I: Human genome maps and I: Human genome maps and localization of disease geneslocalization of disease genes

Loengud ja seminarimaterjal: www.tymri.ut.ee -> õppetöö

User: ML2004Pw: 2004

Kirjandus:

1. T. Strachan and A.P.Read “Human Molecular genetics”

2. A. J.F. Griffiths et al “ Introduction in genetic analysis”

3. Alberts B et al “Molecular Biology of the cell”

4. http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Books

CLONES DISEASES

cDNA Genomic FamiliesChromosomalAbnormalities

ESTs PolymorphicFull-

lengthLargeinsert

STSs HeterogeneityIrregular inheritance

FISHSomatic cell hybridsRadiation hybrids

MultipointLinkage

(CEPH, etc.)

Two-point linakge(Lods, sib pairs,Homozygosoty)

Contig assembly

GENETIC MAPGENETIC MAP

Initial diseaseGene localization

PHYSICAL MAPPHYSICAL MAP

HUMAN GENOME MAPHUMAN GENOME MAP

Sequencing

Gene identification

MOUSE map

Marker-marker framework map

Genetic mapping - the aim is to discover how often 2 loci are separated by meiotic recombination

A2 A2

B2 B2

A2 A1

B2 B1

A1 A1

B2 B1

A2 A1

B1 B1

A2 A1

B2 B1

A1 A1

B1 B1

A1 A1

B2B1

A1 A1

B1 B1

A2 A1

B2 B1

A2 A1

B1 B1

A2 A1

B2B1

A1 A1

B1 B1

A2 A1

B2B1

NR NRNRNRNR R R

Genes A and B with alleles A1, A2 and B1, B2 are segregating in the family

I

II

III

Generation

Recombination fraction between loci A and B: the proportion of children who are recombinant R; the probability that an odd number of crossover events will take place between two loci

*Loci on different chromosomes: r (or =0.5*Loci on the same chromosome or syntetic : r (or <0.5*The closer the loci are, the smaller the value of R

Genetic map unit is 1 cM (centimorgan) = 1% of recombination between two loci

*The mathematical relationship between and genetic map distanceis described by mapping function (Haldane fucntion, Kosambi function)*average 49-55 crossovers per cell (differs between individuals)*chiasmata are more frequent in female meioses (fits Haldane rule that heterogametic sex has the lowere chiasma count)1 female cM=1.68±1.07 Mb1 male cM= 0.92±0.96MbSex-average cM= 1.30±0.80 Mb

Puurand, 2004

Mapping of the genome requires genetic markers:any Mendelian character can be used as a marker

*Genetic map preciseness and quality is increased by :1) dense coverage of markers across genome2) high PIC (polymorphism information content)3) extensive family material - high number of informative meiosis

AA1 1 A2 A3 A4

AA11 A4

A1 A1 A2 A2

A1 A2

AA11 A1 A1 A2

AA11 A1

A1 A2 A1 A2

A1 A2

Uninformative meiosis Informative meiosis

The father has adominant conditionthat he inherited with the marker allele AA11..

Informative meiosisInformative meiosisallows to define thatallows to define thatthe child inherited the child inherited AA11 from the father. from the father.

Marker When used No of loci

Blood groups 1910-1960 ~20

Protein Electromorphs 1960-1975 ~30

HLA tissue types 1970 – 1 haplotype

DNA RFLPs 1975 – <105

DNA minisatellites 1985 – <104

DNAmicrosatellites 1989 – <105

DNA SNPs 1998 – <106

The development of human genetic markers

Mendelian characters are determined by a SINGLE locus genotype

*For human <10 000 Mendelian characters are known (OMIM database)*Character can be either dominant or recessive and is discrete (= Y or N)*Genotype can be either hetero-, homo- or hemizygous (male X and Y loci)

*There are 5 [6]basic Mendelian pedigree patterns:- autosomal dominant inheritance- autosomal recessive inheritance- X-linked dominant inheritance- X-linked recessive inheritance- Y-linked inheritance[-mitochondrial or matrilinear inheritance]

*The mode of inheritance is determined by using several pedigrees*knowledge of the mode of inheritance is the prerequisite for linkage analysis

NON-Mendelian characters multifactorial: (1) continous or quantitative;(2) oligo- or polygenic; (3) environment-dependent expression

Linkage mapping of human disease genes – analysis of the segregation of marker alleles together with a Mendelian disease in human pedigreesIs based on counting recombinant and nonrecombinants

*Usually is not possible to score the recombinants by hand*computerized lod score analysis is used

likelihood that the loci are linked (with ) likelihood that the loci are unlinked (=0.5)Odds of linkage=

Lod score = log10(Odds of linkage)

*lod scores are calculated over the range of values*the most likely is the one with the highest lod score*lod scored can be added up across familiesZ=3 is the threshhold of accepting linkage (= 1000:1 odds)Z<-2 linkage can be rejected (=1:10 odds)

Problems of lod score analysis in humans:

