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Diagnostics and Gene Therapy for

Human Genetic Disorders
Diagnostics and Gene Therapy for Human Genetic Disorders provides an integrative and com-
prehensive source of information blending classical human genetics with the human genome. It
provides a multidisciplinary overview of Mendelian inheritance and multifactorial inheritance, gen-
etic variations, polymorphisms, chromosomal, multifactorial, and mitochondrial disorders.
PCR, electrophoresis, cytogenetics, prenatal, and HPLC based techniques applied for diagnosing
genetic disorders are discussed with applications. Symptoms, etiology, diagnosis, treatment of 14
major and 5 minor genetic disorders are discussed in detail. Methods employed for the preparation
of kits for the diagnosis of diseases are provided. The role of gene therapy in the amelioration of
genetic disorders and the methodology employed are discussed. The success of gene therapy in con-
trolling various disorders such as immune system disorders, neurodegenerative disorders, cardiovas-
cular disorders, eye diseases, and cancer has been described along with type studies.
Features:
1. A blend of classical human genetics with molecular and genome-based applications

2. Techniques applied for the diagnosis of genetic disorders
3. Diagnostics of 19 genetic disorders including symptoms, etiology, diagnosis, and treatment
4. Role of gene therapy in the amelioration of disorders
5. Type studies describing the role of diagnostics in conserving human health
This book attempts to connect the information about classical and modern human genetics, genetic
disorders, and gene therapy to all types of diseases in one place. This work provides a comprehen-
sive source of information that can serve as a reference book for scientific investigations and as a
textbook for graduate students.
ii
iii
Diagnostics and Gene Therapy

for Human Genetic Disorders
K.V. Chaitanya
iv
First edition published 2023

by CRC Press
6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742
and by CRC Press
4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
CRC Press is an imprint of Taylor & Francis Group, LLC
© 2023 K.V. Chaitanya
Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume
responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted
to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission
to publish in this form has not been obtained. If any copyright material has not been acknowledged, please write and let us
know so that we may rectify this in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized
in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying,
microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the
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Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification
and explanation without intent to infringe.
Library of Congress Cataloging‑in‑Publication Data
Names: Chaitanya, K. V., author.
Title: Diagnostics and gene therapy for human genetic disorders / K.V. Chaitanya.
Description: First edition. | Boca Raton : CRC Press, 2023. |
Includes bibliographical references and index.
Identifiers: LCCN 2022035247 (print) | LCCN 2022035248 (ebook) |
ISBN 9781032381640 (hardback) | ISBN 9781032381657 (paperback) |
ISBN 9781003343790 (ebook)
Subjects: LCSH: Genetic disorders. | Genetic disorders–Diagnosis. |
Gene therapy. | Medical genetics.
Classification: LCC RB155.5 .C425 2023 (print) |
LCC RB155.5 (ebook) | DDC 616/.042–dc23/eng/20221021
LC record available at https://lccn.loc.gov/2022035247
LC ebook record available at https://lccn.loc.gov/2022035248
ISBN: 9781032381640 (hbk)
ISBN: 9781032381657 (pbk)
ISBN: 9781003343790 (ebk)
DOI: 10.1201/9781003343790
Typeset in Times
by Newgen Publishing UK
v
Contents
Preface............................................................................................................................................... xv
Acknowledgments...........................................................................................................................xvii
Author Bio.......................................................................................................................................xix
Chapter 1 Genetic Analysis............................................................................................................ 1

1.1 Introduction......................................................................................................... 1
1.2 Monogenic Traits................................................................................................ 1
1.3 Linkage Analysis................................................................................................. 3
1.3.1 Linkage.................................................................................................. 3
1.3.2 Linkage Analysis................................................................................... 3
1.3.3 Polymorphic DNA Markers.................................................................. 4
1.4 Linkage Disequilibrium...................................................................................... 6
1.4.1 Linkage Disequilibrium at One Pair of Loci......................................... 6
1.4.2 Linkage Equilibrium............................................................................. 7
1.4.3 Linkage Disequilibrium at More Than Two Loci.................................. 7
1.4.4 Linkage Disequilibrium Within and Between Populations................... 7
1.4.5 Population Genetics of Linkage Disequilibrium................................... 8
1.4.6 Applications of Linkage Disequilibrium............................................... 9
1.4.7 Linkage Disequilibrium and Human Genome....................................10
1.5 Mendelian Pedigree Patterns............................................................................. 10
1.5.1 Dominance and Recessiveness of Characters...................................... 11
1.5.2 Types of Mendelian Pedigree.............................................................. 11
1.6 Mendelian Pedigree Analysis............................................................................ 13
1.6.1 Pedigree Analysis................................................................................ 13
1.6.2 Patterns Indicating a Recessive Trait................................................... 16
1.6.2.1 Autosomal Recessive........................................................... 16
1.6.3 Patterns Indicating a Dominant Trait................................................... 16
1.6.3.1 Autosomal Dominant Trait................................................... 17
1.6.4 X-linked Recessive.............................................................................. 18
1.6.5 X-Linked Dominant............................................................................ 19
1.6.6 Calculations for the Risk Allele by Pedigree Analysis........................ 19
1.7 Allele Frequency Estimation............................................................................. 20
1.7.1 Determination of Allele Frequency..................................................... 20
1.7.1.1 DNA Pooling........................................................................ 21
1.8 The Hardy-Weinberg Law................................................................................. 22
1.8.1 Infinitely Large Population.................................................................. 23
1.8.2 Random Mating................................................................................... 23
1.8.3 No Mutation........................................................................................ 23
1.8.4 No Immigration................................................................................... 23
1.8.5 No Evolutionary Forces Affecting the Population.............................. 23
1.8.6 Conclusion........................................................................................... 24
1.8.7 Circumstances in which the Hardy-Weinberg Law
Cannot Apply....................................................................................... 24
v
vi
vi Contents
1.9 Multifactorial Inheritance................................................................................. 25

1.9.1 Open Neural Tube Defects (ONTDs).................................................. 26
1.9.2 Hip Dysplasia...................................................................................... 27
1.9.3 Height.................................................................................................. 27
1.9.4 Model for Multifactorial Inheritance................................................... 27
1.10 Heritability........................................................................................................ 28
1.10.1 Types of Heritability............................................................................ 28
1.10.2 Methods of Estimating the Heritability............................................... 29
1.10.3 Heritability and the Environmentability of a Trait.............................. 29
1.10.4 Misconceptions or Misunderstandings on Heritability....................... 30
1.10.5 Heritability of Human Height.............................................................30
1.10.6 Heritability and Human Behavior....................................................... 31
1.10.7 Designs for Estimating the Heritability of Behavior........................... 32
1.10.7.1 Twin Studies...................................................................... 32
1.10.7.2 Adoption Studies............................................................... 32
1.10.7.3 Developmental Models...................................................... 32
1.10.8 Candidate Genes for Studying Heritability......................................... 32
1.11 Polygenic Inheritance........................................................................................ 32
1.11.1 Human Skin Colour............................................................................. 33
1.12 Haplotyping....................................................................................................... 34
1.12.1 The Importance of Haplotype Analysis............................................... 34
1.12.2 Methods of Haplotyping...................................................................... 34
1.12.2.1 Haplotyping for Pedigree.................................................. 34
1.12.2.2 DNA Sample Based Haplotyping Methods....................... 35
1.13 Conclusion........................................................................................................ 35
Chapter 2 Genetic Variations in Human Beings.......................................................................... 39

2.1 Introduction....................................................................................................... 39
2.2 The Origin of Human Beings............................................................................ 39
2.2.1 Out of Africa Theory and Supporting Evidence.................................. 40
2.2.2 Evidence from Population Genetics.................................................... 41
2.3 Genetic Diversity Among Human Beings......................................................... 42
2.3.1 The Salient Features of Human Genetic Diversity.............................. 42
2.3.1.1 From Genetic Research........................................................ 42
2.3.1.2 Findings from Fossil Remains............................................. 43
2.3.1.3 Techniques for the Measurement of Genetic
Diversity............................................................................... 43
2.4 Genetic Polymorphisms.................................................................................... 44
2.4.1 Single Nucleotide Polymorphisms (SNPs)......................................... 45
2.4.1.1 Identification of SNPs.......................................................... 45
2.4.1.2 SNP Genotyping..................................................................48
2.4.1.3 SNP Frequency in Human Genome..................................... 49
2.4.1.4 SNP Based Genome Wide Association Studies
(SNP-GWAS)....................................................................... 49
2.4.2 Short-Tandem-Repeat Polymorphisms (STRPs)................................. 49
2.4.2.1 Minisatellite Repeat Sequences and Their
Functions.............................................................................. 50
2.4.2.2 Microsatellite Repeats.......................................................... 54
2.4.2.3 Alu Insertion Polymorphisms.............................................. 59
vii
Contents vii
2.4.2.4 Hypervariable Region Polymorphism of

Mitochondrial DNA............................................................. 60
2.4.2.5 Y Chromosome Polymorphisms.......................................... 61
2.5 Immunogenic Polymorphisms.......................................................................... 62
2.5.1 Polymorphism in Human Major Histocompatibility
Complex (MHC).................................................................................. 62
2.5.2 Immune Polymorphism....................................................................... 63
2.5.3 Cytokine Polymorphism...................................................................... 64
2.5.3.1 Polymorphisms and cytokine signaling............................... 64
2.6 Trans-Species Polymorphism........................................................................... 65
2.6.1 Trans-Species Polymorphism in Major Histo Compatibility
(MHC) Genes...................................................................................... 66
2.6.2 Trans-Species Polymorphism in Other Genes Related to the
Immune System................................................................................... 66
2.7 Genetic Variations in Drug Metabolizing Enzymes.......................................... 67
2.7.1 Cytochrome P-450............................................................................... 68
2.7.2 Glucose-6-Phosphate Dehydrogenase................................................. 71
2.7.3 N-Acetyltransferase polymorphism.................................................... 71
2.8 Methods of Genotyping.................................................................................... 72
2.8.1 Human HapMap Project...................................................................... 72
2.8.2 Genome Wide Association Study........................................................ 72
2.8.3 Next-Generation Sequencing Technologies for Genotyping............... 74
2.9 Single Stranded Conformational Polymorphism (SSCP)................................. 74
2.9.1 SSCP Procedure.................................................................................. 74
2.9.2 High Throughput Methods for SSCP.................................................. 75
2.9.3 Applications........................................................................................ 75
2.9.4 Limitations.......................................................................................... 75
2.9.5 Modifications of SSCP........................................................................ 75
2.10 Conclusion........................................................................................................ 76
Chapter 3 Genetic Disorders........................................................................................................ 81

3.1 Introduction....................................................................................................... 81
3.2 Classification of Genetic Disorders.................................................................. 81
3.3 Chromosomal Disorders................................................................................... 83
3.3.1 Human Chromosomes......................................................................... 83
3.3.1.1 Human Chromosome 1........................................................ 84
3.3.2 Chromosome Staining and Karyotype................................................ 84
3.3.3 Chromosomal Abnormalities............................................................... 85
3.3.3.1 Factors Responsible for Chromosomal Abnormalities........ 85
3.3.3.2 Types of Chromosomal Abnormalities................................. 85
3.4 Single Gene Disorders...................................................................................... 95
3.4.1 Autosomal Dominant Disorders.......................................................... 96
3.4.1.1 Marfan Syndrome................................................................. 96
3.4.2 Autosomal Recessive Disorders.......................................................... 96
3.4.2.1 Sickle Cell Disease............................................................... 97
3.4.3 Sex-Linked Disorders.......................................................................... 98
3.4.3.1 X-Inactivation....................................................................... 98
3.4.3.2 X-Linked Dominant Disorders............................................. 99
3.4.3.3 X-Linked Recessive Disorders........................................... 100
vii
viii Contents
3.4.3.4 Y-Linked Disorders............................................................ 101

3.5 Mitochondrial Disorders................................................................................. 101
3.5.1 Genetic Diseases due to the Mitochondrial Genome........................ 102
3.6 Multifactorial Disorders.................................................................................. 103
3.7 Molecular Basis of Cancer.............................................................................. 105
3.7.1 Cell Cycle Regulation and the Importance of Apoptosis.................. 105
3.7.2 The p53 Gene and Cancer................................................................. 105
3.7.3 Telomeres, Cell immortalization, and Tumorigenesis....................... 107
3.7.4 Genes Frequently Mutated During Cancer........................................ 108
3.7.4.1 Cellular Oncogenes............................................................ 108
3.7.4.2 Tumor Suppressor Genes................................................... 108
3.7.4.3 DNA Damage Repair Genes.............................................. 109
3.7.5 Mutations in DNA and Cancer.......................................................... 109
3.8 Gene Expression Analysis............................................................................... 110
3.8.1 Gene Expression Profiling of Lung Cancer....................................... 111
3.9 Hematopoietic Disorders................................................................................ 112
3.9.1 Techniques for Studying the Hematopoietic Disorders..................... 113
3.10 Conclusion...................................................................................................... 113
Chapter 4 Techniques for the Diagnosis of Genetic Disorders.................................................. 117

4.1 Introduction..................................................................................................... 117
4.2 Molecular Approach........................................................................................ 117
4.2.1 Nucleic Acid Isolation Methods........................................................ 117
4.2.1.1 DNA Extraction from Buccal Swabs................................. 118
4.2.1.2 DNA Extraction from a Hair Sample................................. 118
4.2.1.3 Extraction of DNA from Urine Samples............................ 119
4.2.1.4 DNA Extraction from a Blood Sample.............................. 119
4.2.2 Restriction Fragment Length Polymorphism.................................... 120
4.2.2.1 Role of RFLPs as Markers for the Identification
of Genetic Disorder............................................................ 120
4.2.2.2 RFLP Procedure................................................................. 121
4.2.2.3 Applications of RFLP........................................................ 122
4.2.3 Hybridization-Based Methods........................................................... 122
4.2.3.1 Membrane-Based Methods................................................ 123
4.2.3.2 Array-Based Methods........................................................ 123
4.2.4 Polymerase Chain Reaction-Based Techniques................................ 124
4.2.4.1 Reverse Transcriptase PCR................................................ 124
4.2.4.2 Real-Time PCR.................................................................. 125
4.2.4.3 Allele-Specific Amplification (ASA) by Amplification
Refractory Mutation System (ARMS) PCR....................... 125
4.2.4.4 Multiplex PCR................................................................... 126
4.2.4.5 Digital PCR........................................................................ 127
4.2.4.6 Nested PCR........................................................................ 129
4.2.4.7 Multiplex Ligation-Dependent Probe Amplification
(MLPA).............................................................................. 130
4.2.5 Electrophoresis Based Methods........................................................ 133
4.2.5.1 Confirmation Sensitive Gel Electrophoresis...................... 133
4.2.5.2 Denaturation Gradient Gel Electrophoresis....................... 135
4.2.5.3 Temporal Temperature Gradient Gel Electrophoresis
(TTGE)............................................................................... 136
ix
Contents ix
4.2.5.4 Single-Strand Conformational Polymorphism

