1. Genetics: From Genes to Genomes
PowerPoint to accompany
Genetics: From Genes to Genomes
Fourth Edition
Leland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. Silver
Prepared by Mary A. Bedell
University of Georgia
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2. 11 Genome-Wide Variation and Trait Analysis CHAPTER OUTLINE PART III
What Genes Are and What They Do
CHAPTER
Genome-Wide Variation and Trait Analysis
CHAPTER OUTLINE
11.1 Genetic Variation Among Individual Genomes
11.2 Single Nucleotide Polymorphisms (SNPs) and Small-Scale-Length Variations
11.3 Deletions or Duplications of a DNA Region
11.4 Positional Cloning: From DNA Markers to Disease-Causing Genes
11.5 Complex Traits
11.6 Genome-Wide Association Studies
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3. Extensive allelic variation distinguishes individuals within a species
1950s – first demonstrations that presumed "wild-type" individuals of the same species produced variant forms of proteins
Gel electrophoresis of proteins
Variety of species (Drosophila to human)
Recent sequencing of individual genomes revealed that humans have staggering degree of sequence variants
Polymorphic locus – locus with two or more alleles that are each present in >1% of a species' members
Genetic variants – alleles of polymorphic loci
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4. Extensive allelic variation distinguishes individuals within a species (cont)
Original views of a locus and alleles have changed with availability of whole genome sequences and evidence for extensive genetic variation
New definition of a locus – any location in the genome that is defined by chromosomal coordinates
Can have multiple genes or no genes
Can be a single base pair or millions of base pairs
New definition of an allele – any variation in the DNA sequence, even if it doesn't have an effect on phenotype
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5. Pairwise comparison of three personal genomes
Single nucleotide polymorphisms in the genomes of three individuals [(Craig Venter, James Watson, and a Chinese man (anonymous, YH)]
Differences across the entire genome
Amino acid-changing substitutions
Fig. 11.2
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6. Categories of genetic variants
Five categories based on size, frequency within individual genomes, and method used for detection
Table 11.1
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7. Single nucleotide polymorphisms (SNPs)
SNPs account for the vast majority of total sequence variation between humans genomes
~ 18 million human SNPs have been identified
Arise from rare mistakes in replication
Per-base mutation rate is < 1 in 30 million per generation
Each SNP can be traced back to a genome change that occurred in a single ancestral genome
Notations for SNP genotypes
Fig. 11.3a,b
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8. The origin of human SNPs is determined by comparison to other species
Comparison of human and chimpanzee genomes reveals the SNPs that occurred since divergence of these species
Human-specific SNP alleles that are shared between individuals indicate recent common ancestry
In the example below:
The first single base change between humans and chimps is not polymorphic in humans
The second single base change is polymorphic between humans
Fig. 11.3c
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9. SNPs in a 400 kb region of human chromosome 7 that contains the CFTR gene
Three tracks of SNPs compared against the RefSeq genome (NCBI Sequence Viewer and UCSC Genome Browser)
SNPs in two individual genomes (Watson and Venter)
SNPs found in sequences from all European subjects (CEU)
Block patterns of SNP similarity and dissimilarity provide the foundation for genome-wide association studies (GWAS)
Fig. 11.3d
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10. SNPs can be genotyped with several different molecular methods
By chance, some SNPs eliminate or create a restriction site
Southern blot analysis of restriction site-altering SNPs (Fig. 11.4)
PCR analysis of restriction site-altering SNPs (Fig. 11.5)
Most SNPs don't alter restriction sites
Allele-specific oligonucleotide hybridization (Fig. 11.6) can detect any SNP
DNA microarrays (described in Chapters 1 and 10) can be used for simultaneous detection of millions of SNPs
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11. Restriction site-altering SNPs detected by Southern blots
In this example, the SNP affects an EcoRI restriction site
