Genetic Disease & Genomic Medicine Pauline Yen ( 蕭百忍 ) Institute of Biomedical Sciences Academia Sinica
Genome Size E. coli: Yeast:
5,000,000 bp 15,000,000 bp
Nematode
100,000,000 bp
Mustard Weed
120,000,000 bp
Fruit Fly
160,000,000 bp
Human
3,000,000,000 bp
Protein-encoding Genes E. Coli (5 Mb)
4,290
Yeast (15 Mb)
6,000
Nematode (100 Mb)
19,500
Mustard Weed (120 Mb) 27,000 Fruit Fly (160 Mb)
13,000
Human (3,000 Mb)
25,000
DNA Content of the Human Genome • Nonrepetitive (60%) – Includes most genes
• Moderately repetitive (25%) – SINES (Short Interspersed Repeated Segments) • Alu repeats: 300 bp, >105 copies
– LINES (Long Interspersed Repeated Segments) • L1 family: >5 kb, ~104 copies
• Highly repetitive (15%) – Satellite DNA at centromeres and telomers
Human Chromosomes
Human Genetic Disorders Category
Frequency (%)
Chromosomal Single-gene Multifactorial Unclassified
0.19 0.36 4.70 0.12
Total
5.37
OMIM Online Mendelian Inheritance in Man This database is a catalog of human genes and genetic disorders
Chromosome Aneuploidy • Trisomy 21 • Trisomy 18 • Trisomy 13
• Klinefelter syndrome – (47, XXY), 1/1000.
• Turner syndrome – (45, X), 1/5,000.
• Trisomy X – (47, XXX), 1/1000.
The cause of chromosome aneuploidy Nondisjunction at meiosis
Down Syndrome Trisomy 21 Incidence of ~ 1/800 Phenotype: Mental retardation Congenital heart disease Premature senility
Autosomal Trisomies Trisomy 21
Trisomy 18
Trisomy 13
(Down Syndrome)
(Edwards Syndrome)
(Patau Syndrome)
~ 40-50 years
< a few months
< a month
Interphase fluorescence in situ hybridization (FISH)
Klinefelter Syndrome (KS) • • • • • • •
Affecting one in 1000 males 47, XXY in 80% of the cases Tall stature Learning & behavior problems Decreased virilization ( 男性化 ) Gynecomastia ( 男乳房增殖 ) Infertility
Turner Syndrome • • • • • • •
Affecting one in 5000 females 45, X and variants Short stature Gonadal dysgenesis Neck-webbing Broad chest with widely spaced nipples Elevated frequency of renal and cardiovascular anomalies
Mammalian X-chromosome Inactivation • In mammalian somatic cells, only one X chromosome is active.. • X-inactivation occurs early in embryogenesis.
Mammalian X-chromosome Inactivation • It is random and heritable. • The inactive X (Xi) is condensed to form the Barr-body. • Xi replicates late in the S phase and is highly methylated. • Not all genes are subjected to inactivation. – 15% of genes on the human X chromosome escape inactivation.
Genomic disorder/ Contiguous gene syndrome A syndrome resulting from a microdeletion of chromosomal DNA extending over two or more contiguous loci.
Unequal crossing over between direct low-copy repeats
(Deletion)
(Duplication)
Prader-Willi Syndrome Lacks paternally derived 15q1113 ~ 1/10,000 to 1/25,000 Phenotype: Mental retardation Short stature Small hands and feet hyperphagia Obesity Hypogonadism
Angelman Syndrome Lacks maternally derived 15q11-13 Phenotype Unusual facial appearance Short stature Severe mental retardation Spasticity Seizures
Genomic Imprinting The phenomenon of different expression of alleles depending on the parent of origin. .
Single Gene Mutations (Medelian Diseases)
• Autosomal dominant
– Gain-of function – Haploid insufficiency
• Autosomal recessive • X-linked
Autosomal Dominant Disorder
Achondroplasia ( 軟骨發育不全 ) Short-limbed dwarfism 1 in 10,000 80% de novo mutation Increased risk with late paternal age G1138A mutation in the FGFR3 gene
Autosomal Dominant Diseases • • • • •
Huntington Disease Familial Hypercholesterolemia Alzheimer Disease Hereditary breast and ovarian cancer Hereditary Nonpolyposis Colon Cancer
Penetrance The fraction of individuals with a genotype known to cause a disease who have any signs or symptoms of the disease.
