Author(s): David Ginsburg, 2009 License: Unless otherwise noted, this material is made available under the terms of the Creative Commons Attribution–Non-commercial–Share Alike 3.0 License: http://creativecommons.org/licenses/by-nc-sa/3.0/ We have reviewed this material in accordance with U.S. Copyright Law and have tried to maximize your ability to use, share, and adapt it. The citation key on the following slide provides information about how you may share and adapt this material. Copyright holders of content included in this material should contact [email protected]with any questions, corrections, or clarification regarding the use of content. For more information about how to cite these materials visit http://open.umich.edu/education/about/terms-of-use. Any medical information in this material is intended to inform and educate and is not a tool for self-diagnosis or a replacement for medical evaluation, advice, diagnosis or treatment by a healthcare professional. Please speak to your physician if you have questions about your medical condition. Viewer discretion is advised: Some medical content is graphic and may not be suitable for all viewers.
Transcript
Author(s): David Ginsburg, 2009
License: Unless otherwise noted, this material is made available under the terms of the Creative Commons Attribution–Non-commercial–Share Alike 3.0 License: http://creativecommons.org/licenses/by-nc-sa/3.0/
We have reviewed this material in accordance with U.S. Copyright Law and have tried to maximize your ability to use, share, and adapt it. The citation key on the following slide provides information about how you may share and adapt this material.
Copyright holders of content included in this material should contact [email protected] with any questions, corrections, or clarification regarding the use of content.
For more information about how to cite these materials visit http://open.umich.edu/education/about/terms-of-use.
Any medical information in this material is intended to inform and educate and is not a tool for self-diagnosis or a replacement for medical evaluation, advice, diagnosis or treatment by a healthcare professional. Please speak to your physician if you have questions about your medical condition.
Viewer discretion is advised: Some medical content is graphic and may not be suitable for all viewers.
Citation Keyfor more information see: http://open.umich.edu/wiki/CitationPolicy
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The Human Genome
David Ginsburg, MD
August 22, 2008
M1 Patients and Populations
Fall 2008
Learning Objectives
UNDERSTAND: • The basic anatomy of the human genome [eg. 3 X109 bp (haploid
genome); 1-2% coding sequence (~20,000 genes); types and extent of DNA sequence variation].
• Recombination and how it allows genes to be mapped • Genetic data for a pedigree, assigning phase, defining haplotypes• Linkage: Use of markers linked to a disease gene for genetic
diagnosis and to identify disease genes (positional cloning)• Distinction between a linked marker and the disease causing
mutation itself• Linkage disequilibrium and haplotype blocks• Genome wide association studies (GWAS) to identify gene variants
contributing to complex diseases/traits
DNA Sequence Variation
• DNA Sequence Variation:– Human to human: ~0.1% (1:1000 bp)
• Human genome = 3X109 bp X 0.1% =~3X106 DNA common variants
– Human to chimp: ~1-2%– More common in “junk” DNA: introns, intergenic regions
• poly·mor·phismPronunciation: "päl-i-'mor-"fiz-&mFunction: noun: the quality or state of existing in or assuming different forms: as a (1) : existence of a species in several forms independent of the variations of sex (2) : existence of a gene in several allelic forms (3) : existence of a molecule (as an enzyme) in several forms in a single species
IV
III
II
I
a/b b/b b/b a/b a/b b/b a/b a/b b/b
b/ba/aa/ba/a
a/b a/ba/b
b/bb/ba/ba/b
a/a
B.
Heteromorphismof chromosome 1
(one copy)
2 normal copiesof chromosome 1
a or b allele of Duffy blood group
Donahue, 1968
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.1
I
II
III
B.
I
II
III
A.+ -
++ -
+ - -
-
+
- +
+ + -
-
1 2
1 2 3 4 5 6 7
1 2 3 4 5 6 7
1 2
1 2 3 4 5 6 7
1 2 3 4 5 6 7
Se/se se/se
Se/se Se/se se/se
se/se se/seSe/Seor
Se/se
se/se
Se/se
se/se
Se/se Se/se
Se/se se/se
se/se
= Myotonic Dystrophy
+ = Secretor Phenotype (Se/Se or Se/se)
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.4
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.4
A A
B B
a a
b b
A A
B B
a a
b b
A A
B B
a a
b b
A A a a
B b B b
LOCIFAR APART
A AB B
a ab b
B B b b
B Bb b
B b B b
LOCICLOSE TOGETHER
A A a a
A Aa a
A Aa a
RECOMBINANTS
MEIOSIS I
Linkage: A/a and B/b tend to be inherited together
the A and B loci are linked.
