbing (ICNC) Disease-interval is flanked by two markers in bold face pointed with arrows (D4S2952 and D4S415). For genotyped individuals, haplotypes are shown beneath each symbol. Position of the HPGD gene is also indicated.
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Table 3.3: Two point and multipoint LOD score results between ICNC locus and chromosome 4 markers in family B Marker Genetic Physical Multipoint Two point LOD score at recombination fraction θ= Distance(cM)* Distance (bp)¶ LOD score 0.00 0.01 0.05 0.10 0.20 0.30 0.40 D4S1603 164.770 163999458 -4.01 -infinity -1.04 -0.41 -0.19 -0.02 0.02 0.02 D4S2398 167.300 165398821 -4.41 -infinity -0.13 0.38 0.45 0.33 0.17 0.06 D4S3337 168.210 166009217 -4.41 0.02 0.01 0.01 0.01 0.01 0.01 0.00 D4S2952 169.131 166675025 -0.53 -infinity -0.22 0.29 0.37 0.28 0.15 0.05 D4S3326 170.540 168626285 3.62 2.50 2.44 2.18 1.85 1.21 0.64 0.20 D4S1502 171.120 168925081 3.62 2.68 2.61 2.34 2.01 1.35 0.75 0.27 D4S2368 171.121 168952654 3.62 2.98 2.91 2.62 2.25 1.52 0.85 0.31 D4S2414 171.180 169783086 3.62 2.80 2.74 2.47 2.13 1.48 0.87 0.36 D4S1597 172.311 170079746 3.61 2.85 2.77 2.48 2.10 1.36 0.69 0.21 D4S2373 174.151 171779246 3.56 2.50 2.44 2.18 1.85 1.21 0.64 0.20 D4S1595 176.600 174554303 3.48 2.50 2.44 2.18 1.85 1.21 0.64 0.20 D4S2431 176.780 175057242 3.47 2.50 2.44 2.18 1.85 1.21 0.64 0.20 HPGD 175647955 D4S415 182.380 178948094 -3.84 -infinity 0.33 0.80 0.82 0.58 0.29 0.08 D4S1529 186.020 181064530 -4.61 -infinity -0.55 0.02 0.17 0.19 0.13 0.06 D4S3041 193.640 184011243 -4.63 0.01 0.01 0.01 0.01 0.01 0.01 0.00 D4S1554 195.790 184925563 -3.01 -infinity -1.13 -0.42 -0.15 0.03 0.06 0.03 *Average-sex distance in cM is according to the Rutgers combined linkage-physical human genome map (Kong et al., 2004) ¶ The physical distances are based according to build 36.1 of the human genome (International Human Genome Sequence Consortium 2001)
Markers in boldface flank the disease associated region for ICNC and markers used in original genome-scan linkage are shown in block
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Figure 3.46: Ideogramatic representation of ICNC disease-associated interval of 12.27-Mb flanked by markers D4S2952 and D4S415 on chromosome 4q32.3-q34.3. Also shown candidate genes located in the linkage interval. Physical (Mb) and genetic (cM) distances are depicted according to the sequence-based physical map (Build 36.1) (International Human Genome Sequence Consortium, 2001) and Rutgers combined linkage-physical map (Build 36.1) (Kong et al., 2004), respectively.
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a) ____ T ____ A ____ I ____ L ____ E ____ P ____ I ____ E ____ K ____ E ____ E
b) ____ T ____ A ____ I ____ L ____ E ____ S/ P ____ I ____ E ____ K ____ E ____ E
c) ____ T ____ A ____ I ____ L ____ E ____ S ____ I ____ E ____ K ____ E ____ E
Figure 3.47: Sequence analysis of the HPGD gene mutation (c.577T>C) in family B with Isolated Congenital Nail Clubbing (ICNC). Partial DNA sequence of the HPGD gene from (a) a homozygous (affected) individual showing a transition (T>C) (b) a heterozygous carrier, and (c) a control individual showing wild type sequence. The arrows indicate position of the mutation. The amino acid sequence above the nucleotide sequence highlight respective translated triplet codons with mutant substituted residue in bold face. Nucleotide and amino acid positions are written according to Refseq NM_000860.3 and NP_000851.2, respectively.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 120 Chapter 3 Ectodermal Dysplasias
Figure 3.48: Tissue transcriptional analysis of the HPGD gene. RT-PCR analysis of the HPGD mRNA from normal human skin and brain showing 355-bp amplified fragment. Lane 1 and 2 (left): brain and skin; Lane 3 (right): a 20 bp DNA ladder.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 121 Chapter 3 Ectodermal Dysplasias
S193P
Hs HPGD 171 MNSGVRLNAICPGFVNTAILESIEK-EENMGQYIEYKDHIKDMIKYYGILDPPLIANGLITL 232 Pt HPGD 171 MNSGVRLNAICPGFVNTAILESIEK-EENMGQYIEYKDHIKDMIKYYGILDPPLIANGLITL 232 Cf HPGD 171 MNSGVRLNAICPGFVNTPILESIEK-EENMGQYIEYKDHIKDMMKFYGILDPSMIASGLITL 232 Bt HPGD 171 MNSGVRLNAICPGFVDTPILKSIEK-EENMGKYIEYMGPIKDMMKYYGILDPSMIANGLITL 232 Mm hpgd 171 MKSGVRLNVICPGFVDTPILESIEK-EENMGQYIEYKDQIKAMMKFYGVLHPSTIANGLINL 232 Rn hpgd 171 MKSGVRLNVICPGFVKTPILESIEK-EENMGQYIEYTDQIKAMMKFYGILDPSAIANGLINL 232 Gg HPGD 171 ENYGVRLNTICPGFVNTPILQSIDK-EENMGQYYSYKDEIKNMMQFYGVMDPSIIAEGLITI 232 Dr LOC565975 171 GNYGVRINALCPAFVDTQLLQTVEH-EETMGKFVKYKDDFKQRMDKYGVLK------221 Dm Pdh 187 QRSGIKFVTVCPGATMTDMFTNFTE-KIIFPETSDETYRILDRLNKQSAAD---VSRCILNV 244 Ce C01G12.5 181 IQHGVRVNSVSPGDIRTGIYETMGMNKESVENIYKFMESRKECCPIGTIAQPVDVANIIVFL 242
Figure 3.49. Partial amino acid sequence comparison of human 15-PGDH with other orthologs. The shaded serine (S) indicates the conserved residue across vertebrate species. The missense mutation (p.S193P) affecting a conserved serine residue in human 15-PGDH is indicated by an arrow. Species abbreviations are as follows: Hs, Homo sapiens; Pt, Pan troglodytes; Cf, Canis lupus familiaris; Bt, Bos taurus; Mm, Mus musculus; Rn, Rattus norvegicus; Gg, Gallus gallus; Dr, Danio rerio; Dm, Drosophila melanogaster; and Ce, Caenorhabditis elegans. The accession numbers for respective proteins are as follows: Hs, NP_000851.2; Pt, XP_001157531.1; Cf, XP_543199.1; Bt, NP_001029591.1; Mm, NP_032304.2; Rn, NP_077366.2; Gg, XP_420526.1; Dr, XP_694331.2; Dm, NP_730153.1; and Ce, NP_497031.1.
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Family C Figures 3.50-3.62 represent electropherograms of ethidium bromide stained 8% non- denaturing polyacrylamide gels (PAGEs) obtained by genotyping microsatellite markers mapped in the disease-interval of family C on chromosome 7p21.1-p15.1. Genetic positions (in centimorgan) for these marker loci were obtained from Rutgers combined linkage-physical map of the human genome (Build 36.1) (Kong et al., 2004). Lane 1:- V-3 (Normal) Lane 4:- IV-2 (Normal) Lane 7: IV-1 (Normal) Lane 2:- V-4 (Affected) Lane 5:- V-6 (Affected) Lane 8: V-2 (Affected) Lane 3:- V-5 (Affected) Lane 6:- V-7 (Normal) Lane 9: V-1 (Normal)
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.50: Allele pattern obtained with marker D7S488 at 31.04-cM on chromosome 7p21.1, showing heterozygous status of the affected individuals (V-4, V- 5, V-6) of family C. This marker defined the telomeric boundary of the disease interval.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.51: Allele pattern obtained with marker D7S815 at 32.52-cM on chromosome 7p21.1-p15.3, showing homozygosity among the affected individuals (V-4, V-5, V-6) of family C.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 123 Chapter 3 Ectodermal Dysplasias
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.52: Allele pattern obtained with marker D7S1802 at 34.63-cM on chromosome 7p15.3, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.53: Allele pattern obtained with marker D7S2562 at 35.75-cM on chromosome 7p15.3, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.54: Allele pattern obtained with marker D7S493 at 36.08-cM on chromosome 7p15.3, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
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V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.55: Allele pattern obtained with marker D7S2190 at 39.92-cM on chromosome 7p15.3, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.56: Allele pattern obtained with marker D7S2525 at 41.05-cM on chromosome 7p15.2, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.57: Allele pattern obtained with marker D7S2848 at 46.61-cM on chromosome 7p15.1, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
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V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.58: Allele pattern obtained with marker D7S2496 at 47.44-cM on chromosome 7p15.1, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.59: Allele pattern obtained with marker D7S2492 at 47.44-cM on chromosome 7p15.1, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.60: Allele pattern obtained with marker D7S632 at 48.65-cM on chromosome 7p15.1, showing homozygosity among the affected individuals (V-4, V- 5, V-6) of family C.
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V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.61: Allele pattern obtained with marker D7S817 at 50.85-cM on chromosome 7p14.3, showing heterozygous status the affected individual (V-6) of family C. This marker defined the centromeric boundary of the disease interval.
V-3 V-4 V-5 IV-2 V-6 V-7 IV-1 V-2 V-1
Figure 3.62: Allele pattern obtained with marker D7S484 at 53.72-cM with similar allele pattern as of D7S817 on chromosome 7p14.3, showing heterozygous status the affected individual (V-6) of family C. This marker defined the centromeric boundary of the disease interval.
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1 2 I
1 2 3 4 II
1 2 3 4 III
1 2 IV
5 6 5 6 D7S2557 27.35 11 12 11 12 D7S507 30.32 4 4 5 4 D7S3051 31.04 9 9 10 9 D7S488 31.04 2 1 1 3 D7S2532 31.19 3 1 1 2 D7S1802 34.63 11 9 9 10 D7S2562 35.75 10 9 9 11 D7S493 36.08 2 1 1 3 D7S1487 38.34 6 5 5 6 D7S2463 38.84 4 3 3 4 D7S2190 39.92 3 2 2 3 D7S2525 41.05 9 8 8 9 D7S2449 41.30 8 7 7 9 D7S1808 42.92 6 5 5 6 D7S2848 46.61 10 9 9 10 D7S2496 47.44 5 4 4 4 D7S2492 47.44 4 4 4 5 D7S2491 48.65 3 3 3 4 D7S632 48.65 6 6 6 7 D7S526 49.27 6 6 6 7 D7S817 50.85 6 6 6 7 D7S484 53.72 2 2 2 3 D7S2250 53.72
1 2 3 4 5 6 7 V
Markers cM Band Telomere 5 6 6 5 6 5 6 5 6 5 6 5 5 6 D7S2557 27.35 11 12 12 11 12 11 12 11 12 11 12 11 11 12 D7S507 30.32 4 4 4 5 4 5 4 5 4 5 4 5 4 4 D7S3051 31.04 9 9 9 10 9 10 9 10 9 10 9 10 9 9 D7S488 31.04 7p21.1 2 1 1 1 1 3 1 1 1 1 1 1 2 1 D7S2532 31.19 3 1 1 1 1 2 1 1 1 1 1 1 3 1 D7S1802 34.63 11 9 9 9 9 10 9 9 9 9 9 9 11 9 D7S2562 35.75 10 9 9 9 9 11 9 9 9 9 9 9 10 9 D7S493 36.08 2 1 1 1 1 3 1 1 1 1 1 1 2 1 D7S1487 38.34 6 5 5 5 5 6 5 5 5 5 5 5 6 5 D7S2463 38.84 4 3 3 3 3 4 3 3 3 3 3 3 4 3 D7S2190 39.92 3 2 2 2 2 3 2 2 2 2 2 2 3 2 D7S2525 41.05 9 8 8 8 8 9 8 8 8 8 8 8 9 8 D7S2449 41.30 8 7 7 7 7 9 7 7 7 7 7 7 8 7 D7S1808 42.92 6 5 5 5 5 6 5 5 5 5 5 5 6 5 D7S2848 46.61 10 9 9 9 9 10 9 9 9 9 9 9 10 9 D7S2496 47.44 5 4 4 4 4 4 4 4 4 4 4 4 5 4 D7S2492 47.44 4 4 4 4 4 5 4 4 4 4 4 4 4 4 D7S2491 48.65 7p15.1 3 3 3 3 3 4 3 3 3 3 3 3 3 3 D7S632 48.65 6 6 6 6 6 7 6 6 6 6 6 6 6 6 D7S526 49.27 6 6 6 6 6 7 6 6 6 6 6 7 6 6 D7S817 50.85 7p14.3 6 6 6 6 6 7 6 6 6 6 6 7 6 6 D7S484 53.72 2 2 2 2 2 3 2 2 2 2 2 3 2 2 D7S2250 53.72 Centromere Figure 3.63: Pedigree and Haplotype Drawing of Family C Segregating A Novel Form of Ectodermal Dysplasia For genotyped individuals, haplotypes of the closely linked microsatellite markers on chromosome 7p21.1-p15.1 are shown beneath each symbol. Disease-interval is flanked by two markers in boldface with arrows (D7S488 and D7S2491) indicating key recombination events.
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Table 3.4: Two point and multipoint LOD score results between ectodermal dysplasia locus and chromosome 7 markers in family C Marker Genetic Physical Multipoint Two point LOD score at recombination fraction θ= Distance(cM)* Distance (bp)¶ LOD score 0.00 0.01 0.05 0.10 0.20 0.30 0.40 D7S2557 27.35cM 15238687 -9.554 -2.87 -1.36 -0.70 -0.43 -0.17 -0.06 -0.01 D7S507 30.32cM 17563591 -13.524 -3.30 -1.63 -0.90 -0.56 -0.21 -0.07 -0.02 D7S3051 31.04cM 18251053 -12.633 -1.89 -0.33 0.25 0.42 0.43 0.29 0.10 D7S488 31.04cM 18355386 -4.064 -2.05 -0.37 0.22 0.40 0.42 0.28 0.10 D7S2532 31.19cM 18779450 3.061 2.68 2.62 2.36 2.04 1.41 0.79 0.24 D7S1802 34.63cM 20671774 3.069 2.68 2.62 2.36 2.04 1.41 0.79 0.24 D7S2562 35.75cM 21451984 3.070 2.93 2.86 2.59 2.25 1.56 0.89 0.29 D7S493 36.08cM 21771614 3.070 2.93 2.86 2.59 2.25 1.56 0.89 0.29 D7S1487 38.34cM 22834085 3.069 2.68 2.62 2.36 2.04 1.41 0.79 0.24 D7S2463 38.84cM 23611298 3.070 2.87 2.80 2.54 2.21 1.55 0.90 0.30 D7S2190 39.92cM 24434845 3.070 2.79 2.72 2.47 2.14 1.50 0.87 0.29 D7S2525 41.05cM 25458363 3.070 2.72 2.66 2.40 2.09 1.46 0.84 0.28 D7S2449 41.30cM 25899305 3.070 2.92 2.86 2.59 2.26 1.58 0.92 0.31 D7S1808 42.92cM 28004027 3.069 2.90 2.83 2.56 2.22 1.54 0.87 0.28 D7S2848 46.61cM 29467861 3.070 2.87 2.80 2.54 2.21 1.55 0.90 0.30 D7S2496 47.44cM 29532991 3.070 2.94 2.87 2.61 2.27 1.60 0.93 0.32 D7S2492 47.44cM 30080279 3.070 1.29 1.26 1.15 1.01 0.70 0.39 0.12 D7S2491 48.65cM 30788339 1.769 0.99 0.97 0.88 0.77 0.53 0.28 0.08 D7S632 48.65cM 30791082 1.769 0.98 0.95 0.87 0.75 0.52 0.28 0.08 D7S526 49.27cM 30915011 1.517 0.99 0.97 0.89 0.78 0.54 0.29 0.08 D7S817 50.85cM 32102921 -6.296 -infini -1.03 -0.39 -0.17 -0.04 -0.02 -0.01 D7S484 53.72cM 35251430 -8.934 -infini -1.03 -0.39 -0.17 -0.04 -0.02 -0.01 D7S2250 53.72cM 35344959 -4.930 -infini -1.07 -0.44 -0.22 -0.08 -0.04 -0.01 * Average-sex distance in cM is according to the Rutgers combined linkage-physical human genome map (Kong et al., 2004) ¶ The physical distances are based according to build 36.1 of the human genome (International Human Genome Sequence Consortium 2001) Markers D7S488 and D7S2491 (in boldface) flank the disease associated region for novel form of ectodermal dysplasia
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p Chromosome 7 q
NPVF, CYSC, NFE2L3, HDAC9, TWISTNB, 7A5, ITGB8, ABCB5, SP8, SP4, HNRPA2B1, CBX3, SNX10, CREB5, CPVL, CHN2, PRR15, TWIST1, DNAH11, RAPGEF5, CDCA7L, IL6, SKAP2, HOXA1, HOXA2, WIPF3, SCRN1, PLEKHA8, FERD3L TOMM7, GPNMB, IGF2BP3, CCDC126, HOXA3, HOXA4, HOXA5, FKBP14, ZNRF2, CRHR2, NUPL2, KLHL7, STK31, TRA2A, NPY, HOXA6, HOXA7, HOXA9, NOD1, GARS, INTM, DFNA5, MPP6, OSBPL3 HOXA10, HOXA11, HOXA13, ADCYAP1R1, AQP1, GHRHR, EVX1, HIBADH, NS5ATP1, NEUROD6,CCDC129, PDE1C D7S488 TAX1BP1, JAZF1 D7S2491 (31.04-cM) (48.65-cM) (Novel ED candidate region) 12.43-Mb Figure 3.64: Ideogramatic representation of a novel ED disease-associated interval of 12.43-Mb flanked by markers D7S488 and D7S2491 on chromosome 7p21.1-p15.1. The candidate genes in the linkage interval are shown in italics. Physical (Mb) and genetic (cM) distances are depicted according to the sequence- based physical map (Build 36.1) (International Human Genome Sequence Consortium, 2001) and Rutgers combined linkage-physical map (Build 36.1) (Kong et al., 2004), respectively.
