Important Genetic Concepts
Gene
Allele
Genotype
Phenotype
Mutation (Chapt. 16)
Classification of Mutations
Mechanisms of Mutagenesis
DNA Repair Pathways
Possible Reading Frames for RNA
A Comprehensive Catalogue of Somatic Mutations
From a Cancer Genome
Nature (December 2009)
Cancer: Malignant Melanoma
Total base substitution mutations: 33,345
Coding mutations: 292
Missense: 172
Nonsense: 15
Silent:105
Total Chromosome mutations: 37
Old
Old
DNA Replication
Template-dependent
Semi-conservative
5’ to 3’ Synthesis
New
Pg. 279
Old
Old
New
Standard Base Pairs
Tautomeric Shift
Base Pairs
A
T
T
G
G
C
G
T
C
A
A
C
Rare tautomers are shown in Red
Affected base pair
Tautomeric Shift of “A”
DNA molecule
with TA CG
transition mutation
Pg. 416
Oxidation of Guanine
( OH)
8-oxo-7,8-dihydro-guanine (8-oxoG)
OH
Produced From Electron Transport
8-oxoG-A Base Pair
8-oxoG
Adenine
Insertion by “Looping Out” of
Newly Synthesized Strand
Deletion by “Looping Out” of Template Strand
Intercalating Agents
Ethidium Bromide
Ethidium Intercalation in DNA
Medical Diagnostics
X-rays, positrons
Radiotherapy
X-rays, gamma rays
Pg. 419
Pyrimidine Dimer
Nucleotide Excision Repair
(NER) Pathway
(NER)
UV
(pyrimidine dimer)
Individuals that are
homozygous for
loss-of-function alleles
of one of the XP genes
Nucleotide Excision Repair
(NER) Pathway
(NER)
UV
(pyrimidine dimer)
Damaged DNA Replicated
Mutations!
Individuals that are
homozygous for
loss-of-function alleles
of one of the XP genes
Xeroderma pigmentosum
Pigmentary changes, premature skin aging, neoplasia
Malignant tumors may develop as early as ages 3-4.
XP is often fatal before the age of 10 (Two-thirds die before 20)
Red Blood Cells
Normal
Sickle Cell Anemia
Sickle Cell Trait
Hemoglobin
ß-globin
(146 amino acids)
ß-globin
(146 amino acids)
Molecular Genetics of Sickle-Cell Anemia
Normal Individuals
Codon #6
Hb-A
Sickle Cell Individuals
Hb-S
E6V
Deoxyhemoglobin
Hb-S
Hb-S
Polymerized
Deoxyhemoglobin
(tactoids)
Molecular Genetics of Sickle-Cell Anemia
HbA Allele
Hb-A
HbS Allele
Hb-S
Codon #6
WT and Mutant ß-globin Proteins
Hb WT Protein
Hb-A
Hb Mutant Proteins
Huntington’s Disease
Mutant Huntingtin Protein Aggregates
Transmission Electron Micrograph
WT Huntingtin-GFP
Mutant Huntingtin-GFP
hh
No Htt Protein Aggregates
No HD
Hh
Htt Protein Aggregates
HD
HH
Htt Protein Aggregates
HD
Trinucleotide Repeat Diseases
Cystic Fibrosis
CFTR Protein
Spectrum of Mutations in CFTR Gene
CFTR Gene
Mutations
Missense mutation
Nonsense mutation
~1200 pathological mutations
Frame-shift mutation
Loss-of-Function Mutations
Deletion, in-frame
Frequency of CF Mutant Alleles
~ 70% of CF Mutant alleles are ∆F508
∆F508 Mutation
Healthy Lung
AA
or
Aa
Lung epithelial
cells
Lung epithelial
cells
Cystic Fibrosis Lung
aa
Lung epithelial
cells
Lung epithelial
cells
