The Development of Personalized Medicine (Molecular Medicine/Precision Medicine)

Science February 2001 Human Genome Project $3 billion 1990 2003

Genes Environment Normal Physiology Disease Chance

The Human Genome Previous Estimates ?100,000 - 35,000 Genes

1,600 genes 397 genes

Fred Sanger 1918 – Nov 2013 Double Nobel Laureate in Chemistry "just a chap who messed about in a lab"

Sanger Institute, Hinxton, Cambridge, UK

X174 bacteriophage 5,386 nucleotides 1977 Fred Sanger

Genomic/cDNA libraries Early 1980s Genomic/cDNA libraries Factor IX gene (34,000 bp – 3+ years)

in 2015 - 1 day Early 1980s Genomic/cDNA libraries Factor IX gene (34,000 bp – 3+ years) in 2015 - 1 day

1983 Sonoma Valley Revelation Dr. Kary Mullis - 1983 Sonoma Valley Revelation (Scientific American April 1990)

Kary Mullis: Nobel Laureate 1993 AIDS denialist Global warming dissenter LSD supporter Polymerase Chain Reaction - PCR (Nucleic acid amplification technique)

Francis Collins National Human Genome Research Institute Craig Ventor Celera Genomics

Dr. Francis Collins Dr. Craig Ventor

Human Genes 20,000 - 25,000 Occupy ~2% of the genome

Human Phenotypic Complexity

Human Phenotypic Complexity Alternative RNA forms - alternative splicing Protein modifications ie - a) Phosphorylation b) Glycosylation

the new genetic knowledge? How have we used the new genetic knowledge?

Initial Genetic Characterizations 1975-1985 Sickle cell anemia Hemophilia Cystic Fibrosis

Sickle Cell Anemia Initial Genetic Characterizations Synthesis of an abnormal form of hemoglobin β globin gene mutation Amino Acid 6 Glutamic Valine Acid

Genetic Knowledge Application Molecular genetic diagnosis Cystic fibrosis Muscular dystrophy Hemochromatosis 2. Production of recombinant therapeutic proteins Hormones - insulin, erythropoietin Clotting factors 3. Gene Therapy

Larger genes responsible for less common single gene Genetic Characterizations: 1985 - 2000 Larger genes responsible for less common single gene (monogenic) disorders

Common Complex Genetic Traits Genetic Characterizations: 2000 - Common Complex Genetic Traits

November 2014

Nature Genetics December 2014

Complex Genetic Diseases Contribution of Genetics Rare monogenic cases (<5%) - strong family history/earlier onset Common forms a) variable onset b) many genes involved c) environmental influences d) chance

The New Age of DNA Sequencing

Standard “Sanger” Sequencing Single template molecule Read lengths - 300-600 nucleotides

Next generation sequencing DNA Sequencing 2010 - Resequencing Deep sequencing Massively parallel DNA sequencing Next generation sequencing

Next Generation Sequencing 100,000 parallel sequencing reactions 25-150 nucleotide read lengths 1-2.5 billion nucleotides/day Resulting Bioinformatic Challenge

1,000 Genomes Sequence Project Nature April 2008 1,000 Genomes Sequence Project UK NHS 100,000 Genomes Project DNA Sequence of James Watson

Illumina launched its HiSeq X Ten Sequencer January 2014 Illumina launched its HiSeq X Ten Sequencer first $1,000 genome at 30x coverage

(single nucleotide variants - SNVs) Human DNA Sequence Variability Single nucleotide polymorphisms - SNPs 3 million SNPs (single nucleotide variants - SNVs) 10,000 SNPs change amino-acid sequences

Genome-Wide Association Analysis (GWAS) ~10,000 subjects Asparagus Anosmia Genome-Wide Association Analysis (GWAS) ~10,000 subjects 535,000 Single nucleotide variants (SNVs)

Freckling

ENCODE Study - Nature 2012 The Role of Regulatory Sequence Variability 80% of genome involved in regulation of gene expression patterns “Junk (intergenic) DNA” critical

Multiple dimmer switch analogy Normal Level of gene expression Disease

Coffee Break

Human Molecular Genetic Pathology Thalassemia Cystic Fibrosis Hemochromatosis Highly prevalent Monogenic traits Significant progress with diagnostic and therapeutic issues

Red Blood Cells (erythrocytes) Anucleate “protein bags” - 70% hemoglobin Synthesized in bone marrow Circulate for 120 days Removal by the reticuloendothelial system liver spleen lymph nodes

Developmental Regulation of Globin Gene Expression

Hemoglobin Tetramer

Thalassemia Pathogenesis Imbalance of Alpha:Beta chain globin synthesis Excess globin chain precipitates Results in 1. RBC precursor death in bone marrow 2. Premature removal of circulating RBCs

Thalassemia Pathogenesis Hemoglobin concentration reduced in all RBCs RBCs Pale - Hypochromic Small - Microcytic

Normal Blood Smear

Thalassemic Blood Smear

Red Blood Cell Effects of Unbalanced Globin Chain Synthesis

Clinical Features of Thalassemia Severe anemia Failure to develop in infants Heart failure Treatment Blood (RBC) transfusions - iron deposition Iron chelation therapy Bone marrow transplant

Pathology of Excessive Iron Deposition Skin - increased pigment Pancreas - late-onset diabetes Liver - cirrhosis/ liver cell cancer Heart - Cardiac failure Pituitary - reduced libido/ impotence Treatment – Phlebotomy/Iron Chelation Therapy

Genetics of Thalassemia Autosomal Recessive Traits Mutational (allelic) heterogeneity

Alpha (chromosome 16) and Beta (chromosome 11) Locus Organization

Beta Thalassemia Mutations + Beta Globin Gene 1 2 3 + 5’ 3’ Transcription Frameshift deletion Deletion Nonsense Poly A site Splicing Beta Thalassemia Mutations + Insertion