Genetics and Genomics in Medicine

CHAPTER 1 Fundamentals of DNA,
Chromosomes, and Cells 1
1.1 The Structure and Function of Nucleic Acids 1
General concepts: the genetic material, genomes, and genes 1
The underlying chemistry of nucleic acids 2
Base pairing and the double helix 3
DNA replication and DNA polymerases 4
Genes, transcription, and the central dogma of molecular biology 6
1.2 The Structure and Function of Chromosomes 7
Why we need highly structured chromosomes, and how
they are organized 7
Chromosome function: replication origins, centromeres,
and telomeres 8
1.3 DNA and Chromosomes in Cell Division
and the Cell Cycle 9
Differences in DNA copy number between cells 9
The cell cycle and segregation of replicated chromosomes
and DNA molecules 10
Mitosis: the usual form of cell division 12
Meiosis: a specialized reductive cell division giving
rise to sperm and egg cells 12
Why each of our gametes is unique 15
Summary 17
Questions 18
Further Reading 18
CHAPTER 2 Fundamentals of Gene
Structure, Gene Expression,
and Human Genome Organization 19
2.1 Protein-Coding Genes: Structure
and Expression 19
Gene organization: exons and introns 20
RNA splicing: stitching together the genetic information
in exons 20
The evolutionary value of RNA splicing 22
Translation: decoding messenger RNA to make a
polypeptide 22
The process of translation 22
Transfer RNA as an adaptor RNA 24
Untranslated regions and 5ʹ cap and 3ʹ poly(A) termini 25
From newly synthesized polypeptide to mature protein 26
Chemical modification 26
Folding 28
Cleavage and transport 28
Binding of multiple polypeptide chains 30
2.2 RNA Genes and Noncoding RNA 30
The extraordinary secondary structure and versatility of RNA 31
RNAs that act as specific regulators: from quirky
exceptions to the mainstream 32
Long ncRNAs 32
Tiny ncRNAs 33
Competing endogenous RNAs 34
2.3 Working Out the Details of Our Genome
and What They Mean 34
The Human Genome Project: working out the details of the
nuclear genome 35
Interrogating our genome 36
What the sequence didn’t tell us 36
Identifying genes and other functionally important DNA
elements through evolutionary conservation 37
Identifying genes and their orthologs 37
Sequence conservation due to selection 38
The ENCODE Project: functional assays to determine what
our genome does 40
Transcript analysis 40
Biochemical signatures 40
The outcome 41
2.4 The Organization and Evolution of the
Human Genome 41
A brief overview of the evolutionary mechanisms that
shaped our genome 41
How much of our genome is functionally significant? 42
Estimating functional constraint 42
The mitochondrial genome: economical usage but limited
autonomy 43
Gene distribution in the human genome 44
The extent of repetitive DNA in the human genome 45
The organization of gene families 46
The significance of gene duplication and repetitive
coding DNA 47
Dosage 47
Novel genetic variants 47
Highly repetitive noncoding DNA in the human genome 50
Transposon-derived repeats in the human genome 50
Summary 52
Questions 54
Further Reading 55
CHAPTER 3 Principles Underlying
Core DNA Technologies 57
3.1 DNA Cloning and PCR 57
DNA cloning: fractionating and purifying DNA by
transforming cells with recombinant DNAs 57
Amplification 58
Vector molecules 58
Physical clone separation 60
Making recombinant DNA 60
DNA libraries and the uses and limitations of DNA cloning 62
The basics of the polymerase chain reaction (PCR) 62
Quantitative PCR and real-time PCR 63
3.2 Principles of Nucleic Acid Hybridization 65
Formation of artificial heteroduplexes 65
Hybridization assays: using known nucleic acids
to find related sequences in a test nucleic acid
population 66
Using high and low hybridization stringency 67
Two classes of hybridization assay 67
Microarray hybridization: large-scale parallel hybridization
to immobilized probes 69
3.3 Principles of DNA Sequencing 70
Dideoxy DNA sequencing 72
Massively parallel DNA sequencing (next-generation
sequencing) 73
Summary 75
Questions 77
Further Reading 78
CHAPTER 4 Principles of Genetic
Variation 79
4.1 Origins of DNA Sequence Variation 80
Genetic variation arising from endogenous errors in
chromosome and DNA function 81
DNA replication errors 81
Chromosome segregation and recombination errors 82
Various endogenous and exogenous sources can
cause damage to DNA by altering its chemical structure 82