Allele 1 has an EcoRI site that is not present in allele 2
After digestion with EcoRI, the two SNP alleles produce different-sized DNA fragments
Fig. 11.4
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12. PCR detection of the sickle cell-causing SNP
The sickle-cell mutation eliminates an MstII restriction site
PCR of the region containing the SNPA produces a 500 bp fragment from both alleles (normal and sickle-cell)
Digestion of the PCR product with MstII produces two smaller fragments from the normal allele, but doesn’t affect the sickle-cell allele
Normal allele (A)
Sickle-cell allele (S)
Fig. 11.5
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13. Short hybridization probes can distinguish single-base mismatches
Hybridization of short (< 40 bases) oligonucleotides to sample (target) DNAs (allele-specific hybridization)
If there is no mismatch between probe and target, hybrid will be stable at high temperature
If there is a mismatch between probe and target, hybrid will not be stable at high temperature
Fig. 11.6a
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14. Korean SNP loci and alleles in the first four exons of the CFTR gene
Standard microarrays, which cost only a few hundred dollars per sample, can detect SNPs alleles at >106 loci Online public SNP database available through NCBI
Fig. 11.7
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15. Genetic variation can be caused by subtraction or addition of short sequences
Deletion-insertion polymorphisms (DIPs)
Short insertions or deletions of a single or a few base pairs
Also called InDels
Detected like SNPs (on microarrays or with allele-specific hybridization) or by PCR and gel electrophoresis
Simple sequence repeats (SSRs)
One-, two-, or three-base sequences repeated times in tandem
Arise because of "stutter" of DNA polymerase during replication of the repeat sequences (Fig. 11.9)
Detected with PCR and gel electrophoresis (Fig )
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16. Size distribution of DIPs between the human RefSeq genome and the Venter genome
292,102 DIPs ranging in length from one base pair to 571 bp Smaller DIPs are much more frequent that larger DIPs
Fig. 11.8a
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17. Distribution of SNPs, DIPs, and SSR variants in the 400 kb that contains the CFTR region
Average frequency over the entire human genome:
SNPs, one every kb
DIPs, one every 10 kb
CA-repeat SSR, one every 30 kb
Fig. 11.8b
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18. SSRs are highly polymorphic because of their potential for faulty replication
Alteration of a 15 (CA) repeat allele to a 17 (CA) repeat allele
(a)
(c) continued
(b)
(d)
(e)
(c)
Fig. 11.9
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19. Detection of SSR polymorphisms by PCR and gel electrophoresis
(a) Determine sequences flanking microsatellites
(c) Analyze PCR products by gel electrophoresis
(b) Amplify alleles by PCR
Fig
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20. Example of a population with three SSR alleles detected by PCR and gel electrophoresis
Fig d
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21. Mutations at the Huntington disease (HD) locus are caused by expansion of an SSR in a coding region
Autosomal dominant disorder Normal allele has < 34 CAG repeats Disease-causing alleles have 42 or more CAG repeats
Triplet repeat expansions in different genes also cause some other neurological disorders
Fig
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22. Deletions or duplications of a DNA region
Minisatellites – repeat unit sizes are 500 bp to 20 kb
Repeat sequences occur at multiple genomic loci
Detected using restriction digests and hybridization of Southern blots with cross-hybridizing minisatellite probe
Ideal for DNA fingerprinting
Copy number variants (CNVs) – large blocks of duplication or deletion with population frequency of < 1%
Copy number polymorphisms (CNPs) if frequency is > 1%
Can affect large blocks (up to 1 Mb) of DNA without having any phenotypic consequences
Detected on arrays (increase or decrease in hybridization)
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23. Minisatellite analysis provides a broad comparison of whole genomes
(a) Digest DNA with restriction enzyme that does not cut inside minisatellite
(b) After electrophoresis, perform Southern blotting and hybridize with probe containing minisatellite sequence
Fig
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24. How many minisatellite loci have to be examined in order to prove identity?