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Table 3.5: Neutral polymorphisms detected in genes sequenced in family C Nucleotide Gene Protein change Location Type change c.393C>T p.A131A Exon 4 Synonymous ITGB8 942T>C p.Y314Y Exon 6 Synonymous Non- HOXA1 c.218G>A p.R72H Exon 1 synonymous HOXA4 c.714A>C p.R238R Exon 2 Synonymous c.30C>G p.S10S Exon 1 Synonymous PRR15 Non- c.343C>T p.S115P Exon 1 synonymous
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Figure 3.65: Sequence analysis of the EDA1 gene mutation in family D with XLHED. Partial DNA sequence of exon 8 of the EDA1 gene from (a) control individual, (b) female carrier and (c) an affected male individual of family D. The arrows in panels (a) and (b) represent the nucleotide position 913 at which insertion of four nucleotides TATA (boxed in panel c) occurred. This insertion mutation is a direct 4-bp tandem repeat.
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Table 3.6: Insertion and deletion mutations reported in the EDA1 gene
S. No Nucleotide change Protein change Location Mutation type Reference 1 c.EDA1 del No protien EDA1 gene Gross Deletion Vincent et al. (2001)
2 c.Ex1del Non-functional Exon 1 Gross deletion Kere et al. (1996)
3 c.77delG p.G26fsX Exon 1 Frameshift and PTC van der Hout et al. (2008)
4 c.245delG p.G82fsX90 Exon 1 Frameshift and PTC Lexner et al. (2008)
5 c.287insC p.fsX16 Exon 1 Frameshift and PTC Kere et al. (1996)
6 c.302_310dup8 p.fsX59 Exon 1 Frameshift and PTC Bayes et al. (1998)
7 c.363insC p.fsX41 Exon 1 Frameshift and PTC Kere et al. (1996)
8 c.494delT p.fsX85 Exon 3 Frameshift and PTC Kere et al. (1996)
9 c.Ex1-3del Non-functional Exons 1-3 Gross deletion Paakkonen et al. (2001)
10 c.Ex3del Non-functional Exon 3 Gross deletion Bayes et al. (1998)
11 c.Ex4-7del Non-functional Exons 4-7 Gross deletion Lexner et al. (2008)
12 c.559_576del18 p.184_189del Exon 5 Inframe deletion van der Hout et al. (2008)
13 c.553_588del36 p.185_196del Exon 5 Inframe deletion Bayes et al. (1998)
14 c.573_574insT p.G192WfsX239 Exon 5 Frameshift and PTC Huang et al. (2006)
15 c.636delT p.T212fsX279 Exon 5 Frameshift and PTC Lexner et al. (2008)
16 c.795_796insTTAT p.G268FfsX274 Exon 8 Frameshift and PTC van der Hout et al. (2008)
17 c.831_841ins10 p.fsX259 Exon 8 Frameshift and PTC Vincent et al. (2001)
18 c.856_875ins19 p.fsX244 Exon 8 Frameshift and PTC Vincent et al. (2001)
19 c.913_914insTATA p.305insIfsX306 Exon 8 Frameshift and PTC Tariq et al. (2007), Present Study
20 c.1117_1138del36 p.E292AfsX300 Exon 8 Frameshift and PTC Li et al. (2008)
21 c.1002_1014del13 p.S335fsX369 Exon 9 Frameshift and PTC Lexner et al. (2008)
22 c.1061_1110del50 p.L354fsX Exon 9 Frameshift and PTC van der Hout et al. (2008)
23 c.1311delC p.fsX374 Exon 9 Frameshift and PTC Vincent et al. (2001)
PTC: Premature termination codon
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Figure 3.66: Sequence analysis of the EDAR gene mutation (c.399_404delGGTCTG) in family E with ARHED. Partial DNA sequence of exon 5 of the EDAR gene from (a) a control individual, (b) a heterozygous carrier and (c) a homozygous (affected) individual of family E. The bar above the wild-type sequence in (a) represents the 6-bp sequence that is deleted in the homozygous state in the affected individual (c). The arrow in (c) represents the site of deletion in affected individual.
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Figure 3.67: Partial transcript representation of the EDAR gene mutation (p.M133_C135delinsI) in family E with ARHED (a) Wild type sequence showing a disulphide bridge between two cystiene residues at position 135 and 148 (in red). (b) Mutant sequence showing insertion of a new isoleucine (I) (in red) with deletion of three residues methionine (M), valine (V) and cystiene (C) (in red) as in (a) and abolishing disulphide bridge between two cystiene residues in EDAR protein.
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Table 3.7: Mutations in the EDAR gene implicated in the pathogenesis of HED
S. No Nucleotide change Protein change Location Mutation type Inheritance Reference
1 c.IVS2-25_52-8del Disturbed splicing of exon 3 Intron 2 Splice site AR Monreal et al. (1999)
2 c.IVS2+1G>A Disturbed splicing of exon 2 Intron 2 Splice site AR Shimomura et al. (2004)
3 c.140G>A p.C47Y Exon 3 Missense AR Chassaing et al. (2006)
4 c.259T>C p.C87R Exon 4 Missense AR Monreal et al. (1999)
5 c.266G>A p.R89H Exon 4 Missense AR Monreal et al. (1999); Chassaing et al. (2006); AD van der Hout et al. (2008) 6 c.Ex4del Truncated protien Exon 4 Gross deletion AR Monreal et al. (1999)
7 c.329A>C p.D110A Exon 4 Missense AR Chassaing et al. (2006); van der Hout et al. (2008)
8 c.399_404delGGTCTG p.M133_C135delinsI Exon 5 Inframe Deletion AR Tariq et al. (2007), Present Study
9 c.442T>C p.C148R Exon 5 Missense AR Chassaing et al. (2006)
10 c.478delC Truncated protien Exon 6 Frameshift and PTC AR RamaDevi et al. (2008)
11 c.IVS6+1G>A Disturbed splicing of exon 6 Intron 6 Splice site AR Chassaing et al. (2006)
12 c.718_721delAAAG Truncated protien Exon 8 Frameshift and PTC AR Naeem et al. (2005)
13 c.1060G>T p.E354X Exon 12 Nonsense AD van der Hout et al. (2008)
14 c.1072C>T p.R358X Exon 12 Nonsense AD Monreal et al. (1999); Lind et al. (2006); van der Hout et al. (2008) 15 c.1118C>T p.T373M Exon 12 Missense AD Valcuende-Cavero et al. (2008)
16 c.1124G>A p.R375H Exon 12 Missense AR Shimomura et al. (2004)
17 c.1129C>T p.L377F Exon 12 Missense AD Chassaing et al. (2006)
18 c.1144G>A p.G382S Exon 12 Missense AR Naeem et al. (2005); RamaDevi et al. (2008)
19 c.1208C>T p.T403M Exon 12 Missense AR Chassaing et al. (2006)
20 c.1237A>C p.T413P Exon 12 Missense AD Chassaing et al. (2006)
21 c.1253T>C p.I418T Exon 12 Missense AD Chassaing et al. (2006)
22 c.1259G>A p.R420Q Exon 12 Missense AD Monreal et al. (1999); Chassaing et al. (2006); van der Hout et al. (2008) 23 c.1302G>T p.W434C Exon 12 Missense AR Chassaing et al. (2006)
AR: Autosomal recessive AD: Autosomal dominant PTC: Premature termination codon
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a
b
c
Figure 3.68: Sequence analysis of the TRPS1 gene mutation in family F with TRPS III. Partial DNA sequence of exon 6 of the TRPS1 gene from an affected individual of the family F, showing heterozygous state of the mutation G>T in 5' 3' direction (a) and C>A in 3' 5' direction (b), and a control individual showing homozygous wild type sequence G (c). The arrows indicate position of the mutation and the sequence underlines highlight the codon containing the mutation.
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Table 3.8: Mutations¶ in exon 6 of the TRPS1 gene associated with TRPS III
Nucleotide change Amino acid change References c.T2681A p.V894D Ludecke et al. (2001) c.A2701C p.T901P Ludecke et al. (2001) c.G2723C p.R908P Ludecke et al. (2001) c.G2723A p.R908Q Ludecke et al. (2001), Hilton et al. (2002), Kobayashi et al. (2002) c.G2755A, c.C2756T p.A919V Ludecke et al. (2001), Hilton et al. (2002) c.G2762T p.G921V Tariq et al. (2008) Present study
¶ All the mutations identified in TRPS1 gene are missense
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ALOPECIAS
Alopecias are genetic hair loss of different etiologies. These may be isolated involving only hair (pure alopecias) or associated with anomalies of other ectodermal appendages in form of ectodermal dysplasias (EDs) and other organs (syndromic alopecias). Alopecias are clinically and genetically heterogeneous group of hair loss with autosomal recessive and dominant mode of inheritance. Alopecias result from abnormalities in hair structure, hair follicle morphogenesis, and hair growth cycling and maintenance. Hair structure, morphogenesis, growth cycle, and physiology are controlled by many genes. However, only few genes responsible for the pathogenesis of alopecias have been identified.
Over the past few years, four genes causing autosomal recessive isolated alopecias have been identified in Pakistani population. These include human hairless (HR) gene at 8p21.3 responsible for atrichia with papular lesions (APL) (Ahmad et al., 1998a), desmoglein 4 (DSG4) gene at 18q12.1 causing LAH1 type hypotrichosis (Kljuic et al., 2003; Rafiq et al., 2004), lipase H (LIPH) gene at 3q27.2 causing LAH2 type hypotrichosis (Aslam et al., 2004; Kazantseva et al., 2006; Ali et al., 2007), P2RY5 gene at 13q14.11-q21.32 causing LAH3 type hypotrichosis (Wali et al., 2007b; Azeem et al., 2008; Shimomura et al., 2008; Pasternack et al., 2008).
Alopecia with mental retardation syndrome (APMR) is an autosomal recessive form of syndromic alopecia. Recently, three APMR loci (APMR1, 2, 3) have been reported in Pakistani population (John et al., 2006a; Wali et al., 2006b; 2007a). However, causative genes for these three APMR conditions have not been identified so far.
In the present study, four consanguineous Pakistani families (G-J) with hereditary autosomal recessive alopecias have been investigated. Candidate gene approach has been used to identify the disease loci. Further, automated DNA sequencing was performed to search for the disease-causing mutations.
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RESULTS
Human Subjects and Clinical Findings
Family G
Family G with hereditary hypotrichosis, belongs to a Pashto speaking tribe of district Parachinar, North West Frontier Province, Pakistan. This large pedigree consists of ten affected individuals (II-4, III-2, III-3, III-4, III-9, IV-1, IV-2, IV-8, IV-9, IV-10) (Figure 4.1). The affected individuals are present in three consecutive generations (III, IV, V). An affected individual (II-4) is deceased. Pedigree analysis strongly suggest autosomal recessive pattern of inheritance of hair loss in the family.
Affected individuals of the family G exhibited typical clinical features of hereditary hypotrichosis. This included sparse to absent scalp hair at birth, which regrew sparsely and slowly after ritual shaving performed a week after birth. Hairs on scalp were thin, slow growing, little curly, brittle, and hypopigmented. Eyebrows, eyelashes, axillary hair, and body hair were sparse in the affected individuals (Figure 4.2). Affected male individuals have sparse beard hair. Teeth, nails, sweating and hearing were normal in the affected individuals. Heterozygous carrier individuals had normal hairs and were clinically indistinguishable from genotypically normal individuals.
Venous blood samples were collected from twenty individuals including nine affected (III-2, III-3, III-4, III-9, IV-1, IV-2, IV-8, IV-9, IV-10) and eleven normal members (III-1, III-5, III-6, III-7, III-8, III-10, IV-3, IV-4, IV-5, IV-6, IV-7) of the family.
Family H
Family H, demonstrating localized autosomal recessive hypotrichosis, belongs to district Rawalpindi, Punjab province, Pakistan. The seven generation pedigree (Figure 4.3) consists of thirteen affected individuals including nine males (V-1, V-3, V-7, V- 8, VI-3, VI-4, VI-5, VI-8, VII-1) and four females (V-9, VI-6, VI-7, VII-5). Two affected individuals (V-3, V-7) are deceased. Two affected individuals (V-1, VI-6) were not present at the time of the study. Pedigree analysis is suggestive of autosomal recessive mode of inheritance and consanguineous loops could account of all the affected persons being homozygous for an abnormal allele.
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Affected individuals of family H showed typical features of the hereditary hypotrichosis. At birth, hairs were present on the scalp but regrew sparsely after ritual shaving, which is usually performed a week after birth. The affected individuals were nearly devoid of normal eyebrows, eyelashes, axillary hair, and body hair (Figure 4.4). Affected male individuals have sparse beard hair; however, hairs were absent on their legs and arms. Teeth, nails, sweating and hearing were normal in all the affected individuals.
Venous blood samples were collected from nine affected (V-8, V-9, VI-3, VI-4, VI-5, VI-7, VI-8, VII-1, VII-5) and four normal members (VI-1, VI-2, VII-2, VII-4) of the family.
Family I
Family I with hereditary hypotrichosis belongs to district Larkana, Sindh province, Pakistan. The five generation pedigree of the family consists of seventeen affected individuals (II-5, IV-2, IV-3, IV-4, IV-6, IV-7, IV-8, IV-9, IV-10, IV-11, IV-15, IV- 16, IV-17, V-1, V-2, V-4, V-5) (Figure 4.5). Affected individuals are present in generation II, IV, and V. Five affected individuals (II-5, IV-7, IV-8, IV-9, IV-10) are deceased while three (IV-16, V-4, V-5) were not present at the time of the study. Pedigree structure strongly supports the autosomal recessive pattern of inheritance.
Affected individuals of the family exhibited typical features of hereditary hypotrichosis. At birth, hairs were present on the scalp but regrew sparsely after ritual shaving, which is usually performed a week after birth. Affected individuals were nearly devoid of normal eyebrows and eyelashes, however, axillary, pubic and mustache hairs were normal in the male affected individuals (Figure 4.6). Teeth, nails and sweat glands were normal in the affected individuals.