(SSCP)................................................................................ 137
4.2.6 DNA Typing and Testing................................................................... 138
4.2.7 HLA Typing....................................................................................... 140
4.3 Cytogenetic Techniques.................................................................................. 142
4.3.1 Classical Cytogenetic Techniques..................................................... 142
4.3.1.1 Specialized Staining Techniques........................................ 143
4.3.2 Molecular Cytogenetic Techniques................................................... 143
4.3.2.1 Fluorescent In Situ Hybridization (FISH).......................... 144
4.3.2.2 Spectral Karyotyping (SKY).............................................. 144
4.3.2.3 Comparative Genomic Hybridization (CGH).................... 144
4.4 Prenatal Genetic Diagnosis............................................................................. 146
4.4.1 Amniocentesis................................................................................... 146
4.4.2 Chorionic Villus Sampling................................................................ 146
4.4.3 Fetal Blood Sampling........................................................................ 146
4.4.4 Prenatal Diagnosis of Genetic Disorders..........................................147
4.5 Denaturing High-Performance Liquid Chromatography................................ 147
4.5.1 Applications...................................................................................... 148
4.6 Conclusion...................................................................................................... 148
Chapter 5 Diagnosis of Genetic Disorders................................................................................. 153

5.1 Introduction..................................................................................................... 153
5.2 The History of Molecular Diagnostics............................................................ 154
5.2.1 Molecular Diagnostic Tests for the Detection of Changes
in Genetic Composition..................................................................... 154
5.2.1.1 Nucleic Acid-Based Diagnostic Tests................................ 154
5.2.1.2 Gene-Based Diagnostic Tests............................................. 155
5.3 Lab on a Chip.................................................................................................. 155
5.3.1 A Brief History of Lab on a Chip...................................................... 156
5.3.2 Materials Used for the Development of LOCs.................................. 156
5.3.3 Operation of a Microfluidic Unit....................................................... 157
5.3.4 Storage of Reagents on a LOC Device.............................................. 157
5.3.5 Microfluidic Platforms Used as LOCs.............................................. 157
5.3.5.1 Lateral Flow Tests.............................................................. 157
5.3.5.2 Linear-Actuated Devices.................................................... 157
5.3.5.3 Centrifugal Microfluidic Devices....................................... 158
5.3.6 LOC Diagnostic Targets.................................................................... 158
5.3.7 Applications and Future perspectives of LOC.................................. 159
5.4 Diagnosis of Genetic Disorders Associated with the Chromosomal
Abnormalities.................................................................................................. 159
5.4.1 Down Syndrome................................................................................ 159
5.4.1.1 Symptoms........................................................................... 160
5.4.1.2 Etiology.............................................................................. 161
5.4.1.3 Diagnosis............................................................................ 161
5.4.1.4 Treatment........................................................................... 161
5.4.2 Turner Syndrome............................................................................... 162
5.4.2.1 Symptoms........................................................................... 163
5.4.2.2 Etiology.............................................................................. 163
5.4.2.3 Diagnosis............................................................................ 164
x
x Contents
5.4.2.4 Treatment........................................................................... 165

5.4.3 Klinefelter Syndrome........................................................................ 165
5.4.3.1 Symptoms........................................................................... 166
5.4.3.2 Etiology.............................................................................. 167
5.4.3.3 Diagnosis............................................................................ 168
5.4.3.4 Treatment........................................................................... 169
5.4.4 Huntington’s Disease......................................................................... 169
5.4.4.1 Symptoms........................................................................... 169
5.4.4.2 Etiology.............................................................................. 170
5.4.4.3 Diagnosis............................................................................ 171
5.4.4.4 Treatment........................................................................... 171
5.4.5 Fragile X Syndrome.......................................................................... 172
5.4.5.1 Symptoms........................................................................... 172
5.4.5.2 Etiology of Fragile X Syndrome........................................ 173
5.4.5.3 Disease Diagnosis.............................................................. 174
5.4.5.4 Treatment........................................................................... 175
5.4.6 Thalassemia....................................................................................... 175
5.4.6.1 Symptoms........................................................................... 176
5.4.6.2 Etiology.............................................................................. 177
5.4.6.3 Diagnosis............................................................................ 179
5.4.6.4 Treatment........................................................................... 179
5.4.7 Sickle Cell Disease............................................................................ 179
5.4.7.1 Symptoms........................................................................... 180
5.4.7.2 Etiology ............................................................................. 180
5.4.7.3 Diagnosis............................................................................ 181
5.4.7.4 Treatment........................................................................... 181
5.4.8 Alzheimer’s Disease.......................................................................... 182
5.4.8.1 Symptoms........................................................................... 182
5.4.8.2 Etiology ............................................................................ 183
5.4.8.3 Diagnosis............................................................................ 184
5.4.8.4 Treatment........................................................................... 185
5.4.9 Schizophrenia.................................................................................... 186
5.4.9.1 Symptoms........................................................................... 186
5.4.9.2 Etiology ............................................................................. 187
5.4.9.3 Diagnosis............................................................................ 188
5.4.9.4 Treatment........................................................................... 188
5.4.10 Cystic Fibrosis................................................................................... 189
5.4.10.1 Symptoms........................................................................... 189
5.4.10.2 Etiology ............................................................................ 190
5.4.10.3 Diagnosis............................................................................ 191
5.4.10.4 Treatment........................................................................... 191
5.4.11 Thrombophilia................................................................................... 192
5.4.11.1 Symptoms........................................................................... 193
5.4.11.2 Etiology.............................................................................. 193
5.4.11.3 Diagnosis............................................................................ 196
5.4.11.4 Treatment........................................................................... 196
5.4.12 Tay Sachs Disease............................................................................. 197
5.4.12.1 Symptoms........................................................................... 198
5.4.12.2 Etiology.............................................................................. 199
5.4.12.3 Diagnosis............................................................................ 200
xi
Contents xi
5.4.12.4 Treatment........................................................................... 201

5.4.13 Muscular Dystrophy.......................................................................... 202
5.4.13.1 Symptoms........................................................................... 203
5.4.13.2 Etiology.............................................................................. 203
5.4.13.3 Diagnosis............................................................................ 204
5.4.13.4 Treatment........................................................................... 205
5.4.14 Ataxia................................................................................................ 206
5.4.14.1 Symptoms........................................................................... 206
5.4.14.2 Etiology.............................................................................. 206
5.4.14.3 Diagnosis............................................................................ 209
5.4.14.4 Treatment........................................................................... 210
5.5 Diagnosis of Rare Genetic Disorders.............................................................. 210
5.5.1 Emanuel Syndrome........................................................................... 210
5.5.1.1 Symptoms........................................................................... 211
5.5.1.2 Etiology.............................................................................. 211
5.5.1.3 Diagnosis............................................................................ 211
5.5.1.4 Treatment........................................................................... 213
5.5.2 Cat Eye Syndrome............................................................................. 213
5.5.2.1 Symptoms........................................................................... 213
5.5.2.2 Etiology.............................................................................. 213
5.5.2.3 Diagnosis............................................................................ 214
5.5.2.4 Treatment........................................................................... 214
5.5.3 Uniparental Disomy.......................................................................... 214
5.5.3.1 Symptoms........................................................................... 215
5.5.3.2 Etiology.............................................................................. 215
5.5.3.3 Diagnosis............................................................................ 215
5.5.3.4 Treatment........................................................................... 216
5.5.4 Jacobsen Syndrome........................................................................... 216
5.5.4.1 Symptoms........................................................................... 216
5.5.4.2 Etiology.............................................................................. 217
5.5.4.3 Diagnosis............................................................................ 218
5.5.4.4 Treatment........................................................................... 218
5.5.5 Kleefstra Syndrome........................................................................... 218
5.5.5.1 Symptoms........................................................................... 218
5.5.5.2 Etiology.............................................................................. 218
5.5.5.3 Diagnosis............................................................................ 219
5.5.5.4 Treatment........................................................................... 219
5.6 Conclusions..................................................................................................... 219
Chapter 6 Gene Therapy............................................................................................................ 225

6.1 Introduction..................................................................................................... 225
6.2 Gene Therapy and Genetic Engineering......................................................... 225
6.2.1 Gene Therapy.................................................................................... 226
6.2.2 Gene Therapy and Hematopoietic Stem Cells..................................226
6.2.3 CAR-T Therapy................................................................................. 227
6.2.4 CRISPR/Cas9.................................................................................... 228
6.2.4.1 CRISPR/Cas9 and Neurodegenerative Disorders.............. 230
6.2.4.2 Limitations of CRISPR/Cas9............................................. 230
xii
xii Contents
6.3 Therapeutic Nucleic Acids and Therapeutic Polynucleotides........................231

6.3.1 DNA-Based Therapeutics.................................................................. 231
6.3.1.1 Plasmids............................................................................. 231
6.3.1.2 Oligonucleotides for Antisense and Antigene
Applications....................................................................... 232
6.3.1.3 DNA Aptamers................................................................... 233
6.3.1.4 DNAzymes......................................................................... 233
6.3.2 RNA Based Therapeutics.................................................................. 235
6.3.2.1 RNA Aptamers................................................................... 235
6.3.2.2 RNA Decoys...................................................................... 235
6.3.2.3 Ribozymes.......................................................................... 236
6.3.2.4 Small Interfering RNAs..................................................... 236
6.3.2.5 MicroRNA (miRNA)......................................................... 237
6.3.2.6 Circular RNA (CircRNA).................................................. 238
6.4 Therapeutic Polynucleotides........................................................................... 238
6.4.1 DNA Vaccines................................................................................... 239
6.5 Therapeutic Genes and Their Role in Cancer Therapy................................... 240
6.5.1 HSV-Thymidine Kinase.................................................................... 240
6.5.2 Cytosine Deaminase.......................................................................... 241
6.5.3 Cytochrome P450 2B1...................................................................... 241
6.5.4 Suicide Genes.................................................................................... 242
6.5.5 Gene Therapy Directed Apoptosis and Cell Control......................... 242
6.5.5.1 P53.....................................................................................242
6.5.5.2 E2F-1.................................................................................. 243
6.5.6 Cytokine Genes................................................................................. 243
6.5.7 Costimulatory Genes......................................................................... 243
6.5.8 Tumor-Associated Antigen Genes..................................................... 244
6.6 Gene Knockin and Gene Knockout................................................................ 245
6.6.1 Gene Targeting.................................................................................. 245
6.6.2 Gene Knockin.................................................................................... 246
6.6.2.1 Knockin Editing................................................................. 247
6.6.3 Gene Knockout.................................................................................. 247
6.6.3.1 Conditional Gene Knockout............................................... 249
6.6.3.2 Other Applications of Gene Knockout............................... 249
6.6.3.3 Limitations......................................................................... 249
6.7 Methods of Gene Delivery.............................................................................. 249
6.7.1 Physical Methods.............................................................................. 250
6.7.1.1 Microinjection.................................................................... 250
6.7.1.2 Particle Bombardment........................................................ 250
6.7.1.3 Electroporation................................................................... 251
6.7.1.4 Sonoporation...................................................................... 251
6.7.1.5 Magnetofection.................................................................. 251
6.7.1.6 Hydroporation.................................................................... 251
6.7.1.7 Photoporation..................................................................... 251
6.7.2 Viral Vector-based Gene Delivery Systems....................................... 252
6.7.2.1 Retroviral Vectors............................................................... 252
6.7.2.2 Adenoviral Vectors............................................................. 253
6.7.2.3 Adeno-Associated Viral Vectors......................................... 253
6.7.2.4 Herpes Simplex Viral Vectors............................................. 253
6.7.2.5 Other Viral Vectors............................................................. 253
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6.7.3 Non-Viral Based Gene Delivery Systems......................................... 254

6.7.3.1 Naked DNA Delivery Systems........................................... 254
6.7.3.2 Polymeric Gene Delivery Systems..................................... 254
6.7.3.3 Liposomal Delivery Systems............................................. 255
6.8 Conclusion...................................................................................................... 255
Chapter 7 Applications of Gene Therapy..................................................................................259

7.1 Introduction..................................................................................................... 259
7.2 Applications of Gene Therapy........................................................................ 259
7.2.1 Prevention of Irradiation Damage to Salivary Glands....................... 259
7.2.2 Autoimmune Disorders..................................................................... 260
7.2.2.1 Gene Therapy for Autoimmune Disorders......................... 260
7.2.2.2 Gene Therapy for Grave’s Disease and Lupus Disease..... 260
7.2.2.3 Sjogren’s Syndrome........................................................... 261
7.2.2.4 Pre-Clinical and Clinical Studies....................................... 261
7.2.3 Systemic Protein Deficiencies........................................................... 262
7.2.4 Spinal Disorders................................................................................ 262
7.2.5 Gene Therapy for Cancer.................................................................. 262
7.2.5.1 Immunotherapy.................................................................. 263
7.2.5.2 Oncolytic Virotherapy........................................................ 263
7.2.5.3 Gene Transfer..................................................................... 264
7.2.6 Gene Therapy for Eye Diseases........................................................ 264
7.2.7 Gene Therapy for Cardiovascular Disorders..................................... 265
7.2.7.1 Gene Therapy for Coronary Artery Disease....................... 266
7.2.7.2 Gene Therapy for Heart Failure......................................... 266
7.2.7.3 Gene Therapy for Vein Graft Stenosis...............................266
7.2.8 Gene Therapy for Neuro Degenerative Disorders............................. 267
7.2.8.1 Gene Therapy for Neurodegenerative Disorders in
Model Organisms............................................................... 267
7.2.9 Gene Therapy and Dermatological Disorders................................... 269
7.3 Difficulties with Gene Therapy....................................................................... 270
7.4 Conclusion...................................................................................................... 271
Chapter 8 Applications of Molecular Diagnostics..................................................................... 275

8.1 Introduction..................................................................................................... 275
8.2 Applications of Diagnostics in Testing for Rare Genetic Disorders............... 276
8.2.1 Targeted Sequencing Panel................................................................ 277
8.2.2 Whole Exome Sequencing................................................................ 277
8.2.3 Whole Genome Sequencing.............................................................. 277
8.3 Applications of Diagnostics in Pharmacogenetics and
Pharmacogenomics......................................................................................... 278
8.3.1 Commercially Available Pharmacogenetic and
Pharmacogenomics Tests..................................................................279
8.3.2 Clinical Significance of Pharmacogenetics and
Pharmacogenomics............................................................................ 279
8.3.2.1 Cancer................................................................................ 279
8.3.2.2 Cardiology.......................................................................... 280
8.3.2.3 Psychiatry........................................................................... 280
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8.4 Applications of Diagnostics in Personalized Medicine.................................. 281