Endogenous chemical damage to DNA 82
Chemical damage to DNA caused by external mutagens 85
4.2 DNA Repair 85
Repair of DNA damage or altered sequence on a single
DNA strand 86
Repair of DNA lesions that affect both DNA strands 87
Repair of DNA interstrand cross-links 89
Undetected DNA damage, DNA damage tolerance,
and translesion synthesis 89
4.3 The Scale of Human Genetic Variation 92
DNA variants, polymorphism, and developing a
comprehensive catalog of human genetic variation 92
Single nucleotide variants and single nucleotide
polymorphisms 94
The imprecise cut-off between indels and copy number
variants 94
Microsatellites and other polymorphisms due to variable
number of tandem repeats 95
Structural variation and low copy number variation 96
Taking stock of human genetic variation 97
4.4 Functional Genetic Variation and Protein
Polymorphism 97
Most genetic variation has a neutral effect on the
phenotype, but a small fraction is harmful 98
Harmful mutations 98
Positive Darwinian selection and adaptive DNA changes
in human lineages 99
Adaptations to altered environments 99
Generating protein diversity by gene duplication
and alternative processing of a single gene 103
Diversity through gene duplication 104
Post-transcriptionally induced variation 104
4.5 Extraordinary Genetic Variation in the
Immune System 104
Pronounced genetic variation in four classes of immune
system proteins 105
Random and targeted post-zygotic (somatic) genetic
variation 106
Somatic mechanisms that allow cell-specific production
of immunoglobulins and T-cell receptors 107
Combinatorial diversity via somatic recombination 107
Additional diversity generation 108
MHC (HLA) proteins: functions and polymorphism 109
Class I MHC proteins 109
Class II MHC proteins 109
MHC restriction 110
MHC polymorphism 110
The medical importance of the HLA system 111
Transplantation and histocompatibility testing 111
HLA disease associations 111
Summary 113
Questions 115
Further Reading 115
CHAPTER 5 Single-Gene Disorders:
Inheritance Patterns, Phenotype
Variability, and Allele Frequencies 117
5.1 Introduction: Terminology, Electronic
Resources, and Pedigrees 118
Background terminology and electronic resources with
information on single-gene disorders 118
Alleles and allele combinations 118
Dominant and recessive phenotypes 118
Electronic information on monogenic disorders 118
Investigating family history of disease and recording
pedigrees 119
5.2 The Basics of Mendelian and
Mitochondrial DNA Inheritance Patterns 120
Autosomal dominant inheritance 120
Autosomal recessive inheritance 121
Consanguinity 121
Disease-related phenotypes in carriers 122
X-linked inheritance and X-chromosome inactivation 122
X-chromosome inactivation 124
X-linked recessive inheritance 124
X-linked dominant inheritance 125
Pseudoautosomal and Y-linked inheritance 126
Pseudoautosomal inheritance 127
Y-linked inheritance 127
Matrilineal inheritance for mitochondrial DNA disorders 128
Variable heteroplasmy and clinical variability 128
5.3 Uncertainty, Heterogeneity, and Variable
Expression of Mendelian Phenotypes 129
Difficulties in defining the mode of inheritance in small
pedigrees 130
New mutations and mosaicism 130
Post-zygotic mutations and mosaicism 131
Heterogeneity in the correspondence between
phenotypes and the underlying genes and mutations 131
Locus heterogeneity 132
Allelic and phenotypic heterogeneity 133
Non-penetrance and age-related penetrance 134
Variable age at onset in late-onset disorders 134
Variable expression of Mendelian phenotypes within
families 135
Imprinting 136
Anticipation 136
5.4 Allele Frequencies in Populations 137
Allele frequencies and the Hardy–Weinberg Law 138
The Hardy–Weinberg Law 138
Applications and limitations of the Hardy–Weinberg Law 139
Nonrandom mating 140
Ways in which allele frequencies change in populations 140
Population bottlenecks and founder effects 141
Mutation versus selection in determining allele frequencies 142
Heterozygote advantage: when natural selection favors
carriers of recessive disease 144
Distinguishing heterozygote advantage from founder effects 145
Summary 145
Questions 147
Further Reading 148
CHAPTER 6 Principles of Gene
Regulation and Epigenetics 149
6.1 Genetic Regulation of Gene Expression 151
Promoters: the major on–off switches in genes 151
Modulating transcription, tissue-specific regulation,
enhancers, and silencers 152
