1. Chapter 8
Gene Mapping

2. two types of gene mapping
Genetic mapping: interlocus distance
Linear description of markers and genes on a given chromosomes with markers closer being inherited together more often.
Physical mapping: location of genes on chromosomes
Cytogenetics
Physical methods

3. Linkage Analysis
linked/unlinked

4. Linkage Analysis Genetic recombination
haplotypes (haploid genotype) of two genes: A1B1/A2B2
recombination → new arrangement of alleles (A1B2/A2B1)
double crossover → no recombination (A1B1/A2B2)

5. Recombination frequency
distance between two loci
1 centiMorgans (cM) ≈ 1% recombination frequency
50 cM apart two syntenic loci → unlinked
alleles on different chromosome → unlinked

6. Linkage analysis of NF1 with a probe on chromosome 17
Recombination frequency
= 1/8 (12.5%)

7. LOD (logarithm of the odds) Scores
For estimation of recombination frequency (q)
A higher number of cases → less misleading linkage results
How to estimate recombination frequency
Establish a pedigree.
Make a number of estimates of recombination frequency.
Calculate a LOD score for each estimate.
The estimate with the highest LOD score will be considered the best estimate.
Meaning of LOD scores
3.0 (1000 times greater in favor of linkage): linked
-2 (100 times greater in favor of unlinkage): unlinked
Likelihood when q = 0.1
LOD = Z = log
Likelihood when q = 0.5

8. Linkage analysis of NF1 with a probe on chromosome 17q
0.1
0.2
0.3
0.4
0.5
LOD -∞
0.32
0.42
0.36
0.22
0.0

9. Linkage analysis of NF1 with a probe on chromosome 17

10. Computer calculation of LOD scores for the linkage analysis of NF1 and the RFLP marker

11. Genetic Markers
new polymorphic makers found by RFLP, STRP, and SNP

12. Genetic Markers properties of marker loci
(1) codominant (RFLP, STRP, SNP)
(2) numerous marker loci
(3) highly polymorphic (eg. microsatellite repeat polymorphism)
two RFLP alleles → uninformative mating
six STRP alleles → informative mating
exceptions: due to recombination

13. Location of disease-causing genes by linkage analysis

14. Linkage Disequilibrium
association of a marker and a disease allele within a family, but no association of a marker and a disease allele between families
independent mutations
recombination even for closely linked loci → resulting in linkage equilibrium

15. Linkage Disequilibrium
The mutation is indicated by a red triangle.
Chromosomal stretches derived from the common ancestor of all mutant chromosomes are shown in yellow, and new stretches introduced by recombination are shown in blue.
Markers that are physically close (that is, in the yellow regions of present-day chromosomes) tend to remain associated with the ancestral mutation even as recombination limits the extent of the region of association over time.

17. Linkage Disequilibrium
Nonrandom Association of Alleles at Linked Loci
linkage disequilibrium (preferential association)
distance (order of genes on chromosome, mapping a gene in 0.1 cM)
evolutionary force (eg. natural selection, genetic drift)
recombination even between closely linked loci if go for long enough generations
eventual linkage equilibrium
resulting in linkage equilibrium (no preferential association between the disease gene and a specific allele at a linked marker locus)

18. Linkage versus Association in Populations
linkage: a positional relationship between two loci
association: a statistical relationship between two traits

19. Linkage versus Association in Populations
ankylosing spondylitis and
HLA-B27 allele

20. Linkage versus Association in Populations
idiopathic hemochromatosis and
HLA-A3 on chromosome 6
type I diabetes and HLA-DQβ allele
False association
ethnic variation
imprecise definition of the disease state
inadequate sample sizes
improper matching of cases and controls

21. Physical Mapping Heteromorphisms
a variation in the appearance of a chromosome
cf. polymorphism
eg. uncoiled region of chromosome 1
not genetic variants, nor diseases (maybe associated)
sometimes useful in mapping genes

22. Physical Mapping Deletions Translocations
narrowing the location of the disease gene
eg. RB, Prader-Willi and Angelman syndrome, Wilms tumor
Translocations
eg. NF1, DMD
interruption of a gene by translocation

