Why learn linkage analysis? - and some basic genetics Kaja Selmer 2013
Outline What is linkage analysis and why learn it? An example of a successful linkage analysis story Basic genetics DNA content and organization The central dogma Meiosis Inheritance of DNA and linkage Genetic variation Genetic disease mapping approaches Summary
Why learn linkage a story about a Norwegian family The index patient Small head Seizures Delayed psychomotor development No language Ataxia Happy demeanor Excessive drooling Flexed arms Converging squinting Hyperkinetic Angelman syndrome was suspected, but genetic tests could not detect any known genetic cause of his symptoms.
A Norwegian family III-2 III-4 III-5
Power analysis Power: Maximum LOD score of 1.5 A bit weak for X-linked disorders
Linkage analysis 48 microsatellite markers on the X-chromosome:
Linkage analysis - results Linkage to Xq24-q27.3 27 Mb region 141 known genes
Identification of candidate genes Which are known to give disease (exclusion) Expression Conservation Bioinformatic gene prioritization tools: Endavour, G2D, Suspects and more..
Linkage analysis Sequencing of nine candidate genes: GRIA3 GLUD2 UBE3A SLC9A1 RBMX FGF13 THOC2 ODZ1 ZNF449
Evaluation of mutation The 6 deleted bp were highly conserved 200 healthy control chromosomes tested Validation in patient cohort: 72 boys with suspected Angelman Syndrome, but where genetic tests failed to diagnose
SLC9A6 mutations Norwegian family 6 bp deletion -> removal of two conserved amino acids Swedish family Nonsense mutation -> stop codon British family Splice site mutation, resulting in a protein without expression of exon 3
SLC9A6 mutations Christianson syndrome X linked severe mental retardation, craniofacial dysmorphology, epilepsy, ophtalmoplegia and cerebellar atrophy in a large South African kindred is localised to Xq24-q27 Christianson et al., J Med Genet, 1999 2 bp deletion -> reading frame shift, leading to a premature stop codon
SLC9A6 > NHE6 - function Lysosome Late endosome H + Na + Na + /H + exchanger -regulates ph in early recycling endosomes -early recycling endosomes have proven important in the plasticity and memory of neurons Nucleus Early endosome Golgi apparatus ER Cell membrane
Outline What is linkage analysis and why learn it? An example of a successful linkage analysis story Basic genetics DNA content and organization The central dogma Meiosis Inheritance of DNA and linkage Genetic variation Genetic disease mapping approaches Summary
DNA deoxyribo nucleic acid Contains the information and instructions on how to make and maintain all living organisms Exists in all cells with a nucleus Units of DNA are called nucleotides, consisting of Nucleobase (Adenine, Guanine, Thymine and Cytosine) Sugar molecule Phosphate group(s) Has an Alpha-helix structure
DNA - organization 23 pairs of chromosomes 22 pairs of autosomes 1 pairs of sex chromosomes (XX, XY) Somatic cells are diploid (have 2 of each chromosome) Gametes (sperm and egg cells) are haploid (have 1 of each chromosome)
From DNA to protein the central dogma DNA RNA PROTEIN
From DNA to protein the genetic code The sequence of triplet codons in the DNA gives the sequence of amino acids in the protein ACCGAAG Arg codon amino acid protein
How is DNA inherited? Sperm cell 23 23 Egg cell 46 Somatic cell
Meiosis and chromosomal crossover Gametes Somatic cell Replication & crossover Meiosis I Meiosis II
Recombination ensures independent assortment of parental alleles
Crossover during meiosis Homologous crossovers ensure DNA recombination between maternal and paternal chromosomes during meiosis Promote independent assortment of parental alleles This assortment is, however, not completely independent, and the recombination fraction between two loci contributes to the measure of genetic distance
Genetic linkage Two loci located close to each other on the same chromosome tend to be inherited together, hence they are linked Linkage analysis is based on this tendency of loci to be inherited together
Outline What is linkage analysis and why learn it? An example of a successful linkage analysis story Basic genetics DNA content and organization The central dogma Meiosis Inheritance of DNA and linkage Genetic variation Genetic disease mapping approaches Summary
Genetic variation A diploid human genome: ~6 000 000 000 bases ~99,9% of sequence identical between 2 given individuals ~98,5% identical between human and chimpanzee Genes ~1,5% (20-25 000)
Genetic variation terms Mutation -> a change of DNA Polymorphism -> variation where minor allele frequency is >1% Variant -> all variation (including mutation and polymorphism)
Human genetic variation Single Nucleotide Polymorphisms (SNPs) Microsatellites (short tandem repeats STRs) Copy number variations (CNVs) Translocations Inversions
Genetic marker A polymorphism (variant) may be used as a genetic markers if it Has a known position in the genome Is known to vary in the population Can be routinly genotyped Genetic markers are used as DNA landmarks To distinguish between individuals To track chromosomal regions through inheritance
Genetic markers Most widely used genetic markers as of today: SNPs Microsatellites Genetic markers are important tools in mapping of genetic disease
Outline What is linkage analysis and why learn it? An example of a successful linkage analysis story Basic genetics DNA content and organization The central dogma Meiosis Inheritance of DNA and linkage Genetic variation Genetic disease mapping approaches Summary
How to find disease genes? Which disease? Which genetic model?
The genetic model Small Genetic impact Large Shot wound Traffic accident Type 1 diabetes Cancer Obesity Epilepsy Huntington disease Cystic fibrosis Large Environmental impact Small Modifisert av Benedicte Lie fra figur i Current opinion in Biotechnology
The genetic model Multifactorial (complex) disease Several genetic and environmental factors interact Monogenic (Mendelian) disease Disruption of one gene alone
Approaches to genetic mapping of disease Multifactorial disease Monogenic disease Syke Friske Association analysis Case/control design Population based Linkage analysis Family design Candidate or whole genome approach possible in both
Summary Linkage analysis is mapping of disease genes in monogenic disorders Reveals information on particular disorders, but also of basic human biology The complete genome is in all cells (with nucleus) Somatic cells are diploid, gametes are haploid Chromosomal crossovers in meiosis promote independent assortment of parental alleles
Summary cont. ~ 99,9% of human DNA sequence is identical between two given individuals Polymorphisms might be used as genetic markers ( e.g. SNPs or microsatellites) Genetic model determines the genetic mapping approach Association analysis in multifactorial disease Linkage analysis in monogenic disease