Enhancers, silencers, and insulator elements 152
Transcription factor binding 153
Genetic regulation during RNA processing: RNA splicing
and RNA editing 154
Regulation of RNA splicing 154
Alternative splicing 155
RNA editing 156
Translational regulation by trans-acting regulatory
proteins 156
Post-transcriptional gene silencing by microRNAs 157
Repressing the repressors: competing endogenous RNAs
sequester miRNA 159
Circular RNAs as abundant miRNA sponges 160
6.2 Chromatin Modification and Epigenetic
Factors in Gene Regulation 160
Changes in chromatin structure producing altered gene
expression 161
Modification of histones in nucleosomes 162
The effect of modified histones and histone variants on
chromatin structure 164
The function of DNA methylation in mammalian cells 165
DNA methylation: mechanisms, heritability, and
global roles during early development and
gametogenesis 166
DNA methylation mechanism 166
DNA methylation in early development and
gametogenesis 166
Noncoding RNAs in epigenetic regulation 169
Cis- and trans-acting regulation 169
Genomic imprinting: differential expression of
maternally and paternally inherited alleles 170
Extent and significance of genome imprinting 171
Establishing sex-specific imprints by differential
methylation 171
X-chromosome inactivation: compensating for sex
differences in gene dosage 173
X-chromosome counting and inactivation choices 174
XIST RNA and initiation of X-inactivation 174
Escaping X-inactivation 175
Epigenomes and dissecting the molecular basis of
epigenetic regulation 175
The International Human Epigenetic Consortium (IHEC) 176
6.3 Abnormal Epigenetic Regulation in
Mendelian Disorders and Uniparental
Disomy 176
Principles of epigenetic dysregulation 176
‘Chromatin diseases’ due to mutations in genes
specifying chromatin modifiers 178
Rett syndrome: a classical chromatin disease 178
Disease resulting from dysregulation of
heterochromatin 179
Inappropriate gene silencing 179
Heterochromatin reduction 179
Uniparental disomy and disorders of imprinting 180
Abnormal gene regulation at imprinted loci 181
Imprinting and assisted reproduction 183
Summary 186
Questions 187
Further Reading 188
CHAPTER 7 Genetic Variation
Producing Disease-Causing
Abnormalities in DNA and
Chromosomes 189
7.1 How Genetic Variation Results in
Disease 190
7.2 Pathogenic Nucleotide Substitutions
and Tiny Insertions and Deletions 190
Pathogenic single nucleotide substitutions within coding
sequences 190
Relative frequencies of silent and amino-acid-replacing
substitutions 191
Conservative substitution: replacing an amino acid by a
similar one 192
Nonconservative substitutions: effects on the
polypeptide/protein 193
Mutations producing premature termination codons
and aberrant RNA splicing 194
Pathogenic splicing mutations 194
Genesis and frequency of pathogenic point mutations 197
Mutation rates in the human genome 197
Total pathogenic load 197
Effect of parental age and parental sex on germ-line
mutation rates 198
Paternal-age-effect disorders and selfish spermatogonial
selection 198
Surveying and curating point mutations that cause
disease 199
Point mutations in coding DNA 199
Point mutations in RNA genes and other noncoding DNA 200
Databases of human pathogenic mutations 200
7.3 Moderate- to Large-Scale Pathogenic
Mutations Triggered by Repetitive DNA 201
Pathogenic expansion of arrays of short tandem
oligonucleotide repeats in coding DNA 202
Pathogenic polyalanine expansion 202
Unstable expansion of CAG repeats encoding
polyglutamine 203
Pathogenic unstable expansion of short noncoding
tandem repeats 204
Pathogenic sequence exchanges between chromatids at
mispaired tandem repeats 205
Pathogenic sequence exchanges between distant repeats
in nuclear DNA and in mtDNA 208
Chromosome microdeletions and microduplications 209
Deletions resulting from direct repeats in mtDNA 212
Intrachromatid recombination between inverted repeats 212
7.4 Chromosome Abnormalities 212
Structural chromosomal abnormalities 213
Large-scale duplications, deletions, and inversions 215
Chromosomal translocations 216
Isochromosomes 218
Chromosomal abnormalities involving gain or loss of
complete chromosomes 218
Polyploidy 218
Aneuploidy 219
Maternal age effects in Down syndrome 220
Mixoploidy 220
7.5 The Effects of Pathogenic Variants on
the Phenotype 220
Mutations affecting how a single gene works:
loss-of-function and gain-of-function 221
Loss-of-function mutations 222
Gain-of-function mutations 222
The effect of pathogenic variants depends on how the
products of alleles interact: dominance and recessiveness
revisited 224
Loss-of-function versus gain-of-function mutations in
recessive and dominant disorders 224
Striking loss of function produced by dominant-negative
effects in heterozygotes 226
Gain-of-function and loss-of-function mutations in the
same gene produce different phenotypes 228
Multiple gene dysregulation resulting from aneuploidies
and point mutations in regulatory genes 228
Segmental aneuploidies 229
Contiguous gene syndromes 230
7.6 A Protein Structure Perspective of
Molecular Pathology 231
Pathogenesis arising from protein misfolding 231
Regulation of protein folding 231
Aberrant protein folding causing disease 231
How protein aggregation can result in disease 232
Sickle-cell anemia: disruptive protein fibers 232
α1-Antitrypsin deficiency: inclusion bodies and cell death 233
Seeding by aberrant protein templates 233
7.7 Genotype–Phenotype Correlations
and Why Monogenic Disorders Are Often
Not Simple 236
The difficulty in getting reliable genotype–phenotype
correlations 236
Exceptional versus general reasons for poor
genotype–phenotype correlations 237
Modifier genes and environmental factors: common
explanations for poor genotype–phenotype correlations 237
Modifier genes: the example of β-thalassemia 237
Environmental factors influencing the phenotype of genetic
disorders 238
Summary 241
Questions 243
Further Reading 244
CHAPTER 8 Identifying Disease
Genes and Genetic Susceptibility
to Complex Disease 247
8.1 Identifying Genes in Monogenic
Disorders 247
Position-dependent strategies 248
The final step: mutation screening 249
Linkage analysis to map genes for monogenic disorders
to defined subchromosomal regions 249
Human genetic maps 249
Principle of genetic linkage 250
Human meiotic recombination frequencies 251
Standard genomewide linkage analyses 252
Autozygosity mapping in extended inbred families 255
Chromosome abnormalities and other large-scale
mutations as routes to identifying disease genes 255
Exome sequencing: let’s not bother getting a position
for disease genes! 256
8.2 Approaches to Mapping and Identifying
Genetic Susceptibility to Complex Disease 259
The polygenic and multifactorial nature of common
diseases 259
Complexities in disease risk prediction 259
Difficulties with lack of penetrance and phenotype
classification in complex disease 261
Phenotype classification and phenocopies 261
Estimating the contribution made by genetic factors to
the variance of complex diseases 262
Family studies 263
Adoption and twin studies 263
Variation in the genetic contribution to disorders 263
Linkage analyses to seek out genes for complex diseases 264
Parametric linkage analyses in Mendelian subsets 264
Nonparametric and affected sib-pair linkage analysis 265
Identifying the disease-susceptibility gene 267
The principle of allelic association 268
Linkage disequilibrium as the basis of allelic associations 269
Sharing of ancestral chromosome segments 272
How genomewide association studies are carried out 273
The transmission disequilibrium test 275
Moving from candidate subchromosomal region to
identify causal genetic variants in complex disease 276
Identifying causal variants 277
The limitations of GWA studies and the issue of missing
heritability 278
The relative contributions of common and rare variants
to complex disease susceptibility 279
The common disease–common variant hypothesis 280
How common deleterious alleles are maintained 280
The common disease–rare variant hypothesis 281
Copy number variants associated with complex diseases 281
CNPs and CNVs in neuropsychiatric disorders 283
Recent explosive growth in human population has meant
that most coding sequence variants are rare variants 283
Massively parallel DNA sequencing to identify rare
sequence variants associated with complex disease 284
De novo sequence variants 285
The overall contribution made by rare variants 285
8.3 Our Developing Knowledge of the
Genetic Architecture of Complex Disease
and the Contributions of Environmental
and Epigenetic Factors 286
The success and the utility of genomewide association
studies 286
The utility of GWA studies 287
Assessment and prediction of disease risk 287