8.4.1 SNP Genotyping and Personalized Medicine................................... 281
8.5 Applications of Diagnostics in Family Risk Assessment............................... 282
8.6 Applications of Diagnostics in Transplantation.............................................. 283
8.6.1 Role of Short-Term Repeats in Organ Transplantation..................... 284
8.7 Applications of Diagnostics in Epigenetics.................................................... 285
8.7.1 Mechanisms of Epigenetic Changes.................................................285
8.7.1.1 DNA Methylation............................................................... 285
8.7.1.2 Histone Modification.......................................................... 286
8.7.1.3 Non-coding RNAs.............................................................. 286
8.7.2 Epigenetic Biomarkers...................................................................... 287
8.7.3 Epigenetics Based Tests Performed in Clinical Laboratories...........287
8.8 Applications of Diagnostics in Forensic Sciences.......................................... 287
8.8.1 Methods in Forensic Sciences for Identification of Humans............ 288
8.8.1.1 Autosomal STR Profiling................................................... 288
8.8.1.2 Y-Chromosome Analysis.................................................... 288
8.8.1.3 Mitochondrial DNA........................................................... 288
8.8.1.4 Autosomal SNPs................................................................ 289
8.8.1.5 Biomarkers in Forensics..................................................... 289
8.9 Conclusion...................................................................................................... 289
Chapter 9 Case Studies.............................................................................................................. 293

9.1 Introduction..................................................................................................... 293
9.2 Factors Responsible for the Occurrence of Genetic Disorders....................... 293
9.3 Genetic Testing............................................................................................... 294
9.4 Case Studies.................................................................................................... 294
9.4.1 Huntington’s Disease......................................................................... 294
9.4.2 Down Syndrome................................................................................ 295
9.4.3 Fragile X Syndrome.......................................................................... 295
9.4.4 Duplication of Chromosomes and Trisomy...................................... 297
9.4.5 Chromosomal Abnormalities and Heteromorphism in
Couples Leading to Recurrent Abortions.......................................... 298
9.4.6 Congenital Anomalies in Newborn Infants....................................... 300
9.4.7 Tree Man Syndrome.......................................................................... 301
9.4.8 Emanuel Syndrome........................................................................... 303
9.4.9 Jacobsen Syndrome........................................................................... 304
9.4.9.1 Case Study.......................................................................... 305
9.4.10 Uniparental Disomy and Chediak-Higashi Syndrome...................... 306
9.4.10.1 Case Study.......................................................................... 306
9.5 Conclusion...................................................................................................... 307
Glossary......................................................................................................................................... 309
Index............................................................................................................................................... 317
xv
Preface
Genetic material DNA is packed in the nucleus of every living as condensed chromatin. In humans,
DNA is further condensed into 46 rod-shaped chromosomes during cell division, with each chromo-
some having a definite size, shape, and a specific number for its identification. There have been
great advances in the field of genetics over the last 25 years. Mendelian genetics, pedigree analysis,
linkage analysis, and allele frequency estimations have been identified to predict the chances of
inheriting a disease, which has arisen due to mutations or lack of gene expression or differences
in the expression of genes within the parents. Epigenetic inheritance and multifactorial inheritance
have further explained the significance of the environment in the expression of a gene. Completion
of the human genome project has provided us with significant technical advances in DNA analysis.
As a result, we can comfortably diagnose a genetic disorder, identify the gene(s) responsible for it,
and check for the possibility of eradicating the disease from that human being through gene therapy.
With more than 6000 genetic disorders having been identified in humans, 600 disorders are currently
being treated.
Diagnostics and Gene Therapy for Human Genetic Disorders provides a blend of classical human
genetics and advanced molecular genetics. The latest happenings in monogenic traits, linkage dis-
equilibrium in the human genome, Mendelian inheritance, and multifactorial inheritance offer a
complete pedigree chart. Genetic variations, polymorphisms, defects in chromosomes, changes
in the genes of nuclear and mitochondrial genomes responsible for chromosomal, multifactorial,
and mitochondrial disorders give a comprehensive and detailed understanding of genetics. HPLC,
PCR, electrophoresis, cytogenetics, and prenatal-based techniques applied to identify changes in
the chromosomes and the DNA will connect the classroom to the laboratory. The significance of
diagnostic kits and methods involved in their preparation will also increase the scope of disease
diagnosis. Symptoms, etiology, diagnosis, and treatment of 14 major genetic disorders will have
a better insight of knowledge into genetics. The role of gene therapy, genome editing, RNAi tech-
nology in the amelioration of genetic disorders will help in providing a better understanding of
the gene therapy tools and their applications. Applications of gene therapy in controlling various
disorders such as immune system disorders, neurodegenerative disorders, cardiovascular disorders,
eye diseases, and cancer will further empathize its role in the treatment of human diseases.
Diagnostics and Gene Therapy for Human Genetic Disorders provides the most up to date infor-
mation, which threads classical human genetics with variations in the genes, polymorphisms, genetic
disorders, disease diagnosis and gene therapy, genome editing applications, making human health
highly susceptible. Through this work, human classical and modern genetics, genetic disorders, and
gene therapy for all types of disorders are provided in one place, which provides a piece of compre-
hensive information for the reader.
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xvi
Acknowledgments
I would like to express my deep gratitude to Professor T. Sekhar, Dr. Nageswara Rao Reddy,
Dr. Utpal Nath, and Professor P.B. Kavi Kishor for their support. I am thankful to my wife Lalita,
my children Abhiram and Aamukta Malyada for their patience. My acknowledgments to genomics
and molecular diagnostics students for the discussions that helped me take the topics collectively
and comprehensively. Finally, I express my deep sense of gratitude to all authors whose works have
been consulted in the writing of this book. I would appreciate any valuable suggestions for further
improvement of this work.
K.V. CHAITANYA
xvii
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newgenprepdf
xix
Author Bio
K.V. Chaitanya is Professor in the Microbiology and Food Science Technology Department, GITAM
University, Visakhapatnam, India. Prof. Chaitanya has a Ph.D. in Life Sciences from Pondicherry
University, Pondicherry. Prof. Chaitanya received a postdoctoral fellowship from the Department of
Biotechnology, Govt of India, to pursue research at the Indian Institute of Science, Bengaluru, India.
He has over 18 years of experience in research and academics in genomics and molecular biology.
He has worked in various capacities in academic and research institutes of international reputation
and has published 60 research articles. He has authored two books on cell and molecular biology
and genomics and has filed five patents to protect the copyright of his experimental products. Prof.
Chaitanya has academic awards and fellowships to his credit.
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1 Genetic Analysis
1.1 INTRODUCTION
Genetic analysis is the name ascribed to the overall understanding of a process by applying the
concepts and thoughts related to classical Mendelian genetics and modern molecular biology.
Identifying the genes and genetic disorders inherited by these genes forms the principal basis of gen-
etic analysis. This subject is under study since prehistoric times when early human beings practiced
selective breeding to improve the productivity of plants and animals. The scientific basis of genetic
analysis has come to light through Mendel’s laws. Gregor Mendel was the first to utilize genetic ana-
lysis to identify the traits inherited by offspring from parents. These traits are controlled by heredi-
tary units called genes, present in every living cell of an organism (both prokaryotic and eukaryotic).
Mendel’s work has contributed to the development of hybrid plants, identifying genetically inherited
disorders, and differential diagnoses of diseases.
Genetic analysis through modern biology is achieved using cytogenetic analysis such as karyo-
typing, fluorescence in situ hybridization (FISH), and molecular techniques such as PCR, DNA
sequencing technology, and microarrays (Espen et al. 2018). These techniques have helped in the
identification of mutations, and changes in the copy numbers. In addition, the development of
reverse genetics has helped in the detection of codons and genetic code. Genetic linkage studies
have analyzed the spatial arrangement of genes on chromosomes.
1.2 MONOGENIC TRAITS
A trait is a distinct variant of an organism’s phenotypic character, which can be inherited or environ-
mentally determined, or a combination of both. For example, eye color is a character or abstraction
of an attribute, while blue, brown, and hazel are traits. The human genetic traits are classified into
simple monogenic and complex polygenic traits.
The monogenic trait is encoded by a single gene, which means a single gene determines a spe-
cific trait. The wild-type allele is normal and healthy whereas the mutated or diseased allele gives
a particular disease to the phenotype. Monogenic disorders occur due to mutations in a single
gene, which minimizes the function and stability of the corresponding protein by altering its three-
dimensional structure (Andreas, 1999). These mutations include point mutations (changes in the
single nucleotides that change the amino acid sequence), insertions, deletions in the DNA sequence
that encodes the protein, and changes in the non-coding DNA, interfering with the gene splicing.
Thus, monogenic traits are strongly influenced by variation within a single gene, recognized by their
classic patterns of inheritance within families. As monogenic traits form the primary basis for trad-
itional genetics, it has become clear that conditions whose legacies strictly confirm the Mendelian
principles are relatively rare.
DOI: 10.1201/9781003343790-1 1
2 Diagnostics and Gene Therapy for Human Genetic Disorders
Multiple different genes encode a polygenic trait. Each of them might have several possible
different alleles, whose combinations can give rise to a range of possible phenotypes. For example,
at least three genes control human eye color. Each gene has several different alleles, resulting in
many different possible eye colours such as brown, hazel, green, blue, grey, purple, and amber.
Complex traits arise from variations within multiple genes and their interactions with behavioral
and environmental factors. These traits do not readily follow the predictable patterns of inheritance.
There is a significant distinction between a monogenic and polygenic trait, which is overly sim-
plistic. Monogenic traits are also influenced by variation in multiple genes called modifier genes. On
the other hand, variations in a single gene can predominantly influence complex traits.
The evolution of single-gene traits as versions of complex traits has been illustrated by: (1) widely
different phenotypes accounting for allelic variations in a single gene; (2) the blurring of predicted
relationships between genotype and phenotype in several monogenic disorders; and (3) modifier
genes and non-genetic factors contributing to the phenotypes of monogenic disorders. The com-
plexity of the relationships between genotype and phenotype, leading to three different monogenic
disorders, cystic fibrosis, Hartnup phenotype, and the PiZZ form of α-1 antitrypsin deficiency, has
been demonstrated in Figure 1.1. Cystic fibrosis (MIM 219700) occurs due to both allelic variation
at the CFTR locus and the expression of a key modifier locus, explaining the difference between the
pancreatic-sufficient and insufficient forms of the disease. The Hartnup phenotype (MIM 234500),
in which a mutation at the major locus accounts for amino acid transport disorder in the kidneys and
intestines. There is also evidence for a multifactorial threshold phenomenon affecting the overall
homeostasis of plasma amino acid levels. The latter accounts for the difference between a patient
having only the variant Hartnup amino acid transport phenotype (a biochemical trait) and having
Hartnup disease, a pellagra-like clinical entity. The PiZZ form of α-1 antitrypsin deficiency, where
some of the variations in the clinical phenotype involving the lungs, is explained by environmental
factors such as smoking, whereas inter-individual differences involving the liver are related to the
handling of mutant protein by the chaperones and proteases.
FIGURE 1.1 Influence of genotype and other non-genetic factors on the phenotype of various monogenic
disorders.
Genetic Analysis 3
The above figure indicates the estimated relative importance of background genotype and
environment as the major contributors to the phenotype of several monogenic diseases. The
equation VP =VG + VE implies that variations in genotype and environment contribute to the
variation in phenotype. A detailed classification of monogenic traits and their disorders is
provided in Chapter 3.
1.3 LINKAGE ANALYSIS
1.3.1 Linkage
Two genetic loci are considered to be in linkage if the alleles at these loci segregate together more
often. These two loci locations are so close on the same chromosome that the chances of their sep-
aration by a crossover event (also denoted as a recombination) during meiosis are remote. The prob-
ability of inheritance of any two alleles at two randomly selected loci together is 0.5. If two loci are
closely linked, then the chance of crossover or recombination occurrence is <0.5. The probability
of recombination taking place is linked to the distance between any two loci. The recombination
fraction [θ] is a measure of the genetic distance between two loci. The distance between two loci is
measured in centimorgans. One centimorgan is the genetic distance between two loci with a recom-
bination frequency of 1%. Although the centimorgan is not an exact measure of physical distance, it
typically equates to a physical space of one million base pairs. So, two loci close to the F8 gene with
a 5% probability of recombination would be five centimorgans apart: approximately 5 million base
pairs. Linkage analysis aims to identify the appropriate markers that co-segregate with the gene of
interest, tracking the gene within a family without knowing the mutation (Jurg et al. 2015). By def-
inition, this marker must co-segregate with the gene of interest and so be present in affected family
members but absent in unaffected family members.
1.3.2 Linkage Analysis
Linkage analysis is a method applied to establish the carrier status of female ‘at-risk’ carriers and
prenatal diagnoses. In many cases, linkage analysis replaces mutational analysis. In a small number
of families in whom the mutation cannot be identified, linkage analysis remains the only method
for the genetic diagnosis of carriers. The principle of linkage analysis is straightforward. All human
chromosomes come in pairs, with one inherited from the mother and one from the father. Each pair
of chromosomes contains the same genes in the same order, but their sequences are not identical.
It is easy to determine whether a particular sequence of offspring comes from a mother or father.
These sequence variations are called maternal and paternal alleles. In the case of a diseased gene,
the alternative alleles will be normal alleles and the diseased allele is distinguished by looking for
the occurrence of the disease in a family tree or pedigree. Genetic markers are DNA sequences that
show polymorphism (variations in size or sequence) in the population. They are present in every
human genome and can be typed (identification of the allele) using techniques such as polymerase
chain reaction. This ability to determine the parental origin of a DNA sequence allows us to show
whether recombination has occurred or not. Recombination occurs in germ cells that make eggs and
sperm. In these cells, the maternal and paternal chromosomes pair up and exchange their fragments.
After recombination, the chromosomes contain a mixture of maternal and paternal alleles, placed in
the germinal cells and passed to the next generation (Pulst, 1999).
Recombination occurs randomly. During this process, if there is a considerable distance between
two DNA sequences on a chromosome, there is a good chance that recombination will occur between
them, and the maternal and paternal alleles will be mixed up (A and C in Figure 1.2). In contrast,
if two DNA sequences are very close together, they will rarely recombine. As a result, the maternal
and paternal alleles will tend to stay together (A and B in Figure 1.2). Diseased genes are mapped by
measuring the recombination against a panel of different markers spread over the entire genome. In
FIGURE 1.2 Linkage analysis: Top panel shows paternal (ABC alleles) and maternal (abc alleles)
chromosomes aligned in a germ cell, a cell that gives rise to eggs or sperm. The middle panel shows the physical
process of recombination, which involves the crossing over of DNA strands between the paired chromosomes.
The bottom panel shows what happens when the crossover is resolved. The maternal and paternal alleles are
mixed (recombined), and these mixed chromosomes are passed to the sperm or eggs. If A is the diseased gene
and B and C are genetic markers, recombination is likely to occur much more frequently between A and C than
between A and B. This allows the disease gene to be mapped relative to markers B and C.
most cases, recombination will occur frequently, indicating that the disease gene and marker are far
apart. A few markers will tend not to recombine with the diseased gene due to their proximity. These
markers are said to be linked to it. Ideally, close markers have identified that flank, with the diseased
gene, and have defined a candidate region of the genome between 1 and 5 million bp in length. The
gene responsible for the disease lies somewhere in this region.
1.3.3 Polymorphic DNA Markers

