Genetics: From genes to genomes - Chapter 11: What genes are and what they do

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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PowerPoint to accompanyGenetics: From Genes to GenomesFourth EditionLeland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. SilverPrepared by Mary A. BedellUniversity of Georgia*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display*PART IIICopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11Genome-Wide Variation and Trait Analysis11.1 Genetic Variation Among Individual Genomes11.2 Single Nucleotide Polymorphisms (SNPs) and Small-Scale-Length Variations11.3 Deletions or Duplications of a DNA Region11.4 Positional Cloning: From DNA Markers to Disease-Causing Genes11.5 Complex Traits11.6 Genome-Wide Association StudiesWhat Genes Are and What They DoCHAPTER OUTLINECHAPTERExtensive allelic variation distinguishes individuals within a species1950s – first demonstrations that presumed "wild-type" individuals of the same species produced variant forms of proteinsGel electrophoresis of proteinsVariety of species (Drosophila to human)Recent sequencing of individual genomes revealed that humans have staggering degree of sequence variantsPolymorphic locus – locus with two or more alleles that are each present in >1% of a species' membersGenetic variants – alleles of polymorphic lociCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*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 variationNew definition of a locus – any location in the genome that is defined by chromosomal coordinatesCan have multiple genes or no genesCan be a single base pair or millions of base pairsNew definition of an allele – any variation in the DNA sequence, even if it doesn't have an effect on phenotypeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Pairwise comparison of three personal genomesSingle nucleotide polymorphisms in the genomes of three individuals [(Craig Venter, James Watson, and a Chinese man (anonymous, YH)]Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.2Differences across the entire genomeAmino acid-changing substitutionsCategories of genetic variantsFive categories based on size, frequency within individual genomes, and method used for detectionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Table 11.1Single nucleotide polymorphisms (SNPs)SNPs account for the vast majority of total sequence variation between humans genomes~ 18 million human SNPs have been identifiedArise from rare mistakes in replicationPer-base mutation rate is 106 lociOnline public SNP database available through NCBI Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.7Genetic variation can be caused by subtraction or addition of short sequencesDeletion-insertion polymorphisms (DIPs)Short insertions or deletions of a single or a few base pairsAlso called InDelsDetected like SNPs (on microarrays or with allele-specific hybridization) or by PCR and gel electrophoresisSimple sequence repeats (SSRs)One-, two-, or three-base sequences repeated 15-100 times in tandemArise because of "stutter" of DNA polymerase during replication of the repeat sequences (Fig. 11.9)Detected with PCR and gel electrophoresis (Fig. 11.10)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Size distribution of DIPs between the human RefSeq genome and the Venter genome292,102 DIPs ranging in length from one base pair to 571 bpSmaller DIPs are much more frequent that larger DIPsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.8aDistribution of SNPs, DIPs, and SSR variants in the 400 kb that contains the CFTR regionAverage frequency over the entire human genome:SNPs, one every kbDIPs, one every 10 kbCA-repeat SSR, one every 30 kb Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.8bSSRs are highly polymorphic because of their potential for faulty replicationAlteration of a 15 (CA) repeat allele to a 17 (CA) repeat alleleCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.9(c) continued(d)(e)(a)(b)(c)Detection of SSR polymorphisms by PCR and gel electrophoresis(a) Determine sequences flanking microsatellitesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.10(b) Amplify alleles by PCR(c) Analyze PCR products by gel electrophoresisExample of a population with three SSR alleles detected by PCR and gel electrophoresisCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.10dMutations at the Huntington disease (HD) locus are caused by expansion of an SSR in a coding regionAutosomal dominant disorderNormal allele has 1%Can affect large blocks (up to 1 Mb) of DNA without having any phenotypic consequencesDetected on arrays (increase or decrease in hybridization)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Minisatellite analysis provides a broad comparison of whole genomesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.12(a) Digest DNA with restriction enzyme that does not cut inside minisatellite(b) After electrophoresis, perform Southern blotting and hybridize with probe containing minisatellite sequenceHow many minisatellite loci have to be examined in order to prove identity?DNA fingerprinting developed by Alec Jeffreys in 1985Probability of two individuals with identical genotypes at loci with two equally prevalent alleles One locus, probability = 37.5%10 unlinked loci, probability = 37.510 = 0.005% (1 in 20,000)24 unlinked loci, probability = 37.524 = 1 in 17 billionTotal human population = 8 billionTherefore, if 24 minisatellite loci analyzed, there is virtually no chance of two different individuals (except identical twins) having identical genotypesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*DNA fingerprint analysis confirmed that Dolly was cloned from an adult udder cellUsing DNA fingerprinting in forensics:Men accused of rapeSince 1993, > 150 men imprisoned for rape have been released from jail because of DNA fingerprint analysisPlant DNA as murder evidenceIdentification of skeletal remainsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.13Chromosomal locations of CNPs or CNVs identified in multiple individualsResults of DNA microarray analysis of 88 samplesArray has several hundred thousand non-polymorphic oligonucleotide probes (NPOs) spaced evenly across entire genomeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.14aChromosomal locations of CNPs or CNVs identified in multiple individuals (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.14bAs of June 2009, > 6000 human CNVs have been identified> 99% of all CNVs are inherited and are not derived from new mutation Some CNVs are associated with a phenotypeCNVs in the olfactory receptor (OR) family (Fig. 11.15a)Humans have ~ 1000 OR paralogsLarge differences in numbers of OR paralogs in different peopleCNVs and mental disease (Fig. 11.15b)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 retardationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Positional cloning: From DNA markers to disease-causing genesPositional cloning – using knowledge of a specific chromosomal region to identify genes responsible for disease phenotypesLinkage analysis with DNA markersHuntington disease (HD) locus was the first human disease gene to be successfully mapped by positional cloningIn some cases, a causative gene can be identified without mappingExample: hemophilia A (Fig. 11.16) - knowledge of the biochemical functions involved in blood clotting led to identification of factor VIII as the defective gene productCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*How geneticists identified and cloned the hemophilia A geneBased on knowledge of blood clotting process (see Fig 11.16b), factor VIII was tested as a candidate for causing hemophilia ACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.16dReverse translation of purified factor VIII led to cloning of the factor VIII geneSequencing of the factor VIII gene from affected and unaffected people revealed the causative mutation Positional cloning: From phenotype to chromosomal location to guilty geneFirst goal is to identify DNA markers that shows linkage to the disease locusGenotype all members of disease-carrying families with a series of DNA markersLocations of thousands of DNA markers in human genome are already knownBasically this is a series of two-point crosses between the trait and each marker Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.17Positional cloning: From phenotype to chromosomal location to guilty gene (cont)Identify candidate genes in the smallest genetically-defined area that must contain the disease locusCompare structure and expression of candidate genes in many diseased vs. nondiseased individualsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.17Huntington disease (HD) was the first human disease gene to be mapped by positional cloningDetection of linkage between the DNA marker G8 and the HD locusSegregation of the G8 DNA marker (four alleles - A, B, C, and D) in a large Venezuelan pedigree affected with HDCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.18Complexities that alter traditional Mendelian ratiosMost human traits don't have single-gene inheritance patternsLinkage mapping and positional cloning can be done with complex traits, but it is more difficult and the mapping strategy is different from single-gene traitsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Table 11.2Inheritance of breast cancerAutosomal 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 penetrantOnly 66% of women with a mutant BRCA1 allele will develop cancerWhen mapping traits with incomplete penetrance, DNA analysis should be done only with affected individualsWith 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 traitsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Incomplete penetrance and genetic heterogeneity in the inheritance of breast cancerCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.19Haplotyping allows the world population to be seen as one giant pedigreeHaplotypes –unique combinations of common SNP alleles over extended regions of the genomeInheritance of blocks of DNA that remain intact over many generationsExtended versions of alleles that cover regions containing multiple genesA small number of "tag SNPs" can be used to obtain a nearly complete whole-genome profile of individualsDNA microarray with 500,000 tag SNPs will cover the entire human genomeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Genome-wide association studies (GWAS)A GWAS doesn't depend on traditional pedigree analysis but is computationally intensiveThousands of individuals make up a study groupEach individual is observed or tested for expression of one or more traits of interestDNA microarrays are used to obtain whole-genome profiles for each member of the study populationGenotypes 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 traitReveals genomic regions that harbor alleles associated with the traitCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*GWAS of body mass index (BMI)P values for all SNPs tested for association with BMI across all chromosomesEach dot represents a single SNP testLowest P values are shown as the highest dotsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.20aGWAS of BMI (cont)Fine-scale mapping of two BMI-associated regionsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*Fig. 11.20bThe open source human genomeAll published research with SNPs uses standardized nomenclatureAll newly-obtained information is deposited in freely- available, public databases maintained by the NIH29 interlinked databases at NCBIExamplesdbSNP – comprehensive catalog of all human SNPsdbGap – database of results obtained in various GWASsOMIM – online compendium of annotated records of each heritable trait and gene in humansNumerous software tools to query and retrieve genetic dataCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 11*

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