Key Concepts: Linkage and Recombination
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.2
I
II
A
D N
a A A
N N
A
N
A
DN
a A
N N
a A
N
a
D
A
N N
a A
N
A
N
A
N
A
D
A
D
7654321
1 2
Marker= A or aDisease allele = DNormal allele = N
Linkage between Marker A/a and Disease D
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.3
I
II
III
1 2
1 2 3
4.7 kb
3.0 kb
5 64 7 8 9
1 2 3 4 5 6 7 8 9
3.0 kb= NEUROFIBROMATOSIS (NF1)
1.7 kbEcoRI
*PROBE
EcoRI EcoRI
recombinant
Linkage between NF and RFLP marker
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.5
3.5
3.0
2.5
2.0
1.5
1.0
0.5
00.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
=
LOD
0
0.05
3.16
0.15
2.86
0.20
2.57
0.3
1.82
0.4
0.89
0.10
3.08
Z
3.165
0.059
LOD
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.6
DISEASE
FUNCTION
GENE
MAP
DISEASE
FUNCTION
GENE
MAP
FUNCTIONAL CLONING POSITIONAL CLONING
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.15
I
II
III
IV
V
AD AA AA AA AB AA AC AB AB AB AB AA AA AA CD BC AC AC CD AA AA AA AB
Gusella, et al. A polymorphic DNA marker genetically linked to Huntington's disease. Nature 306:234-238, 1983.
The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72:971-983, 1993.
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.26
Positional Cloning
GENETIC MARKERS
FAMILIES
MUTATION IDENTIFICATION
FINE GENETIC MAPPING
MUTATION SEARCH
PhysicalMapping
andCloning
YACs andBACs
TranscriptIdentification
CytogenicAbnormality
Positional CandidateApproach
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.31
Image of Cystic Fibrosis
carrier screening
advertisement removed
Positional Cloning Approach
Image of positional cloning
procedure removed
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E
Types of DNA Sequence Variation• RFLP: Restriction Fragment Length Polymorphism• VNTR: Variable Number of Tandem Repeats
– or minisatellite– ~10-100 bp core unit
• SSR : Simple Sequence Repeat – or STR (simple tandem repeat) – or microsatellite– ~1-5 bp core unit
• SNP: Single Nucleotide Polymorphism– Commonly used to also include rare variants
• Insertions or deletions– INDEL – small (few nucleotides) insertion or deletion
23 pairs of chromosomes made of 3 billion base pairs
30%70%
~20,000 genes~20,000 genesExtragenic DNAExtragenic DNA
Repetitive Repetitive sequencessequences
Control regionsControl regions Spacer DNA Spacer DNA
between genesbetween genes Function mostly Function mostly
unknownunknown
Source Undetermined
Characteristics of the Human Genome Sequence
• 99% of euchromatin is covered, 2.85 Gb• Error rate: <<1:100,000 bp• <350 unclonable gaps• All data is freely accessible without restriction• Humans have fewer genes than expected
– ~20,000 from prev. estimates of 100,000)– ? human genes make more proteins
Levy, et al. Levy, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413:488-494, 2001.
University Of California Santa Cruz
National Center for Biotechnology
Learning Objectives
UNDERSTAND: • The basic anatomy of the human genome [eg. 3 X109 bp (haploid
genome); 1-2% coding sequence (~20,000 genes); types and extent of DNA sequence variation].
• Recombination and how it allows genes to be mapped • Linkage: Use of markers linked to a disease gene for genetic
diagnosis and to identify disease genes (positional cloning)• Distinction between a linked marker and the disease causing
mutation itself• Genetic data for a pedigree, assigning phase, defining haplotypes• Linkage disequilibrium and haplotype blocks• Genome wide association studies (GWAS) to identify gene variants
contributing to complex diseases/traits
A A
B B
a a
b b
A A
B B
a a
b b
A A
B B
a a
b b
A A a a
B b B b
LOCIFAR APART
A AB B
a ab b
B B b b
B Bb b
B b B b
LOCICLOSE TOGETHER
A A a a
A Aa a
A Aa a
RECOMBINANTS
MEIOSIS I
Linkage: A/a and B/b tend to be inherited together
the A and B loci are linked.
Key Concepts: Linkage and Recombination
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.2
DP DQ DRCYP21
C4B
CYP21P
C4A
C2 B C A
CLASS ICLASS II CLASS III
~4000 kb
TNFHFEBA
TELOMERECENTROMERE
The HLA (MHC) Locus
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.12
A2, A24, B7, B35, DR1, DR13
A.
I.
II.
1 2
1 2 3 4 5
A1, A29, B7, B8, DR3, DR4
A2, A29, B7, B35,
DR4, DR13
A24, A29, B7,
DR1, DR4
A1, A24, B7, B8,
DR1, DR3
A2, A29, B7, B35,
DR4, DR13
A2, A29, B7, B35,
DR1, DR4
B.
I.
II.