Huntington Disease • Late childhood to late adulthood onset • Progressive loss of movement and cognition • Viable expression • Reduced penetrance • Presymptomatic counseling • Triplet repeat (CAG) expansion
Triplet Repeat Diseases Disease
Repeat
Gene
Location
Huntington CAG huntingtin coding
Mechanism of Disease poly-glutamines
Repeat number Normal Unstable Affected <36
>35
Myotonic Dystrophy 11000
CTG DMPK CCTG ZNF9
3’ UTR intron
toxic RNA
<30 ~ 40
50-80
Fragile X
CGG
FMR1
5’ UTR
reduced expression
<60
60-200
Friedreich
AAG
frataxin
intron
RNA processing <34 reduced expression
36-100
80–2000 75>200 >100
Myotonic dystrophy • Autosomal Dominant • 1 in 7400 live births • Variable expression in clinical severity and age of onset – Myotonia (delayed muscle relaxation due to repetitive action potentials in myofibers) – Muscular dystrophy – Cardiac conduction defects – Cataracts – Hypogonadism – Frontal balding
Anticipation The progressively earlier appearance and increased severity of a disease in successive generations Grandma: bilateral cataracts Ma:facial weakness, ptosis, myotonia, cataract Child: congenital myotonic dystrophy
RNA gain-of function hypothesis Mutant DMPK transcripts sequester nuclear factors required for proper muscle development and maintenance.
Autosomal Recessive Disorder
Sickle Cell Anemia Red Blood Cells
β−Globin Gene
Tay-Sachs Disease
Affects 1 in 3900 Ashkenazi Jews. Defective in hexosaminidase A. Inability to degrade GM2 ganglioside. Appears normal until 3-6 months of age. Progressive neurological deterioration. Death at 2 to 4 years.
Consanguineous mating
1/16
1/32
X-linked Disorder
X-linked Diseases • • • • •
Hemophilia Color-blind G6PD deficiency Duchenne muscular dystrophy Mental Retardation
Female is the better fitted sex
Mutations in the same gene may cause different phenotypes
Androgen Receptor A transcription factor that mediates the action of androgens in the development, differentiation, and function of male reproductive and accessory sex tissues.
Androgen Receptor • Kennedy spinal and bulbar muscular atrophy – – – –
facial fasciculations ( 成束 ) muscle weakness and wasting late onset triplet repeat (CAG) expansion
Androgen insensitivity syndrome/ Testicular feminization syndrome •1/20,000 live birth •46 XY •Absence of functional AR •Normal female external genitalia •Blind vagina and no uterus •Testes within the abdomen or in the inguinal canal
Genes also affect personality and behavior
Nurturing Behavior in Mice
Wild-type mother: Groups and crouches over her pups. FosB mutant mother: Leaves pups scattered and untended. Pups will eventually die. (Cell 86:297, 1996)
Type of mutations in human genetic diseases • Nucleotide substitution – – – –
Missense mutation Nonsense mutation RNA processing mutation Regulatory mutation
• Deletion and insertion – – – –
Small Larger gene deletion, inversion, duplication Insertion of L1 or Alu element Expansion of trinucleotide repeat sequence
Genomic Medicine • Practice of medicine using knowledge gained from gene discovery via genome research: To enhance the diagnostic skills To develop new therapeutic options To improve the overall quality of health care
• Identification of Molecular Defects • Prenatal Diagnosis – Down syndrome, Tay-Sachs disease.
• New Born Screening – Phenylketonuria
• Treatment – – – – –
Dietary Restriction Enzyme Therapy Gene Therapy Organ Transplantation Surgical Prevention
Multifactorial Disorders
Disorders that are determined by a combination of multiple factors, genetic and possibly also environmental.