Linkage analysis aims to identify an appropriate DNA marker co-segregating with the gene of
interest and can track the gene within a family without knowing the mutation. The markers com-
monly used to track a gene within a family are polymorphic. There are two major types of poly-
morphic markers or polymorphisms.
a) Single Nucleotide Polymorphisms (SNPs)

b) Short Tandem Repeat (STRs) or Variable Number Tandem Repeats (VNTRs)
Genetic Analysis 5
a) Single Nucleotide Polymorphisms [SNPs]: are single nucleotide changes that usually
occur in the DNA, which will not change the amino acid sequence of the corresponding protein
of interest. The location of SNPs is throughout the human genome as intragenic polymorphisms
occurring within a gene, usually in the introns or rarely in the 5’ and 3’ untranslated regions (UTRs).
Extragenic polymorphisms are closely linked to a gene. Historically, SNPs are often designated by
a restriction endonuclease or an enzyme that digests the DNA before agarose gel electrophoresis
and southern blotting hybridization. For example, within the F8 gene, the enzyme Bcl I identified an
intragenic polymorphism located within intron 18 by cutting the DNA into two fragments of 0.8 kb
and 1.1 kb. Similarly, the enzyme Bgl I identified an SNP located within intron 25 of the same F8
gene by cutting the DNA into two sequences of 5 kb and 20 kb, The enzyme Bgl II identified an SNP
located close to, but not a part of the F8 gene, gives rise to two fragments of 5.8 kb and 2.8 kb. The
common feature is that when digested with Bgl II, Bcl I and Bgl I, the DNA fragments generated
are of different lengths. The polymorphisms giving rise to these differing fragments are known
as restriction fragment length polymorphisms or RFLPs. Southern blotting is rarely performed
today. Detection of SNPs is by PCR either with sequence analysis or by the resolution of the DNA
fragments on agarose gel electrophoresis.
b) Short Tandem Repeat (STRs) or Variable Number of Tandem Repeats (VNTRs): Short
Tandem Repeats are extremely resourceful DNA markers that are highly polymorphic and inherited
in a strict Mendelian fashion. Areas of repetitive DNA occur throughout the genome where the
repeating unit is tiny, either 1-6 nucleotides (minisatellite) or 2-3 nucleotides (microsatellite).
Microsatellite repeats are comprised of dinucleotide repeat sequences such as CA in which the
repeated sequence occurs multiple times (CACACACACA) and trinucleotide repeats (ATT)n.
Minisatellite repeats contain tetranucleotide repeats [GATA]n or hexanucleotide repeats (CAATAC)
n. These are highly polymorphic within a population and can be used for bone marrow transplant
engraftment, forensics, identity testing, paternity testing, and so forth. STRs are widely used in gen-
etic linkage studies. The reason for this lies in the greater chance that a particular individual may
be heterozygous for a specific marker. Although the number of repeat sequences may change, this
change is predicted to occur every 100 generations.
Genetic linkage analysis is a powerful tool for detecting the chromosomal location of diseased
genes. It is based on the observation that genes that reside physically close on a chromosome
remain linked during meiosis. For most neurological disorders whose underlying biochemical
defect was not known, identifying the chromosomal location of the diseased gene was the first
step in its eventual isolation. Genes that have been isolated in this way include examples from
all types of neurological disorders such as Alzheimer’s, Parkinson’s, or ataxia, and diseases of
ion channels leading to periodic paralysis or hemiplegic migraine, or tumor syndromes such as
neurofibromatosis types 1 and 2 (Scriver et al. 1999). With the advent of new genetic markers
and automated genotyping, rapid genetic mapping is possible in a minimum amount of time.
Generated genetic linkage maps for the human genome provide the basis for constructing phys-
ical maps and permit the rapid mapping of disease traits. After establishing the chromosomal
location of a diseased phenotype, genetic linkage analysis helps determine the diseased pheno-
type due to single or multiple gene mutations. Mutations in other genes may also give rise
to an identical or similar phenotype. Often it is found that mutations in different genes can
cause similar phenotypes. Examples are autosomal dominant spinocerebellar ataxias, caused by
mutations in different genes but with very similar phenotypes. In addition to providing novel,
genotype-based classifications of neurologic diseases, genetic linkage analysis can also aid
in disease diagnosis. However, in contrast to direct mutational analysis such as detecting an
expanded CAG repeat in the Huntington gene, diagnosis using flanking markers requires the
analysis of several family members.
1.4 LINKAGE DISEQUILIBRIUM
Linkage disequilibrium is a nonrandom association of alleles at two or more loci. Lewontin and
Kojima coined the word in 1960. Initially, linkage disequilibrium was not the primary concern of the
population geneticists due to the non-availability of data for the study. Its importance to evolutionary
biology and human genetics was unrecognized outside population genetics. However, the import-
ance of linkage disequilibrium gained widespread interest in the 1980s after the role of linkage dis-
equilibrium in gene mapping became evident and large-scale surveys of closely linked loci became
feasible. It also plays a vital role in evolutionary biology and human genetics, as there are so many
factors that affect and are affected by linkage disequilibrium. Linkage disequilibrium provides infor-
mation about past events and constrains the potential response to natural and artificial selection.
Linkage disequilibrium throughout the genome reflects the population history, the breeding system,
and the pattern of geographic subdivision (Montgomery, 2008). Linkage disequilibrium in each
genomic region demonstrates the history of natural selection, gene conversion, mutation, and other
forces that cause gene-frequency evolution. How these factors affect linkage disequilibrium between
a particular pair of loci or in a genomic region depends on local recombination rates. The population
genetics theory of linkage disequilibrium is well developed and widely used to provide insight into
evolutionary history and as the basis for mapping genes in human beings and other species.
1.4.1 Linkage Disequilibrium at One Pair of Loci

Linkage disequilibrium between alleles at two loci has been defined in many ways, but all definitions
depend on the quantity:
DAB =pAB –p A pB (1)
DAB refers to the difference between the frequency of gametes carrying a pair of alleles A and
B at two loci (pAB) and the frequency product of those alleles (pA and pB). The initial definition
was in terms of gamete frequencies because it allowed the possibility that the loci are on different
chromosomes. The second definition is for the loci on the same chromosome. In this case, the allele
pair AB is called a haplotype, and pAB is the haplotype frequency. As defined, DAB characterizes a
population, and in practice, DAB is estimated from the allele and haplotype frequencies of a sample.
Standard sampling theory must be applied to find the confidence intervals of estimated values.
The quantity, DAB is the coefficient of linkage disequilibrium. It is defined for a specific pair
of alleles, A and B, and does not depend on how many other alleles are at the two loci. Each pair
of alleles has its own D. Constriction of the values for different pairs of alleles is due to the allele
frequencies at both loci, and the haplotype frequencies must add up to a value of 1. If both loci are
diallelic, as is the case with all SNPs, the constraint is strong enough that only one value of D is
needed to characterize linkage disequilibrium between those loci. In fact,
DAB= –DAb= –DaB =Dab
Here, a and b are the other alleles. In this case, the D is used without a subscript. Thus, the sign
of D is arbitrary and depends on which pair of alleles one starts with.
If either locus contains more than two alleles, no single statistical method can quantify the
overall linkage disequilibrium between them. A statistical method is needed when both the loci
have numerous alleles, as with loci in the major histocompatibility complex of vertebrates, with
more than hundreds of alleles. On the other hand, there might be no single pair of alleles with a
particular interest, whether more linkage disequilibrium between one loci pair than another pair or
more linkage disequilibrium between a loci pair in one species than in one species another one (John
and William, 2018).
Genetic Analysis 7
1.4.2 Linkage Equilibrium
If D =0, there is linkage equilibrium (Le), which has similarities with Hardy Weinberg equilibrium
in implying statistical independence. When genotypes at a single locus are at H, an allele present
on one chromosome is independent of whether it is present on its homolog. Consequently, the fre-
quency of the AA homozygote is the square of the frequency of A (pAA =pA2), and the frequency
of the Aa heterozygote is twice the product of pA and pa, the two being necessary to allow for both
Aa and aA. Here, the essential feature of Hardy Weinberg equilibrium is that regardless of the initial
genotype frequencies, equilibrium is established in one generation of random mating. Any initial
deviation from H will disappear immediately. Significant departures from Hardy Weinberg equilib-
rium indicate that something interesting is going on, as in the case of extensive inbreeding, strong
selection, or genotyping error. Le is like Hardy Weinberg equilibrium as it also implies that alleles
at different loci are randomly associated. The frequency of the AB haplotype is the product of the
allele frequencies (pApB). Linkage equilibrium, however, differs from Hardy Weinberg equilibrium
because it is not established in one generation of random mating. Instead, D decreases at a rate that
depends on the recombination frequency c, between the two loci:
DAB (t +1) =(1 – c) DAB (t) (2)
Where t is time in generations. Even for unli nked loci (c =0.5), D decreases only by a factor
of half each generation, as proved by Weinberg in 1909. The general formula was obtained first
by Jennings. Although linkage equilibrium will be reached, it will occur slowly in closely linked
loci. This is the basis for the use of linkage disequilibrium. Other population genetic forces such as
selection, gene flow, genetic drift, and mutation, all affect D. So, substantial linkage disequilibrium
will persist under many conditions. When surveying the number of polymorphic loci in a genome
is possible, the extent of linkage disequilibrium in a genome can be quantified with great preci-
sion, allowing a fine-scale analysis of the forces governing the genomic variation. The co-efficient
of linkage disequilibrium and related quantities are descriptive. Their magnitude does not indi-
cate whether there is a statistically significant association between alleles in haplotypes. Standard
statistical tests, including the chi-squared and Fisher’s exact test, are commonly used to test the
significance.
1.4.3 Linkage Disequilibrium at More Than Two Loci

When more than two loci are considered together, a common practice is to distinguish the allelic
pairs that show high levels of linkage disequilibrium from those that do not. The result is a graph
introduced by Miyashita and Langley (1998) to describe the patterns of linkage disequilibrium in
Drosophila melanogaster. A 216 kb segment in the class II region of the major histocompatibility
complex in humans comprises non-overlapping sets of loci in strong linkage disequilibrium. Each
group is called a haplotype block, and boundaries were associated with hotspots of recombination.
Similar patterns were found in other regions in the human genome, leading to the hypothesis that
most of the human genome had a block-like pattern of linkage disequilibrium. Haplotype blocks in
humans vary in size from a few kb to more than 100 kb.
1.4.4 Linkage Disequilibrium Within and Between Populations

When data for more than one population is available, linkage disequilibrium between a pair of loci
can be partitioned into contributions within and between populations. Ohta (1982) first suggested this
partitioning is like Wright’s partitioning of deviations from Hardy Weinberg equilibrium frequen-
cies into FIS, the average deviation within populations, and FST, the average deviation attributable
to differences in allele frequency among populations. Ohta partitioned DIT, the total disequilibrium
in a subdivided population, into DIS, the average disequilibrium within subpopulations, and DST
contributing to the overall disequilibrium caused by the differences in allele frequencies within
the subpopulations. Computer programs such as Genepop are available to calculate DIS and DST.
These statistics are widely applied in the analysis of data from non-human populations but rarely
for human populations. This rarity in application is probably due to the focus on the humans of each
population, whereas the focus on other species is often on the overall pattern of linkage disequilib-
rium. Natural selection favoring adaptations to local conditions will increase DST whenever alleles
at different loci are favored. Partitioning overall linkage disequilibrium is an appropriate first step
when determining the differences in linkage disequilibrium resulting only from differences in allele
frequency or from other factors that vary among populations.
1.4.5 Population Genetics of Linkage Disequilibrium

1. Natural selection
Initial interest in linkage disequilibrium has been increased because of the questions raised on the
operation of natural selection. If alleles at two loci are in linkage disequilibrium and they both affect
reproductive fitness, the response to selection on one locus might be accelerated or impeded by
selection affecting the other. One line of research in this area concerns the effect of linkage disequi-
librium on long-term trends in evolution. Kimura (1965) and Nagylaki (1974) showed that unless
interacting loci are very closely linked or selection is very strong, recombination dominates, and as
a good approximation, linkage disequilibrium can be ignored. This theory supports Fisher’s depic-
tion of natural selection as steadily increasing the average fitness of a population. This theory also
shows that when selection is strong, and fitness interactions among loci are complex, average fitness
might not increase in every generation because linkage disequilibrium constrains how haplotype
frequencies respond to a selection. In this case, linkage must be accounted for explicitly before even
qualitative predictions can be made. In some cases, selection alone can increase linkage disequi-
librium when fitness values are more than multiplicative, which means that the average fitness of
an individual carrying the AB haplotype exceeds the product of the average fitness of individuals
carrying A alone or B alone. This pattern is easiest to see with diallelic loci in haploid organisms.
If the relative fitness (w) of the ab, Ab, and aB haplotypes are wAb and waB, then selection will
increase linkage disequilibrium only if wAB > wAbwaB. If both A and B are maintained by balan-
cing selection, then linkage disequilibrium can persist indefinitely. Further, when more than two loci
interact in this way, large blocks of linkage disequilibrium can be maintained by selection, where an
individual locus is not an appropriate unit of selection. Interest in this theory decreased during the
1970s, after discovering linkage disequilibrium that was not detected between alleles distinguishable
by protein electrophoresis. This theory might become popular again or perhaps be re-invented as
studies find increasing evidence of intragenic interactions that can create strong epistasis in fitness.
2. Genetic Drift
Genetic drift alone can generate linkage disequilibrium between the closely linked loci. Even if two
loci are in linkage equilibrium, sampling only a few individuals will create some linkage disequilib-
rium. First results obtained during the late 1960s suggested that a genetic drift balanced by a muta-
tion and recombination would maintain only low levels of linkage disequilibrium. The expectation
of D2 is small even if there is no recombination because the flux of mutations at both loci tends to
eliminate most linkage disequilibrium. To attend to this, genetic drift was largely ignored as a cause
of linkage disequilibrium. However, the expectation of D2 does not tell the whole story because it
includes cases in which one or both loci are monomorphic (when D is necessarily 0). The expectation
of D2 when both loci are polymorphic cannot be calculated analytically, but simulations show that
Genetic Analysis 9
much more significant values are seen. Genetic drift surprisingly interacts with selection. Selection
affecting closely linked loci becomes slightly weakened because drift creates small amounts of
linkage disequilibrium that, on average, reduces the response to selection. This effect, known as the
Hill–Robertson effect, is relatively weak when only two loci are considered (Felsenstein, 1965).
Nevertheless, it is much stronger per locus when many selected loci are closely linked.
3. Inbreeding, Inversions, and Gene Conversion
Other forces that create linkage disequilibrium are inbreeding, inversions, and gene conversion.
Inbreeding creates linkage disequilibrium for the same reason as population subdivision. Because
of recent common ancestry, inbreeding augments the covariance between alleles at different loci.
Genomic inversions significantly reduce the recombination between the inverted and non-inverted
segments as the recombination produces aneuploid gametes. Consequently, the inverted and original
segments become equivalent to almost completely isolated subpopulations between which linkage
disequilibrium accumulates. Drosophila geneticists have long appreciated this fact.
Gene conversion affects the linkage disequilibrium at a pair of loci in the same way that reciprocal
recombination does. This equivalence can be seen by considering a pair of diallelic loci A/a and B/
b. Gene conversion at the B/b locus will result in an individual with haplotype phase AB/ab who
will produce Ab or aB gametes depending on whether B converts b or the reverse. However, gene
conversion differs from recombination when more than two loci are considered together. Reciprocal
crossing over affects linkage disequilibrium between all pairs of loci on opposite sides where the
crossing over has taken place. By contrast, gene conversion affects loci only within the conversion
track, which is relatively short. Loci that are not within the track are unaffected. For example, three
loci A/a, B/b, and C/c are located on a chromosome in order, where B/b is in a conversion track. The
linkage disequilibrium between A/a, B/b, and B/b, C/c is affected by the conversion, but the linkage
disequilibrium between A/a and C/c is not. Several methods for inferring the relative rates of gene
conversion and recombination are based on this idea.
1.4.6 Applications of Linkage Disequilibrium