23. Physical Mapping Dosage Mapping Using Deletions and Duplications
a deletion on a chromosome → reduction of the gene product
adenylate kinase (a deletion on chromosome 9)
red cell acid phosphatase (a deletion on chromosome 2)
a duplication → increase of the gene product
superoxide dismutase-1 (SOD-1, 21q)

24. Somatic Cell Hybridization
cell fusion (by polyethylene glycol or Sendai virus)
→ culture the hybrid cells in HAT (hypoxanthine, aminophterine, thymidine) media
→ The hybrid cells will lost some of human chromosomes.
→ analyzing which chromosome contains the gene in question (DNA segment)
Detection of genes
protein electrophoresis
southern blotting
PCR

25. Scheme for generating human-rodent somatic cell hybrids

26. Detection of genes in hybrid cells

27. Detection of genes in hybrid cells

28. Radiation Hybrid Mapping
irradiation of x-ray or γ-ray to a somatic cell hybrid containing a single human chromosome
→ double-strand breaks in chromosomes
→ fusion of human chromosome with rodent chromosomes
→ detection of human chromosomes by screening Alu sequence and a specific combination of human loci
→ estimate the relative distances between the loci

29. Generation of a radiation hybrid mapping panel

30. Comparison of the steps in functional and positional gene cloning

31. Positional cloning
locating the disease gene by canvasing the region around a linked marker
Process
Select a tightly linked polymorphic marker (a probe)
pick up overlapping segments of DNA from a genomic library
use the overlapped segment as a probe to pick up next overlapping segment
repeat until the disease gene is reached
chromosome walking
bidirectional walking

32. The general approach to positional cloning

33. The scale of positional cloning
YAC : 50-1,000 Kb
BAC , PAC: Kb
Plasmid : ~10Kb
Cosmid : ~5Kb

34. Useful tools for positional cloning
sequence-taged sites (STS)
sequences of a few hundred bp whose chromosome location is known
used as signposts
contig maps

35. Determination of disease gene - How to know getting to the disease genes
Analysis of cross-species conservation
exposure a probe to denatured DNA from several species
Identification of CG islands
unmethylated CpG repeats in 5′region
Exon identification techniques
exon trapping: genomic DNA insertion in plasmid → expression
direct cDNA selection: hybridization of genomic DNA
Computer analysis of DNA sequence
sophisticated computer algorithms (eg. GRAIL)
computerized databases of known DNA sequences
expressed sequence tags (both ends of cDNA clones , locations, now over 4.5 million ESTs)
similarity searching for human genes in other organisms

36. Screening for mutations in the sequence
for small mutations (eg. missence mutations)
single-strand conformation polymorphism (SSCP)
denaturing gradient gel electrophoresis (DGGE)
direct DNA sequencing
*distinguish disease-causing mutations from polymorphism by comparing the DNA of patients (a new mutation) with the DNA of their unaffected parents
for a submicorscopic deletion
(a) small deletion: conventional southern blotting
(b) larger deletion: restriction digestion with enzymes cleaving infrequently → pulsed field gel electrophoresis → southern blotting

37. Test for gene expression
Northern blotting
microarray for mRNA
injection of the normal version of the DNA sequence into a defective cell

38. Examples of genetic mapping
Marfan syndrome
collagen genes
→ linkage analysis
→ negative
fibrillin-1 gene (chromosome 15)
→ linkage/mutation analysis
→ positive
→ finding mutation (missence) in patients

39. Quantitative trait locus
A quantitative trait locus (QTL)
To find a region of DNA that is associated with a particular phenotypic trait
the genetic architecture of a trait ← the number of QTLs
eg. height controlled by many genes of small effect, or by a few genes of large effect
Identification of candidate genes underlying a trait
Identification of a DNA region → DNA sequencing → compared to a database of DNA for known genes
classical QTL analyses/gene expression profiling (i.e. DNA microarrays) → expression QTLs (e-QTLs)
cis- and trans-controlling elements for the expression of disease-associated genes
a cross-validation of genes within the interacting loci with metabolic pathway and scientific literature databases

40. A QTL for osteoporosis on the human chromosome 20

41. A genome-wide scan for QTL of osteoporosis