Venous blood samples were collected from eighteen individuals including nine affected (IV-2, IV-3, IV-4, IV-6, IV-11, IV-15, IV-17, V-1, V-2) and nine normal members (III-2, III-4, III-5, III-8, III-12, IV-1, IV-5, IV-12, V-3) of the family.
Family J
Family J belongs to district Larkana, Sindh, Pakistan. The family pedigree consists of six affected individuals including three males (IV-3, V-3, VI-3) and three females (V- 2, VI-4, VII-1) (Figure 4.7) and segregates atrichia with papular lesions in autosomal recessive pattern. The affected individuals are present in generation IV, V, VI, and
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VII. An affected individual (VI-3) is deceased while another affected individual (IV- 3) was not present at the time of the study.
Clinical features of the affected individuals in family J included absent scalp hair; absent eyebrows and eyelashes; absent armpit, axillary, and body hair (Figure 4.8). Remnants of the papular lesions were present on scalp and extremities of the body in the affected individuals. Affected individuals showed no abnormality with respect to nails, dentition, and skin. Based on the clinical features of the affected individuals of the family J, the appropriate diagnosis for this family was congenital atrichia with papular lesions (APL).
Venous blood samples were collected from twelve individuals including four affected (V-2, V-3, VI-4, VII-1) and eight normal members (IV-6, V-1, V-2, V-6, VI-1, VI-2, VI-5, VII-2) of the family.
Linkage and Mutational Analysis
Family G
Family G was tested for linkage to known genes involved in hereditary alopecias including HR at 8p21.3, DSG4 at 18q11.2-q12.1, LIPH at 3q27.3, P2RY5 at 13q14.11- q32.21, APMR1 at 3q26.33-q27.2, APMR2 at 3q26.2-q26.31, and APMR3 at 18q11.2-q12.2. Microsatellite markers linked to these loci were genotyped in all the twenty DNA samples of the family. Haplotype analysis showed that the family was linked to the P2RY5 gene at LAH3 locus (D13S1312, D13S168, D13S284, D13S1807) on chromosome 13q14.11-q32.21. To further confirm the linkage of the family at LAH3 locus, ten additional markers (D13S325, D13S328, D13S126, D13S153, D13S1325, D13S1245, D13S169, D13S256, D13S1301, D13S176) from the mapped region were genotyped in the affected and normal individuals of the family. Genotyping results showed that five polymorphic microsatellite markers (D13S328, D13S284, D13S1245, D13S169, D13S1807) were homozygous in affected and heterozygous in normal individuals of the family (Figure 4.9-4.13).
After establishing linkage to LAH3 locus, the P2RY5 gene [GenBank accession # NM_005767] was sequenced in two affected (IV-1, IV-8) and one normal (IV-3) individuals of the family. Sequence analysis of the P2RY5 gene revealed a novel homozygous missense mutation (c.742A>T) in the affected individuals (Figure 4.14). This transition results in the exchange of a small polar asparagine (AAT) residue with
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aromatic hydrophobic nonpolar tyrosine (TAT) at position 248 (p.N248Y). This mutation was found in homozygous state in all the affected individuals of the family. The mutation was present in heterozygous state in all the phenotypically normal individuals of the family, confirming their obligate heterozygous carrier status (Figure 4.14). To exclude the possibility that this particular genetic change is not a non- pathogenic polymorphism, a panel of 150 unaffected unrelated ethnically control individuals (300 chromosomes) was screened and this mutation was not identified outside the family (Figure 4.14).
Multiple sequence alignment (MSA) of P2Y5 protein sequences from different vertebrate species revealed that asparagine at position 248 is a conserved residue (http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene) (Figure 4.15).
To date, 13 novel mutations have been reported in the P2RY5 gene (Table 4.1).
Family H
Family H is the original family which was used to map a novel locus for autosomal recessive hereditary hypotrichosis (LAH3) (Wali et al., 2007b; Wali, 2008). In the present study, sequencing of the P2RY5 gene was performed in this family. Upon sequence analysis, a 4-bp recurrent insertion mutation was detected at cDNA position 69, designated as c.69insCATG. The mutation results in a frameshift and premature termination codon at amino acid position 52 of the P2Y5 protein (p.24insHfsX52). The mutation was present in homozygous state in all the affected individuals and in heterozygous state in all the phenotypically normal obligate carriers of the family (Figure 4.16).
Family I
Family I was tested for linkage to two known genes involved in hereditary alopecias (HR at 8p21.3 and DSG4 at 18q11.2-q12.1). Ten DNA samples including six affected (IV-2, IV-3, IV-6, IV-11, IV-13, V-1) and four normals (III-2, III-4, III-12, IV-12) of the family were genotyped. Genotyping results showed that five polymorphic microsatellite markers (D18S1108, D18S478, D18S847, D18S36, D18S536) were homozygous in affected and heterozygous in normal individuals (Figure 4.17-4.21), thus establishing linkage of the family to the DSG4 gene at LAH1 locus on chromosome 18q11.2-q12.1.
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After establishing linkage of the family to LAH1 locus, the coding region and splice junctions of the human desmoglein 4 (DSG4) gene [GenBank accession # NM_177986] were PCR amplified and sequenced in the family. Four exons (5–8) failed to get amplified from the genomic DNA of the affected individuals. However, PCR amplification of all the exons of the DSG4 gene from normal individuals showed that exons 5-8 were missing only in the affected members of the family. Therefore, a primer set, encompassing exons 4-8, was designed from intron 4 and 8 of the DSG4 gene to search for the mutation as reported earlier (Kljuic et al., 2003; Rafiq et al., 2004; Moss et al., 2004). The primer sequences used were:
5’-AAACATGGCAGACTGAACCC-3’ (Forward)
5’-CAGTATGCAAGGTTCTCAGC-3’ (Reverse)
The PCR results showed amplification of a 563-bp DNA fragment only from DNA of the affected individuals. Sequence analysis of the amplified product revealed the recurrent mutation (c.Ex5_8del) in the affected individuals of the family (Figure 4.22).
To date, only nine different mutations have been reported in the DSG4 gene (Table 4.2).
Family J
Family J was tested for linkage to human hairless (HR) gene on chromosome 8p21.3. Five microsatellite markers (D8S258, D8S298, D8S1786, D8S1733, D8S1739), linked to HR gene, were genotyped in all the family members including four affected (V-2, V-3, VI-4, VII-1) and six normals (IV-5, V-1, V-6, VI-2, VI-5, VII-2). Genotyping results showed that the markers were homozygous in all the affected and heterozygous in normal individuals of the family (Figure 4.23-4.25), thus establishing linkage of the family to the HR gene on chromosome 8p21.3. After establishing linkage to HR gene, the coding region and splice junction sites of the gene were sequenced bidirectionally in affected and normal individuals of the family. Sequence analysis with control sequence of the hairless gene [Ensembl accession ID: Human: ENSG00000168453] revealed no functional sequence variant causing disease phenotype in this family. However, this cannot exclude the presence of the mutation in the regulatory sequences of the gene.
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DISCUSSION
For the study, presented here, four families (G, H, I, J) with hereditary alopecias were studied from remote regions in Pakistan. Clinical features of the three families (G, H, I) were almost similar. These included sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse body and pubic hair. Affected male individuals in these three families had sparse to normal beard hairs. The fourth family (J) represented a case of congenital atrichia with papular lesions. Affected individuals in this family had total alopecia including absence of hair on scalp and rest of the body.
Genetic mapping by genotyping microsatellite markers, linked to genes involved in hereditary alopecias, showed linkage of two families (G and H) to LAH3 locus on chromosome 13q14.11-q32.21. In family I linkage was established to LAH1 locus on chromosome 18q11.2-q12.1. The fourth family (J) showed linkage to hairless (HR) gene on chromosome 8p21.3.
In families G and H, which showed linkage to LAH3 locus, the P2RY5 gene was sequenced in the affected and normal individuals. In family G, a novel missense mutation c.742A>T (p.N248Y) in the P2RY5 gene was detected in the affected individuals. This mutation is located in the sixth transmembrane domain (TM6: 228- 253 amino acids) of the P2Y5 protein (http://au.expasy.org/uniprot/P43657). In family H, a recurrent frameshift mutation (c.69insCATG) in the P2RY5 gene was detected resulting in 52 amino acids truncated P2Y5 protein (p.24insHfsX52). This mutation was previously reported by Shimomura et al. (2008) in a Pakistani family with autosomal recessive woolly hair. Pasternack et al. (2008) have experimentally proved that truncated P2Y5 protein accumulate in the endoplasmic reticulum in contrast to wild type P2Y5, which co-localized at the plasma membrane with cadherins (Pasternack et al., 2008).
The P2Y5, a member of the G protein-coupled receptors (GPCRs), was recently deciphered as key regulator of the hair follicle development (Shimomura et al., 2008; Pasternack et al., 2008). The P2RY5 gene encodes 344 amino acids of P2Y5 protein (Herzog et al., 1996). This contains four potential extracellular domains, four cytoplasmic domains and seven predicted hydrophobic transmembrane regions (http://au.expasy.org/uniprot/P43657). The P2Y5 was originally reported to bind extracellular nucleotides (Herzog et al., 1996; Webb et al., 1996). However,
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Pasternack et al. (2008) found that oleoyl-L-alpha-lysophosphatidic acid (LPA), a bioactive lipid and product of the lipase H, serves as a ligand for the P2Y5. The novel missense mutation (p.N248Y), identified in family G, would probably affect the binding of P2Y5 to its ligand LPA. The LPA, a mixture of various fatty acids, is involved in many cellular processes including cellular proliferation, cell migration, apoptosis and smooth muscle contraction. Most of the LPA actions are mediated through at least seven G protein- coupled receptors (GPCRs). Recent studies on hereditary human hair loss revealed that genetic defects in phospholipase-H (LIPH) gene result in hair growth defects in Russian and Pakistani families (Kazantseva et al., 2006; Ali et al., 2007; Jelani et al., 2008; Naqvi et al., 2008). Interestingly, cellular distribution of both P2Y5 and LIPH are similar and express in inner root sheath (IRS) of the hair follicles. Although the precise molecular mechanism defining the role of P2Y5 and LIPH in growth of the hair is not known but taken together the mutational and functional studies of the LIPH and P2RY5 genes suggest an integrated model in which these two genes along with the LPA are involved in regulation of hair growth.
In family I, sequence analysis of the DSG4 gene at LAH1 locus in the affected individuals revealed a recurrent intragenic large in-frame deletion mutation (c.Ex5- 8del) reported by Kljuic et al. (2003), Rafiq et al. (2004) and Moss et al. (2004). This mutation begins 35-bp upstream of exon 5 and ends 289-bp downstream of exon 8. This generates an in-frame deletion creating a predicted protein with missing amino acids from 125 to 335. The amino acid sequence in this region is believed to be critical in cadherin–cadherin interaction and dimerization, necessary for proper cell adhesion (Boggon et al., 2002; He et al., 2003). The recurrent deletion in the DSG4 gene, detected in this family, is reported previously in seven families from different ethnic groups of Pakistani population (Kljuic et al., 2003; Rafiq et al., 2004; Moss et al., 2004; John et al., 2006b). This suggests that this deletion is an ancestral founder mutation widely dispersed during evolution. Missense mutations in the same region of the dsg4 have been demonstrated in the lah/lah mouse (Kljuic et al., 2003) and the lah/lah rat (Jahoda et al., 2004). A missense mutation (p.A129S) in the DSG4 gene has also been reported in an Iraqi family with LAH1 (Messenger et al., 2005). Recently, Wajid et al. (2007) reported a single base pair deletion (c.87delG) resulting
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in frameshift and premature stop codon, which leads to absence of functional DSG4 protein in a Pakistani family with LAH1 type hypotrichosis.
The DSG4 gene consists of 16 exons encoding 1040 amino acids protein. Structurally DSG4, a member of classical cadherins, contains a number of domains: a signal sequence; a propeptide of around 130 residues; a single transmembrane domain; an extracellular domain of five tandem repeated elements; and an N-terminal cytoplasmic domain (Yap et al., 1997). The DSG4 gene is expressed in the suprabasal epidermis, matrix, precortex, and hair follicle (Kljuic et al., 2003). Interestingly, DSG4 is the only desmosomal cadherin associated with a human hair loss (Huber, 2003; McGrath and Wessagowit, 2005).
In family J, sequence analysis of the entire coding region and splice junction sites of the human hairless (HR) gene failed to detect any functional pathogenic sequence variant. However, presence of a mutation in the regulatory sequences of the gene cannot be ruled out.
Hairless (HR) gene consists of 18 coding exons encoding 1189 amino acids protein, a putative zinc-finger transcription factor. Hairless protein with a single zinc-finger domain is highly expressed in brain and skin. At least two different isoforms of HR protein are known resulting from the alternative splicing of the HR mRNA (Cichon et al., 1998).
To date, 38 different mutations in the HR gene have been reported causing APL from different populations around the world, thereby establishing the molecular basis of this condition (Ahmad et al., 1998a,b; 1999a,b; Cichon et al., 1998; Zlotogorski et al., 1998, 2002; Kruse et al., 1999; Sprecher et al., 1999a,b; Aita et al., 2000; Paradisi et al., 2003; Ahsoor et al., 2005; John et al., 2005; Wali et al., 2006a; Betz et al., 2007; Michailidis et al., 2007; Kim et al., 2007; Kraemer et al., 2008; Roelandt et al., 2008).
In human, the HR gene appears to function in the cellular transition to the first adult hair cycle and in its absence hair growth completely ceases, a new hair is never induced, and the result is a complete form of inherited alopecia (Ahmad et al., 1998b; Zlotogorski et al., 1998). Potter et al. (2001) demonstrated that the HR protein functions as a nuclear receptor corepressor. This protein interacts with and influences the transcriptional activity of several nuclear factors, including thyroid hormone
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receptor, retinoic acid receptor-related orphan receptor-α and vitamin D receptor (VDR) (Potter et al., 2001; Hsieh et al., 2003). Thompson et al. (2006) demonstrated that the HR protein represses expression of WISE protein, an inhibitor of Wnt signaling. This leads to a model in which HR controls the timing of Wnt signaling required for hair cycling.
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Figures and Tables of Chapter 4
Figure 4.1: Pedigree of family G with hereditary autosomal recessive hypotrichosis. Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate consanguineous marriages.
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a b
Figure 4.2: Clinical features of family G with hereditary hypotrichosis. Affected individuals IV-1 (a) and IV-8 (b) showing typical features of hypotrichosis with sparse, brittle and curly scalp hair, and sparse to absent eyebrows and eyelashes.
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I I-1 I-2
II II-1 II-2 II-3 II-4 II-5 II-6
III III-1 III-2 III-3 III-4 III-5 III-6
IV
IV-1 IV-2 IV-3 IV-4 IV-5 IV-6
V
V-1 V-2 V-3 V-4 V-5 V-6 V-7 V-8 V-9
VI VI-1 VI-2 VI-3 VI-4 VI-5 VI-6 VI-7 VI-8
VII VII-1 VII-2 VII-3 VII-4 VII-5
Figure 4.3: Pedigree of family H with hereditary autosomal recessive hypotrichosis. Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate consanguineous marriages. This family was earlier used for mapping the LAH3 locus on chromosome 13q14.11-q32.21 (Wali et al., 2007b).
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Figure 4.4: Clinical features of family H with hereditary hypotrichosis. Affected individual VI-5 showing typical features of hypotrichosis with sparse scalp hair and sparse eyebrows and eyelashes (Adopted from Wali et al., 2007b).
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1 2 I
1 2 3 4 5 6 7 8 9 10 II
5 7 8 9 10 11 12 1 2 3 4 6 III
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 IV
1 2 3 4 5 6 V
Figure 4.5: Pedigree of family I with hereditary autosomal recessive hypotrichosis. Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate consanguineous marriages.
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a b
c
Figure 4.6: Clinical features of family I with hereditary hypotrichosis. a and b: An affected individual (IV-6) showing typical features of hypotrichosis with sparse, curly scalp hair and sparse eyebrows and eyelashes. c: An affected individual (V-4) showing sparse scalp hair, sparse eyebrows and eyelashes.
