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Linkage mapping in dominant and recessive disorders including autozygosity mapping Describe the principles underlying this approach, methods employed and potential pitfalls. Ana Terron-Kwiatkowski MRCPath part I course London 2010. Linkage mapping in AD and AR disorders.
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Linkage mapping in dominant and recessive disorders including autozygosity mapping Describe the principles underlying this approach, methods employed and potential pitfalls Ana Terron-Kwiatkowski MRCPath part I course London 2010
Linkage mapping in AD and AR disorders Principles of linkage mapping Meiotic recombination Genetic markers Identification of genes by linkage mapping Linkage mapping in AD and AR disorders –parametric methods - - Two-point mapping - Multi-point mapping Autozygosity mapping in AR disorders Limitations, potential errors and pitfalls of linkage mapping Linkage mapping in common disorders - non-parametric approaches- - Affected sib-pair mapping - Homozygous haplotype mapping
Linkage mapping in AD and AR disorders: principles Mapping disease locus to a chromosomal region Based on co-inheritance of a genetic trait with a particular allele of a polymorphic DNA marker of known chromosomal location
Linkage mapping: meiotic recombination Meiotic crossover: During prophase of meiosis I pairs of homologous chromosomes synapse and exchange segments = recombination
Linkage mapping: meiotic recombination Probability of two loci on different chromosomes are inherited together is 50% (recombination fraction 0.5) Probability of two loci on same chromosome (syntenic) are inherited together depends on their genetic distance * Recombination will rarely separate two loci that lie close together * Set of alleles on the same small chromosomal segment - haplotype - tend to be transmitted as a block through a pedigree * Two loci that show 1% recombination are defined as being 1cM apart * More than one crossover may occur if loci are located far apart * Max recombination fractionis 0.5
Linkage mapping: genetic markers Marker: any polymorphic Mendelian character that can be used to follow a chromosomal segment through a pedigree Linkage analysis requires informative meiosis: possible to identify whether the gamete is recombinant or not Meiosis not informative with given marker: - parent homozygous for marker - 1/2 cases where both parents have same heterozygous genotype Heterozygosity of marker: - chance that a randomly selected person will be heterozygous Polymorphic Information Content (PIC) - heterozygous cases can be uninfomative
Identification of genes by linkage mapping GW linkage analysis in one or more families in which the disease is segregating: LOD score regions Fine linkage mapping: narrow down chromosomal region More families analyzed: candidate region Candidate genes within region Potential function Expression pattern Identify mutations in affected individuals
Linkage mapping in AD and AR disorders Straightforward linkage analysis is parametric = a model is proposed to explain the inheritance pattern observed in a pedigree and then tested Requirements: - Large families: No. meiosis The resolution of mapping depends on the No meiosis: the more meiosis analyzed, the greater the chance of a recombination event narrowing down the linked region - Clear/ definite diagnosis - Pattern of inheritance - Penetrance - Frequency of gene Linkage mapping is most appropriate and productive in finding loci associated with diseases showing Mendelian inheritance e.g. Cystic Fibrosis, Marfan syndrome
Linkage mapping: two-point mapping Linkage between a genetic marker and disease No linkage when recombination fraction is 0.5 Not always easy to determine recombination fraction () in a pedigree Phase-unknown = alleles inherited from each parent cannot be established LOD score analysis is best to analyze data in complex pedigrees Odds of linkage = loci are linked (recombination fraction ) / loci are not linked (recombination 0.5) LOD score = log10 (odds) Most likely is value at which LOD score is max LOD scores can be added up across families Complex pedigrees: computer programs to calculate probabilities Linkage LOD scores > 3; corresponds to 1000:1 odds; p=0.05 of statistical significance Exclusion = LOD scores <-2
Linkage mapping: multi-point mapping More efficient than 2-point mapping Can locate disease locus within a framework of markers (of known order and chromosomal location) Useful to establish the chromosomal order of a set of linked loci Advantages: - Easier to detect double recombinants - Overcome problems due to limited informativeness of markers Data analysis using computer programs: - LINKAGE: able to handle very large pedigrees analysis time exponentially with No possible haplotypes - GENEHUNTER: able to analyze many loci computation time increases with size of pedigree Genetic maps show order and probability that markers will be separated by recombination (cM) Physical maps show features along the chromosome and distances between markers in Kb or Mb
Homozygosity mapping in rare AR disorders Rare recessive diseases: not many families with multiple affected individuals Homozygosity mapping: seek regions of homozygosity in multiple consanguineous families with at least one affected child - children from 1st cousin marriage: 1/16 genome homozygous - children from 2nd cousin marriage: 1/64 genome homozygous Within a limited population it is assumed that all affected individuals inherited the same disease causing allele - possibility of finding a gene in a heterogeneous disorder
