Allele Important Genetic Concepts Genotype Phenotype Gene

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 DNA Replication Pg. 279 Template-dependent Semi-conservative 5’ to 3’ Synthesis Old New

Standard Base Pairs Tautomeric Shift Base Pairs AT G C T G G T AC CA Rare tautomers are shown in Red

Tautomeric Shift of “A” DNA molecule with TA CG transition mutation Affected base pair Pg. 416

Oxidation of Guanine 8-oxo-7,8-dihydro-guanine (8-oxoG) ( OH) OHProduced 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

(NER) Individuals that are homozygous for loss-of-function alleles of one of the XP genes UV Nucleotide Excision Repair (NER) Pathway (pyrimidine dimer)

(NER) Damaged DNA Replicated Mutations! UV Nucleotide Excision Repair (NER) Pathway (pyrimidine dimer) Individuals that are homozygous for loss-of-function alleles of one of the XP genes

Xeroderma pigmentosum

Sickle Cell Anemia Sickle Cell Trait Normal Red Blood Cells

Hemoglobin ß-globin (146 amino acids) ß-globin (146 amino acids)

Molecular Genetics of Sickle-Cell Anemia Hb-A Hb-S Codon #6 Normal Individuals Sickle Cell Individuals E6V

Hb-S Deoxyhemoglobin

Polymerized Deoxyhemoglobin (tactoids)

Molecular Genetics of Sickle-Cell Anemia Hb A Allele Hb S Allele Hb-A Hb-S Codon #6

Hb-A WT and Mutant ß-globin Proteins Hb Mutant Proteins Hb WT Protein

Huntington’s Disease

Mutant Huntingtin Protein Aggregates Transmission Electron Micrograph

WT Huntingtin-GFPMutant Huntingtin-GFP

hhHhHH No Htt Protein Aggregates Htt Protein Aggregates No HDHD

Trinucleotide Repeat Diseases

Cystic Fibrosis

CFTR Protein

Missense mutation Nonsense mutation Frame-shift mutation Deletion, in-frame CFTR Gene Mutations Loss-of-Function Mutations ~1200 pathological mutations Spectrum of Mutations in CFTR Gene

Frequency of CF Mutant Alleles ~ 70% of CF Mutant alleles are ∆F508

∆F508 Mutation

Healthy Lung Lung epithelial cells Lung epithelial cells AA or Aa

Lung epithelial cells Lung epithelial cells Cystic Fibrosis Lung aa

Erythropoietin Receptor Mutation And Olympic Glory Seven Olympic medals Eero Mäntyranta

(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 Hb S Hb A Pg. 644 MstII cut sites

“Prodrug” Tamoxifen Bioactive form of Tamoxifen OH Cytochrome P450 (CYP2D6)

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) Sperm cells Embryos (4-cell stage) Oocytes

Pre-implantation Genetic Diagnosis (PGD) 8-cell Embryo

Removal viral genes Splice in WT Allele Viral Vector for Gene Therapy WT Allele aaaaa

Gene Therapy Vectors Retroviruses (e.g. MLV, HIV) Adenoviruses Herpes viruses Cell Host Range Dividing vs. non-dividing cells Overall efficiency of transfer “Evolved” virus

Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-Linked Adrenoleukodystrophy Science 6 November 2009: Vol pp Cartier et al (26 authors!)

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

Thymidine Kinase Cytosine Deaminase Nitroimidazole reductase GanciclovirPhosphoganciclovir 5-fluorocytosone5-fluorouracil CB1954Alkylating agent EnzymeProdrugToxic Metabolite Gene Therapy for Cancer

M-Phase Chromosome Interphase (G1, S, G2) Chromosomes

Giemsa Stain

Human Euploid Karyotypes FemaleMale Fig. 7-6

Homologous Chromosomes PM P = Paternal M = Maternal

2001

Human Genome Information

“Raw” DNA sequence Where are the genes?

3000 bp scanned for ORFs 5’ 3’ Three Reading Frames Three Reading Frames

Y58 Mbp X 22 1 ChromosomeDNA sizeORF #ChromosomeDNA SizeORF # 155 Mbp Mbp 47 Mbp Mbp Mbp Mbp Mbp Mbp Mbp Mbp Mbp 243 Mbp Mbp 191 Mbp Mbp Mbp 159 Mbp Mbp 140 Mbp Mbp 134 Mbp Mbp Mbp

X Chromosome 155 Million base pairs (bp) 1606 Genes Muscular Dystrophy Adrenoleukodystrophy Hemophilia A Hemophilia B Green Color Blindness Red Color Blindness X-linked Traits

Y Chromosome 58 Million base pairs (bp) 344 Genes