*long generation time

*inability to control matings

*inability to control environmental exposure

*errors in genotyping and misdiagnosis

*computational difficulties

*locus heterogeneity

*limited resolution of the map

*lod score mapping is limited to Mendelian characters

Types of Maps Features Resolution 1. Cytogenetic Chromosome Banding maps Several Mb

2.Chromosome a) Somatic cell hybrid panels Several Mb

Breakpoint maps b) Radiation hybrids maps >0.5 Mb

3. Restriction map Rare-cutter (e.g. Not-I) maps <0.5 Mb

4. Clone contig map a) overlapping YAC clones 0.1-1Mb

b) overlapping cosmid clones ~40 kb

5. STS-maps typed by PCR; requires prior ~100 kb

sequence information for PCR primers

6. EST-maps sequencing 200-300 bp from ~40 kb

a cDNA clone, mapping back to other maps

7. DNA sequence map 1kb

Types of physical maps available for human genome

Localizationof the mapped gene

Principle of the Radiation Hybrid Panel mapping

*Mapping is by PCR typing or Southern blot hybridization of the studied gene*The higher the initial radiation dose, the higher resolution mapping

Different radiation hybrid cell lines

The mappingfunction Dis measured incentiRays (cR)

Clone contig maps: contig=contigous DNA without any gaps across the whole chromosome or selected genomic region

Overlap between the clones can be detected using STSs content mapping, repetitive DNA fingerprinting (long insert clones like YACs) or RFLP, microsatellite typing, and FISH analysis (shorter insert clones like cosmids or PACs)

Clone (cosmid, BAC, PAC, YAC)

STS and EST

STS- sequence tagged-siteSTS- sequence tagged-site„foot-print“ of a genomic region: short DNA stretches, amplifiable by adefined unique pair of primersApplied for: cchracterization and mapping of genomic clones into the context of the particular genomic region or contig

EST - expressed sequence tagEST - expressed sequence tagSource: various cDNA librariesMethod:1) cDNAs from a library are cloned into vectors; 2) 200-300 bp of each of cDNAs are sequenced random; 3) a public EST-database is formed, where scientists can identify and derive the clones containing the cDNAs of interest; 4) EST Initiative usually also tries maps the ESTsto the genome map

Publicly available genome databasesPublicly available genome databases

NCBI: http://www.ncbi.nlm.hih.gov/ENSEMBL: http://www.ensembl.org/

Organisms: human, mouse, rat, fruitfly, zebrafish, C.elegans, etc.Information: 1) genome maps – genetic, physical2) coding sequence (transcript maps, ESTs etc.)3) marker databases and maps (SNPs, mikrosatellites, RFLPs etc.)4) Gene information (genomic structure, mRNA, peptide, gene family,polymorphisms, function, diseases, etc.)5) polymorphism information6) homology maps (e.g. Mouse and Human)7) links to other databases (PubMed, OMIM, SNP databases, clone availability etc.)

Functional cloning

Identification of disease genes: position-independent strategies

Knowledge of thedefective protein

product

Gene specific Oligonucleotides

(AspartylglucoseAminuria, AGU

in Finns and AGAlocus )

Use of specificAntibodies

(PhenylketonuriaAnd phenylalanineHydrpxylase, PAH)

Identification of a genethrough its normal

funtion

Functional„rescue“ incell lines or/transgenic mice(Fanconi’s anemiaGroup C)

SubstractionCloning(Dystrophin and DMD gene)

Identification of disease genes: position-dependent strategies

Step 1. Positional cloning

Define the candidate Region

High-resolutionMap of the candidate region

Linkage mappingChromosomal

aberrations in patients

Polymorphismscreening

Genetic and physical mapping

Linkage disequilibrium mapping

Candidate gene search and analysisSearch for transcripts

Search thedatabases

Application of chromosomalApplication of chromosomalAberrations for mappingAberrations for mappingthe disease locus:the disease locus:3 individuals among the Finnish AGU (aspartyl-glucoseaminuria) patientswere characterized byaberrant karyotype andsimultaneously either under-(patients a,b) or over- (patient c)expression of the AGA protein.Patients a and b missed one telomeric segment of chr. 4q,patient c had an extra copy of thisRegion translocated to chr.21p.Thus, the AGA gene couldBe mapped 4q33->tel

Step 2. Positional candidate cloning

Define the candidate gene (s)

Confirming a candidate gene

Expression patternAnd function

Homology to relevantHuman gene or EST

Homology to a relevantGene in a model organism

Mutation screening

Difficulties:*Locus heterogneity

*mutational homogeneity*neutral versus pathogenic

mutations*other types of mutation than SNPs

Restoration of Normal phenotype

Mouse model of thedisease

Understand the functionof the gene

Mapping human traits using isolates

Given the current size of world’s population, the human genome is LESS diverse than might be expected:

1. Recent divergence from other primates

2. Relatively small size of human population over most of its history

Major waves of human migrations:

1. 100 000 years ago out of Africa

2. 50 000 - 30 000 to new regions, as Americas and Australia

3. 10 000 ya with spread of agriculture after the last glacial period.