Autozygosity mapping in rare AR disorders Homozygosity mapping - a single affected child of 2nd cousin marriage will contribute a value of 1.8 to the lod score = log10(64) Small number inbred families can generate significant LOD scores The more polymorphic and densely packed markers used are, the smaller number of families required The rarer the disease causing allele is, the more likely homozygosity is due to identity by descent (IBD) - homozygosity = autozygosity - Homozygosity mapping has been used to find responsible genes in heterogeneous AR hearing loss, Fanconi anemia and Charcot-Marie-Tooth disease
Homozygosity mapping revealed a locus for AR nonsyndromic hearing impairment DFNB35 AR nonsyndromic hearing impairment - genetically heterogeneous disorder: 67 loci mapped, 24 genes identified Collin et al. 2008; Am J Hum Genetics 82:125-138
Homozygosity mapping using SNP arrays Identification of additional disease-causing genes hindered by lack of families suitable for traditional linkage mapping. Advantage of smaller pedigrees and high-density SNP arrays genotyping for homozygosity mapping in small consanguineous families Bardet-Biedl syndrome (BBS) extremely heterogeneous human obesity syndrome. Autosomal recessive. 9 genes previously mapped and identified. - Affimetrix SNP array- 14 chromosomal regions with linkage based on homozygosity of > 25 consecutive SNPs - TRIM32 gene and mutation in family identified within 2.4Mb in 9q. No mutation in other 90 BBS probands 400 STRP failed to detect shared homozygous regions
Linkage mapping limitations and pitfalls Parametric linkage analysis: - Requires a genetic model - mode of inheritance, gene frequencies, penetrance - Depends on No informative meiosis - limited resolution Limitations of LOD score analysis: - Limits of resolution achievable with markers (estimated 1-10 cM) - no recombination observed - Computational limits of what pedigrees can be analyzed Errors: - Errors in genotyping and misdiagnoses spurious recombinants - Multi-point analysis may show such errors as close recombinants - very unlikely Locus heterogeneity: difficult to map AD or AR diseases (autozygosity mapping)
Linkage mapping in common diseases Straightforward parametric linkage analysis not appropriate for finding genes involved in common diseases: - Not precise model (mode of inheritance) - Difficult diagnosis - often subtle phenotypic changes - Incomplete penetrance, genetic heterogeneity and polygenic inheritance Linkage analysis on ‘affected-only’ - late onset Alzheimer’s disease to chromosome 19- APOE gene More stringent disease phenotype nearly Mendelian inheritance pattern e.g. early age onset familial breast cancer - BRCA1 + BRCA2 cancer families with extreme polyposis - APC gene
Affected sib-pair analysis in common diseases Non-parametric approach: - Model-free - Detection of inheritance of a chromosomal region in a non-random fashion Analysis of affected sib-pairs (ASP) - Many affected sib-pairs compared - No of marker alleles or haplotype marker alleles inherited - Random inheritance predicts siblings share 0,1,2 alleles with freq 1/4, 1/2, 1/4 - For AD traits, all affected siblings are expected to share 1 parental haplotype - For AR traits, sibs should share 2 parental haplotypes - Complex disorders: chromosomal regions whose inheritance deviates from expected 1:2:1 or 1:1 ratios - Statistical analysis (NPL –non-parametic LOD score; MLS - max LOD score –) Used in identification of susceptibility locus for diabetes type I upstream of insulin gene
Genome-wide scan linkage analysis for Parkinson’s disease • Monogenically inherited forms of Parkinson’s: • - AR early onset : PARK2 (parkin gene) -6q25; • PARK9, PARK6 (PINK1), PARK7 on 1p36, 1p36-p35 • - AD: PARK1/PARK4 (α synyclein) - 4q21 • PARK5 - 4p14 • Remaining loci mapped: 1p32 (PARK10), 2p13 (PARK3), 12p11 (PARK8), 2q36 (PARK11) • Genetic model of common forms of familial Parkinson’s unknown
GW screen for Parkinson’s susceptibility loci - Model-free affected-sibling pair method - 199 multiplex pedigrees (USA and Europe) - 391 microsatellite markers: average marker density 10cM, heterozygosity 75% Genome-wide scan linkage analysis for Parkinson’s disease - 6 chromosomal regions implicated - None statistical significant linkage p<0.05 - 2 regions: 2p11-q12 and 5q23 excess allele sharing also reported in independent family datasets - Replication studies essential Martinez et al. J Med Genet 2004; 41:900-907
Homozygosity Haplotype mapping Homozygosity haplotype (HH): Non-parametric method Reduced haplotype described by homozygous SNPs only Mutation inherited from common ancestor - chromosomal segment identical by descent (IBD) Applicability and effectiveness of HH: - Monogenic disorders with high-density SNP data - Susceptibility genes inherited by common ancestor - Identifying potential linkage for monogenic disorders - Use affected-only or family based case-controls
Homozygosity Haplotype mapping Schneider crystalline corneal dystrophy Illumina HapMap300 318,237 SNPs Search candidate regions common HH HH very efficient and easy to implement Advantage: high computational efficiency Not necessary to genotype many family members more distantly related subjects are theoretically more informative Errors identifying candidate regions establish ‘cutoff’ value Genotyping errors 0.1% (Mendelian compatible)
References Human Molecular Genetics. Strachan & Read 2004. Elahi, Kumm and Ronaghi. Global Genetic Analysis. Biochemistry and Molecular Biology 2004; 37:11-27. Chiang et al. Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11). PNAS 2006; 103:6287-92. Miyazawa et al. Homozygosity Haplotype allows genomewide search for autosomal segments shared among patients. Am J Hum Genet 2007; 1090-1102. Jiang et al. Application of homozygosity haplotype analysis to genetic mapping with high-density SNP genotype data. PLoS ONE 2009; 4:e5280.