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1 2 I
1 2 3 4 II
1 2 3 4 5 6 7 8 III
6 7 8 1 2 3 4 5 IV
1 2 3 4 5 6 V
1 2 3 4 5 VI
1 2 VII
Figure 4.7: Pedigree of family J segregating congenital atrichia with papular lesions (APL). Circles and squares represent females and males, respectively. Filled symbols represent affected individuals. Crossed symbols represent deceased individuals. Double lines indicate consanguineous marriages.
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a b
Figure 4.8: Clinical features of the family J with congenital atrichia and papular lesions (APL). a and b: An affected individual (V-3) showing absent scalp hair, and absent eyebrows and eyelashes.
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Family G Figures 4.9-4.13 represent electropherograms of ethidium bromide stained 8% non- denaturing polyacrylamide gels (PAGEs) obtained by genotyping microsatellite markers linked to P2RY5 gene on chromosome 13q14.11-q32.21 in family G. Genetic positions (in centimorgan) for these marker loci were obtained from Rutgers combined linkage-physical map of the human genome (build 36.1) (Kong et al., 2004). Lane 1: III-1 (Normal) Lane 6: III-6 (Normal) Lane 11: IV-4 (Normal) Lane 16: IV-9 (Affected) Lane 2: III-2 (Affected) Lane 7: III-9 (Affected) Lane 12: IV-5 (Normal) Lane 17: IV-10 (Affected) Lane 3: III-3 (Affected) Lane 8: IV-1(Affected) Lane 13: IV-6 (Normal) Lane 18: III-7 (Normal) Lane 4: III-4 (Affected) Lane 9: IV-2 (Affected) Lane 14: IV-7 (Normal) Lane 19: III-8 (Normal) Lane 5: III-5 (Normal) Lane 10: IV-3 (Normal) Lane 15: IV-8 (Affected) Lane 20: III-10 (Normal)
III-1 III-2 III-3 III-4 III-5 III-6 III-9 IV-1 IV-2 IV-3 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10 III-7 III-8 III-10
Figure 4.9: Allele pattern obtained with marker D13S328 at 50.18-cM on chromosome 13q14.13-q14.2, showing homozygosity among the affected individuals (III-2, III-3, III-4, III-9, IV-1, IV-2, IV-2, IV-8, IV-9, IV-10) of family G.
III-1 III-2 III-3 III-4 III-5 III-6 III-9 IV-1 IV-2 IV-3 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10 III-7 III-8 III-10
Figure 4.10: Allele pattern obtained with marker D13S284 at 54.17-cM on chromosome 13q14.3, showing homozygosity among the affected individuals (III-2, III-3, III-4, III-9, IV-1, IV-2, IV-2, IV-8, IV-9, IV-10) of family G.
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III-1 III-2 III-3 III-4 III-5 III-6 III-9 IV-1 IV-2 IV-3 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10 III-7 III-8 III-10
Figure 4.11: Allele pattern obtained with marker D13S1245 at 54.74-cM on chromosome 13q21.1, showing homozygosity among the affected individuals (III-2, III-3, III-4, III-9, IV-1, IV-2, IV-2, IV-8, IV-9, IV-10) of family G.
III-1 III-2 III-3 III-4 III-5 III-6 III-9 IV-1 IV-2 IV-3 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10 III-7 III-8 III-10
Figure 4.12: Allele pattern obtained with marker D13S169 at 55.14-cM on chromosome 13q21.1, showing homozygosity among the affected individuals (III-2, III-3, III-4, III-9, IV-1, IV-2, IV-2, IV-8, IV-9, IV-10) of family G.
III-1 III-2 III-3 III-4 III-5 III-6 III-9 IV-1 IV-2 IV-3 IV-4 IV-5 IV-6 IV-7 IV-8 IV-9 IV-10 III-7 III-8 III-10
Figure 4.13: Allele pattern obtained with marker D13S1807 at 55.5-cM on chromosome 13q21.1, showing homozygosity among the affected individuals (III-2, III-3, III-4, III-9, IV-1, IV-2, IV-2, IV-8, IV-9, IV-10) of family G.
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Figure 4.14: Sequence analysis of the P2RY5 gene mutation (c.742A>T) in family G with LAH3. Partial DNA sequence of the P2RY5 gene from (a) a homozygous affected individual showing a transition (A>T) (b) a heterozygous carrier and (c) a control individual showing wild type sequence. The arrows indicate position of the mutation and the sequence underlines highlight the codon containing the mutation.
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p.N248Y
Hs P2RY5 LTKPVTLSRS-KINKTKVLKMIFVHLIIFCFCFVPYNINLILYS Pt P2RY5 LTKPVTLSRS-KINKTKVLKMIFVHLIIFCFCFVPYNINLILYS Bt P2RY5 LNKPVTLSRS-KINKTKVLRMIFVHLVIFCFCFVPYNINLILYS Mm P2ry5 LNKPVTLSRS-KMNKTKVLKMIFVHLVIFCFCFVPYNINLILYS Rn P2ry5 LNKPVTLSRS-KMNKTKVLKMIFVHLVIFCFCFVPYNINLILYS Gg P2RY5 LNKPLTLSRN-KLSKKKVLKMIFVHLVIFCFCFVPYNITLILYS Dr zgc:153784 LRRPNTVSRGGKLNKKKILRMIIVHLFIFCFCFIPYNVNLVFYS
Figure 4.15. Partial amino acid sequence comparison of human P2Y5 with other orthologs. The shaded asparagine (N) indicates the conserved residue across vertebrate species. The missense mutation (p.N248Y) affecting a conserved asparagine residue in human P2Y5 is indicated by an arrow. Boldface residues show the sixth transmembrane domain (TM6: 228-253 a. a) of the P2Y5. Species abbreviations are as follows: Hs, Homo sapiens; Pt, Pan troglodytes; Bt, Bos taurus; Mm, Mus musculus; Rn, Rattus norvegicus; Gg, Gallus gallus; Dr, Danio rerio. The accession numbers for respective proteins are as follows: Hs, NP_000851.2; Pt, XP_001157531.1; Bt, NP_001029591.1; Mm, NP_032304.2; Rn, NP_077366.2; Gg, XP_420526.1; Dr, XP_694331.2.
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a
c
b
Figure 4.16: Sequence analysis of the P2RY5 gene mutation (c.69insCATG) in family H with LAH3. Partial DNA sequence of the P2RY5 gene from (a) a homozygous (affected) individual showing 4-bp insertion sequence (underlined) (b) a heterozygous carrier individual (c) a control individual showing wild type sequence. Point of insertion is indicated by an arrow in the control sequence.
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Table 4.1: Mutations reported in the P2RY5 gene
S. No Nucleotide change Protein change Mutation type Phenotype Reference
1 c.8G>C p.S3T Missense LAH3 Azeem et al. (2008)
2 c.36insA p.D13RfsX16 Frameshift and PTC LAH3 Azeem et al. (2008)
3 c.160insA p.N54TfsX58 Frameshift and PTC LAH3 Azeem et al. (2008)
4 c.69insCATG p.24insHfsX52 Frameshift and PTC LAH3, ARWH Shimmomura et al. (2008); Azeem et al. (2008); Present study
5 c.172-175delAACT;177delG p.N58-L59delinsCfsX90 Complex deletion LAH3, ARWH Shimmomura et al. (2008); Unpublished data
6 c.188A>T p.D63V Missense LAH3, ARWH Shimmomura et al. (2008); Azeem et al. (2008); Unpublished data
7 c.436G>A p.G146R Missense LAH3 Azeem et al. (2008); Unpublished data
8 c.562A>T p.I188F Missense ARWH Shimmomura et al. (2008)
9 c.565G>A p.E189K Missense LAH3, ARWH Shimmomura et al. (2008); Azeem et al. (2008); Unpublished data 10 c.463C>T¶ p.Q155X Nonsense ARHS Pasternack et al. (2008)
11 c.373-374delAA¶ p.K125NfsX162 Frameshift and PTC ARHS Pasternack et al. (2008)
12 c.742A>T p.N248Y Missense LAH3 Present Study
13 c.830C>T p.L277P Missense LAH3 Unpublished data
PTC: Premature termination codon
ARWH: Autosomal recessive woolly hair
ARHS: Autosomal recessive hypotrichosis simplex
¶ These mutations were identified in Saudi Arabian families while the rest were reported in Pakistani population
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Family I Figures 4.17-4.21 represent electropherograms of ethidium bromide stained 8% non- denaturing polyacrylamide gels (PAGEs) obtained by genotyping microsatellite markers linked to desmoglein 4 (DSG4) gene on chromosome 18q12.1 in family I. Genetic positions (in centimorgan) for these marker loci were obtained from Rutgers combined linkage-physical map of the human genome (build 36.1) (Kong et al., 2004). Lane 1: IV-6 (Affected) Lane 6: III-2 (Normal) Lane 2: III-4 (Normal) Lane 7: III-12 (Normal) Lane 3: IV-11 (Affected) Lane 8: IV-15(Affected) Lane 4: IV-3 (Affected) Lane 9: IV-12 (Normal) Lane 5: IV-4 (Affected) Lane 10: V-2 (Affected)
IV-6 III-4 IV-11 IV-3 IV-4 III-2 III-12 IV-15 IV-12 V-2
Figure 4.17: Allele pattern obtained with marker D18S1108 at 50-cM on chromosome 18q11.2, showing homozygosity among the affected individuals (IV-6, 1V-11, IV-3, 1V-4, 1V-15, V-2) of family I.
IV-6 III-4 IV-11 IV-3 IV-4 III-2 III-12 IV-15 IV-12 V-2
Figure 4.18: Allele pattern obtained with marker D18S478 at 54.85-cM on chromosome 18q12.1, showing homozygosity among the affected individuals (IV-6, 1V-11, IV-3, 1V-4, 1V-15, V-2) of family I.
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IV-6 III-4 IV-11 IV-3 IV-4 III-2 III-12 IV-15 IV-12 V-2
Figure 4.19: Allele pattern obtained with marker D18S847 at 57.41-cM on chromosome 18q12.1, showing homozygosity among the affected individuals (IV-6, 1V-11, IV-3, 1V-4, 1V-15, V-2) of family I.
IV-6 III-4 IV-11 IV-3 IV-4 III-2 III-12 IV-15 IV-12 V-2
Figure 4.20: Allele pattern obtained with marker D18S36 at 59.09-cM on chromosome 18q12.1, showing homozygosity among the affected individuals (IV-6, 1V-11, IV-3, 1V-4, 1V-15, V-2) of family I.
IV-6 III-4 IV-11 IV-3 IV-4 III-2 III-12 IV-15 III-12 V-2
Figure 4.21: Allele pattern obtained with marker D18S536 at 60.9-cM on chromosome 18q12.1, showing heterozygous status of the affected individuals (IV-3, 1V-4, V-2) of family I.
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A
B
Intron 4
AATTCCTTTGGTGGAAAAAGA Wild type sequence
Intron 8
GGAGTTCACAGTGCAATAAAT Wild type sequence
Break point of deletion
Mutant sequence AATTCCTTTGGTGCAATAAAT
Figure 4.22: A: Sequence analysis of the DSG4 gene in family I with autosomal recessive hereditary hypotrichosis (LAH1). Partial DNA sequence of the DGS4 gene from an affected individual of family I showing homozygous deletion mutation encompassing exons 5 through 8 (c.Ex5_8del). An arrow represents the nucleotides flanking the deletion between introns 4 and 8 of the DSG4 gene. B: Wild type and mutant sequences of the DSG4 gene. The arrows represent point of deletions in intron 4 and 8, and homozygous mutant sequence.
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Table 4.2: Mutations reported in the DSG4 gene causing autosomal recessive hereditary hypotrichosis (LAH1) and autosomal recessive monilethrix
S. No Nucleotide change Protein change Location Mutation type Population Phenotype Reference
1 c.87delG* p.K30RfsX83 Exon 3 Frameshif and PTC Pakistan LAH1 Wajid et al. (2007)
2 c.385G>C* p.A129S Exon 5 Missense Iraq LAH1 Messenger et al. (2005)
3 c.Ex5-8del* Inframe deletion Spanning intron 4-to-intron 8Gross deletion Pakistan LAH1 Kljuic et al. (2003); Rafiq et al. (2004); Moss et al. (2004); John et al. (2006b) Present study
4 c.216+1G>T¶ Abnormal mRNA splicing Intron 3 splice donor site Splice site Iran Monilethrix Schaffer et al. (2006); Zlotogorski et al. (2006) 5 c.574T>C¶ p.S192P Exon 6 Missense Japan Monilethrix Shimomura et al. (2006)
6 c.763delT¶ p.S255LfsX256 Exon 7 Frameshif and PTC Iraq, Iran Monilethrix Zlotogorski et al. (2006)
7 c.799C>G* ¶ p.P267R Exon 7 Missense Iraq Monilethrix Zlotogorski et al. (2006); c.800C>G ¶ Schaffer et al. (2006)
8 c.865C>T¶ p.R289X Exon 8 Nonsense Morocco Monilethrix Zlotogorski et al. (2006)
9 c.2039insT¶ p.W681LfsX684 Exon 13 Frameshif and PTC Japan Monilethrix Shimomura et al. (2006)
PTC: Premature stop codon
* Homozygous mutations
¶ Allelic to compound heterozygous mutations
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Family J Figures 4.23-25 represent electropherograms of ethidium bromide stained 8% non- denaturing polyacrylamide gels (PAGEs) obtained by genotyping microsatellite markers tightly linked to human hairless (HR) gene on chromosome 8p21.3 in family J. Genetic positions (in centimorgan) for these marker loci were obtained from Rutgers combined linkage-physical map of the human genome (build 36.1) (Kong et al., 2004). Lane 1: V-3 (Affected) Lane 5: IV-5 (Normal) Lane 8: VII-1 (Affected) Lane 2: V-1 (Normal) Lane 6: VI-4 (Affected) Lane 9: VI-5 (Normal) Lane 3: VI-2 (Normal) Lane 7: V-6 (Normal) Lane 10: V-2 (Affected) Lane 4: VII-2 (Normal)
V-3 V-1 VI-2 VII-2 IV-5 VI-4 V-6 VII-1 VI-5 V-2
Figure 4.23: Allele pattern obtained with marker D8S258 at 37.51-cM on chromosome 8p21.3, showing homozygosity among the affected individuals (V-3, VI- 4, VII-1, V-2) of family J.
V-3 V-1 VI-2 VII-2 IV-5 VI-4 V-6 VII-1 VI-5 V-2
Figure 4.24: Allele pattern obtained with marker D8S298 at 40.11-cM on chromosome 8p21.3, showing homozygosity among the affected individuals (V-3, VI- 4, VII-1, V-2) of family J.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 167 Chapter 4 Alopecias
V-3 V-1 VI-2 VII-2 IV-5 VI-4 V-6 VII-1 VI-5 V-2
Figure 4.25: Allele pattern obtained with marker D8S1786 at 41.41-cM on chromosome 8p21.3, showing homozygosity among the affected individuals (V-3, VI- 4, VII-1, V-2) of family J.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 168 Chapter 5 General Conclusion
General Conclusion Pakistan lies in the northwestern part of the Indian subcontinent. It is bounded in the north and the northwest by Afghanistan, on the northeast by Jammu and Kashmir, on the east and southeast by India, on the south by the Arabian Sea, and on the west by Iran. There are various ethnic groups in Pakistan with different cultures, socioeconomic conditions, religion, geography and linguistics like Urdu (official), Punjabi, Sindhi, Pushtu, Baluchi and Brahvi. Consanguineous marriage pattern in Pakistani population along with other factors such as religion, ethnicity, socioeconomic conditions, language, geography etc resulted in the genetically isolated groups having extended and multigenerational pedigrees with several cases of rare genetic diseases. These extended pedigrees are used by molecular geneticists for mapping monogenic autosomal recessive genetic disorders. In the study, presented in this thesis, ten consanguineous families with different human skin disorders were studied from different areas of Pakistan. This included four families from Punjab province, two from North West Frontier Province, two from Sindh province, and two from Azad Jammu and Kashmir. Genetic mapping studies in these families led to the localization of three novel ectodermal dysplasia loci. These included an ectodermal dysplasia of hair-nail-teeth type locus on chromosome 18q22.1-q22.3, isolated congenital nail clubbing (ICNC) locus on chromosome 4q32.3-q34.3, and a novel form of ectodermal dysplasia locus on chromosome 7p21.1-p15.1. Screening of candidate genes in the genetic interval of the ICNC detected a novel missense mutation (p.S193P) in the HPGD gene in the affected individuals. DNA sequence analysis has identified four novel and two recurrent mutations in families with various forms of ectodermal dysplasia and autosomal recessive hypotrichosis. In two families with hypohidrotic ectodermal dysplasia (HED) two novel mutations p.305insIfsX306 and p.M133_C135delinsI were identified in the EDA1 and EDAR genes, respectively. In a family with trichorhinophalangeal syndrome type III (TRPS III) a novel missense mutation (p.G921V) was detected in the TRPS1 gene. This is a sixth novel mutation in the TRPS1 gene causing TRPS III phenotype. In two families with autosomal recessive hypotrichosis a novel missense (p.N248Y) and recurrent insertion (p.24insHfsX52) mutations were identified in the
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 169 Chapter 5 General Conclusion
P2RY5 gene. In the third family with autosomal recessive hypotrichosis a recurrent mutation involving a large deletion (c.Ex5-8del) in the DSG4 gene was detected in the affected individuals. This is the eighth Pakistani family which showed the same mutation.