DNA fingerprinting developed by Alec Jeffreys in 1985
Probability of two individuals with identical genotypes at loci with two equally prevalent alleles
One locus, probability = 37.5%
10 unlinked loci, probability = = 0.005% (1 in 20,000)
24 unlinked loci, probability = = 1 in 17 billion
Total human population = 8 billion
Therefore, if 24 minisatellite loci analyzed, there is virtually no chance of two different individuals (except identical twins) having identical genotypes
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25. DNA fingerprint analysis confirmed that Dolly was cloned from an adult udder cell
Using DNA fingerprinting in forensics:
Men accused of rape
Since 1993, > 150 men imprisoned for rape have been released from jail because of DNA fingerprint analysis
Plant DNA as murder evidence
Identification of skeletal remains
Fig
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26. Chromosomal locations of CNPs or CNVs identified in multiple individuals
Results of DNA microarray analysis of 88 samples Array has several hundred thousand non-polymorphic oligonucleotide probes (NPOs) spaced evenly across entire genome
Fig a
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27. Chromosomal locations of CNPs or CNVs identified in multiple individuals (cont)
As of June 2009, > 6000 human CNVs have been identified
> 99% of all CNVs are inherited and are not derived from new mutation
Fig b
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28. Some CNVs are associated with a phenotype
CNVs in the olfactory receptor (OR) family (Fig a)
Humans have ~ 1000 OR paralogs
Large differences in numbers of OR paralogs in different people
CNVs and mental disease (Fig b)
Very long (> 1 Mb) deletions or duplication anywhere in the genome are associated with 30% increased risk of psychiatric disorder
Deletions in some specific genomic regions are directly associated with autism, schizophrenia, or mental retardation
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29. Positional cloning: From DNA markers to disease-causing genes
Positional cloning – using knowledge of a specific chromosomal region to identify genes responsible for disease phenotypes
Linkage analysis with DNA markers
Huntington disease (HD) locus was the first human disease gene to be successfully mapped by positional cloning
In some cases, a causative gene can be identified without mapping
Example: hemophilia A (Fig ) - knowledge of the biochemical functions involved in blood clotting led to identification of factor VIII as the defective gene product
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30. How geneticists identified and cloned the hemophilia A gene
Based on knowledge of blood clotting process (see Fig b), factor VIII was tested as a candidate for causing hemophilia A
Reverse translation of purified factor VIII led to cloning of the factor VIII gene
Sequencing of the factor VIII gene from affected and unaffected people revealed the causative mutation
Fig d
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31. Positional cloning: From phenotype to chromosomal location to guilty gene
First goal is to identify DNA markers that shows linkage to the disease locus
Genotype all members of disease-carrying families with a series of DNA markers
Locations of thousands of DNA markers in human genome are already known
Basically this is a series of two-point crosses between the trait and each marker
Fig
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32. Positional cloning: From phenotype to chromosomal location to guilty gene (cont)
Identify candidate genes in the smallest genetically- defined area that must contain the disease locus Compare structure and expression of candidate genes in many diseased vs. nondiseased individuals
Fig
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33. Huntington disease (HD) was the first human disease gene to be mapped by positional cloning
Detection of linkage between the DNA marker G8 and the HD locus Segregation of the G8 DNA marker (four alleles - A, B, C, and D) in a large Venezuelan pedigree affected with HD
Fig
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34. Complexities that alter traditional Mendelian ratios
Most human traits don't have single-gene inheritance patterns
Linkage mapping and positional cloning can be done with complex traits, but it is more difficult and the mapping strategy is different from single-gene traits
Table 11.2
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35. Inheritance of breast cancer
Autosomal dominant inheritance of two unlinked disease loci that predispose to breast cancer (see Figure 11.19)
BRCA1 (chromosome 17) and BRCA2 (chromosome 13)
BRCA1 and BRCA2 mutations are incompletely penetrant
Only 66% of women with a mutant BRCA1 allele will develop cancer
When mapping traits with incomplete penetrance, DNA analysis should be done only with affected individuals
With completely penetrant traits, both affected and non- affected individuals have informative genotypes
More families are needed for linkage analysis of incompletely penetrant traits than with completely penetrant traits
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36. Incomplete penetrance and genetic heterogeneity in the inheritance of breast cancer
Fig
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37. Haplotyping allows the world population to be seen as one giant pedigree
Haplotypes –unique combinations of common SNP alleles over extended regions of the genome
Inheritance of blocks of DNA that remain intact over many generations
Extended versions of alleles that cover regions containing multiple genes
A small number of "tag SNPs" can be used to obtain a nearly complete whole-genome profile of individuals
DNA microarray with 500,000 tag SNPs will cover the entire human genome
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38. Genome-wide association studies (GWAS)
A GWAS doesn't depend on traditional pedigree analysis but is computationally intensive
Thousands of individuals make up a study group
Each individual is observed or tested for expression of one or more traits of interest
DNA microarrays are used to obtain whole-genome profiles for each member of the study population
Genotypes at each tag SNP are tested for association with each trait
Only a small number of tag SNPs will show a significant association with a trait
Reveals genomic regions that harbor alleles associated with the trait
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39. GWAS of body mass index (BMI)
P values for all SNPs tested for association with BMI across all chromosomes
Each dot represents a single SNP test
Lowest P values are shown as the highest dots
Fig a
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40. GWAS of BMI (cont) Fine-scale mapping of two BMI-associated regions
Fig b
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41. The open source human genome
All published research with SNPs uses standardized nomenclature
All newly-obtained information is deposited in freely- available, public databases maintained by the NIH
29 interlinked databases at NCBI
Examples
dbSNP – comprehensive catalog of all human SNPs
dbGap – database of results obtained in various GWASs
OMIM – online compendium of annotated records of each heritable trait and gene in humans
Numerous software tools to query and retrieve genetic data
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