1. Mutation and Gene Mapping
Mutation has a unique role in creating a linkage disequilibrium. When a mutant allele, M, first
appears on a chromosome, it is in low frequency, pM = 1/(2N) (N is the population size) and is in
perfect linkage disequilibrium with the alleles at other loci that are on the chromosome carrying
the first copy of M. perfect linkage disequilibrium means that D′ =1. If D′ =1, only three of
the four possible haplotypes are present in the population. Perfect linkage disequilibrium will per-
sist until recombination involving an M‑bearing chromosome creates a non-ancestral haplotype.
Consequently, loci closely linked to M will remain in perfect linkage disequilibrium for a long time
and in strong linkage disequilibrium for an even longer time. The persistence of strong linkage dis-
equilibrium between a mutant allele and the loci closely linked to it has many practical implications.
Rare marker alleles in strong linkage disequilibrium with a monogenic disease locus must be closely
linked to the causative locus.
A relatively simple mathematical theory indicates how close these two are. This method, known
as linkage disequilibrium mapping, has been successfully used with several diseases. The same idea
underlies the association mapping of complex diseases. Closely linked polymorphic SNPs tend to
be in strong linkage disequilibrium with one another. The fine-scale pattern of linkage disequilib-
rium in humans confirms that the human genome comprises haplotype blocks within which most
or all SNPs are in high linkage disequilibrium. These high levels of linkage disequilibrium among
SNPs are assumed to be true for alleles that increase the risk of complex inherited diseases. This
idea, combined with the development of efficient methods for surveying large numbers of SNPs,
has led to the many recent genome-wide association (GWA) studies that have detected SNPs sig-
nificantly associated with breast cancer, colorectal cancer, and type 2 diabetes, and heart disease.
However, one potential problem in genome-wide association studies is creating linkage disequilib-
rium by unrecognized population subdivision. Several methods have been proposed to account for
such linkage disequilibrium. Although genome-wide association studies have successfully found
new causative alleles, the overall proportion of risk accounted for is often relatively low. Alleles
accounting for a more significant proportion of risk might be found in more extensive studies. It
is unclear whether most causative variants will ultimately be found this way. The reason is that
GWA studies are more effective in finding causative alleles that are relatively high frequency. Other
methods might be needed for low-frequency causative alleles.
2. Detection of Natural Selection
Strong positive selection quickly increases the frequency of an advantageous allele, with the result
that linked loci remain in unusually strong linkage disequilibrium with that allele. Recently, methods
have been developed to detect unusually low heterozygosity regions that indicate past hitch-hiking
events. If an advantageous allele has not gone to fixation, variability at linked markers will be lower
on chromosomes bearing that allele than on other chromosomes. Several tests of neutrality are based
on this idea. One class of methods assumes that a potentially advantageous allele at a locus has been
identified and tests whether there is significantly more linkage disequilibrium with that allele than
with other alleles at the same locus. The second class of methods assumes only that the potentially
selected locus has been identified and tests whether patterns of haplotype variation at that locus are
consistent with neutrality.
3. Estimation of Allele Age
Strong linkage disequilibrium with an allele in a relatively large region indicates that not much time
has passed since the allele arose by a mutation. If the mutant allele has reached a relatively high
frequency in a short time, it is likely to have done so under the effect of positive selection. This ten-
dency provides the basis for the selection. In addition to it, linkage disequilibrium can indicate the
point in time when an allele arose as a mutation. This approach is straightforward, which takes a
reasonable estimate of an allele age, but it does not consider the stochastic nature of recombination
and genetic drift and exaggerates the accuracy of the resulting estimates.
1.4.7 Linkage Disequilibrium and Human Genome

Linkage disequilibrium is one of the major processes studied in human population genetics. After
resolving the technical problem of efficiently genotyping 500,000 SNPs, which considerably
declined the cost of genotyping, there is an increased scope for many very large-scale genome-wide
association studies to be carried out. Soon the new technologies will allow large-scale re-sequencing
studies, including the 1000 Genomes Project, enabling more data for these studies. The only limiting
factor will be the availability of people willing to participate in the genome-wide association studies
and the resources needed for the accurate clinical assessment (Erlich et al. 2009).
1.5 MENDELIAN PEDIGREE PATTERNS

The most uncomplicated genetic characters are those whose presence or absence depends on the
genotype at a single locus. Sometimes, a particular genotype at a single locus is necessary and suf-
ficient for that character to be expressed, given the organism’s typical genetic and environmental
Genetic Analysis 11
background. Such characters are called Mendelian. In humans, more than 10,000 Mendelian
characters have been identified. If a family is affected by a disease or a disorder, the pattern of dis-
ease transmission from one generation to another can be established by accurate family history.
Additionally, family history can also help to exclude a few genetic diseases. Most genes have one
or more versions due to mutations or polymorphisms, referred to as alleles. Individuals may carry a
normal, diseased, or rare allele, depending on the impact of the mutation or polymorphism and the
population frequency of that allele.
1.5.1 Dominance and Recessiveness of Characters

A character is considered dominant if manifested in the heterozygote and is considered reces-
sive if it is not manifested in the heterozygote. Dominance and recessiveness are the properties
of characters, not genes. Most of the dominant human syndromes are known only in heterozy-
gote individuals. Sometimes homozygotes have also been described, born from the mating of two
heterozygotes, where people are much more severely affected. Examples are achondroplasia (short-
limbed dwarfism) and Type 1 Waardenburg syndrome (deafness with pigmentary abnormalities).
Nevertheless, achondroplasia and Waardenburg syndrome are described as dominant because these
terms also describe phenotypes seen in heterozygotes. When this uncertainty does not exist, the con-
dition is named semi-dominant, where the heterozygote has an intermediate phenotype, reserving
the dominant conditions. The homozygote is indistinguishable from the heterozygote, as exem-
plified in the case of Huntington’s disease (an adult-onset progressive neurological deterioration).
Males are always hemizygous for the loci on the X and Y chromosomes, where they have only a
single copy of each gene. In this case, the question of dominance or recessiveness does not arise in
males for both X and Y-linked characters. Autosomal characters in males and females, and X-linked
characters in females can be dominant or recessive.
1.5.2 Types of Mendelian Pedigree

No human being has two genetically different Y chromosomes (except in the rare case of XYY
males, where the two Y chromosomes are duplicates). There are five archetypal Mendelian pedigrees
(i) Autosomal dominant, (ii) autosomal recessive, (iii) X-linked recessive, (iv) X-linked dominant,
and (v) Y-linked.
i. Autosomal Dominant
Autosomal dominance is a pattern of inheritance that occurs if the gene of question is located on
one of the 1-22 or non-sex chromosomes. “Dominant” means a single copy of the disease-associated
mutation is sufficient to cause the disease. Huntington’s disease and neurofibromatosis-1 are typ-
ical examples of autosomal dominant genetic disorders. Autosomal dominance is one of the several
ways that a trait or a disorder can be passed down through families. It describes a trait or disorder
in which the phenotype is expressed in those who have inherited only one copy of a particular gene
mutation (heterozygotes), explicitly referring to a gene on one of the 22 pairs of autosomes. One of
the parents may often have the disease. Inheriting a disease, condition, or trait depends on the type
of chromosome affected. It also depends on whether the trait is dominant or recessive. In most of the
cases, the abnormal gene dominates. Each child’s risk is independent of whether their sibling has the
disorder or not. If the first child has the disorder, the next child has the same 50% risk of inheriting
the disorder. Children who do not inherit the abnormal gene will not develop or pass on the disease.
If someone has an abnormal gene inherited in an autosomal dominant manner, the parents should
also be tested for the abnormal gene.
ii. Autosomal Recessive
The autosomal recessive inheritance gene in question is located on one of the 1-22 autosomes or
non-sex chromosomes. Autosomes do not affect an offspring’s gender. “Recessive” means that two
copies of the gene are necessary to have the trait or disorder, with one copy inherited from the
mother and the other from the father. If only one recessive gene is present, it is considered a “carrier”
for the trait or disease, which will not generate any health problems. Carrier genes do not show any
signs of the disease or condition. Most people even do not know that they are recessive gene carriers
until they have a child with that disease. Once parents have a child with a recessive trait or disease,
there is a 25% chance that another child will be born with the same trait or disorder with each sub-
sequent pregnancy. This means a three out of four or a 75 % chance that another child will not have
the trait or disease. The birth of a child with a recessive condition is often a total surprise to a family.
In most cases, there is no previous family history of a recessive condition. It is estimated that all
people carry about five or more recessive genes that cause genetic diseases or conditions. Examples
of autosomal recessive disorders include cystic fibrosis, sickle cell anemia, and Tay-Sachs disease.
iii. X-linked Dominant
Sex-linked dominance is a rare way that a trait or disorder can be passed down through families.
A single abnormal gene on the X chromosome can cause a dominant sex-linked disease. Dominant
inheritance occurs when an abnormal gene from one parent can cause disease, even though a
matching gene from the other parent is normal. The abnormal gene dominates the gene pair. In the
case of X-linked dominant disorder, if a father carries the abnormal gene on the X chromosome, all
his daughters will inherit the disease, and none of his sons will have the disease because daughters
always inherit their father’s X chromosome. If the mother carries the abnormal X gene, half of all
their children (daughters and sons) will inherit the disease tendency.
If there are four children (two males and two females), and the mother is affected with one
abnormal X, and she has the disease, but the father is not, the statistical expectation is
• Two children (one girl and one boy) with the disease and
• Two children (one girl and one boy) without the disease.
If there are four children (two males and two females) and the father is having the affected
abnormal gene on the X chromosome, and he has the disease, but the mother is not, the statistical
expectation is:
• Two girls with the disease

• Two boys without the disease.
Males are more severely affected than females because, in each affected female, there is one
normal allele producing a regular gene product and one mutant allele producing the non-functioning
product, while in each affected male, there is only the mutant allele with its non-functioning product
and the Y chromosome, no specific gene product at all. Affected females are more prevalent in the
general population because the female has two X chromosomes, which could carry the mutant allele,
while the male only has one X chromosome as a target for the mutant allele. When the disease is no
more deleterious in males than in females, females are about twice as likely to be affected as males.
iv. X-linked Recessive
A mode of inheritance in which a mutation in a gene on the X-chromosome causes the pheno-
type to be expressed in males who are hemizygous for the gene mutation (they have only one X
Genetic Analysis 13
chromosome) and in females who are homozygous for the gene mutation (they have a copy of the
gene mutation on each of their two X chromosomes). Carrier females who have only one copy of the
mutation do not usually express the phenotype, although differences in X-chromosome inactivation
can lead to varying degrees of clinical expression in carrier females. Examples of X-linked recessive
conditions include red-green color blindness and hemophilia A:
Red-green color blindness: Red-green color blindness means that a person cannot distinguish
the shades of red and green. Their visual acuity is normal. There are no severe complications.
However, affected individuals may not be considered for certain occupations involving transporta-
tion or the armed forces where color recognition is required. Males are affected 16 times more often
than females because the gene is located on the X chromosome.
Hemophilia: Hemophilia A is a disorder where the blood cannot clot properly due to a clotting
factor deficiency known as Factor VIII. This results in abnormally heavy bleeding that will not stop,
even from a small cut. People with hemophilia A bruise easily and can have internal bleeding into
their joints and muscles. The occurrence of hemophilia A (Factor VIII deficiency) and hemophilia B
(Factor IX deficiency) combined is one in 10,000 live male births, with hemophilia A accounting for
80 percent of all cases. Treatment is available by infusion of Factor VIII (blood transfusion). Female
carriers of the gene may show some mild signs of Factor VIII deficiency, such as bruising easily or
taking longer than usual to stop bleeding when cut. However, not all female carriers present these
symptoms. One-third of all hemophilia cases are due to new mutations in the family (not inherited
from the mother).
V. Y-linked
Although a few Y-linked characters have been described, no Y-linked diseases are known, apart
from disorders of male sexual function. Conceivably such a disease may exist undiscovered, but
this is unlikely for two reasons. (1) The pedigree pattern would be strikingly noticeable, especially
in societies that trace the family through the male line, yet they have not been noted. (2) The Y-
chromosome cannot carry any genes whose function is essential for health because females are
perfectly normal without any Y-linked genes. Thus, any Y-linked genes must code either for non-
essential characters or male-specific functions, and defects are unlikely to cause diseases apart from
defects of male sexual function.
1.6 MENDELIAN PEDIGREE ANALYSIS

1.6.1 Pedigree Analysis
A pedigree is a family relationship chart that uses symbols to represent people and lines to
represent genetic relationships. These diagrams make it easier to visualize the relationships within
families, particularly extensive families. Pedigrees are often used to determine the dominance and
recessiveness of genetic diseases. An example of a pedigree chart showing the four generations of
a family with four individuals affected by color blindness is displayed in Figure 1.3. Controlled
crosses cannot be made in human beings. Geneticists must emphasize the family records hoping
that available informative matings can be used to deduce the dominance and distinguish the auto-
somal form of X-linked inheritance. The investigator traces the history of some variant phenotypes
back through the family’s history and draws up a family chart or pedigree using the standard
symbols. Standard symbols used for the pedigree analysis are listed in Table 1.1. Clues in the
pedigree must be interpreted differently. The interpretation depends on one contrasting pheno-
type, such as a rare disorder, or where both of the phenotypes of a pair are common morphs of a
polymorphism.
In this pedigree chart, the unaffected founding mother, I-1, and affected founding father, I-2
are the parents of two affected daughters, II-1 and II-2. The affected founding daughter II-2 and
the unaffected male II-3 who “marries into the family” have two offspring, an unaffected daughter
FIGURE 1.3 Pedigree chart of a family affected with color blindness. Circles represent females and squares
represent males. A dark circle or a dark square represents an individual affected by the trait. Individuals are
represented by the Roman numeral, which stands for the family’s generation. The female at the upper left is
an individual I-1. Founder parents in this family are the female I-1 and the male I-2 in the first generation at
the top. A male and female in this pedigree are directly connected by a horizontal line, have mated, and had
children. These three pairs have mated in this tree: I-1 & I-2, II-2 & II-3, III-2 & III-3. Vertical lines connect
parents to their children. For instance, the females II-1 and II-2 are daughters of I-1 and II-2. The founding
family consists of the two founding parents and their children, II-1 and II-2.
TABLE 1.1
Symbols Used for the Pedigree Analysis
S. No Symbol Representation in Pedigree Analysis
1. Male
2. Female
3. Mating
4. Parents and Children in order of birth.