New insights into biological pathways in complex
diseases may offer new approaches in classifying and
treating disease 289
The pathogenesis of inflammatory bowel disease 289
Connections between different disease pathways 293
Protective factors and the basis of genetic resistance to
infectious disease 293
Gene–gene interaction (epistasis) in complex disease 295
Gene–environment interactions in complex disease 296
A plethora of ‘environmental factors’ 296
Prospective cohort studies 297
Epigenetics in complex disease and aging: significance
and experimental approaches 297
Experimental investigations 300
Epigenetic changes during aging 300
Epigenetic changes in monozygotic twins 301
The developmental origins of adult health and disease 301
Transgenerational epigenetic effects 302
Summary 302
Questions 305
Further Reading 306
CHAPTER 9 Genetic Approaches
to Treating Disease 309
9.1 An Overview of Treating Genetic
Disease and of Genetic Treatment of Disease 310
Three different broad approaches to treating genetic
disorders 310
Augmentation therapy for genetic deficiencies 310
Applicability of molecular augmentation therapy 311
Treatment for disorders producing positively harmful effects 312
Treatment by altering disease susceptibility 312
Very different treatment options for different inborn
errors of metabolism 313
Two broad phenotype classes 313
Augmentation therapy 313
Treating or preventing harmful effects of elevated metabolites 314
Mixed success in treatment 314
Genetic treatment of disease may be conducted at many
different levels 316
9.2 Genetic Inputs into Treating Disease
with Small Molecule Drugs and Therapeutic
Proteins 317
Small molecule drugs 318
New approaches 318
An overview of how genetic differences affect the
metabolism and performance of small molecule drugs 319
Different stages at which genetic variation influences drug
metabolism 319
Phase I and phase II reactions in drug metabolism 320
Phenotype differences arising from genetic variation in drug
metabolism 320
Genetic variation in cytochrome P450 enzymes in
phase I drug metabolism 321
Genetic variation in CYP2D6 and its consequences 322
Genetic variation in other cytochrome P450 enzymes 323
Genetic variation in enzymes that work in phase II drug
metabolism 324
Altered drug responses resulting from genetic variation
in drug targets 325
When genotypes at multiple loci in patients are important
in drug treatment: the example of warfarin 326
Translating genetic advances: from identifying novel
disease genes to therapeutic small molecule drugs 327
Cystic fibrosis: not an easy prospect 328
Familial hypercholesterolemia: new and valuable drugs 330
Marfan syndrome: advantages of a mouse model 331
Tuberous sclerosis: from a biological pathway to a
promising drug 331
Translating genomic advances and developing generic
drugs as a way of overcoming the problem of too few
drug targets 332
Translating genomic advances 332
Developing generic drugs 332
Developing different drugs: therapeutic recombinant
proteins produced by genetic engineering 333
Genetically engineered therapeutic antibodies with
improved therapeutic potential 333
Genetically engineered antibodies 334
Intrabodies 335
9.3 Principles of Gene and Cell Therapy 336
Two broad strategies in somatic gene therapy 337
The delivery problem: designing optimal and safe
strategies for getting genetic constructs into the cells
of patients 338
Efficiency and safety aspects 338
Different ways of delivering therapeutic genetic constructs,
and the advantages of ex vivo gene therapy 342
In vivo and ex vivo gene therapy 342
Nonviral systems for delivering therapeutic genetic
constructs: safety at the expense of efficiency 343
Viral delivery of therapeutic gene constructs: relatively
high efficiency but safety concerns 345
Integrating and non-integrating viral vectors 345
The importance of disease models for testing potential
therapies in humans 346
When rodent disease models can be inadequate 347
9.4 Gene Therapy for Inherited Disorders
and Infectious Disease: Practice and Future
Directions 350
Multiple successes for ex vivo gene augmentation therapy
targeted at hematopoietic stem cells 350
Safety issues in gammaretroviral integration 351
Increased safety profiles using lentiviral vectors 353
In vivo gene therapy: approaches, barriers, and recent
successes 354
Delivery using adenovirus and adeno-associated virus