1 2
1 2 3 4 5
A29, B7, DR4 A1, B8, DR3
A2, B35, DR13 A24, B7, DR1
A29, B7, DR4A2, B35, DR13
A1, B8, DR3 A24, B7, DR1
A29, B7, DR4 A2, B35, DR1
A29, B7, DR4 A24, B7, DR1
A29, B7, DR4 A2, B35, DR13
Assigning Phase
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.13
A2HFE
HFEHFEA3
OTHER
A2
A3
A2
A1
A24
A11
A32
OTHERS
A1
A3
A11
A24
A32
A28
A26
A2
TIME
HEMOCHROMATOSIS CHROMOSOMES NORMAL CHROMOSOMES
10%
23%
14%
8%
5%
5%
39%
X
X
X
70%
30%
Linkage Disequilibrium
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 9.14
These three SNPs could theoretically occur in 8 different haplotypes
…C…A…A…
…C…A…G…
…C…C…A…
…C…C…G…
…T…A…A…
…T…A…G…
…T…C…A…
…T…C…G…
But in practice, only two are observed
…C…A…A…
…C…A…G…
…C…C…A…
…C…C…G…
…T…A…A…
…T…A…G…
…T…C…A…
…T…C…G…
…C…A…A…
…C…A…G…
…C…C…A…
…C…C…G…
…T…A…A…
…T…A…G…
…T…C…A…
…T…C…G…
These three variants are said to be in linkage disequilibrium
A high frequency of a specific gene mutation in a A high frequency of a specific gene mutation in a population founded by a small ancestral grouppopulation founded by a small ancestral group
Founder Effect
Professor Marninalia (wikimedia)
Regents of The University of Michigan
Regents of The University of Michigan
Hb S only occurs on 4 haplotypes…only occurred 4 times in history
Could we use this approach to find human disease genes (identify specific haplotypes present more often in patients than in controls)?
– > 10 million cases in the US> 10 million cases in the US– Leading cause of blindness among the elderlyLeading cause of blindness among the elderly
• Common variant in complement factor H (CFH) geneCommon variant in complement factor H (CFH) gene
– Tyr402HisTyr402His– His allele = 2-4 X increase risk of AMDHis allele = 2-4 X increase risk of AMD– Accounts for 20-50% of AMD riskAccounts for 20-50% of AMD risk
Nature Genetics, 2006, 38:320-323.
• Odds ratio 1.7• Replicated multiple times
Image of journal article on variant of
transcription factor 7 like 2 removed
Images of journal articles about genome-wide
association studies removed
Source Undetermined
How do we distinguish a disease causing mutation from a silent sequence variation?
• Obvious disruption of gene– large deletion or rearrangement– frameshift– nonsense mutation
• Functional analysis of gene product– expression of recombinant protein– transgenic mice
• Most (nearly all) previous “candidate” gene association studies are wrong
• Most common variants have only modest effects on risk (<< 2 fold)
• For type II diabetes, variants identified to date only account for ~5% of overall risk– NOT useful clinically (yet)
• Other diseases may be different– AMD– Thrombosis
• For the future:– ? More predictive, combinatorial analyses– ? Identify new drug targets
GWAS Lessons (continued)
• common alleles with small effects: – ? Clinical diagnostic utility– New biologic pathways- ?significance– New drug targets- ? value
• Should we be looking for large effect, rare variants instead?– Deep resequencing of extremes of the distribution– New technologies (massively parallel sequencing)
Personalized Medicine:Personalized Medicine:Potential Impact of GenomicsPotential Impact of Genomics
• Cytochrome P450– warfarin– Multiple other drugs:
• antidepressants, tamoxifen, PPIs
• VKORC1– warfarin
Sadler, Nature 427:493, 2004.
Image of Warfarin mechanism of
action removed
Pharmacogenomics for warfarin dosingPharmacogenomics for warfarin dosing
• Clinical value unprovenClinical value unproven• Practical limitations:Practical limitations:
– VKORC1/CYP2C9 genotype only accounts for ~30% of variance in VKORC1/CYP2C9 genotype only accounts for ~30% of variance in warfarin requirementwarfarin requirement
Sconce et al., Sconce et al., Blood,Blood, 2005, 106: 2329-33 (Both Images) 2005, 106: 2329-33 (Both Images)
The Future…The Future…
• Will be differentWill be different– New/improved anticoagulantsNew/improved anticoagulants– Cheaper/faster DNA testing/sequencingCheaper/faster DNA testing/sequencing– Complex computational analysis- Complex computational analysis-
combinatorial risk factorscombinatorial risk factors– Much larger data sets– health system wideMuch larger data sets– health system wide– Data to support genotype-specific Data to support genotype-specific
therapy/prophylaxistherapy/prophylaxis
New Sequencing Technologies
Source Undetermined
Slide 6: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.1
Slide 7: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.4
Slide 8: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.2
Slide 9: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.3
Slide 10: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.5
Slide 11: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.6
Slide 12: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.15
Slide 13: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.26
Slide 14: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 9.31
Slide 16: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E
Slide 18: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.22
Slide 19: Adapted from ASCO teaching slides
Slide 20: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 10.3
Slide 21: Source Undetermined
Slide 22: Source Undetermined; Andre Karwath (wikipedia); U.S. Federal Government (wikimedia)
Slide 24: Source Undetermined
Slide 26: National Center for Biotechnology, http://www.ncbi.nlm.nih.gov/
Slide 27: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E
Slide 28: University Of California Santa Cruz, http://genome.ucsc.edu
Slide 29: Levy, et al. Levy, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413:488-494, 2001.
Slide 30: University Of California Santa Cruz, http://genome.ucsc.edu
Slide 31: National Center for Biotechnology, http://www.ncbi.nlm.nih.gov/Omim/mimstats.html
Slide 41: Regents of The University of Michigan
Slide 42: Regents of The University of Michigan
Slide 46: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 10.3