1 Boy and 1 Girl.
Genetic Analysis 15
TABLE 1.1 (Continued)

Symbols Used for the Pedigree Analysis
S. No Symbol Representation in Pedigree Analysis
5. Dizygotic or non-identical twins
6. Monozygotic twins
7. Unspecified Sex
8. Abortion or Still Birth (Sex unspecified)
9. Propositus
10. Carrier of sex-linked recessive
11. Death
12. Affected Individuals
13. Heterozygotes
14. Consanguineous Marriage

III-1 and an affected son III-2. Finally, this affected male III-2 and the unaffected female III-3 who
“marries in” have an unaffected son, IV-1. The genetic disorders of human beings can be either auto-
somal or X-linked.
1.6.2 Patterns Indicating a Recessive Trait

If any affected founding daughter has two unaffected parents, the disease must be autosomal reces-
sive. The disease must be recessive if any affected individual has two unaffected parents. At least
one parent must have an allele for the disease (Figure 1.4).
1.6.2.1 Autosomal Recessive
An affected individual must inherit a recessive allele from both parents, so both parents must have
that allele. If the father had a recessive X-linked allele, he must have been affected by only one X-
linked allele. If an affected founding son has two unaffected parents, we cannot determine if the dis-
ease is autosomal, or X-linked (Figure 1.5). If the trait is autosomal, both parents can be unaffected
carriers of the disease. If the trait is X-linked, the son must have inherited his allele from his mother
only, and his father can be unaffected.
1.6.3 Patterns Indicating a Dominant Trait

The disease must be dominant if every affected child of the non-founding parent has an affected
parent. The unaffected mother, who is marrying, does not carry an allele for the disease. So, the
affected child inherits an allele only from the affected father. No child could be affected by a single
autosomal recessive allele or X-linked recessive allele, so the trait is dominant (Figure 1.6).
FIGURE 1.4 Pedigree patterns that indicate a recessive Trait.
FIGURE 1.5 Pedigree of an autosomal recessive disorder.

Genetic Analysis 17
FIGURE 1.6 Pedigree patterns that indicate a dominant trait.
FIGURE 1.7 Pedigree of a dominant phenotype determined by a dominant allele, A with all genotypes
deduced.
1.6.3.1 Autosomal Dominant Trait

In autosomal dominant disorders, the normal allele is a recessive one, and the abnormal allele or the
diseased allele is dominant. It might seem paradoxical that a rare disorder can be dominant. It can
also be considered that dominance and recessiveness reflect how different alleles act, which is not
defined in terms of predominance in the population. During the pedigree analysis, the main clue for
identifying an autosomal dominant disorder is the phenotype that tends to appear in every generation
of the pedigree and affected parents (both father and mother) transmit the phenotype to both sons
and daughters. Here, the phenotype appears in every generation as an abnormal allele, carried by an
individual who might have inherited it from his parent in the previous generation. Mendelian ratios
need not be necessarily observed in all families. In recessive disorders, individuals bearing one copy
of the rare allele (A/a) are much more common than those bearing two copies (A/A). So, most of the
affected people are heterozygotes. Virtually all matings involving dominant disorders are A/a × a/a.
Therefore, when the progeny of such matings is totaled, a 1:1 ratio is expected of unaffected (a/a)
to affected individuals (A/a). In relatively rare cases, there is a possibility that abnormal alleles can
also arise due to mutations (Figure 1.7).
Huntington’s disease is an example of an autosomal dominant disorder resulting in neural degen-

eration, leading to convulsions and premature death. It is a late-onset disease, where the symptoms
do not appear till the person has begun to have children. Each child, a carrier of the abnormal allele,
has a 50% chance of inheriting the allele associated with the disease. This tragic pattern has led to
finding ways of identifying people carrying this abnormal allele before they experience the onset of
the disorder. The discovery of the molecular nature of a mutant allele and neutral mutations on the
DNA close to the affected allele can be considered markers on the specific chromosome and have
revolutionized the diagnosis of this disease.
1.6.4 X-linked Recessive
If an affected non-founding son has two unaffected parents, then the disease must be X-linked
recessive. The father, who is marrying, does not have any disease alleles since he is marrying into
the family. So, the affected son inherits an allele only from his unaffected mother. A male cannot
be affected by a single autosomal recessive allele but can be affected by a single X-linked recessive
allele (Figure 1.8). The alleles determine a few phenotypes on the X-chromosome’s differential
regions, related to the sex determination. Phenotypes inheriting the X-linked recessiveness display
the following patterns in the pedigree analysis.
1. Male phenotypes are more X-linked recessive than female phenotypes because a female
phenotype is the result of mating by a father and mother, with the father bearing the allele
(X A /X a × X a/Y), whereas a male with the X-linked phenotype can be produced when only the
mother carries the allele. Almost all individuals showing the recessive phenotype are males.
2. None of the offspring of an affected male are affected, but all his daughters will be hetero-
zygous carriers as the females receive one of their X chromosomes from their fathers. As a
result, half of the sons born to these carrier daughters will be affected.
The best-known example of X-linked recessive is hemophilia, a malady in which a person’s

blood fails to clot. Most famous cases of hemophilia have been reported from the pedigree of the
European royal families. The original hemophilia allele in the pedigree arose from a spontaneous
mutation in the reproductive cells of either Queen Victoria’s parents or Queen Victoria herself.
Alexis, the son of the last czar of Russia, inherited the allele from Queen Victoria, the grandmother
of his mother, Alexandra. Duchenne dystrophy is another lethal X-linked recessive disorder, where
the phenotype is the atrophy of muscles. The onset of the disease generally occurs before six years
of age, with confinement to a wheelchair by 12 and death by 20 years. The gene responsible for the
disorder has been isolated and shown to encode a muscular protein known as dystrophin. A sporadic
X-linked recessive phenotype testicular feminization syndrome occurs at a frequency of about
1 in 65,000 males. People afflicted with this syndrome are chromosomally males (44A+XY) but
develop as females. They have female external genitalia, a blind vagina without a uterus. Testes may
FIGURE 1.8 Pedigree of an X-linked Recessive inheritance.

Genetic Analysis 19
be present either in the labia or in the abdomen. These people are sexually sterile, and the condition
cannot be reversed even by injecting the androgen (male hormone). Hence, it is also called androgen
insensitivity syndrome. One of the main reasons for the sensitivity is the allele that codes for a
malfunctioning androgen receptor protein. As a result, androgen will not affect the target organs
involved in the maleness. In humans, femaleness results when the male determining system is not
functional.
1.6.5 X-Linked Dominant
The phenomenon of X-chromosome inactivation complicates the mechanisms of X-linked domin-
ance in human beings. When an affected daughter of non-founding parents has an affected father,
it is difficult to determine whether the dominant disease is autosomal or X-linked (Figure 1.9). The
affected father can transmit either an autosomal dominant allele or an X-linked dominant allele to his
daughter. Hypophosphatemia, a type of vitamin D-resistant rickets, is an example of X-linked dom-
inant inheritance. The pedigree of rare X-linked dominant males and females shows the following
characteristics:
1. Affected males transmit the condition to all their daughters but none of their sons.
2. Females married to unaffected males pass the condition on to half their sons and daughters.
1.6.6 Calculations for the Risk Allele by Pedigree Analysis

After confirming the diseased allele in a family, the probability of the prospective parents having
a child with the respective disorder is calculated by using simple gene patterns. For example, if a
married couple finds out that each had an uncle with a severe autosomal recessive Tay-Sachs disease
(a disorder that leads to the destruction of the brain and spinal cord), the pedigree will be as
mentioned in Figure 10.
FIGURE 1.9 Pedigree of an X-linked dominant inheritance.
FIGURE 1.10 Pedigree of a married couple, each having an uncle with Tay-Sachs disease.
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Around the memories of Bradshaw and his illustrious brethren his
deathless soldiery still pitch their invincible tents, still keep their long-
resounding march, sure warders against obloquy and oblivion.
After the death of Cromwell, Milton continued faithful to

republicanism, and on the very eve of the Restoration published his
last political tract, showing a short and easy way to establish a
Christian commonwealth. He had long ago quarreled with the
Presbyterians in discipline, and separated from the Independents in
doctrine. For many years he did not go within any church and had
become a Unitarian. He had begun “Paradise Lost” in 1658, and
after the Restoration, with a broken fortune, but with a constancy
which nothing could break, shattered in health, blind, and for a time
in danger, he continued the composition of it. It was complete in
1665, when Elwood, the Quaker, had the reading of it, and it was
published in 1667.
The translation of the Bible had to a very great extent Judaized the
Puritan mind. England was no longer England, but Israel. Those
fierce enthusiasts could always find Amalek and Philistia in the men
who met them in the field, and one horn or the other of the beast in
every doctrine of their theological adversaries. The spiritual
provincialism of the Jewish race found something congenial in the
Anglo-Saxon intellect. This element of the Puritan character appears
in Milton also, as in that stern sonnet:
Avenge, O Lord, thy slaughtered saints, whose bones

Lie scattered on the Alpine mountains cold,
Even them who kept thy truth so pure of old
When all our fathers worshipped stocks and stones.
In Milton’s prose there is a constant assertion of himself as a man

set apart to a divine ministry. He seems to translate himself out of
Hebrew into English. And yet so steeped was he in Greek culture
that it is sometimes hard to say whether he would rather call himself
the messenger of Jehovah or the son of Phœbus. Continually the
fugitive mists of dialectics are rent, and through them shine down
serene and solemn peaks that make us feel that we are encamped
about by the sacred mounts of song, but whether of Palestine or of
Greece is doubtful. We may apply to Milton what Schiller says of the
poet, “Let the kind divinity snatch the suckling from his mother’s
breast, nourish him with the milk of a better age, and let him come to
maturity beneath a distant Grecian sky. Then when he has become a
man let him return, a foreign shape, into his century, not to delight it
with his apparition, but terrible, like Agamemnon’s son, to purify it.”
I said that Milton had a sublime egotism. The egotism of a great

character is inspiration because it generalizes self into universal law.
It is a very different thing from the vulgar egotism of a little nature
which contracts universal Law into self. The one expands with a
feeling that it is a part of the law-making power, the other offers an
amendment in town-meeting as if it came from Sinai. Milton’s superb
conception of himself enters into all he does; if he is blind, it is with
excess of light—it is a divine favor, an overshadowing with angel’s
wings. Phineus and Tiresias are admitted among the prophets
because they, too, had lost their sight. There is more merit in the
blindness of Mæonides than in his “Iliad” or “Odyssey.” If the
structure of his mind is undramatic, why, then the English drama is
barbarous, and he will write a tragedy on a Greek model with blind
Samson for a hero.
It results from this that no great poet is so uniformly self-conscious

as he. Dante is individual rather than self-conscious, and the cast-
iron Dante becomes pliable as a field of grain at the breath of
Beatrice, and his whole nature, rooted as it is, seems to flow away in
waves of sunshine. But Milton never lets himself go for a moment.
As other poets are possessed by their theme, so is he always self-
possessed, his great theme being Milton, and his duty being that of
interpreter between John Milton and the world. I speak it reverently—
he was worth translating.
We should say of Shakspeare that he had the power of transforming

himself into everything, and of Milton that he had that of transforming
everything into himself. He is the most learned of poets. Dante, it is
true, represents all the scholarship of his age, but Milton belonged to
a more learned age, was himself one of the most learned men in it,
and included Dante himself among his learning. No poet is so
indebted to books and so little to personal observation as he. I
thought once that he had created out of his own consciousness
those exquisite lines in “Comus”:
A thousand fantasies
Begin to throng into my memory
Of calling shapes, and beckoning shadows dire,
And airy tongues that syllable men’s names
On sands and shores and desert wildernesses.
But I afterwards found that he had built them up out of a dry

sentence in Marco Polo’s “Travels.” The wealth of Milton in this
respect is wonderful. He subsidizes whole provinces of learning to
spend their revenues upon one lavish sentence, and melts history,
poetry, mythology, and philosophy together to make the rich Miltonic
metal of a single verse.
The first noticeable poem of Milton is his “Hymn of the Nativity,” and
the long-enwoven harmony of the versification is what chiefly
deserves attention in it. It is this which marks the advent of a new
power into English poetry.
In Spenser meaning and music are fused together; in Shakspeare

the meaning dominates always (and I intend the sentiment as
included in the word meaning); but in Milton the music is always a
primary consideration. He is always as much musician as poet. And
he is a harmonist, not a melodist. He loves great pomps and
sequences of verse, and his first passages move like long
processions, winding with sacred chant, and priestly robes rich with
emblematic gold, and waving of holy banners, along the echoing
aisles of some cathedral. Accordingly, no reader of Milton can fail to
notice that he is fond of lists of proper names which can have only
an acquired imaginative value, and in that way serve to excite our
poetic sensibility, but which also are of deep musical significance.
This was illustrated by reading various passages from “Paradise

Lost.”
Another striking peculiarity of Milton is the feeling of spaciousness

which his poetry gives us, and that not only in whole paragraphs, but
even in single words. His mind was one which demanded illimitable
room to turn in. His finest passages are those in which the
imagination diffuses itself over a whole scene or landscape, or where
it seems to circle like an eagle controlling with its eye broad sweeps
of champaign and of sea, bathing itself in the blue streams of air, and
seldom drawn earthward in the concentrated energy of its swoop.
This shows itself unmistakably in the epithets of his earlier poems. In

“Il Penseroso,” for example, where he hears
The far-off curfew sound

Over some wide-watered shore
Swinging slow with sullen roar;
where he sees
Gorgeous Tragedy
In sceptered pall come sweeping by,
or calls up the great bards who have sung
Of forests and enchantments drear