vectors 354
Amenability of disorders to in vivo gene therapy 354
Two recent examples of successful in vivo gene therapy 355
Complex disease applications: the example of Parkinson
disease 355
RNA targeting therapies: gene silencing by RNA
interference and modulation of RNA splicing 357
Gene-silencing therapy using RNA interference 357
Modulation of splicing 357
Future prospects and new approaches: therapeutic
stem cells, cell reprogramming, and genome editing 359
Therapeutic embryonic stem cells 362
Therapeutic induced pluripotent stem cells 362
Therapeutic cell reprogramming by transdifferentation 363
Therapeutic genome editing 363
The prospect of germ-line gene therapy to prevent
mitochondrial DNA disorders 366
Summary 366
Questions 369
Further Reading 370
CHAPTER 10 Cancer Genetics
and Genomics 373
10.1 Fundamental Characteristics and
Evolution of Cancer 373
The defining features of unregulated cell growth
and cancer 373
Cancer as a battle between natural selection operating
at the level of the cell and of the organism 375
The balance between cell proliferation and cell death 376
Why we do not all succumb to cancer 377
Cancer cells acquire several distinguishing biological
characteristics during their evolution 377
The initiation and multi-stage nature of cancer evolution
and why most human cancers develop over many decades 380
Clonal expansion and successive driver mutations 381
Cancer development through accelerated mutation 382
Mutation accumulation and age of cancer onset 382
Intratumor heterogeneity arises through cell infiltration,
clonal evolution, and differentiation of cancer stem cells 383
10.2 Oncogenes and Tumor Suppressor Genes 385
Two fundamental classes of cancer gene 385
Viral oncogenes and the natural roles of cellular oncogenes 387
Oncogene activation: chromosomal rearrangements
resulting in increased gene expression or gain-of-function
mutations 388
Activation by gene amplification 388
Translocation-induced gene activation 389
Gain-of-function mutations 391
Tumor suppressor genes: normal functions, the two-hit
paradigm, and loss of heterozygosity in linked markers 391
Familial cancers and the two-hit paradigm 391
Loss of heterozygosity 393
The key roles of gatekeeper tumor suppressor genes in
suppressing G1–S transition in the cell cycle 394
The additional role of p53 in activating different apoptosis
pathways to ensure that rogue cells are destroyed 395
Rare familial cancers and the need for a revision of the
classical two-hit tumor suppressor paradigm 397
Haploinsufficiency and gain-of-function mutations 398
A revised model for tumor suppression 398
The significance of miRNAs and long noncoding RNAs in
cancer 398
10.3 Genomic Instability and Epigenetic
Dysregulation in Cancer 399
An overview of genome and epigenome instability
in cancer 399
Different types of chromosomal instability in cancer 402
Chromothripsis 402
Telomeres and chromosome stability 403
Deficiency in mismatch repair results in unrepaired
replication errors and global DNA instability 404
The mechanism of mismatch repair 405
Consequences of defective mismatch repair 405
Epigenetic dysregulation in cancer and its effects on gene
expression and genome stability 406
Aberrant DNA methylation 407
Genome–epigenome interactions 407
10.4 New Insights from Genomewide Studies
of Cancers 408
Genomewide gene expression screens to enable clinically
useful gene expression signatures 408
Clinical applications 409
Genome sequencing reveals extraordinary mutational
diversity in tumors and insights into cancer evolution 411
Mutation number 411
Mutational processes and cancer evolution 412
Intertumor and intratumor heterogeneity 413
Defining the landscape of driver mutations in cancer
and the quest to establish a complete inventory of
cancer-susceptibility genes 414
Cancer gene and driver mutation distribution 416
Novel cancer-susceptibility genes 417
Non-classical cancer genes linking metabolism to the
epigenome 417
10.5 Genetic Inroads into Cancer Therapy 418
Treatment or prevention? 419
The efficacy of cancer therapy 419
The different biological capabilities of cancer cells afford
many different potential therapeutic entry points 420
Targeted cancer therapy after genetic studies define a
precise molecular focus for therapy 422
The molecular basis of tumor recurrence and the evolution
of drug resistance in cancers 423