Genetic consequences of a bottleneck accompanied by isolation:*Less diversity*Inbreeding*Genetic drift - random enrichment of recessive and neutral alleles

bottleneck

time

Populationgenetic diversitybefore bottleneck

Populationgenetic diversityafter bottleneck

Use of population isolates for mapping human traits

Peltonen et al., 2000

I. Examples of exploited isolated populations with High frequency of certain Mendelian disorders :

*Finns, Amish, Sardinians, Bedouins

II for mapping complex diseases, it might be useful to study very young isolates (10-20 generations):

*eastern Finland (Kuusamo), Costa Rica, Quebec, Newfoundland

These population isolates are have:

(a) Reduced genetic complexity

(b) Uniform environmental and cultural feature in the isolate

Peltonen et al., 20002000-4000 years ago

16th centuryTwo waves of settlement by

founder effect:>30 Finnish Mendelian diseases

Population isolates have been used with great success for identifying single gene defects.

Linkage analysis needs: (a) reliable diagnosis; (b) pattern of inheritance

Due to founder effect and genetic drift, the “disease allelechromosomes” possess strong haplotype signatures:The younger the mutation and the lower the recombination rate,

the longer the “disease” haplotype around the mutation

Novel mutation (M) is in absolute allelic association with certain haplotypic pattern of all polymorphic markers (P ) of the same chromosome

Present chromo-somes

M P PPP

Other chromosomes in populations with random distribution of alleles at loci P

Recombinations in population history

“disease” chromosome

Simple versus Complex disease mapping: are the isolates also here useful?

In practise, most successes in mapping complex disease loci in population isolates have depended on large pedigrees with proven or predicted genealogical ties between affected individuals.

Other strategies - genome scans to monitor intrafamilial association and linkage disequilibrium in population isolates have been less successful

Important! Subdivision of patient populations by qualitative clinical criteria minimizes genetic heterogeneity

Peltonen et al., 2000

Icelandic experiment:

*Iceland was founded 9th-10th century by limited numbers of founders from Scandinavia*minimal immigration during 1100 years*most of 275 000 Icelanders are descendents of original settlers*A tradition of recording family trees - genealogy of Icelanders traces back >1000 years*reduced genetic heterogeneity due to founder effect and inbreeding*deCODE project: cross-populations databases of linked genealogical, patients and genotyping records

Example of the mapping strategy of a Mendelian disease gene Example of the mapping strategy of a Mendelian disease gene In population isolate :vLINCLIn population isolate :vLINCL

NCL( neuronal ceroid lipofuscinosis) - a group of neurodegenerative Disorders of childhood with an incidence of 1:12 500 births

vLINCL(Finnish variant for late infantile NCL)vLINCL(Finnish variant for late infantile NCL)affects children at the age 4-7 yo. with first symptoms of clumsiness,followed by progressive visual failure, mental and motor deterioration

Enriched in Southern Ostrobothnia region of FinlandMost Finnish patients probably share a mutation, which was introduced20-30 generations ago (500-750 ya), i.e. during the period of inner migrationIn Finland from coast to the inland. Mutation was spread and clustered in the area probably due to low number of the founders, followed by demographic expansion and relativelystrong isolation

vLINCL 1

Linkage with polymorphic markersD13D160 and D13S162 at chromosome 13q : critical region 4 cM

II.Physical mapAcross the region

using FISH-methods:

I.Clones: previouslyAvailable and isolated

During the project

III.New polymorphic markers

1. Genetic mapping

1. Refined chromo-Somal region 13q32

2. Exclusion ofCandidate genes

By position

3. Contig across critical region

IV.Identification of novel candidate genesby the searches in EST database and

cDNA library screening

2. LD mapping:Haplotype analysis

Narrowed downcritical region 200 kb

Disease mutation identification in patients versus controls

cDNA clone assambly for putative CLN5 gene:ESTs, RACE, library cDNAs

RNA expression pattern by Northern and RT-PCR

Tissue expression analysis: mutationmutationverificationverification and disease pathology study

vLINCL 2

Physical mapping of vLINCL candidate region by FISH onmetaphase chromosomes,mechanically streched chromosomesand DNA fibers

vLINCL 3

vLINCL 4

CLN5 gene:CLN5 gene: putative transmembrane protein with no homology to previously reported genes (Savukoski et al., 1998)

3 different haplotypic backgrounds with 3 different mutations:1) a 2 -bp deletion in exon 4 (FinMajor), in the highest risk area with the carrier frequency 1:24, in en extended high-risk area 1:100, not present elswere in Finland2) a nonsense mutation (FinMinor) - transversion in exon 1, present in only one family, not present elsewere in Finland nor Europe3) a missense mutation (Dutch mutation) -transversion in exon 4

CLN5 gene:CLN5 gene: lysosomally targeted glycoprotein(Isosomppi et al., 2002)

1) expressed in embryonic human brain at the beginning of cortical neurogenesis2) transfection experiments: WT is lysosomally targeted and partially secreted into culture medium3) transient localization in ER and Golgi reflects intracellular traffiking of CLN5 to lysosomes4) CLN5 is N-glycosylated ->soluble protein5) FINMajor mutation -> protein expressed but not targeted to lysosomes