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 170 Chapter 6 References
LITERATURE REFERENCES
Abecasis GR, Cherny SS, Cookson WO, Cardon LR (2002). Merlin-rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 30: 97-101
Adaimy L, Chouery E, Megarbane H, Mroueh S, Delague V, Nicolas E, Belguith H, de Mazancourt P, Megarbane A (2007). Mutation in WNT10A is associated with an autosomal recessive ectodermal dysplasia: the odonto-onycho-dermal dysplasia. Am J Hum Genet 81: 821-828
Ahmad W, Faiyaz ul Haque M, Brancolini V, Tsou HC, ul Haque S, Lam H, Aita VM, Owen J, deBlaquiere M, Frank J, Cserhalmi-Friedman PB, Leask A, McGrath JA, Peacocke M, Ahmad M, Ott J, Christiano AM (1998a). Alopecia universalis associated with a mutation in the human hairless gene. Science 279: 720-724
Ahmad W, Irvine AD, Lam H, Buckley C, Bingham EA, Panteleyev AA, Ahmad M, McGrath JA, Christiano AM (1998b). A missense mutation in the zinc-finger domain of the human hairless gene underlies congenital atrichia in a family of Irish travellers. Am J Hum Genet 63: 984-991
Ahmad W, Panteleyev AA, Christiano AM (1999b). The molecular basis of congenital atrichia in humans and mice: mutations in the hairless gene. J Investig Dermatol Symp Proc 4: 240-243
Ahmad W, Zlotogorski A, Panteleyev AA, Lam H, Ahmad M, ul Haque MF, Abdallah HM, Dragan L, Christiano AM (1999a). Genomic organization of the human hairless gene and identification of a mutation underlie congenital atrichia in an Arab Palestinian family. Genomics 56: 141-148
Aita VM, Ahmad W, Panteleyev AA, Kozlowska U, Kozlowska A, Gilliam TC, Jablonska S, Christiano AM (2000). A novel missense mutation (C622G) in the zinc-finger domain of the human hairless gene associated with congenital atrichia with papular lesions. Exp Dermatol 9: 157-162
Akahoshi K, Sakazume S, Kosaki K, Ohashi H, Fukushima Y (2003). EEC syndrome type 3 with a heterozygous germline mutation in the P63 gene and B cell lymphoma. Am J Med Genet A 120: 370-373
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 171 Chapter 6 References
Alasti F, Sadeghi A, Sanati MH, Farhadi M, Stollar E, Somers T, Van Camp G (2008). A mutation in HOXA2 is responsible for autosomal-recessive microtia in an Iranian family. Am J Hum Genet 82: 982-991
Ali G, Chishti MS, Raza SI, John P, Ahmad W (2007). A mutation in the lipase H (LIPH) gene underlie autosomal recessive hypotrichosis. Hum Genet 121: 319- 325
Amorosi S, D'Armiento M, Calcagno G, Russo I, Adriani M, Christiano AM, Weiner L, Brissette JL, Pignata C (2008). FOXN1 homozygous mutation associated with anencephaly and severe neural tube defect in human athymic Nude/SCID fetus. Clin Genet 73: 380-384
Anggård E (1966). The biological activities of three metabolites of prostaglandin E 1. Acta Physiol Scand 66: 509-510
Anggård E, Larsson C, Samuelsson B (1971). The distribution of 15-hydroxy prostaglandin dehydrogenase and prostaglandin-delta 13-reductase in tissues of the swine. Acta Physiol Scand 81: 396-404
Ashoor GG, Greenstein RM, Lam H, Martinez-Mir A, Zlotogorski A, Christiano AM (2005). Novel compound heterozygous nonsense mutations in the hairless gene causing atrichia with papular lesions. J Dermatol Sci 40: 29-33
Aslam M, Chahrour MH, Razzaq A, Haque S, Yan K, Leal SM, Ahmad W (2004). A novel locus for autosomal recessive form of hypotrichosis maps to chromosome 3q26.33-q27.3. J Med Genet 41: 849-852
Azeem Z, Jelani M, Naz G, Tariq M, Wasif N, Kamran-Ul-Hassan Naqvi S, Ayub M, Yasinzai M, Amin-Ud-Din M, Wali A, Ali G, Chishti MS, Ahmad W (2008). Novel mutations in G protein-coupled receptor gene (P2RY5) in families with autosomal recessive hypotrichosis (LAH3). Hum Genet 123: 515-519
Bal E, Baala L, Cluzeau C, El Kerch F, Ouldim K, Hadj-Rabia S, Bodemer C, Munnich A, Courtois G, Sefiani A, Smahi A (2007). Autosomal dominant anhidrotic ectodermal dysplasias at the EDARADD locus. Hum Mutat 28: 703- 709
Banner DW, D'Arcy A, Janes W, Gentz R, Schoenfeld HJ, Broger C, Loetscher H, Lesslauer W (1993). Crystal structure of the soluble human 55 kd TNF receptor-
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 172 Chapter 6 References
human TNF beta complex: implications for TNF receptor activation. Cell 73: 431- 445
Barbareschi M, Cambiaghi S, Crupi AC, Tadini G (1997). Family with 'pure' hair- nail ectodermal dysplasia. Am J Med Genet 72: 91-93
Basel-Vanagaite L, Attia R, Ishida-Yamamoto A, Rainshtein L, Ben Amitai D, Lurie R, Pasmanik-Chor M, Indelman M, Zvulunov A, Saban S, Magal N, Sprecher E, Shohat M (2007). Autosomal recessive ichthyosis with hypotrichosis caused by a mutation in ST14, encoding type II transmembrane serine protease matriptase. Am J Hum Genet 80: 467-477
Basu S, Stjernschantz J (1997). Delta13-reductase dependent metabolism of prostaglandins in the mammalian brain and eye. Prostaglandins Leukot Essent Fatty Acids 57: 305-310
Bayés M, Hartung AJ, Ezer S, Pispa J, Thesleff I, Srivastava AK, Kere J (1998). The anhidrotic ectodermal dysplasia gene (EDA) undergoes alternative splicing and encodes ectodysplasin-A with deletion mutations in collagenous repeats. Hum Mol Genet 7: 1661-1669
Benke PJ, Hajianpour MJ (1985). Alopecia universalis-mental retardation is an autosomal recessive syndrome disorder. Am J Hum Genet 37: A44
Bennett RL, Steinhaus KA, Uhrich SB, O'Sullivan CK, Resta RG, Lochner-Doyle D, Markel DS, Vincent V, Hamanishi J (1995). Recommendations for standardized human pedigree nomenclature. Pedigree standardization task force of the national society of genetic counselors. Am J Hum Genet 56: 745-752
Bergmann C, Senderek J, Anhuf D, Thiel CT, Ekici AB, Poblete-Gutierrez P, van Steensel M, Seelow D, Nürnberg G, Schild HH, Nürnberg P, Reis A, Frank J, Zerres K (2006). Mutations in the gene encoding the Wnt-signaling component R-spondin 4 (RSPO4) cause autosomal recessive anonychia. Am J Hum Genet 79: 1105-1109
Betz RC, Indelman M, Pforr J, Schreiner F, Bauer R, Bergman R, Lentze JM, Nothen MM, Cichon S, Sprecher E (2007). Identification of mutations in the human hairless gene in two new families with congenital atrichia. Arch Dermatol Res 299: 157-161
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 173 Chapter 6 References
Betz RC, Lee YA, Bygum A, Brandrup F, Bernal AI, Toribio J, Alvarez JI, Kukuk GM, Ibsen HH, Rasmussen HB, Wienker TF, Reis A, Propping P, Kruse R, Cichon S, Nothen MM (2000). A gene for hypotrichosis simplex of the scalp maps to chromosome 6p21.3. Am J Hum Genet 66: 1979-1983
Blaydon DC, Ishii Y, O'Toole EA, Unsworth HC, Teh MT, Rüschendorf F, Sinclair C, Hopsu-Havu VK, Tidman N, Moss C, Watson R, de Berker D, Wajid M, Christiano AM, Kelsell DP (2006). The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat Genet 38: 1245-1247
Boggon TJ, Murray J, Chappuis-Flament S, Wong E, Gumbiner BM, Shapiro L (2002). C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science 296: 1308-1313
Bosley TM, Alorainy IA, Salih MA, Aldhalaan HM, Abu-Amero KK, Oystreck DT, Tischfield MA, Engle EC, Erickson RP (2008). The clinical spectrum of homozygous HOXA1 mutations. Am J Med Genet A 146: 1235-1240
Breyer MD, Breyer RM (2000). Prostaglandin E receptors and the kidney. Am J Physiol Renal Physiol 279: F12-23
Bühler EM, Bühler UK, Beutler C, Fessler R (1987). A final word on the tricho-rhino- phalangeal syndromes. Clin Genet 31: 273-275
Byrne C, Hardman M, Nield K (2003). Covering the limb--formation of the integument. J Anat 202: 113-123
Calzavara-Pinton P, Carlino A, Benetti A, De Panfilis G (1991). Pili torti and onychodysplasia. Report of a previously undescribed hidrotic ectodermal dysplasia. Dermatologica 182: 184-187
Celli J, Duijf P, Hamel BC, Bamshad M, Kramer B, Smits AP, Newbury-Ecob R, Hennekam RC, Van Buggenhout G, van Haeringen A, Woods CG, van Essen AJ, de Waal R, Vriend G, Haber DA, Yang A, McKeon F, Brunner HG, van Bokhoven H (1999). Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99: 143-153
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 174 Chapter 6 References
Chassaing N, Bourthoumieu S, Cossee M, Calvas P, Vincent MC (2006). Mutations in EDAR account for one-quarter of non-ED1-related hypohidrotic ectodermal dysplasia. Hum Mutat 27: 255-259
Chavan AJ, Ensor CM, Wu P, Haley BE, Tai HH (1993). Photoaffinity labeling of human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase with [alpha-32P]2N3NAD+. Identification of a peptide in the adenine ring binding domain. J Biol Chem 268: 16437-16442
Chen H, Lun Y, Ovchinnikov D, Kokubo H, Oberg KC, Pepicelli CV, Gan L, Lee B, Johnson RL (1998). Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome. Nat Genet 19: 51-55
Chen T, Li Q, Xu J, Ding K, Wang Y, Wang W, Li S, Shen Y (2007). Mutation screening of BMP4, BMP7, HOXA4 and HOXB6 genes in Chinese patients with hypospadias. Eur J Hum Genet 15: 23-28
Chuong CM, Nickoloff BJ, Elias PM, Goldsmith LA, Macher E, Maderson PA, Sundberg JP, Tagami H, Plonka PM, Thestrup-Pederson K, Bernard BA, Schröder JM, Dotto P, Chang CM, Williams ML, Feingold KR, King LE, Kligman AM, Rees JL, Christophers E (2002). What is the 'true' function of skin? Exp Dermatol 11: 159-187
Chuong CM, Widelitz RB, Ting-Berreth S, Jiang TX (1996). Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance. J Invest Dermatol 107: 639-646
Cichon S, Anker M, Vogt IR, Rohleder H, Putzstuck M, Hillmer A, Farooq SA, Al- Dhafri KS, Ahmad M, Haque S, Rietschel M, Propping P, Kruse R, Nothen MM (1998). Cloning, genomic organization, alternative transcripts and mutational analysis of the gene responsible for autosomal recessive universal congenital alopecia. Hum Mol Genet 7: 1671-1679
Clark RA (1985). Cutaneous tissue repair: basic biologic considerations. I. J Am Acad Dermatol 13: 701-725
Clarke A, Phillips DI, Brown R, Harper PS (1987). Clinical aspects of X-linked hypohydrotic ectodermal dysplasia. Arch Dis Child 10: 989-996
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 175 Chapter 6 References
Coggins KG, Latour A, Nguyen MS, Audoly L, Coffman TM, Koller BH (2002). Metabolism of PGE2 by prostaglandin dehydrogenase is essential for remodeling the ductus arteriosus. Nat Med 8: 91-92
Cottingham Jr RW, Indury RM, Schaffer AA (1993). Faster sequential genetic linkage computation. Am J Hum Genet 53: 252-263
Cui CY, Durmowicz M, Tanaka TS, Hartung AJ, Tezuka T, Hashimoto K, Ko MS, Srivastava AK, Schlessinger D (2002). EDA targets revealed by skin gene expression profiles of wild-type, Tabby and Tabby EDA-A1 transgenic mice. Hum Mol Genet 11: 1763-1773
Davalos NO, Garcia-Vargas A, Pforr J, Davalos IP, Picos-Cardenas VJ, Garcia-Cruz D, Kruse R, Figuera LE, Nothen MM, Betz RC (2005). A non-sense mutation in the corneodesmosin gene in a Mexican family with hypotrichosis simplex of the scalp. Br J Dermatol 153: 1216-1219
Dawber R, de Berker D, Baran, R (2001). In. Diseases of the nails and their management. 3rd edition. Oxford: Blackwell Science
Döffinger R, Smahi A, Bessia C, Geissmann F, Feinberg J, Durandy A, Bodemer C, Kenwrick S, Dupuis-Girod S, Blanche S, Wood P, Rabia SH, Headon DJ, Overbeek PA, Le Deist F, Holland SM, Belani K, Kumararatne DS, Fischer A, Shapiro R, Conley ME, Reimund E, Kalhoff H, Abinun M, Munnich A, Israël A, Courtois G, Casanova JL (2001). X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling. Nat Genet 27: 277-285
Dreyer SD, Zhou G, Baldini A, Winterpacht A, Zabel B, Cole W, Johnson RL, Lee B (1998). Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail patella syndrome. Nat Genet 19: 47-50
Ellis RWB, Van Creveld S (1940). A syndrome characterized by ectodermal dysplasia, polydactyly, chondro-dysplasia and congenital morbus cordis: report of three cases. Arch Dis Child 15: 65-84
Ensor CM, Tai HH (1994). Bacterial expression and site-directed mutagenesis of two critical residues (tyrosine-151 and lysine-155) of human placental NAD(+)-
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 176 Chapter 6 References
dependent 15-hydroxyprostaglandin dehydrogenase. Biochim Biophys Acta 1208: 151-156
Ensor CM, Tai HH (1995). 15-Hydroxyprostaglandin dehydrogenase. J Lipid Mediat Cell Signal 12: 313-319
Ensor CM, Tai HH (1996). Site-directed mutagenesis of the conserved serine 138 of human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine results in an inactive enzyme. Biochem Biophys Res Commun 220: 330- 333
Eteson DJ, Clark RD (1988). A new autosomal dominant tricho-dental dysplasia. March of Dimes Birth Defects Conference, Baltimore, 10-13 July
Frank J, Pignata C, Panteleyev AA, Prowse DM, Baden H, Weiner L, Gaetaniello L, Ahmad W, Pozzi N, Caerhalmi-Friedman PB, Aita VM, Uyttendaele H, Gordon D, Ott J, Brissette JL, Christiano AM (1999). Exposing the human nude phenotype. Nature 398: 473-474
Freire-Maia N (1971). Ectodermal dysplasias. Hum Hered 21: 309-312
Freire-Maia N (1977). Ectodermal dysplasias revisited. Acta Genet Med Gemellol 26: 121-131
Freire-Maia N, Lisboa-Costa T, Pagnan NA (2001). Ectodermal dysplasias: how many? Am J Med Genet 104: 84
Freire-Maia N, Pinheiro M (1982). Carrier detection in Christ-Siemens-Touraine syndrome (X-linked hypohidrotic ectodermal dysplasia). Am J Hum Genet 34: 672-674
Freire-Maia N, Pinheiro M (1988). Ectodermal dysplasias--some recollections and a classification. Birth Defects Orig Artic Ser 24: 3-14
Fuchs E (2007). Scratching the surface of skin development. Nature 445: 834-842
Galdzicka M, Patnala S, Hirshman MG, Cai JF, Nitowsky H, Egeland JA, Ginns EI (2002). A new gene, EVC2, is mutated in Ellis-van Creveld syndrome. Mol Genet Metab 77: 291-295
Gehring WJ (1993). Exploring the homeobox. Gene 135: 215-221
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 177 Chapter 6 References
Gentile M, Fiorente P, Buonadonna AL, Macina F, Cariola F (2003). A novel mutation in exon 7 in a family with mild tricho-rhino-phalangeal syndrome type I. Clin Genet 63: 166-167