Where more is meant than meets the ear.
Milton seems to produce his effects by exciting or dilating our own
imaginations; and this excitement accomplished, he is satisfied.
Shakspeare, on the other hand, seldom leaves any work to be done
by the imagination of his readers; and after we have enjoyed the
total effect of a passage, we may always study the particulars with
advantage. Shakspeare never attaches any particular value to his
thoughts, or images, or phrases, but scatters them with a royal
carelessness. Milton seems always to respect his; he lays out broad
avenues for the triumphal processions of his verse; covers the
ground with tapestry inwoven with figures of mythology and
romance; builds up arches rich with historic carvings for them to
march under, and accompanies them with swells and cadences of
inspiring music. “Paradise Lost” is full of what may be called vistas of
verse. Notice, for example, how far off he begins when he is about to
speak of himself—as at the beginning of the third book and of the
seventh. When you read “Paradise Lost” the feeling you have is one
of vastness. You float under a great sky brimmed with sunshine, or
hung with constellations; the abyss of space is around you; thunders
mutter on the horizon; you hear the mysterious sigh of an unseen
ocean; and if the scene changes, it is with an elemental movement
like the shifting of mighty winds. Of all books it seems most purely
the work of a disembodied mind. Of all poets he could most easily
afford to be blind; of all, his poetry owes least to the senses, except
that of hearing; everything, except his music, came to him through a
mental medium, and perhaps even that may have been intellectual—
as in Beethoven, who composed behind the veil of deafness.
Milton is a remarkable instance of a great imaginative faculty fed by

books instead of Nature. One has only to read the notes of the
commentators upon his poems to see how perfectly he made
whatever he took his own. Everything that he touches swells and
towers into vastness. It is wonderful to see how, from the most
withered and juiceless hint that he met in his reading, his grand
images “rise like an exhalation”; how from the most hopeless-looking
leaden box that he found in that huge drag-net with which he
gathered everything from the waters of learning, he could conjure a
tall genius to do his bidding.
That proud consciousness of his own strength, and confidence at the
same time that he is the messenger of the Most High, never forsake
him. It is they which give him his grand manner, and make him speak
as if with the voice of a continent. He reverenced always the
sacredness of his own calling and character. As poet, full of the lore
of antiquity, and, as prophet, charged to vindicate the ways of God, it
seems to me that I see the majestic old man laying one hand upon
the shoulder of the Past, and the other upon that of the Future, and
so standing sublimely erect above that abject age to pour his voice
along the centuries. We are reminded of what is told of Firdusi,
whose father on the night he was born dreamed he saw him
standing in the middle of the earth and singing so loud and clear that
he was heard in all four quarters of the heavens at once.
I feel how utterly inadequate any single lecture must be on such a

theme, and how impossible it is to say anything about Milton in an
hour. I have merely touched upon three or four points that seemed to
me most characteristic of his style, for our concern with him is solely
as a poet. Yet it would be an unpardonable reticence if I did not say,
before I close, how profoundly we ought to reverence the grandeur
of the man, his incorruptible love of freedom, his scholarly and
unvulgar republicanism, his scorn of contemporary success, his faith
in the future and in God, his noble frugality of life.
The noise of those old warfares is hushed; the song of Cavalier and
the fierce psalm of the Puritan are silent now; the hands of his
episcopal adversaries no longer hold pen or crozier—they and their
works are dust; but he who loved truth more than life, who was
faithful to the other world while he did his work in this; his seat is in
that great cathedral whose far-echoing aisles are the ages
whispering with blessed feet of the Saints, Martyrs, and Confessors
of every clime and creed; whose bells sound only centurial hours;
about whose spire crowned with the constellation of the cross no
meaner birds than missioned angels hover; whose organ music is
the various stops of endless changes breathed through by endless
good; whose choristers are the elect spirits of all time, that sing,
serene and shining as morning stars, the ever-renewed mystery of
Creative Power.
LECTURE VIII
BUTLER
(Friday Evening, February 2, 1855)
VIII
Neither the Understanding nor the Imagination is sane by itself; the
one becomes blank worldliness, the other hypochondria. A very little
imagination is able to intoxicate a weak understanding, and this
appears to be the condition of religious enthusiasm in vulgar minds.
Puritanism, as long as it had a material object to look forward to, was
strong and healthy. But Fanaticism is always defeated by success;
the moment it is established in the repose of power, it necessarily
crystallizes into cant and formalism around any slenderest threads of
dogma; and if the intellectual fermentation continue after the spiritual
has ceased, as it constantly does, it is the fermentation of
putrefaction, breeding nothing but the vermin of incoherent and
destructively-active metaphysic subtleties—the maggots, as Butler,
condensing Lord Bacon, calls them, of corrupted texts. That wise
man Oliver Cromwell has been reproached for desertion of principles
because he recognized the truth that though enthusiasm may
overturn a government, it can never carry on one. Our Puritan
ancestors came to the same conclusion, and have been as unwisely
blamed for it. While we wonder at the prophetic imagination of those
heroic souls who could see in the little Mayflower the seeds of an
empire, while we honor (as it can only truly be honored—by
imitating) that fervor of purpose which could give up everything for
principle, let us be thankful that they had also that manly English
sense which refused to sacrifice their principles to the fantasy of
every wandering Adoniram or Shear-Jashub who mistook himself for
Providence as naturally and as obstinately as some lunatics suppose
themselves to be tea-pots.
The imaginative side of Puritanism found its poetical expression in

Milton and its prose in Bunyan. The intellectual vagaries of its
decline were to have their satirist in Butler. He was born at
Strensham in Worcestershire in 1612, the son of a small farmer who
was obliged to pinch himself to afford his son a grammar-school
education. It is more than doubtful whether he were ever at any
university at all. His first employment was as clerk to Mr. Jeffereys, a
Worcestershire justice of the peace, called by the poet’s biographers
an eminent one. While in this situation he employed his leisure in
study, and in cultivating music and painting, for both of which arts he
had a predilection. He next went into the family of the Countess of
Kent, where he had the use of a fine library, and where he acted as
amanuensis to John Selden—the mere drippings of whose learning
were enough to make a great scholar of him. After this he was
employed (in what capacity is unknown) in the house of Sir Samuel
Luke, an officer of Cromwell, and a rigid Presbyterian. It was here
that he made his studies for the characters of Sir Hudibras and his
squire, Ralpho, and is supposed to have begun the composition of
his great work. There is hardly anything more comic in “Hudibras”
itself than the solemn Country Knight unconsciously furnishing
clothes from his wardrobe, and a rope of his own twisting, to hang
himself in eternal effigy with. Butler has been charged with
ingratitude for having caricatured his employer; but there is no hint of
any obligation he was under, and the service of a man like him must
have been a fair equivalent for any wages.
On the other hand, it has been asserted that Butler did not mean Sir
Samuel Luke at all, but a certain Sir Henry Rosewell, or a certain
Colonel Rolle, both Devonshire men. And in confirmation of it we are
told that Sir Hugh de Bras was the tutelary saint of Devonshire.
Butler, however, did not have so far to go for a name, but borrowed it
from Spenser. He himself is the authority for the “conjecture,” as it is
called, that his hero and Sir Samuel Luke were identical. At the end
of the first canto of part first of “Hudibras” occurs a couplet of which
the last part of the second verse is left blank. This couplet, for want
of attention to the accent, has been taken to be in ten-syllable
measure, and therefore an exception to the rest of the poem. But it is
only where we read it as a verse of four feet that the inevitable
rhyme becomes perfectly Hudibrastic. The knight himself is the
speaker:
’Tis sung there is a valiant Mameluke

In foreign lands yclept (Sir Sam Luke)
To whom we have been oft compared
For person, parts, address and beard.
Butler died poor, but not in want, on the 25th of September, 1680, in
his sixty-eighth year.
Butler’s poem is commonly considered the type of the burlesque—

that is, as the representative of the gravely ludicrous, which seems
to occupy a kind of neutral ground between the witty and the
humorous. But this is true of the form rather than the matter of the
poem. Burlesque appears to be wit infused with animal spirits—
satire for the mere fun of the thing, without any suggestion of
intellectual disapproval, or moral indignation. True wit is a kind of
instantaneous logic which gives us the quod erat demonstrandum
without the intermediate steps of the syllogism. Coleridge, with
admirable acuteness, has said that “there is such a thing as scientific
wit.” Therefore pure wit sometimes gives an intellectual pleasure
without making us laugh. The wit that makes us laugh most freely is
that which instantly accepts another man’s premises, and draws a
conclusion from them in its own favor. A country gentleman was
once showing his improvements to the Prince de Ligne, and, among
other things, pointed out to him a muddy spot which he called his
lake. “It is rather shallow, is it not?” said the Prince. “I assure you,
Prince, a man drowned himself in it.” “Ah, he must have been a
flatterer, then,” answered De Ligne. Of the same kind is the story told
of one of our old Massachusetts clergymen, Dr. Morse. At an
association dinner a debate arose as to the benefit of whipping in
bringing up children. The doctor took the affirmative, and his chief
opponent was a young minister whose reputation for veracity was
not very high. He affirmed that parents often did harm to their
children by unjust punishment from not knowing the facts in the
case. “Why,” said he, “the only time my father ever whipped me was
for telling the truth.” “Well,” retorted the doctor, “it cured you of it,
didn’t it?” In wit of this sort, there is always a latent syllogism.
Then there is the wit which detects an unintentional bit of satire in a

word of double meaning; as where Sir Henry Wotton takes
advantage of the phrase commonly used in his day to imply merely
residence, and finds an under meaning in it, saying that
“ambassadors were persons sent to lie abroad for the service of their
prince.”
On the other hand I think unconsciousness and want of intention, or

at least the pretense of it, is more or less essential to the ludicrous.
For this reason what may be called the wit of events is always
ludicrous. Nothing can be more so, for example, than the Pope’s
sending a Cardinal’s hat to John Fisher, Bishop of Rochester, which
arrived in England after Henry VIII had taken off that prelate’s head.
So, when Dr. Johnson said very gravely one day, that he had often
thought that if he had a harem he would dress all the ladies in white
linen, the unintentional incongruity of the speech with the character
of the great moralist threw Boswell into an ecstasy of laughter. Like
this is the ludicrousness of Pope Paul III writing to the Council of
Trent “that they should begin with original sin, observing yet a due
respect unto the Emperor.”
Captain Basil Hall, when he traveled in this country, found the

Yankees a people entirely destitute of wit and humor. Perhaps our
gravity, which ought to have put him on the right scent, deceived
him. I do not know a more perfect example of wit than something
which, as I have heard, was said to the captain himself. Stopping at
a village inn there came up a thunderstorm, and Captain Hall,
surprised that a new country should have reached such perfection in
these meteorological manufactures, said to a bystander, “Why, you
have very heavy thunder here.” “Well, yes,” replied the man, “we du,
considerin’ the number of inhabitants.” Here is another story which a
stage-driver told me once. A wag on the outside of the coach called
to a man by the roadside who was fencing some very poor land: “I
say, mister, what are you fencing that pasture for? It would take forty
acres on’t to starve a middle-sized cow.” “Jesso; and I’m a-fencing of
it to keep eour kettle eout.”
Now in the “forty acre” part of this story we have an instance of what
is called American exaggeration, and which I take to be the symptom
of most promise in Yankee fun. For it marks that desire for intensity
of expression which is one phase of imagination. Indeed many of
these sayings are purely imaginative; as where a man said of a
painter he knew, that “he painted a shingle so exactly like marble
that when it fell into the river it sunk.” A man told me once that the
people of a certain town were so universally dishonest that “they had
to take in their stone walls at night.” In some of these stories
imagination appears yet more strongly, and in that contradictory
union with the understanding lies at the root of highest humor. For
example, a coachman driving up some steep mountains in Vermont
was asked if they were as steep on the other side also. “Steep!
chain-lightnin’ couldn’t go down ’em without the breechin’ on.” I
believe that there is more latent humor among the American people
than in any other, and that it will one day develop itself and find
expression through Art.
If we apply the definitions we have made to Butler’s poem, we shall

find that it is not properly humorous at all; that the nearest approach
to the humorous is burlesque. Irony is Butler’s favorite weapon. But
he always has an ulterior object. His characters do not live at all, but
are only caricatured effigies of political enemies stuffed with bran
and set up as targets for his wit. He never lets us forget for a
moment that Presbyterian and Independent are primarily knaves and
secondarily men. The personality never by accident expands into
humanity. There is not a trace of imagination or of sympathy in his
poem. It is pure satire, and intellectual satire only. There is as much
creativeness in Trumbull’s “McFingal,” or Fessenden’s “Terrible
Tractoration” as in “Hudibras.” Butler never works from within, but
stands spectator covering his victims with merciless ridicule; and we
enjoy the fun because his figures are as mere nobodies as Punch
and Judy, whose misfortunes are meant to amuse us, and whose
unreality is part of the sport. The characters of truly humorous writers
are as real to us as any of our acquaintances. We no more doubt the
existence of the Wife of Bath, of Don Quixote and Sancho, of
Falstaff, Sir Roger de Coverley, Parson Adams, the Vicar, Uncle
Toby, Pickwick or Major Pendennis, than we do our own. They are
the contemporaries of every generation forever. They are our
immortal friends whose epitaph no man shall ever write. The only
incantation needed to summon them is the taking of a book from our
shelf, and they are with us with their wisdom, their wit, their courtesy,
their humanity, and (dearer than all) their weaknesses.
But the figures of Butler are wholly contemporaneous with himself.

