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The Development of Molecular Medicine
Molecular Medicine
Personalized Medicine
Precision Medicine
Cambridge
February 1953
“My Dear Michael,
Jim Watson and I have probably made a most important discovery........
….....Our structure is very beautiful”
Letter auctioned at Christie's New York on 10 April 2013 for $6,059,750, the
largest amount ever paid for a letter at auction
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
Early 1980s
Genomic/cDNA libraries
Factor IX gene (34,000 bp – 3+ years)
Early 1980s
Genomic/cDNA libraries
Factor IX gene (34,000 bp – 3+ years)
in 2017 - 1 day
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
Occupy ~2% of the genome
Functions of Human Gene Products
Human Phenotypic Complexity
Human Phenotypic Complexity
1. Alternative RNA forms
- alternative splicing
2. Protein modifications
ie a) Phosphorylation
b) Glycosylation
How have we used
the new genetic knowledge?
Initial Genetic Characterizations
1975-1985
Sickle cell anemia
Hemophilia
Cystic Fibrosis
Initial Genetic Characterizations
Sickle Cell Anemia
Synthesis of an abnormal form of hemoglobin
β globin gene mutation
Amino Acid 6
Glutamic
Acid
Valine
Genetic Knowledge Application
1. Molecular genetic diagnosis
Cystic fibrosis
Muscular dystrophy
Hemochromatosis
2. Production of recombinant therapeutic proteins
Hormones - insulin, erythropoietin
Clotting factors
3. Gene Therapy
Genetic Characterizations: 1985 - 2000
Larger genes responsible for
less common single gene
(monogenic) disorders
Genetic Characterizations: 2000 -
Common Complex Genetic Traits
November 2014
Nature Genetics
December 2014
Asparagus Anosmia
Genome-Wide Association Analysis (GWAS)
~10,000 subjects
535,000 Single nucleotide variants (SNVs)
Complex Human Traits: Results from Genome-Wide Association Studies
Approx Number
of Loci
Approx Number
of Participants
1. Educational Attainment
2. Height
93
584
294,000
253,000
3. Crohn's Disease
4. Rheumatoid Arthritis
61
74
6,000 / 15,000
14,000 / 44,000
5. Mean red cell volume
6. Platelet count
42
50
48,000
44,000
Pickrell et. al. Nature Genetics July 2016
Complex Genetic Diseases
Contribution of Genetics
1. Rare monogenic cases (<5%)
- strong family history/earlier onset
2. 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
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
Nature April 2008
DNA Sequence of James Watson
1,000 Genomes Sequence Project
UK NHS 100,000 Genomes Project
January 2014
Illumina launched its HiSeq X Ten
Sequencer
first $1,000 genome at 30x coverage
Human DNA Sequence Variability
Single nucleotide polymorphisms - SNPs
3 million SNPs
(single nucleotide variants - SNVs)
10,000 SNPs change amino-acid sequences
The Role of Regulatory Sequence Variability
ENCODE Study - Nature 2012
80% of genome involved in regulation of
gene expression patterns
“Junk (intergenic) DNA” critical
Level of gene expression
Multiple dimmer switch analogy
Normal
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 Globin Gene
1
5’
2
3
3’
+
+
Transcription
Frameshift deletion
Nonsense
Deletion
Splicing
Poly A site
Insertion
Beta Thalassemia Mutations