Giansanti JS, Long SM, Rankin JL (1974). The "tooth and nail" type of autosomal dominant ectodermal dysplasia. Oral Surg Oral Med Oral Pathol 37: 576-582
Gonzalez-Huerta LM, Cuevas-Covarrubias SA, Messina-Baas OM (2007). Tricho- rhino-phalangeal type I syndrome and mental retardation: identification of a novel mutation in the TRPS1 gene. J Dermatol Sci 48: 61-63
Gudbjartsson DF, Thorvaldsson T, Kong A, Gunnarsson G, Ingolfsdottir A (2005). Allegro version 2. Nat Genet 37: 1015-1016
Hamm H, Karl S, Bröcker EB (2000). Isolated congenital nail dysplasia: a new autosomal dominant condition. Arch Dermatol 136: 1239-1243
Harrison S, Sinclair R (2004). Hypotrichosis and nail dysplasia: a novel hidrotic ectodermal dysplasia. Austral J Dermatol 45: 103-105
Hart TC, Bowden DW, Bolyard J, Kula K, Hall K, Wright JT (1997). Genetic linkage of the tricho-dento-osseous syndrome to chromosome 17q21. Hum Mol Genet 6: 2279-2284
He W, Cowin P, Stokes DL (2003). Untangling desmosomal knots with electron tomography. Science 302: 109-113
Headon DJ, Emmal SA, Ferguson BM, Tucker AS, Justice MJ, Sharpe PT, Zonana J, Overbeek PA (2001). Gene defect in ectodermal dysplasia implicates a death domain adapter in development. Nature 414: 913-916
Headon DJ, Overbeek PA (1999). Involvement of a novel Tnf receptor homologue in hair follicle induction. Nat Genet 22: 370-374
Healy E, Holmes SC, Belgaid CE, Stephenson AM, Mclean WH, Rees JL, Munro CS (1995). A gene for monilethrix is closely linked to the type II keratin gene cluster at 12q13. Hum Mol Genet 4: 2399-2402
Herzog H, Darby K, Hort YJ, Shine J (1996). Intron 17 of the human retinoblastoma susceptibility gene encodes an actively transcribed G protein-coupled receptor gene. Genome Res 6: 858-861
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 178 Chapter 6 References
Hilton MJ, Sawyer JM, Gutiérrez L, Hogart A, Kung TC, Wells DE (2002). Analysis of novel and recurrent mutations responsible for the tricho-rhino-phalangeal syndromes. J Hum Genet 47: 103-106
Höhl W, Stahl B, Mundkowski R, Hofmann U, Meese CO, Kuhlmann U, Schlegel W (1993). Mass determination of 15-hydroxyprostaglandin dehydrogenase from human placenta and kinetic studies with (5Z, 8E, 10E, 12S)-12-hydroxy-5,8,10- heptadecatrienoic acid as substrate. Eur J Biochem 214: 67-73
Hooper JD, Clements JA, Quigley JP, Antalis TM (2001). Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes. J Biol Chem 276: 857-860
Horev L, Djabali K, Green J, Sinclair R, Martinez-Mir A, Ingber A, Christiano AM, Zlotogorski A (2003). De novo mutations in monilethrix. Exp Dermatol 12: 882- 885
Horsfall Jr FL (1936). Congenital familial clubbing of the fingers and toes. Can Med Assoc J 34: 145-149
Hsieh JC, Sisk JM, Jurutka PW, Haussler CA, Slater SA, Haussler MR, Thompson CC (2003). Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling. J Biol Chem 278: 38665-38674
Hu JC, Simmer JP (2007). Developmental biology and genetics of dental malformations. Orthod Craniofac Res 10: 45-52
Huang C, Yang Q, Ke T, Wang H, Wang X, Shen J, Tu X, Tian J, Liu JY, Wang QK, Liu M (2006). A novel de novo frame-shift mutation of the EDA gene in a Chinese Han family with hypohidrotic ectodermal dysplasia. J Hum Genet 51: 1133-1137
Huber O (2003). Structure and function of desmosomal proteins and their role in development and disease. Cell Mol Life Sci 60: 1872-1890
Hudson CD, Witkop CJ (1975). Autosomal dominant hypodontia with nail dysgenesis. Report of twenty-nine cases in six families. Oral Surg Oral Med Oral Pathol 39: 409-423
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 179 Chapter 6 References
Hymowitz SG, Compaan DM, Yan M, Wallweber HJ, Dixit VM, Starovasnik MA, de Vos AM (2003). The crystal structure of EDA-A1 and EDA-A2: Splice Variants with distinct receptor specificity. Structure 11: 1513-1520
Indelman M, Bergman R, Lurie R, Richard G, Miller B, Petronius D, Ciubutaro D, Leibu R, Sprecher E (2002). A missense mutation in CDH3, encoding P-cadherin, causes hypotrichosis with juvenile macular dystrophy. J Invest Dermatol 119: 1210-1213
Indelman M, Eason J, Hummel M, Loza O, Suri M, Leys MJ, Bayne M, Schwartz FL, Sprecher E (2007). Novel CDH3 mutations in hypotrichosis with juvenile macular dystrophy. Clin Exp Dermatol 32: 191-196
Indelman M, Hamel CP, Bergman R, Nischal KK, Thompson D, Surget MO, Ramon M, Ganthos H, Miller B, Richard G, Lurie R, Leibu R, Russell-Eggitt I, Sprecher E (2003). Phenotypic diversity and mutation spectrum in hypotrichosis with juvenile macular dystrophy. J Invest Dermatol 121: 1217-1220
Indelman M, Leibu R, Jammal A, Bergman R, Sprecher E (2005). Molecular basis of hypotrichosis with juvenile macular dystrophy in two siblings. Br J Dermatol 153: 635-638
International Human Genome Sequence Consortium (2001). Initial sequence and analysis of the human genome. Nature 409: 860-921
Islam M, Lurie AG, Reichenberger E (2005). Clinical features of tricho-dento-osseous syndrome and presentation of three new cases: an addition to clinical heterogeneity. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 100: 736-742
Itin PH, Fistarol SK (2004). Ectodermal dysplasias. Am J Med Genet 131C: 45-51
Itoh S, Kanno S, Gai Z, Suemoto H, Kawakatsu M, Tanishima H, Morimoto Y, Nishioka K, Hatamura I, Yoshida M, Muragaki Y (2008). Trps1 plays a pivotal role downstream of Gdf5 signaling in promoting chondrogenesis and apoptosis of ATDC5 cells. Genes Cells 13: 355-363
Jahoda CA, Kljuic A, O'Shaughnessy R, Crossley N, Whitehouse CJ, Robinson M, Reynolds AJ, Demarchez M, Porter RM, Shapiro L, Christiano AM (2004). The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene. Genomics 83: 747-756
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 180 Chapter 6 References
Jajic Z, Jajic I, Nemcic T (2001). Primary hypertrophic osteoarthropathy: clinical, radiologic, and scintigraphic characteristics. Arch Med Res 32: 136-142
Jakobsson PJ, Thorén S, Morgenstern R, Samuelsson B (1999). Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci USA 96: 7220-7225
Jamora C, DasGupta R, Kocieniewski P, Fuchs E (2003). Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422: 317-22 Erratum in: Nature (2003). 424: 974
Jelani M, Chisthi MS, Ahmad W (2008). A novel splice-site mutation in the CDH3 gene in hypotrichosis with juvenile macular dystrophy. Clin Exp Dermatol (In press)
Jelani M, Wasif N, Ali G, Chishti MS, Ahmad W (2008). A novel deletion mutation in LIPH gene causes autosomal recessive hypotrichosis (LAH2). Clin Genet 74: 184- 188
John P, Ali G, Chishti MS, Naqvi SM, Leal SM, Ahmad W (2006a). Localization of a novel locus for alopecia with mental retardation syndrome to chromosome 3q26.33-q27.3. Hum Genet 118: 665-667
John P, Aslam M, Rafiq MA, Amin-ud-din M, Haque S, Ahmad W (2005). Atrichia with papular lesions in two Pakistani families resulting from mutations in the human hairless gene. Arch Dermatol Res 297: 226-230
John P, Tariq M, Arshad Rafiq M, Amin-Ud-Din M, Muhammad D, Waheed I, Ansar M, Ahmad W (2006b). Recurrent intragenic deletion mutation in desmoglein 4 gene underlies autosomal recessive hypotrichosis in two Pakistani families of Balochi and Sindhi origins. Arch Dermatol Res 298: 13135-13137
Jörnvall H, Persson B, Krook M, Atrian S, Gonzàlez-Duarte R, Jeffery J, Ghosh D (1995). Short-chain dehydrogenases/reductases (SDR). Biochemistry 34: 6003- 6013
Jumlongras D, Bei M, Stimson JM, Wang WF, DePalma SR, Seidman CE, Felbor U, Maas R, Seidman JG, Olsen BR (2001). A nonsense mutation in MSX1 causes Witkop syndrome. Am J Hum Genet 69: 67-74
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 181 Chapter 6 References
Kaiser FJ, Brega P, Raff ML, Byers PH, Gallati S, Kay TT, de Almeida S, Horsthemke B, Lüdecke HJ (2004). Novel missense mutations in the TRPS1 transcription factor define the nuclear localization signal. Eur J Hum Genet 12: 121-126
Kajii T, Fernandez Gonzalez I, Matsuura S (1994). Tricho-rhino-phalangeal syndrome type III. Am J Med Genet 49: 349-350
Kapadia H, Mues G, D'Souza R (2007). Genes affecting tooth morphogenesis. Orthod Craniofac Res 10: 237-244
Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C, Wu W (2004). R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev Cell 7: 525-534
Kazantseva A, Goltsov A, Zinchenko R, Grigorenko AP, Abrukova AV, Moliaka YK, Kirillov AG, Guo Z, Lyle S, Ginter EK, Rogaev EI (2006). Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH. Science 314: 982-985
Kere J, Srivastava AK, Montonen O, Zonana J, Thomas N, Ferguson B, Munoz F, Morgan D, Clarke A, Baybayan P, Chen EY, Ezer S, Saarialho-Kere U, de la Chapelle A, Schlessinger D (1996). X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat Genet 13: 409 -416
Kersey PJ (1987). Tricho-dental syndrome: a disorder with a short hair cycle. Br J Dermatol 116: 259-263
Kim H, Wajid M, Kraemer L, Shimomura Y, Christiano AM (2007). Nonsense mutations in the hairless gene underlie APL in five families of Pakistani origin. J Dermatol Sci 48: 207-211
Kim KA, Zhao J, Andarmani S, Kakitani M, Oshima T, Binnerts ME, Abo A, Tomizuka K, Funk WD (2006). R-Spondin proteins: a novel link to beta-catenin activation. Cell Cycle 5: 23-26
Kljuic A, Bazzi H, Sundberg JP, Martinez-Mir A, O'Shaughnessy R, Mahoney MG, Levy M, Montagutelli X, Ahmad W, Aita VM, Gordon D, Uitto J, Whiting D, Ott J, Fischer S, Gilliam TC, Jahoda CA, Morris RJ, Panteleyev AA, Nguyen VT, Christiano AM (2003). Desmoglein 4 in hair follicle differentiation and epidermal
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 182 Chapter 6 References
adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris. Cell 113: 249-260
Kobayashi H, Hino M, Shimodahira M, Iwakura T, Ishihara T, Ikekubo K, Ogawa Y, Nakao K, Kurahachi H (2002). Missense mutation of TRPS1 in a family of tricho- rhino-phalangeal syndrome type III. Am J Med Genet 107: 26-29
Kömüves LG, Shen WF, Kwong A, Stelnicki E, Rozenfeld S, Oda Y, Blink A, Krishnan K, Lau B, Mauro T, Largman C (2000). Changes in HOXB6 homeodomain protein structure and localization during human epidermal development and differentiation. Dev Dyn 218: 636-647
Kong A, Gudbjartsson DF, Sainz J, Jonsdottir GM, Gudjonsson SA, Richardsson B, Sigurdardottir S, Barnard J, Hallbeck B, Masson G, Shlien A, Palsson ST, Frigge ML, Thorgeirsson TE, Gulcher JR, Stefansson K (2002). A high-resolution recombination map of the human genome. Nat Genet 31: 241-247
Kong X, Murphy K, Raj T, He C, White PS, Matise TC (2004). A combined linkage- physical map of the human genome. Am J Hum Genet 75: 1143-1148
Kools P, Van Imschoot G, van Roy F (2000). Characterization of three novel human cadherin genes (CDH7, CDH19, and CDH20) clustered on chromosome 18q22- q23 and with high homology to chicken cadherin-7. Genomics 68: 283-295
Koppinen P, Pispa J, Laurikkala J, Thesleff I, Mikkola ML (2001). Signaling and subcellular localization of the TNF receptor Edar. Exp Cell Res 269: 180-192
Korge BP, Hamm H, Jury CS, Traupe H, Irvine AD, Healy E, Birch-MacHin M, Rees JL, Messenger AG, Holmes SC, Parry DA, Munro CS (1999). Identification of novel mutations in basic hair keratins hHb1 and hHb6 in monilethrix: implications for protein structure and clinical phenotype. J Invest Dermatol 113: 607-612
Kraemer L, Wajid M, Shimomura Y, Christiano AM (2008). Mutations in the hairless gene underlie APL in three families of Pakistani origin. J Dermatol Sci 50: 25-30
Krebsová A, Hamm H, Karl S, Reis A, Hennies HC (2000). Assignment of the gene for a new hereditary nail disorder, isolated congenital nail dysplasia, to chromosome 17p13. J Invest Dermatol 115: 664-667
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 183 Chapter 6 References
Krook M, Ghosh D, Duax W, Jörnvall H (1993). Three-dimensional model of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase and relationships to the NADP(+)-dependent enzyme (carbonyl reductase). FEBS Lett 322: 139-142
Krumlauf R (1994). Hox genes in vertebrate development. Cell 78: 191-201
Kruse R, Cichon S, Anker M, Hillmer AM, Barros-Núñez P, Cantú JM, Leal E, Weinlich G, Schmuth M, Fritsch P, Ruzicka T, Propping P, Nöthen MM (1999). Novel Hairless mutations in two kindreds with autosomal recessive papular atrichia. J Invest Dermatol 113: 954-959
Kumar A, Eby MT, Sinha S, Jasmin A, Chaudhary PM (2001). The ectodermal dysplasia receptor activates the nuclear factor-kappaB, JNK, and cell death pathways and binds to ectodysplasin A. J Biol Chem 276: 2668-2677
Kunath M, Lüdecke HJ, Vortkamp A (2002). Expression of Trps1 during mouse embryonic development. Gene Expr Patterns 2: 119-122
Lamartine J (2003). Towards a new classification of ectodermal dysplasias. Clin Exp Dermatol 28: 351-355
Lander ES, Botstein D (1987). Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children. Science 236: 1567-1570
Leech SN, Moss C (2007). A current and online genodermatosis database. Br J Dermatol 156: 1115-1148
Levy-Nissenbaum E, Betz RC, Frydman M, Simon M, Lahat H, Bakhan T, Goldman B, Bygum A, Pierick M, Hillmer AM, Jonca N, Toribio J, Kruse R, Dewald G, Cichon S, Kubisch C, Guerrin M, Serre G, Nothen MM, Pras E (2003). Hypotrichosis simplex of the scalp is associated with nonsense mutations in CDSN encoding corneodesmosin. Nat Genet 34: 151-153
Lexner MO, Bardow A, Juncker I, Jensen LG, Almer L, Kreiborg S, Hertz JM (2008). X-linked hypohidrotic ectodermal dysplasia. Genetic and dental findings in 67 Danish patients from 19 families. Clin Genet 74: 252-259
Li M, Xu TY, Yang LJ, Zhu XH (2008). A novel 22-bp deletion mutation in a Chinese family with X-linked hypohidrotic ectodermal dysplasia. Arch Dermatol Res 300: 389-391
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 184 Chapter 6 References