Erythropoietin Receptor Mutation
And Olympic Glory
Eero Mäntyranta
Seven Olympic medals
(Epo)
Erythropoietin (Epo)
Red Blood Cell Development
Genetic Testing
Disease-related Diagnosis
Current disease
Risk of future disease
Carrier status
Pharmacogenomics
Efficacy of therapeutic drug treatment
Tissue Typing
Transplantation
Genetic Testing for Sickle Cell Anemia
HbS
HbA
MstII cut sites
Pg. 644
Tamoxifen
Cytochrome P450
(CYP2D6)
“Prodrug”
OH
Bioactive form
of Tamoxifen
Cytochrome P450 (CYP2D6)
~ 10% Caucasians are “poor” metabolizers
Genetic Testing for Tissue Typing
Silent Mutations mmmmmmm
Missense
Early Embryonic Development (~ 7 days)
In Vitro Fertilization (IVF)
Oocytes
Sperm cells
Embryos (4-cell stage)
Pre-implantation Genetic Diagnosis (PGD)
8-cell Embryo
Viral Vector for Gene Therapy
Removal viral genes
Splice in WT Allele
WT Allele aaaaa
Gene Therapy Vectors
Retroviruses (e.g. MLV, HIV)
Adenoviruses
Herpes viruses
“Evolved” virus
Cell Host Range
Dividing vs. non-dividing cells
Overall efficiency of transfer
Hematopoietic Stem Cell Gene Therapy with
a Lentiviral Vector in X-Linked Adrenoleukodystrophy
Cartier et al (26 authors!)
Science 6 November 2009: Vol. 326. pp. 818 - 823
X-Linked Adrenoleukodystrophy (ALD)
Monogenic
Recessive
Loss-of-function mutations in ABCD1 gene
Defective transport of “very long chain fatty acids” to peroxisomes
Multifocal demyelination of CNS cells
1/17,000 newborn boys are affected
Myelinated Neuron
Schwann Cell
Myelin
Hematopoietic Stem Cells (HSCs)
Brains of ALD Patients
Untreated
HSC Therapy
Gene Therapy for Cancer
Gene Therapy for Cancer
Enzyme
Prodrug
Toxic Metabolite
Thymidine Kinase
Ganciclovir
Phosphoganciclovir
Cytosine Deaminase
5-fluorocytosone
5-fluorouracil
Nitroimidazole reductase
CB1954
Alkylating agent
Interphase (G1, S, G2)
Chromosomes
M-Phase
Chromosome
Giemsa Stain
Human Euploid Karyotypes
Female
Fig. 7-6
Male
Homologous Chromosomes
P
M
P = Paternal
M = Maternal
2001
Human Genome Information
http://www.ncbi.nlm.nih.gov/
http://www.genome.gov/
http://genomics.energy.gov/
“Raw” DNA sequence
Where are
the genes?
3000 bp scanned for ORFs
Three
Reading
Frames
5’
3’
3’
5’
Three
Reading
Frames
Chromosome
DNA size
ORF #
Chromosome
DNA Size
1
247 Mbp
2
ORF #
3380
14
106 Mbp
1453
243 Mbp
2204
15
100 Mbp
1202
3
200 Mbp
1760
16
89 Mbp
1318
4
191 Mbp
1361
5
181 Mbp
1536
17
79 Mbp
1714
6
171 Mbp
1959
18
76 Mbp
517
7
159 Mbp
1764
19
64 Mbp
1992
8
146 Mbp
1247
20
62 Mbp
857
9
140 Mbp
1435
21
47 Mbp
425
10
135 Mbp
1305
11
134 Mbp
2051
22
50 Mbp
835
12
132 Mbp
1629
X
155 Mbp
1606
13
114 Mbp
649
Y
58 Mbp
397
X Chromosome
155 Million base pairs (bp)
1606 Genes
X-linked Traits
Muscular Dystrophy
Adrenoleukodystrophy
Hemophilia A
Hemophilia B
Green Color Blindness
Red Color Blindness
Y Chromosome
58 Million base pairs (bp)
344 Genes