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Human Molecular Genetics Institute of Medical Genetics Yaoqin Gong 2003 Human Gene Mapping Introduction Common methods Strategies Examples Introduction Gene mapping: determine the gene location on specific chromosomal region Genetic map (linkage map) - a map of genetic loci based on recombination frequencies. Physical map - a map of the physical distance between genetic loci measured in base pairs. Historically, genetic maps have been made first, and subsequently correlated with physical maps. Why map a disease gene ? Can lead to a genetic test provide more info to people at risk First step in cloning the gene Tells you sequence of the protein it encodes, which may give clues to the pathology and etiology of the disease May enable protein therapy (e.g. Factor VIII) or gene therapy Things human geneticists can’t do Establish true-breeding lab strains Perform testcrosses or backcrosses Score lots of progeny from the same mating Human Gene Mapping Gene mapping before 1967 was limited to the X chromosome First autosomal gene mapped was Duffy blood group locus Heterochromatic region on chromosome 1 Human Gene Mapping I 2 1 II a/b b/b a/b a/b b/b a/b a/b b/b a/b a/b b/b III IV a/a b/b 5 a/a a/b a/b a/b a/a a/b Solid symbols indicate the presence of the heterochomatin Duffy blood group locus is on chromosome 1 b/b Methods used in human gene mapping In Situ Hybridization Somatic Cell Hybrids Chromosomal abnormalities Dosage effect analysis Linkage analysis Fluorescent in situ hybridization If a gene is cloned, it can be directly mapped to a chromosomal locus Requirements : Karyotype & Labeled Gene probe fluorescent spots appear in the same location on homologous chromosomes Fluoresence In Situ Hybridization FISH Somatic cell hybrid mapping Based on ability to fuse somatic cells of different species, e.g., human and rodent Somatic cell hybrids typically retain only small number of human chromosomes Isolate stable partial hybrids using selectable markers Human-rodent cell hybrids Basic discovery: cells of different species can be fused if brought close together (e.g. with Sendai virus). Nuclear fusion may follow. Over many cell cycles, rodent cells will lose some human chromosomes at random before becoming stable. Human-rodent cell hybrids Rodent cells are chosen that are deficient for enzymes of interest. Those that carry a human chromosome that bears the human homolog of that gene will be complemented. Identify human chromosome by process of elimination. Somatic cell hybrid mapping Somatic Hybrid Mapping Probe1 Probe2 Probe3 1 - Chromosome 2 3 4 + + + + - 5 + Probe1 -- maps to chromosome 2 Probe2 -- maps to chromosomes 3 Probe3 -- maps to chromosome 2, 3, and 5, --possible paralogs, pseudogene, or low-copy repeat Somatic Hybrid Mapping EXP + + WIL1 WIL6 WIL7 wil14 SIR3 … % discord 1 + + 2 + + + 3 + + + 4 + + 5 + + + 6 + + + 7 + + + 8 + + + + - 9 10 11 12 13 - - - - - + + - - + + - + - + - + + + + + + 14 15 16 + - + - + - + + - - + 17 18 19 20 21 22 + - - - + + - + + + + + - - + + - - - - + + + + + + X + 0 32 17 24 31 21 21 31 21 24 30 21 21 28 14 24 21 28 17 34 41 21 27 + + + Radiation Hybrid Mapping Fragment human chromosomes with X-rays. Allow uptake of chromosome fragments into rodent cells. Most will be incorporated into rodent genome, but are still recognizable by their banding. Find complemented cells and correlate with human chromosome fragments that are present. The more closely linked two loci are, the more likely they will end up in the same hybrid cell. Radiation hybrid mapping is a method for high-resolution mapping. Radiation hybrid mapping Chromosome aberration Mapping Chromosome aberrations provide shortcuts to mapping Deletions are particularly useful Deletion mapping the Y chromosome Identify males with cytologically aberrant Y chromosomes Test each male for presence or absence of sequence known to map to the Y chromosome Order the results into a conservative map (the deletion map) Deletion mapping the Y chromosome DMD: Another monument to deletion cloning Duchenne’s muscular dystrophy known to be X-linked Regional mapping accomplished by two chromosome aberrations: deletion of DMD and several other genes Translocation interrupting the DMD gene DMD: Another monument to deletion cloning DMD: Another monument to deletion cloning Patient B.B. Rare cytological detectable deletion Width of Xp21 significantly reduced Multiple genetic disorders DMD Chronic granulamatous disease (CGC) Retinitis pigmentosa - Deletion 10 mb in length DMD: Another monument to deletion cloning Normal X DMD Patient B.B. CGD RB Dosage Effect Analysis SOD 1.5 : 1 Dosage Effect Analysis Autosomal probe X-chromosomal probe Linkage Analysis Principle Human genetic maps Mapping the disease genes Linkage and recombination Two classes of progeny Parental types – progeny that result from gametes with the same combination of alleles as the parental gametes Recombinant (nonparental) types - arise from crossover between linked genes on homologous chromosomes (OR from independent segregation of chromosomes in a pattern that separates the two parental chromosomes) Recombination Frequency (RF) RF = number of recombinants total number of progeny x 100% RF 50% RF is significantly < 50% no linkage linkage RF is significantly > 50% impossible Genetic Map Unit One genetic map unit is the distance between genes that gives one recombinant out of 100 meioses. A recombination frequency of 0.01 (1%) = 1 map unit (m.u.) = 1 centiMorgan (cM) In humans, 1 cM 1 Mb (megabase). Because many chromosomes are > 50 Mb in size, two distant genes on the same chromosome can behave as if unlinked. (The maximum possible RF is 50%.) Three-point testcross Definition: Crossing a triple heterozygote to a triply recessive tester (I.e. A/a • B/b • C/c x a/a • b/b • c/c) Purpose: To determine linkage and (if applicable) order of genes Note: In writing out genotypes, can omit alleles from tester for this analysis because they will all be “abc” (i.e. aBC = aaBbCc) P aaBBCC F1 F2: aBC Abc abC ABc abc ABC aBc AbC Total x AaBbCc x 580 592 45 40 89 94 3 5 1448 AAbbcc aabbcc Parental Classes (highest 2 classes) Recombinant Classes Double Recombinants (not always present) Determining map distances (A - C) aBC Abc abC ABc abc ABC aBc AbC Total 580 592 45 40 89 94 3 5 1448 Analyze each set of 2 loci at a time: AC Ac aC ac 94 + 5 = 99 592 + 40 = 632 580 + 45 = 625 89 + 3 = 92 RF = 99 + 92 = 13.2% 1448 (significantly < 50% linked and distance = 13.2 cM) Determining Map Distances (B - C) aBC Abc abC ABc abc ABC aBc AbC Total 580 592 45 40 89 94 3 5 1448 Analyze each set of 2 loci at a time: BC Bc bC bc 580 + 94 = 674 40 + 3 = 43 45 + 5 = 50 592 + 85 = 677 RF = 43 + 50 = 6.4% 1448 (significantly < 50% linked and distance = 6.4 cM) Determining Map Distances (A - B) aBC Abc abC ABc abc ABC aBc AbC Total 580 592 45 40 89 94 3 5 1448 Analyze each set of 2 loci at a time: AB Ab aB ab 40 + 94 = 134 592 + 5 = 597 580 + 3 = 583 45 + 89 = 134 RF = 134 + 134 = 18.5% 1448 (significantly < 50% linked and distance = 18.5 cM) Ordering Loci AC = 13.2 cM BC = 6.4 cM AB = 18.5 cM A C 13.2 18.5 B 6.4 Problem: 13.2 + 6.4 = 19.6 18.5 (because double crossover class was counted as non-recombinant when actually there were two crossovers) a a C C B B A A c c b b a a C c B B A A C c b b Double Cross-Over Recombinant Recombinant Human Linkage Maps Polymorphic Markers CEPH families Linkage Maps LOD Score Polymorphic Markers Types: RFLP, STR, SNP Known location Highly polymorphic: Heterozygosity> 75% CEPH Families Human Linkage Map LOD Score It is the logarithm of the odds ratio It is a statistical measure of likelihood that two genes are linked at a particular distance. LOD Score Test of linkage (H0 : =1/2 ; H1: <1/2 ) Z= LOD score = log10 {(L =RF)/(L =1/2)} Z > 3: accept linkage Z < -2: reject linkage 2 < Z < 3 : uncertain (collect more data) d 1 D d 1 2 d 1 d 1 D 2 d 1 D 2 d 1 d 1 d 1 D 2 d 1 d 1 d 1 D 2 d 1 d 1 d 1 d 2 d 1 d 1 1 1 D 1 d 1 D 2 d 1 d 1 Here: 2 recombinants out of 10 meiosis: RF = Z () = N log (2) + R log + (N-R) log (1- ) Z = 10 log (2) + 2 log (0.2) + 8 log (0.8) = 0.837 Recall that if = 0 and no recombinants are observed: Z = 10 log (2) = 3.01 Thus: the denser the map the higher the power to detect linkage. Mapping disease genes by Linkage Analysis Find a large, multigenerational, affected family Test linkage of the disease to a mapped polymorphism Determine the odds of obtaining the observed pedigree assuming a given amount of linkage, and compare to the odds assuming no linkage Mapping disease genes by Linkage Analysis Disease locus Dd dd Dd dd dd Dd Dd Dd dd Marker 1 12 34 13 23 24 14 13 14 23 Marker 2 12 34 13 14 23 24 13 14 23 Marker 1 : linked to disease locus Marker 2 : unlinked to disease locus Mapping BDB by Linkage Analysis Disease locus Dd dd Dd dd Dd Dd dd dd Dd Dd Dd dd Dd Dd dd Dd dd Dd D9S938 21 34 23 33 23 22 14 13 24 22 22 42 23 21 31 21 14 24 D9S123 13 22 12 24 13 14 32 12 12 13 14 23 12 13 23 13 32 12 D9S938 : tightly linked to disease locus D9S123 : observed 1 recombinant out of 16, RF=1/16=6.25% Summary for common methods Method Known genes Known protein Disease genes In situ hybridization Yes No No Somatic cell hybrid Yes Yes No Chromosome aberration No No Yes Dosage effect Yes Yes No Linkage analysis Yes Yes Yes Mapping strategies Known genes Disease genes Mapping a Known Gene In Situ Hybridization NO YES Somatic cell hybrid mapping YES Specific chromosome Linkage Fine mapping NO Linkage analysis Mapping COL9A3 by somatic cell hybrid analysis Design primers which specifically amplify human COL9A3, not mouse col9a3 PCR test H M H+M control Mapping COL9A3 by somatic cell hybrid analysis Detect different cell lines Cell lines Human chromosomes retained PCR result A 1, 7, 15, 18, 21, X - B 3, 5, 15, 20, 21, 22 + C 3, 8, 18, 21, 22, X - D 2, 7, 15, 18, 21, 22 - E 2, 6, 14, 17, 19, 20 + COL9A3 is on chromosome 20 Mapping COL9A3 by linkage analysis Screening for polymorphism within COL9A3 gene Select polymorphic markers at 10cM on chromosome 20 Genotyping CEPH families Linkage analysis Mapping COL9A3 by linkage analysis Intragenic polymorphism Bam HI : GGATCC GGATCC CCTAGG Allele 1 GAATCC CTTAGG Allele 2 with or without Bam HI site RFLP Mapping COL9A3 by linkage analysis Detect intragenic polymorphism by PCR-RFLP 100 Genomic DNA GGATCC CCTAGG PCR 150 250bp product Bam HI 250 150 100 22 12 11 Gel electrophoresis Mapping COL9A3 by linkage analysis Genotyping chromosome 20 markers 1 2 3 4 5 6 Genomic DNA PCR 8 7 6标 5记 4 3 2 1 PCR products Denaturing PAGE 13 24 55 36 18 57 Mapping COL9A3 by linkage analysis Analyze CEPH families 12 Chooce the families with heterozygote parents Mapping COL9A3 by linkage analysis COL9A3 12 22 12 12 22 12 22 12 22 22 D20S111 23 14 21 31 21 24 24 21 21 24 Mapping COL9A3 by linkage analysis COL9A3 12 22 12 12 22 12 22 12 22 22 D20S115 13 24 12 14 32 14 34 12 12 34 D20S123 13 24 12 14 32 14 34 12 32 34 D20S143 13 24 12 14 32 14 34 12 32 34 D20S145 13 24 12 34 32 14 34 12 32 34 Mapping COL9A3 by linkage analysis D20S115 13 24 12 14 32 14 34 12 12 34 COL9A3 12 22 12 12 22 12 22 12 22 22 D20S123 13 24 12 14 32 14 34 12 32 34 D20S143 13 24 12 14 32 14 34 12 32 34 D20S145 13 24 12 34 32 14 34 12 32 34 Mapping a disease gene Collect families Chromosomal analysis NO YES Candidate chromosome linkage Fine mapping Candidate genes NO Genome scanning Candidate region YES Mutation detection EPPK I 1 2 II 1 2 3 4 5 6 7 9 8 III 1 2 3 4 5 6 7 8 Mapping EPPK gene Chromosomal analysis NO Candidate genes EPPK -Candidate gene analysis II 1 2 3 4 5 6 III 1 D17S579 5/5 D12S90 3/4 2 4/3 4/5 4/3 2/2 4/2 4/1 1/2 5/3 5/4 5/3 1/2 3/2 5/2 5/2 Linked to D17S579 which is tightly linked to KRT9 gene EPPK -Mutation Detection C544T EPPK -Mutation Detection BDB BDB D9S1820 65 36 56 46 64 34 63 24 43 46 63 56 64 44 D9S1795 52 22 22 23 52 22 52 22 22 25 52 52 25 25 D9S1842 42 33 23 32 43 33 43 33 33 34 43 43 35 35 D9S1781 54 55 45 14 51 51 55 55 55 55 55 55 51 51 D9S1815 53 34 34 52 55 35 53 36 63 65 53 54 41 51 D9S1832 72 58 28 26 72 72 75 21 15 17 75 78 86 56