Lind LK, Stecksén-Blicks C, Lejon K, Schmitt-Egenolf M (2006). EDAR mutation in autosomal dominant hypohidrotic ectodermal dysplasia in two Swedish families. BMC Med Genet 7: 80
Liu F, Chu EY, Watt B, Zhang Y, Gallant NM, Andl T, Yang SH, Lu MM, Piccolo S, Schmidt-Ullrich R, Taketo MM, Morrisey EE, Atit R, Dlugosz AA, Millar SE (2008). Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis. Dev Biol 313: 210-224
Locksley RM, Killeen N, Lenardo MJ (2001). The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104: 487-501
Loomis CA, Harris E, Michaud J, Wurst W, Hanks M, Joyner AL (1996). The mouse Engrailed-1 gene and ventral limb patterning. Nature 382: 360-363
Lüdecke HJ, Schaper J, Meinecke P, Momeni P, Gross S, von Holtum D, Hirche H, Abramowicz MJ, Albrecht B, Apacik C, Christen HJ, Claussen U, Devriendt K, Fastnacht E, Forderer A, Friedrich U, Goodship TH, Greiwe M, Hamm H, Hennekam RC, Hinkel GK, Hoeltzenbein M, Kayserili H, Majewski F, Mathieu M, McLeod R, Midro AT, Moog U, Nagai T, Niikawa N, Orstavik KH, Plöchl E, Seitz C, Schmidtke J, Tranebjaerg L, Tsukahara M, Wittwer B, Zabel B, Gillessen-Kaesbach G, Horsthemke B (2001). Genotypic and phenotypic spectrum in tricho-rhino-phalangeal syndrome types I and III. Am J Hum Genet 68: 81-91
Maas SM, de Jong TP, Buss P, Hennekam RC (1996). EEC syndrome and genitourinary anomalies: an update. Am J Med Genet 63: 472-478
Malik TH, Shoichet SA, Latham P, Kroll TG, Peters LL, Shivdasani RA (2001). Transcriptional repression and developmental functions of the atypical vertebrate GATA protein TRPS1. EMBO J 20: 1715-1725
Mazzarella R, Schlessinger D (1998). Pathological consequences of sequences duplications in the human genome. Genome Res 8: 1007-1021
McGrath JA, Wessagowit V (2005). Human hair abnormalities resulting from inherited desmosome gene mutations. Keio J Med 54: 72-79
Mégarbané H, Haddad M, Delague V, Renoux J, Boehm N, Mégarbané A (2004). Further delineation of the odonto-onycho-dermal dysplasia syndrome. Am J Med Genet A 129: 193-197
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 185 Chapter 6 References
Messenger AG, Bazzi H, Parslew R, Shapiro L, Christiano AM (2005). A missense mutation in the cadherin interaction site of the desmoglein 4 gene underlies localized autosomal recessive hypotrichosis. J Invest Dermatol 125: 1077-1079
Michailidis E, Theos A, Zlotogorski A, Martinez-Mir A, Christiano AM (2007). Atrichia with papular lesions resulting from novel compound heterozygous mutations in the human hairless gene. Pediatr Dermatol 24: E79-82
Momeni P, Glöckner G, Schmidt O, von Holtum D, Albrecht B, Gillessen-Kaesbach G, Hennekam R, Meinecke P, Zabel B, Rosenthal A, Horsthemke B, Lüdecke HJ (2000). Mutations in a new gene, encoding a zinc-finger protein, cause tricho- rhino-phalangeal syndrome type I. Nat Genet 24: 71-74
Monreal AW, Ferguson BM, Headon DJ, Street SL, Overbeek PA, Zonana J (1999). Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia. Nat Genet 22: 366-369
Moss C, Martinez-Mir A, Lam H, Tadin-Strapps M, Kljuic A, Christiano AM (2004). A recurrent intragenic deletion in the desmoglein 4 gene underlies localized autosomal recessive hypotrichosis. J Invest Dermatol 123: 607-610
Myers KA, Farquhar DR (2001). The rational clinical examination. Does this patient have clubbing? JAMA 286: 341-347
Naeem M, Jelani M, Lee K, Ali G, Chishti MS, Wali A, Gul A, John P, Hassan MJ, Leal SM, Ahmad W (2006b). Ectodermal dysplasia of hair and nail type: mapping of a novel locus to chromosome 17p12-q21.2. Br J Dermatol 155: 1184-1190
Naeem M, John P, Ali G, Ahmad W (2007). Pure hair-nail ectodermal dysplasia maps to chromosome 12p11.1-q21.1 in a consanguineous Pakistani family. Clin Exp Dermatol 32: 502-505
Naeem M, Muhammad D, Ahmad W (2005). Novel mutations in the EDAR gene in two Pakistani consanguineous families with autosomal recessive hypohidrotic ectodermal dysplasia. Br J Dermatol 15: 346-350
Naeem M, Wajid M, Lee K, Leal SM, Ahmad W (2006a). A mutation in the hair matrix and cuticle keratin KRTHB5 gene causes ectodermal dysplasia of hair and nail type. J Med Genet 43: 274-279
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 186 Chapter 6 References
Nakamura T, de Vega S, Fukumoto S, Jimenez L, Unda F, Yamada Y (2008). Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number. J Biol Chem 283: 4825-4833
Naqvi SK, Raza SI, Naveed AK, John P, Ahmad W (2008). A novel deletion mutation in the phospholipase H (LIPH) gene in a consanguineous Pakistani family with autosomal recessive hypotrichosis (LAH2). Br J Dermatol (In press)
Narumiya S, Sugimoto Y, Ushikubi F (1999). Prostanoid receptors: structures, properties, and functions. Physiol Rev 79: 1193-1226
Nehls M, Pfeifer D, Schorpp M, Hedrich H, Boehm T (1994). New member of the winged-helix protein family disrupted in mouse and rat nude mutations. Nature 372: 103-107
Niikawa N, Kamei T (1986). The Sugio-Kajii syndrome, proposed tricho-rhino- phalangeal syndrome type III. Am J Med Genet 24: 759-760
Nomura T, Lu R, Pucci ML, Schuster VL (2004). The two-step model of prostaglandin signal termination: in vitro reconstitution with the prostaglandin transporter and prostaglandin 15 dehydrogenase. Mol Pharmacol 65: 973-978
Nothen MM, Cichon S, Vogt IR, Hemmer S, Kruse R, Knapp M, Holler T, Faiyaz ul Haque M, Haque S, Propping P, Ahmad M, Rietschel M (1998). A gene for universal congenital alopecia maps to 8p21-22. Am J Hum Genet 62: 386-390
O’Connell JR, Weeks DE (1998). PedCheck: a program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet 63: 259-266
Okita RT, Okita JR (1996). Prostaglandin-metabolizing enzymes during pregnancy: characterization of NAD(+)-dependent prostaglandin dehydrogenase, carbonyl reductase, and cytochrome P450-dependent prostaglandin omega-hydroxylase. Crit Rev Biochem Mol Biol 31: 101-126
Ota T, Gilks CB, Longacre T, Leung PC, Auersperg N (2007). HOXA7 in epithelial ovarian cancer: interrelationships between differentiation and clinical features. Reprod Sci 14: 605-614
Pääkkönen K, Cambiaghi S, Novelli G, Ouzts LV, Penttinen M, Kere J, Srivastava AK (2001). The mutation spectrum of the EDA gene in X-linked anhidrotic ectodermal dysplasia. Hum Mutat 17: 349
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 187 Chapter 6 References
Paradisi M, Chuang GS, Angelo C, Pedicelli C, Martinez-Mir A, Christiano AM (2003). Atrichia with papular lesions resulting from a novel homozygous missense mutation in the hairless gene. Clin Exp Dermatol 28: 535-538
Pasternack SM, von Kügelgen I, Aboud KA, Lee YA, Rüschendorf F, Voss K, Hillmer AM, Molderings GJ, Franz T, Ramirez A, Nürnberg P, Nöthen MM, Betz RC (2008). G protein-coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth. Nat Genet 40: 329-334
Pichaud F, Frendo JL, Delage-Mourroux R, de Vernejoul MC, Moukhtar MS, Jullienne A (1995). Sequence of a novel mRNA coding for a C-terminal-truncated form of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase. Gene 162: 319-322
Pignata C, Fiore M, Guzzetta V, Castaldo A, Sebastio G, Porta F, Guarino A (1996). Congenital Alopecia and nail dystrophy associated with severe functional T-cell immunodeficiency in two sibs. Am J Med Genet 65: 167-170
Pinheiro M, Freire-Maia N (1979). Christ-Siemens-Touraine syndrome--a clinical and genetic analysis of a large Brazilian kindred: I. Affected females. Am J Med Genet 4: 113-122
Pinheiro M, Freire-Maia N (1987). Trichodysplasia-xeroderma: an autosomal dominant condition. Clin Genet 31: 337-342
Pinheiro M, Freire-Maia N (1992). Hair-nail dysplasia--a new pure autosomal dominant ectodermal dysplasia. Clin Genet 41: 296-298
Pinheiro M, Freire-Maia N (1994). Ectodermal dysplasias: a clinical classification and a causal review. Am J Med Genet 53: 153-162
Pispa J, Thesleff I (2003). Mechanisms of ectodermal organogenesis. Dev Biol 262: 195-205
Potter GB, Beaudoin GM 3rd, DeRenzo CL, Zarach JM, Chen SH, Thompson CC (2001). The hairless gene mutated in congenital hair loss disorders encodes a novel nuclear receptor corepressor. Genes Dev 15: 2687-2701
Price JA, Bowden DW, Wright JT, Pettenati MJ, Hart TC (1998b). Identification of a mutation in DLX3 associated with tricho-dento-osseous (TDO) syndrome. Hum Mol Genet 7: 563-569
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 188 Chapter 6 References
Price JA, Wright JT, Kula K, Bowden DW, Hart TC (1998a). A common DLX3 gene mutation is responsible for tricho-dento-osseous syndrome in Virginia and North Carolina families. J Med Genet 35: 825-828
Priolo M, Laganà C (2001). Ectodermal dysplasias: a new clinical-genetic classification. J Med Genet 38: 579-585
Priolo M, Silengo M, Lerone M, Ravazzolo R (2000). Ectodermal dysplasias: not only 'skin' deep. Clin Genet 58: 415-430
Qumsiyeh MB (1992). EEC syndrome (ectrodactyly, ectodermal dysplasia and cleft lip/palate) is on 7p11.2-q21.3. Clin Genet 42: 101
Rafiq MA, Ansar M, Mahmood S, Haque S, Faiyaz-ul-Haque M, Leal SM, Ahmad W (2004). A recurrent intragenic deletion mutation in DSG4 gene in three Pakistani families with autosomal recessive hypotrichosis. J Invest Dermatol 123: 247-248
Rafiq MA, Ansar M, Pham T, Amin-ud-Din M, Anwar M, Haque S, Chahrour MH, Yan K, Leal SM, Ahmad W (2004). Localization of a novel locus for hereditary nail dysplasia to chromosome 17q25.1-17q25.3. Clin Genet 66: 73-78
Rafiq MA, Faiyaz-Ul-Haque M, Ud Din MA, Malik S, Sohail M, Anwar M, Haque S, Paterson AD, Tsui LC, Ahmad W (2005). A novel locus of ectodermal dysplasia maps to chromosome 10q24.32-q25.1. J Invest Dermatol 124: 338-342
Rafique MA, Ansar M, Jamal SM, Malik S, Sohail M, Faiyaz-Ul-Haque M, Haque S, Leal SM, Ahmad W (2003). A locus for hereditary hypotrichosis localized to human chromosome 18q21.1. Eur J Hum Genet 11: 623-628
RamaDevi AR, Reddy EC, Ranjan S, Bashyam MD (2008). Molecular genetic analysis of patients from India with hypohidrotic ectodermal dysplasia reveals novel mutations in the EDA and EDAR genes. Br J Dermatol 158: 163-167
Reisler TT, Patton MA, Meagher PP (2006). Further phenotypic and genetic variation in ADULT syndrome. Am J Med Genet A 140: 2495-2500
Roelandt T, Kraemer L, Van Neste D, Lissens W, Roseeuw D, Christiano AM, Hachem JP (2008). Novel mutation in the human hairless gene once more erroneously diagnosed and treated as 'alopecia areata'. Br J Dermatol 158: 834- 835
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 189 Chapter 6 References
Ross FP, Christiano AM (2006). Nothing but skin and bone. J Clin Invest 116: 1140- 1149
Rossi A, Devirgiliis V, Panasiti V, Borroni RG, Carlesimo M, Gentile M, Cariola F, Calvieri S (2007). Missense mutation in exon 7 of TRPS1 gene in an Italian family with a mild form of trichorhinophalangeal syndrome type I. Br J Dermatol 157: 1021-1024
Rozen S, Skaletsky HJ (2000). Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana, NJ: pp. 365-386
Ruiz-Perez VL, Ide SE, Strom TM, Lorenz B, Wilson D, Woods K, King L, Francomano C, Freisinger P, Spranger S, Marino B, Dallapiccola B, Wright M, Meitinger T, Polymeropoulos MH, Goodship J (2000). Mutations in a new gene in Ellis-van Creveld syndrome and Weyers acrodental dysostosis. Nat Genet 24: 283-286 Erratum in: Nat Genet (2000). 25: 125
Ruiz-Perez VL, Tompson SW, Blair HJ, Espinoza-Valdez C, Lapunzina P, Silva EO, Hamel B, Gibbs JL, Young ID, Wright MJ, Goodship JA (2003). Mutations in two nonhomologous genes in a head-to-head configuration cause Ellis-van Creveld syndrome. Am J Hum Genet 72: 728-732
Salinas CF, Spector M (1980). Tricho-dental syndrome. In: Brown AC, Crounse RG (eds.): Hair, Trace Elements, and Human Illness. New York: Praeger. pp. 240- 256
Sambrook J, Russell DW (2001). Preparation and analysis of eukaryotic genomic DNA. In: Molecular Cloning: A laboratory manual. Vol. 1. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. pp. 6.4-6.11
Samman DP, Fenton AD (1995). Nail disorders associated with general medical conditions. In: Samman’s The Nails in Disease. 5th edition. Butterworth- Heinemann Ltd. Oxford. pp. 113-114
Sankhyan N, Kaushal RK, Sarin S (2006). Ectodermal dysplasia, ectrodactyly, cleft lip/palate syndrome without ectrodactyly. Dermatol Online J 12: 5
Sartaj R, Sharpe P (2006). Biological tooth replacement. J Anat 209: 503-509
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 190 Chapter 6 References
Sassá Benedete AP, Sobral AP, Lima DM, Kamibeppu L, Soares FA, Lourenço SV (2008). Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: immunolocalization according to the odontogenesis phases. Pediatr Dev Pathol 11: 206-212
Schaffer JV, Bazzi H, Vitebsky A, Witkiewicz A, Kovich OI, Kamino H, Shapiro LS, Amin SP, Orlow SJ, Christiano AM (2006). Mutations in the desmoglein 4 gene underlie localized autosomal recessive hypotrichosis with monilethrix hairs and congenital scalp erosions. J Invest Dermatol 126: 1286-1291
Schneider P, Street SL, Gaide O, Hertig S, Tardivel A, Tschopp J, Runkel L, Alevizopoulos K, Ferguson BM, Zonana J (2001). Mutations leading to X-linked hypohydrotic ectodermal dysplasia affect three major Functional domains in the tumor necrosis factor family member ectodysplasin-A. J Biol Chem 276: 18819- 18827
Schoon DD (2005). In: Nail structure and product chemistry. Fingernail anatomy. 2nd edition Thomson Delmar Learning, Canada: pp. 1-18
Schughart K, Bieberich CJ, Eid R, Ruddle FH (1991). A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs. Development 112: 807-811
Segre JA (2006). Epidermal barrier formation and recovery in skin disorders. J Clin Invest 116: 1150-1158
Segre JA, Nemhauser JL, Taylor BA, Nadeau JH, Lander ES (1995). Positional cloning of the nude locus: genetic, physical, and transcription maps of the region and mutations in the mouse and rat. Genomics 28: 549-559