The basis of tumor recurrence 423
The evolution of drug resistance 424
Combinatorial drug therapies 424
Summary 424
Questions 427
Further Reading 427
CHAPTER 11 Genetic Testing from
Genes to Genomes, and the Ethics of
Genetic Testing and Therapy 431
11.1 An Overview of Genetic Testing 432
Evaluating genetic tests 432
Genetic testing: direct genotyping assays, mutation
scanning, downstream assays, and indirect linkage analyses 432
Indirect linkage analyses 433
Different levels at which genetic testing can be carried out 434
11.2 The Technology of Genetic Testing for
Chromosome Abnormalities and
Large-Scale DNA Changes 435
Detecting aneuploidies with the use of quantitative
fluorescence PCR 436
Principles of quantitative fluorescence PCR 436
Autosomal aneuploidies 436
Sex chromosome aneuploidies 437
Noninvasive fetal aneuploidy screening 438
Detecting large-scale DNA copy number changes with
the use of microarray-based genomic copy number analysis 438
Array comparative genome hybridization (aCGH) 438
SNP microarray hybridization 439
Unclassified variants and incidental findings 440
The need for conventional karyotyping and chromosome
FISH (fluorescence in situ hybridization) 441
Chromosome FISH 441
DNA technologies for detecting pathogenic changes in
copy number of specific DNA sequences 442
Multiplex ligation-dependent probe amplification (MLPA) 445
11.3 The Technology of Genetic Testing for
Small-Scale DNA Changes 446
Scanning for undefined point mutations in single and
multiple genes, and in whole exomes and genomes 447
Gene-specific microarray-based mutation scanning 447
Multiplex mutation scanning: multiple genes to whole
exomes 447
Interpreting sequence variants and the problem of
variants of uncertain clinical significance 451
Mutation interpretation and databases 452
Variants of uncertain clinical significance 453
Scanning for possible pathogenic changes in cytosine
methylation patterns 454
Technologies for genotyping a specific point mutation or SNP 455
Multiplex genotyping of specific disease-associated
variants as a form of mutation scanning 456
11.4 Genetic Testing: Organization of
Services and Practical Applications 458
Genetic testing of affected individuals and their close
relatives 458
Cascade testing 459
Traditional prenatal diagnosis uses fetal tissue samples
recovered by an invasive procedure 460
Preimplantation genetic testing often analyzes a single
cell in the context of assisted reproduction (in vitro
fertilization) 460
Noninvasive prenatal testing (NIPT) and whole fetal
genome screening 464
Technological breakthroughs 464
From fetal aneuploidy screening to genome screening 465
Pre-symptomatic and predictive testing for single-gene
disorders in asymptomatic individuals 465
Pre-symptomatic testing without medical intervention 466
An overview of the different types and levels of genetic
screening 466
Maternal screening for fetal abnormalities 467
Newborn screening to allow early medical intervention 469
Benefits versus disadvantages 470
Carrier screening for disease prevention in serious
autosomal recessive disorders 470
Screening for β-thalassemia 470
Community screening for Tay–Sachs disease 471
New genomic technologies are beginning to transform
cancer diagnostics 471
Multiplex testing using targeted genome sequencing 472
Noninvasive cancer testing 473
Genetic testing for complex disease and
direct-to-consumer genetic testing 474
11.5 Ethical Considerations and Societal
Impact of Genetic Testing and Genetic
Approaches to Treating Disease 474
Consent issues in genetic testing 475
Consent issues in testing children 475
Problems with sharing of genetic information and the
limits on confidentiality 476
Ethical and societal issues in prenatal diagnosis and testing 477
Preimplantation genetic diagnosis (PGD) 477
Restrictions on genetic testing as a result of gene patenting 477
Genetic discrimination and ethical, societal, and practical
issues raised by clinical genomewide sequencing 478
Incidental findings 479
Neonatal genome sequencing 481
The ethics of genetic manipulation of the germ line to
prevent disease or to enhance normal human traits 482
Genetic enhancement and designer babies 482
Ethical considerations and societal sensitivity to
three-person IVF treatment for mitochondrial DNA disorders 483
Summary 484
Questions 486
Further Reading 487
Glossary 489
Index 503