Mendelian vs Common Disesae -single genes
-polygenic
-high penetrance
-low penetrance
-low frequency alleles
-high frequency alleles
-little environmental impact -large environmental impact -tidy pedigrees
-untidy pedigrees
-easy to discern genetic role -difficult to discern genetic role -gene isolation tractable
-difficult to isolate genes
-? public health impact
-tremendous impact
(LDLR, CCR5)
Common Diseases With a Strong Genetic Component • • • • • •
Alzheimer Disease Infectious Diseases Cancer Osteoporosis Cirrhosis Glaucoma
• Age related Hearing Loss • Schizophrenia • Bipolar Disease • Depression • Obesity
Genetics interacts with environment to result in disease • Risk of lung cancer is increased with smoking, but the great majority of smokers avoid it. • Alcohol increases risk of cirrhosis, but a minority of alcholics develop cirrhosis • Only 10% of those exposed to TB ever develop clinical infection • Outcome of head trauma is highly variable
Polymorphism A variation in DNA sequence within a population
Restriction Fragment Length Polymorphism
Variable Number Tandem Repeat (VNTR) Short Tandem Repeat (STR)
Individual Identification Locus A
B
Allele 1 2 3 4 1 2 3 4 5
Frequency 0.25 0.17 0.43 0.15 0.14 0.26 0.08 0.32 0.20
Locus C
Allele 1 2 3
Frequency 0.3 0.38 0.32
D
1 2 3 4 5 6
0.13 0.06 0.19 0.21 0.29 0.12
The frequency of A1B3C2D4 = 0.0016 , one in 625. A4B3C3D2=0.0002, one in 5,000.
SNP Single-nucleotide polymorphism Common, single-base-pair variations in DNA
Finding Disease Susceptibility Genes Case-Control Association Study SNP Genotyping
….GCCGTTGAC…. ….GCCATTGAC…. Case
Screen for disease susceptibility
….GCCATTGAC…. ….GCCATTGAC…. Contr
Common disease susceptibility (polygenic; complex inheritance) Common late-onset Alzheimer’s disease ApoE on 19q13 Susceptibility gene locus on 12q Non-insulin-dependent diabetes mellitus Susceptibility gene loci on 12q, 2q Migraine Susceptibility gene loci on 19p13; Xq24
Pharmacogenetics A special area of biochemical genetics that deals with the contribution of genetics to variation in drug response.
Drug Efficacy 30% patients do not respond to statin (used to lower cholesterol) 35% patients do not respond to betablockers (used to lower blood pressure) 50% patients do not respond to some older (tricyclic) antidepressants
The Acetylation Polymorphism Arylamine N-acetyltransferase Rapid acetylator Slow acetylator: Asian: 5-20% African: 50% Caucasian: 65% Affects the disposition of many drugs. isoniazid – tuberculosis hydralazine - hypertension
Impact of Adverse Drug Reactions (ADRs) The overall incidence of serious ADRs is 6.7% of hospitalized patients The overall incidence of fatal ADRs is 0.32% of hospitalized patients => the 4th ~6th leading cause of death • 5~9% of all hospital costs are related to ADRs Drugs withdrawn from major markets: 34 since 1990 Ref: Need et al. Nature Genetics 37: 671, 2005
Pharmacogenomic Study
Poor response
Treat with alternative dose/drug
Genotyping before prescribing
Adverse drug reactions
Treat with alternative drug
Treat with conventional drug or dose Annu. Rev. Genomics & Hum.
Susceptibility to HIV infection
Science 307:1422, 2005
Research Theme Genetic epidemiology
ENU mouse
Novel disease genes
Bioinformatics
Proteomics
System biology
Diagnostics Preventive medicine Pharmacogenetics Gene therapy
Proteins, pathways, biology Drug therapy
Search for Novel Genes Associated with Human Diseases- Approaches • Genetic epidemiology: map and identify novel disease genes • Mouse mutant: screen phenotypes resembling human diseases to map and identify novel disease genes • Bioinformatics: linked genes, comparative genomics • Proteomics: interacting proteins, protein expression profile
DNA microarray (gene chip) DNA clones
test
reference reverse Transcription Label with fluor dyes
PCR amplification purification Robotic printing
hybridize target to microarray
excitation laser 2
laser 1
emission
computer analysis
Gene expression pattern in human cancer
Genetics verses
Environment
Teratogen An agent that produces or raises the incidence of congenital malformations.
Thalidomide • Used as a tranquilizer and antiemetic in early pregnancy in the early 1960s • Produced babies with “seal limbs”
Retinoic Acid Embryopathy (Accutane Embryopathy) • Taking Accutane beyond the 15th day following conception. • Craniofacial, cardiovascular and central nervous abnormalities. • Subnormal intelligence.