Shimomura Y, Sakamoto F, Kariya N, Matsunaga K, Ito M (2006). Mutations in the desmoglein 4 gene are associated with monilethrix-like congenital hypotrichosis. J Invest Dermatol 126: 1281-1285
Shimomura Y, Sato N, Miyashita A, Hashimoto T, Ito M, Kuwano R (2004). A rare case of hypohidrotic ectodermal dysplasia caused by compound heterozygous mutations in the EDAR gene. J Invest Dermatol 123: 649-655
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 191 Chapter 6 References
Shimomura Y, Wajid M, Ishii Y, Shapiro L, Petukhova L, Gordon D, Christiano AM (2008). Disruption of P2RY5, an orphan G protein-coupled receptor, underlies autosomal recessive woolly hair. Nat Genet 40: 335-339
Shimomura Y, Wajid M, Shapiro L, Christiano AM (2008). P-cadherin is a p63 target gene with a crucial role in the developing human limb bud and hair follicle. Development 135: 743-753
Smith CA, Farrah T, Goodwin RG (1994). The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76: 959-962
Smith WL, DeWitt DL, Garavito RM (2000). Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 69: 145-182
Sobel E, Lange K (1996). Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics. Am J Hum Genet 58: 1323-1337
Sprecher E, Bergman R, Richard G, Lurie R, Shalev S, Petronius D, Shalata A, Anbinder Y, Leibu R, Perlman I, Cohen N, Szargel R (2001). Hypotrichosis with juvenile macular dystrophy is caused by a mutation in CDH3, encoding P- cadherin. Nat Genet 29: 134-136
Sprecher E, Bergman R, Szargel R, Friedman-Birnbaum R, Cohen N (1999a). Identification of a genetic defect in the hairless gene in atrichia with papular lesions: evidence for phenotypic heterogeneity among inherited atrichias. Am J Hum Genet 64: 1323-1329
Sprecher E, Ishida-Yamamoto A, Mizrahi-Koren M, Rapaport D, Goldsher D, Indelman M, Topaz O, Chefetz I, Keren H, O'brien TJ, Bercovich D, Shalev S, Geiger D, Bergman R, Horowitz M, Mandel H (2005). A mutation in SNAP29, coding for a SNARE protein involved in intracellular trafficking, causes a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma. Am J Hum Genet 77: 242-251
Sprecher E, Lestringant GG, Szargel R, Bergman R, Labay V, Frossard PM, Friedman-Birnbaum R, Cohen N (1999b). Atrichia with papular lesions resulting from a nonsense mutation within the human hairless gene. J Invest Dermatol 113: 687-690
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 192 Chapter 6 References
Srivastava AK, Pispa J, Hartung AJ, Du Y, Ezer S, Jenks T, Shimada T, Pekkanen M, Mikkola ML, Ko MS, Thesleff I, Kere J, Schlessinger D (1997). The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains. Proc Natl Acad Sci USA 94: 13069-13074
Stelnicki EJ, Kömüves LG, Kwong AO, Holmes D, Klein P, Rozenfeld S, Lawrence HJ, Adzick NS, Harrison M, Largman C (1998). HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development. J Invest Dermatol 110: 110-115
Stimson JM, Sivers JE, Hlava GL (1997). Features of oligodontia in three generations. J Clin Pediatr Dent 21: 269-275
Strachen T, Read AP (2004). In: Human Molecular Genetics. Molecular Pathology. 3rd edition. New York: Garland Science, pp. 562-585
Tai HH (1976). Enzymatic synthesis of (15s)-[15-3h] prostaglandins and their use in the development of a simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase. Biochemistry 15: 4586-4592
Tai HH, Cho H, Tong M, Ding Y (2006). NAD+-linked 15-hydroxyprostaglandin dehydrogenase: structure and biological functions. Curr Pharm Des 12: 955-962
Tai HH, Ensor CM, Tong M, Zhou H, Yan F (2002). Prostaglandin catabolizing enzymes. Prostaglandins Other Lipid Mediat 68-69: 483-493
Tariq M, Ahmad S, Ahmad W (2008). A novel missense mutation in the TRPS1 gene underlies trichorhinophalangeal syndrome type III. Br J Dermatol 159: 476-478
Tariq M, Chishti MS, Ali G, Ahmad W (2008). A novel locus for ectodermal dysplasia of hairs, nails and teeth type maps to chromosome 18q22.1-22.3. Ann Hum Genet 72: 19-25
Tariq M, Wasif N, Ahmad W (2007). A novel deletion mutation in the EDAR gene in a Pakistani family with autosomal recessive hypohidrotic ectodermal dysplasia. Br J Dermatol 157: 207-209
Thompson CC, Sisk JM, Beaudoin GM 3rd (2006). Hairless and Wnt signaling: allies in epithelial stem cell differentiation. Cell Cycle 5: 1913-1917
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 193 Chapter 6 References
Tucker A, Sharpe P (2004). The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet 5: 499-508
Uppal S, Diggle CP, Carr IM, Fishwick CW, Ahmed M, Ibrahim GH, Helliwell PS, Latos-Bieleńska A, Phillips SE, Markham AF, Bennett CP, Bonthron DT (2008). Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic osteoarthropathy. Nat Genet 40: 789-793
Urlaub G, Mitchell PJ, Ciudad CJ, Chasin LA (1989). Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol Cell Biol 9: 2868-2880
Valcuende-Cavero F, Martinez F, Pérez-Pastor G, Oltra S, Ferrer I, Tomás-Cabedo G, Moreno-Presmanes M (2008). Autosomal-dominant hypohidrotic ectodermal dysplasia caused by a novel mutation. J Eur Acad Dermatol Venereol 22: 1508- 1510 van Bokhoven H, Hamel BC, Bamshad M, Sangiorgi E, Gurrieri F, Duijf PH, Vanmolkot KR, van Beusekom E, van Beersum SE, Celli J, Merkx GF, Tenconi R, Fryns JP, Verloes A, Newbury-Ecob RA, Raas-Rotschild A, Majewski F, Beemer FA, Janecke A, Chitayat D, Crisponi G, Kayserili H, Yates JR, Neri G, Brunner HG (2001). p63 gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype- phenotype correlation. Am J Hum Genet 69: 481-492 van Bokhoven H, Jung M, Smits AP, van Beersum S, Rüschendorf F, van Steensel M, Veenstra M, Tuerlings JH, Mariman EC, Brunner HG, Wienker TF, Reis A, Ropers HH, Hamel BC (1999). Limb mammary syndrome: a new genetic disorder with mammary hypoplasia, ectrodactyly, and other Hand/Foot anomalies maps to human chromosome 3q27. Am J Hum Genet 64: 538-546 van der Hout AH, Oudesluijs GG, Venema A, Verheij JB, Mol BG, Rump P, Brunner HG, Vos YJ, van Essen AJ (2008). Mutation screening of the Ectodysplasin-A receptor gene EDAR in hypohidrotic ectodermal dysplasia. Eur J Hum Genet 16: 673-679 van Steensel M, Smith FJ, Steijlen PM, Kluijt I, Stevens HP, Messenger A, Kremer H, Dunnill MG, Kennedy C, Munro CS, Doherty VR, McGrath JA, Covello SP, Coleman CM, Uitto J, McLean WH (1999). The gene for hypertrichosis of Marie
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 194 Chapter 6 References
Unna maps between D8S258 and D8S298: exclusion of the hr gene by cDNA and genomic sequencing. Am J Hum Genet 65: 413-419
Van Steensel MA, Happle R, Steijlen PM (2000). Molecular genetics of the hair follicle: the state of the art. Proc Soc Exp Biol Med 223: 1-7 van Steensel MA, Koedam MI, Swinkels OQ, Rietveld F, Steijlen PM (2001). Woolly hair, premature loss of teeth, nail dystrophy, acral hyperkeratosis and facial abnormalities: possible new syndrome in a Dutch kindred. Br J Dermatol 145: 157-161 van Steensel MA, Steijlen PM, Bladergroen RS, Vermeer M, van Geel M (2005). A missense mutation in the type II hair keratin hHb3 is associated with monilethrix. J Med Genet 42: e19
Vincent MC, Biancalana V, Ginisty D, Mandel JL, Calvas P (2001). Mutational spectrum of the ED1 gene in X-linked hypohidrotic ectodermal dysplasia. Eur J Hum Genet 9: 355-363
Vogt BR, Traupe H, Hamm H (1988). Congenital atrichia with nail dystrophy, abnormal facies, and retarded psychomotor development in two siblings: a new autosomal recessive syndrome? Pediatr Dermatol 5: 236-242
Wajid M, Bazzi H, Rockey J, Lubetkin J, Zlotogorski A, Christiano AM (2007). Localized autosomal recessive hypotrichosis due to a frameshift mutation in the desmoglein 4 gene exhibits extensive phenotypic variability within a Pakistani family. J Invest Dermatol 127: 1779-1782
Wali A (2008). Identification of genes involved in hereditary alopecias. PhD Thesis. Quaid-I-Azam University, Islamabad, Pakistan
Wali A, Ali G, John P, Lee K, Chishti MS, Leal SM, Ahmad W (2007a). Mapping of a gene for alopecia with mental retardation syndrome (APMR3) on chromosome 18q11.2-q12.2. Ann Hum Genet 71: 570-577
Wali A, Ansar M, Khan MN, Ahmad W (2006a). Atrichia with popular lesions resulting from a novel insertion mutation in the human hairless gene. Clin Exp Dermatol 31: 695-698
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 195 Chapter 6 References
Wali A, Chishti MS, Ayub M, Yasinzai M, Kafaitullah K, Ali G, John P, Leal SM, Ahmad W (2007b). Localization of a novel autosomal recessive hypotrichosis locus (LAH3) to chromosome 13q14.11-q21.32. Clin Genet 72: 23-29
Wali A, John P, Gul A, Lee K, Chishti MS, Ali G, Hassan MJ, Leal SM, Ahmad W (2006b). A novel locus for alopecia with mental retardation syndrome (APMR2) maps to chromosome 3q26.2-q26.31. Clin Genet 70: 233-239
Wang PG, Gao M, Cui Y, Xiao FL, Zhu YG, Ren YQ, Yan KL, Du WH, Zhang KY, Yang S, Huang W, Zhang XJ (2007). A new clinical variant of hereditary localized alopecia: report of a Chinese family mapped to chromosome 2p25.1- 2p23.2. J Invest Dermatol 127: 1776-1779
Webb TE, Kaplan MG, Barnard EA (1996). Identification of 6H1 as a P2Y purinoceptor: P2Y5. Biochem Biophys Res Commun 219: 105-110
Wedden SE, Pang K, Eichele G (1989). Expression pattern of homeobox-containing genes during chick embryogenesis. Development 105: 639-650
Weeks DE, Sobel E, O’Connell JR, Lange K (1995). Computer programs for multilocus haplotyping of general pedigrees. Am J Hum Genet 56: 1506-1507
Wehrle-Haller B, Imhof BA (2003). Integrin-dependent pathologies. J Pathol 200: 481-487
Wicomb GM, Stephen LX, Beighton P (2004). Dental implications of Tooth-Nail dysplasia (Witkop syndrome): a report of an affected family and an approach to dental management. J Clin Pediatr Dent 28: 107-112
Winter H, Rogers MA, Gebhardt M, Wollina U, Boxall L, Chitayat D, Babul-Hirji R, Stevens HP, Zlotogorski A, Schweizer J (1997b). A new mutation in the type II hair cortex keratin hHb1 involved in the inherited hair disorder monilethrix. Hum Genet 101: 165-169
Winter H, Rogers MA, Langbein L, Stevens HP, Leigh IM, Labreze C, Roul S, Taieb A, Krieg T, Schweizer J (1997a). Mutations in the hair cortex keratin hHb6 cause the inherited hair disease monilethrix. Nat Genet 16: 372-374
Wisniewski SA, Kobielak A, Trzeciak WH, Koblielak K (2002). Recent advances in understanding of the molecular basis of anhidrotic ectodermal dysplasia:
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 196 Chapter 6 References
discovery of a ligand, ectodysplasin and its two receptors. J Appl Genet 43: 97- 107
Witkop CJ (1965). Genetic disease of the oral cavity. In: Tiecke RW (ed) Oral pathology. Blakiston Division, McGraw-Hill, New York: pp. 810-814
Wu HH, Wang NM, Lin CY, Tsai HD (2008). Genetic alterations of HOXA10 and their effect on the severity of endometriosis in a Taiwanese population. Reprod Biomed Online 16: 416-424
Yan M, Wang LC, Hymowitz SG, Schilbach S, Lee J, Goddard A, de Vos AM, Gao WQ, Dixit VM (2000). Two-amino acid molecular switch in an epithelial morphogen that regulates binding to two distinct receptors. Science 290: 523-527
Yan M, Zhang Z, Brady JR, Schilbach S, Fairbrother WJ, Dixit VM (2002). Identification of a novel death domain-containing adaptor molecule for ectodysplasin-A receptor that is mutated in crinkled mice. Curr Biol 12: 409-413
Yang S, Gao M, Cui Y, Yan KL, Ren YQ, Zhang GL, Wang PG, Xiao FL, Du WH, Liang YH, Sun LD, Xu SJ, Huang W, Zhang XJ (2005). Identification of a novel locus for Marie Unna hereditary hypotrichosis to a 17.5 cM interval at 1p21.1- 1q21.3. J Invest Dermatol 125: 711-714
Yap AS, Brieher WM, Gumbiner BM (1997). Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol 13: 119-146
Zhou H, Yan F, Tai HH (2001). C-Terminal region of human NAD+-dependent 15- hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates. Eur J Biochem 268: 3368-3374
Zirbel GM, Ruttum MS, Post AC, Esterly NB (1995). Odonto-onycho-dermal dysplasia. Br J Dermatol 133: 797-800
Zlotogorski A, Ahmad W, Christiano AM (1998). Congenital atrichia in five Arab Palestinian families resulting from a deletion mutation in the hman hairless gene. Hum Genet 103: 400-404
Zlotogorski A, Marek D, Horev L, Abu A, Ben-Amitai D, Gerad L, Ingber A, Frydman M, Reznik-Wolf H, Vardy DA, Pras E (2006). An autosomal recessive form of monilethrix is caused by mutations in DSG4: clinical overlap with localized autosomal recessive hypotrichosis. J Invest Dermatol 126: 1292-1296
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 197 Chapter 6 References
Zlotogorski A, Panteleyev AA, Aita VM, Christiano AM (2002). Clinical and molecular diagnostic criteria of congenital atrichia with papular lesions. J Invest Dermatol 118: 887-890
Zonana J (1993). Hypohidrotic (anhidrotic) ectodermal dysplasia: molecular genetic research and its clinical applications. Semin Dermatol 12: 241-246
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 198 Chapter 6 References
ELECTRONIC DATABASE INFORMATION
1) Current and Online Genodermatoses Database: http://www.blackwell- synergy.com/loi/bjd
2) Ensembl Genome Browser: http://www.ensembl.org/Homo_sapiens
3) ExPASy Proteomics Server: http://www.expasy.org/uniprot
4) GenBank Accession Numbers:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=
5) Genome Database: http://www.gdb.org
6) HomoloGene: Discover homologs:
http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene
7) http://www.sweating.ca/sweat_glands_intro.html
8) Human Gene Mutation Database Website: http://www.hgmd.cf.ac.uk
9) Human Genome Variation Society recommendations: http://www.hgvs.org/mutnomen/
10) Online Mendelian Inheritance in Man (OMIM):
http://www.ncbi.nlm.nih.gov/omim/
11) USCS Genome Bioinformatics website, March 2006: http://genome.ucsc.edu/cgi-bin/hgGateway
Genetic Studies of Human Hereditary Skin Disorders: Ectodermal Dysplasias and Alopecias 199