They are dead things nailed to his age, like crows to a barn-door, for
an immediate in terrorem purpose, to waste and blow away with time
and weather. The Guy Fawkes of a Fifth of November procession
has as much manhood in it.
Butler, then, is a wit—in the strictest sense of the word—with only

such far-off hints at humor as lie in a sense of the odd, the droll, or
the ludicrous. But in wit he is supreme. “Hudibras” is as full of point
as a paper of pins; it sparkles like a phosphorescent sea, every
separate drop of which contains half a dozen little fiery lives. Indeed,
the fault of the poem (if it can be called a fault) is that it has too much
wit to be easy reading.
Butler had been a great reader, and out of the dryest books of school
divinity, Puritan theology, metaphysics, medicine, astrology,
mathematics, no matter what, his brain secreted wit as naturally as a
field of corn will get so much silex out of a soil as would make flints
for a whole arsenal of old-fashioned muskets, and where even
Prometheus himself could not have found enough to strike a light
with. I do sincerely believe that he would have found fun in a joke of
Senator—well, any senator; and that is saying a great deal. I speak
of course, of senators at Washington.
Mr. Lowell illustrated his criticism by copious quotations from

“Hudibras.” He concluded thus:
It would not be just to leave Butler without adding that he was an
honest and apparently disinterested man. He wrote an indignant
satire against the vices of Charles the Second’s court. Andrew
Marvel, the friend of Milton, and the pattern of incorruptible
Republicanism, himself a finer poet and almost as great a wit as
Butler—while he speaks contemptuously of the controversialists and
satirists of his day, makes a special exception of “Hudibras.” I can
fancy John Bunyan enjoying it furtively, and Milton, if he had had
such a thing as fun in him, would have laughed over it.
Many greater men and greater poets have left a less valuable legacy
to their countrymen than Butler, who has made them the heirs of a
perpetual fund of good humor, which is more nearly allied to good
morals than most people suspect.
LECTURE IX
POPE
(Thursday Evening, February 6, 1855)
IX
There is nothing more curious, whether in the history of individual
men or of nations, than the reactions which occur at more or less
frequent intervals.
The human mind, both in persons and societies, is like a pendulum

which, the moment it has reached the limit of its swing in one
direction, goes inevitably back as far on the other side, and so on
forever.
These reactions occur in everything, from the highest to the lowest,

from religion to fashions of dress. The close crop and sober doublet
of the Puritan were followed by the laces and periwigs of Charles the
Second. The scarlet coats of our grandfathers have been displaced
by as general a blackness as if the world had all gone into mourning.
Tight sleeves alternate with loose, and the full-sailed expanses of
Navarino have shrunk to those close-reefed phenomena which, like
Milton’s Demogorgon, are the name of bonnet without its
appearance.
English literature, for half a century from the Restoration, showed the
marks of both reaction and of a kind of artistic vassalage to France.
From the compulsory saintship and short hair of the Roundheads the
world rushed eagerly toward a little wickedness and a wilderness of
wig. Charles the Second brought back with him French manners,
French morals, and French taste. The fondness of the English for
foreign fashions had long been noted. It was a favorite butt of the
satirists of Elizabeth’s day. Everybody remembers what Portia says
of the English lord: “How oddly is he suited! I think he bought his
doublet in Italy, his round hose in France, his bonnet in Germany,
and his behavior everywhere.”
Dryden is the first eminent English poet whose works show the
marks of French influence, and a decline from the artistic toward the
artificial, from nature toward fashion. Dryden had known Milton, had
visited the grand old man probably in that “small chamber hung with
rusty green,” where he is described as “sitting in an elbow-chair,
neatly dressed in black, pale but not cadaverous”; or had found him
as he “used to sit in a gray, coarse cloth coat, at the door of his
house near Bunhill Fields, in warm, sunny weather, to enjoy the fresh
air.” Dryden undertook to put the “Paradise Lost” into rhyme, and on
Milton’s leave being asked, he said, rather contemptuously, “Ay, he
may tag my verses if he will.” He also said that Dryden was a “good
rhymist, but no poet.” Dryden turned the great epic into a drama
called “The State of Innocence,” and intended for representation on
the stage. Sir Walter Scott dryly remarks that the costume of our first
parents made it rather an awkward thing to bring them before the
footlights. It is an illustration of the character of the times that Dryden
makes Eve the mouthpiece of something very like obscenity. Of the
taste shown by such a travesty nothing need be said.
In the poems of Dryden nothing is more striking than the alternations

between natural vigor and warmth of temperament and the merest
common-places of diction. His strength lay chiefly in the
understanding, and for weight of sterling sense and masculine
English, and force of argument, I know nothing better than his prose.
His mind was a fervid one, and I think that in his verse he sometimes
mistook metrical enthusiasm for poetry. In his poems we find wit,
fancy, an amplitude of nature, a rapid and graphic statement of the
externals and antitheses of character, and a dignified fluency of
verse rising sometimes to majesty—but not much imagination in the
high poetic meaning of the term.
I have only spoken of his poems at all because they stand midway
between the old era, which died with Milton and Sir Thomas Browne,
and the new one which was just beginning. In the sixty years
extending from 1660 to 1720, more French was imported into the
language than at any other time since the Norman Conquest. What
is of greater importance, it was French ideas and sentiments that
were coming in now, and which shaped the spirit and, through that,
the form of our literature.
The condition of the English mind at the beginning of the last century
was one particularly capable of being magnetized from across the
Channel. The loyalty of everybody, both in politics and religion, had
been dislocated. A generation of materialists was to balance the
over-spiritualism of the Puritans. The other world had had its turn
long enough, and now this world was to have its chance. There
seems to have been a universal skepticism, and in its most
dangerous form—that is, united with a universal pretense of
conformity. There was an unbelief that did not believe even in itself.
Dean Swift, who looked forward to a bishopric, could write a book
whose moral, if it had any, was that one religion was about as good
as another, and accepted a cure of souls when it was doubtful if he
thought men had any souls to be saved, or, at any rate, that they
were worth saving if they had. The answer which Pulci’s Margutte
makes to Morgante, when he asks him if he believed in Christ or
Mahomet, would have expressed well enough the creed of the
majority of that generation:
Margutte answered then, To tell thee truly,

My faith in black’s no greater than in azure;
But I believe in capons, roast meat, bouilli,
And above all in wine—and carnal pleasure.
It was impossible that anything truly great—great, I mean, on the

moral and emotional as well as on the intellectual sides—could be
produced in such a generation. But something intellectually great
could be, and was. The French mind, always stronger in the
perceptive and analytic than in the imaginative faculty, loving
precision, grace, and fineness, had brought wit and fancy, and the
elegant arts of society, to the perfection, almost, of science. Its ideal
in literature was to combine the appearance of carelessness and
gayety of thought with intellectual exactness of statement. Its
influence, then, in English literature will appear chiefly in neatness
and facility of expression, in point of epigrammatic compactness of
phrase, and these in conveying conventional rather than universal
experiences; in speaking for good society rather than for man.
Thus far in English poetry we have found life represented by

Chaucer, the real life of men and women; the ideal or interior life as it
relates to this world, by Spenser; what may be called imaginative life,
by Shakspeare; the religious sentiment, or interior life as it relates to
the other world, by Milton. But everything aspires toward a
rhythmical utterance of itself, and accordingly the intellect and life, as
it relates to what may be called the world, were waiting for their poet.
They found or made a most apt one in Alexander Pope.
He stands for perfectness of intellectual expression, and it is a

striking instance how much success and permanence of reputation
depend upon conscientious and laborious finish as well as upon
natural endowments.
I confess that I come to the treatment of Pope with diffidence. I was

brought up in the old superstition that he was the greatest poet that
ever lived, and when I came to find that I had instincts of my own,
and my mind was brought in contact with the apostles of a more
esoteric doctrine of poetry, I felt that ardent desire for smashing the
idols I had been brought up to worship, without any regard to their
artistic beauty, which characterizes youthful zeal. What was it to me
that Pope was a master of style? I felt, as Addison says in his
“Freeholder” in answering an argument in favor of the Pretender
because he could speak English and George I could not, “that I did
not wish to be tyrannized over in the best English that was ever
spoken.” There was a time when I could not read Pope, but disliked
him by instinct, as old Roger Ascham seems to have felt about Italy
when he says: “I was once in Italy myself, but I thank God my abode
there was only nine days.”
But Pope fills a very important place in the history of English poetry,
and must be studied by every one who would come to a clear
knowledge of it. I have since read every line that Pope ever wrote,
and every letter written by or to him, and that more than once. If I
have not come to the conclusion that he is the greatest of poets, I
believe I am at least in a condition to allow him every merit that is
fairly his. I have said that Pope as a literary man represents
precision and grace of expression; but, as a fact, he represents
something more—nothing less, namely, than one of those external
controversies of taste which will last as long as the Imagination and
Understanding divide men between them. It is not a matter to be
settled by any amount of argument or demonstration. Men are born
Popists or Wordsworthians, Lockists or Kantists; and there is nothing
more to be said of the matter. We do not hear that the green
spectacles persuaded the horse into thinking that shavings were
grass.
That reader is happiest whose mind is broad enough to enjoy the

natural school for its nature and the artificial for its artificiality,
provided they be only good of their kind. At any rate, we must allow
that a man who can produce one perfect work is either a great
genius or a very lucky one. As far as we who read are concerned, it
is of secondary importance which. And Pope has done this in the
“Rape of the Lock.” For wit, fancy, invention, and keeping, it has
never been surpassed. I do not say that there is in it poetry of the
highest order, or that Pope is a poet whom any one would choose as
the companion of his best hours. There is no inspiration in it, no
trumpet call; but for pure entertainment it is unmatched.
The very earliest of Pope’s productions gives indications of that

sense and discretion, as well as wit, which afterwards so eminently
distinguished him. The facility of expression is remarkable, and we
find also that perfect balance of metre which he afterwards carried
so far as to be wearisome. His pastorals were written in his sixteenth
year, and their publication immediately brought him into notice. The
following four verses from the first Pastoral are quite characteristic in
their antithetic balance:
You that, too wise for pride, too good for power,
Enjoy the glory to be great no more,
And carrying with you all the world can boast,
To all the world illustriously are lost.
The sentiment is affected, and reminds one of that future period of

Pope’s correspondence with his friends, where Swift, his heart
corroding with disappointed ambition at Dublin, Bolingbroke raising
delusive turnips at his farm, and Pope pretending to disregard the
lampoons which embittered his life, played together the solemn farce
of affecting to despise the world which it would have agonized them
to be forgotten by.
In Pope’s next poem, the “Essay on Criticism,” the wit and poet
become apparent. It is full of clear thoughts compactly expressed. In
this poem, written when Pope was only twenty-one, occur some of
those lines which have become proverbial, such as:
A little learning is a dangerous thing;
For fools rush in where angels fear to tread;
True Wit is Nature to advantage dressed,

What oft was thought, but ne’er so well expressed;
For each ill author is as bad a friend.

In all these we notice that terseness in which (regard being had to
his especial range of thought) Pope has never been equaled. One
cannot help being struck also with the singular discretion which the
poem gives evidence of. I do not know where to look for another
author in whom it appeared so early; and considering the vivacity of
his mind and the constantly besetting temptation of his wit, it is still
more wonderful. In his boyish correspondence with poor old
Wycherly, one would suppose him to be a man and Wycherly the
youth. Pope’s understanding was no less vigorous than his fancy
was lightsome and sprightly.
I come now to what in itself would be enough to have immortalized

him as a poet, the “Rape of the Lock,” in which, indeed, he appears
more purely as a poet than in any other of his productions.
Elsewhere he has shown more force, more wit, more reach of
thought, but nowhere such a truly artistic combination of elegance
and fancy. His genius has here found its true direction, and the very
same artificiality which in his Pastorals was unpleasing heightens the
effect and adds to the general keeping. As truly as Shakspeare is the
poet of man as God made him, dealing with great passions and
innate motives, so truly is Pope the poet of society, the delineator of
manners, the exposer of those motives which may be called
acquired, whose spring is in institutions and habits of purely worldly
origin.
The whole poem more truly deserves the name of a creation than
anything Pope ever wrote. The action is confined to a world of his
own, the supernatural agency is wholly of his own contrivance, and
nothing is allowed to overstep the limitations of the subject. It ranks
by itself as one of the purest works of human fancy. Whether that
fancy be truly poetical or not is another matter. The perfection of
form in the “Rape of the Lock” is to me conclusive evidence that in it
the natural genius of Pope found fuller and freer expression than in
any other of his poems. The others are aggregates of brilliant
passages rather than harmonious wholes.
Mr. Lowell gave a detailed analysis of the poem, with extracts of

some length.

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Diagnostics and Gene Therapy for Human Genetic

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The Gale Encyclopedia of Genetic Disorders 5th Edition

Diagnostics and Gene Therapy for

1. A blend of classical human genetics with molecular and genome-​based applications

Diagnostics and Gene Therapy

First edition published 2023

Chapter 1 Genetic Analysis............................................................................................................ 1

1.9 Multifactorial Inheritance................................................................................. 25

Chapter 2 Genetic Variations in Human Beings.......................................................................... 39

2.4.2.4 Hypervariable Region Polymorphism of

Chapter 3 Genetic Disorders........................................................................................................ 81

3.4.3.4 Y-​Linked Disorders............................................................ 101

Chapter 4 Techniques for the Diagnosis of Genetic Disorders.................................................. 117

4.2.5.4 Single-​Strand Conformational Polymorphism

Chapter 5 Diagnosis of Genetic Disorders................................................................................. 153

5.4.2.4 Treatment........................................................................... 165

5.4.12.4 Treatment........................................................................... 201

Chapter 6 Gene Therapy............................................................................................................ 225

6.3 Therapeutic Nucleic Acids and Therapeutic Polynucleotides........................231

6.7.3 Non-​Viral Based Gene Delivery Systems......................................... 254

Chapter 7 Applications of Gene Therapy..................................................................................259

Chapter 8 Applications of Molecular Diagnostics..................................................................... 275

8.4 Applications of Diagnostics in Personalized Medicine.................................. 281

Chapter 9 Case Studies.............................................................................................................. 293

1.3.3 Polymorphic DNA Markers

a) Single Nucleotide Polymorphisms (SNPs)

1.4.1 Linkage Disequilibrium at One Pair of Loci

DAB =​pAB –​p A pB (1)

DAB=​ –​DAb=​ –​DaB =​Dab

DAB (t +​1) =​(1 – c) DAB (t) (2)

1.4.3 Linkage Disequilibrium at More Than Two Loci

1.4.4 Linkage Disequilibrium Within and Between Populations

1.4.5 Population Genetics of Linkage Disequilibrium

3. Inbreeding, Inversions, and Gene Conversion

1.4.6 Applications of Linkage Disequilibrium

2. Detection of Natural Selection

3. Estimation of Allele Age

1.4.7 Linkage Disequilibrium and Human Genome

1.5 MENDELIAN PEDIGREE PATTERNS

1.5.1 Dominance and Recessiveness of Characters

1.5.2 Types of Mendelian Pedigree

ii. Autosomal Recessive

iii. X-​linked Dominant

• Two girls with the disease

iv. X-​linked Recessive

1.6 MENDELIAN PEDIGREE ANALYSIS

S. No Symbol Representation in Pedigree Analysis

4. Parents and Children in order of birth.

TABLE 1.1 (Continued)

1. A blend of classical human genetics with molecular and genome-based applications

3.4.3.4 Y-Linked Disorders............................................................ 101

4.2.5.4 Single-Strand Conformational Polymorphism

6.7.3 Non-Viral Based Gene Delivery Systems......................................... 254

1.3.3 Polymorphic DNA Markers

1.4.1 Linkage Disequilibrium at One Pair of Loci

DAB =pAB –p A pB (1)

DAB= –DAb= –DaB =Dab

DAB (t +1) =(1 – c) DAB (t) (2)

1.4.3 Linkage Disequilibrium at More Than Two Loci

1.4.4 Linkage Disequilibrium Within and Between Populations

1.4.5 Population Genetics of Linkage Disequilibrium

1.4.6 Applications of Linkage Disequilibrium

1.4.7 Linkage Disequilibrium and Human Genome

1.5 MENDELIAN PEDIGREE PATTERNS

1.5.1 Dominance and Recessiveness of Characters

1.5.2 Types of Mendelian Pedigree

iii. X-linked Dominant

iv. X-linked Recessive

1.6 MENDELIAN PEDIGREE ANALYSIS

5. Dizygotic or non-identical twins

10. Carrier of sex-linked recessive

1.6.2 Patterns Indicating a Recessive Trait

1.6.3 Patterns Indicating a Dominant Trait

1.6.3.1 Autosomal Dominant Trait

The best-known example of X-linked recessive is hemophilia, a malady in which a person’s

FIGURE 1.8 Pedigree of an X-linked Recessive inheritance.

1.6.6 Calculations for the Risk Allele by Pedigree Analysis

FIGURE 1.9 Pedigree of an X-linked dominant inheritance.