Hunting Disease Genes in the
Wilds of the Genome -- I
HMGP
Richard A. Spritz, M.D.
March 29, 2011
richard.spritz@ucdenver.edu
303-724-3107
Why Find Disease Genes?
Disease Gene Identification—
“Functional Cloning” vs. “Positional Cloning”
Positional Cloning: Determine a Disease
Gene’s Genomic Position, and then
Identify the Gene
Obviated by
Human Genome
Project
Gene Mapping Technology
Polymorphic DNA Markers
•
•
•
•
You can only track/measure differences
between people and through families
Polymorphic DNA markers constitute any
scoreable differences at known genomic
positions
Surrogates for disease mutations; some
polymorphisms cause disease; most don’t
Most commonly used marker types:
– microsatellites
– single-nucleotide polymorphisms (SNPs)
– copy-number variations (CNVs)
The First Goal of the HGP was to
Assemble a High-Density Genome Map
of Polymorphic Markers
Genetic Linkage Studies
•Search for regions of genome that are
systematically co-inherited along with
disease on passage through families
•Requires families with multiple affected
relatives (multiplex families)
•Best at detecting genes with Mendelian
effects (uncommon alleles with strong
effects)
•Unit of genetic linkage is LOD (“Log of the
Odds) score (>3)
Genetic linkage—
Two loci close together on a chromosome
tend not to be separated by recombination
Principle of genetic linkage—
Loci close by on a chromosome tend not to be
separated by recombination vs. loci far apart
Loci on the same chromosome
Loci on different
chromosomes
Very close Nearby Far Apart
Freq. of crossover Rare
between 2 loci
Some
Frequent
-
Linkage
Tight
Some
Absent
Absent
0%
1-49%
50%
Recombination
50%
• Unit of genetic “distance” is centiMorgan
(cM) = 1% recombination/meiosis; ~ 1 Mb
“Genetic linkage analysis”
Co-segregation of disease gene in “multiplex
families” with alleles of polymorphic DNA
“markers” (initially RFLPs)
Restriction Fragment Length
Polymorphism (RFLP)
EcoRI
Allele 1
AGAGCCTCAACTTGAATTCGTTTAGTAA
Allele 2
AGAGCCTCAACTTGAATTTGTTTAGTAA
Restriction enzyme EcoRI cuts at sequence
5’-GAATTC-3’
Allele 1 has an EcoRI cut site; Allele 2 does not
• This RFLP is assaying a SNP
“Microsatellites” (SSLPs; STRPs, SSRs)
[multi-allelic; ~ 1/30,000 bp; mostly used for
linkage analysis, forensics]
ggctgcacacacacacacacacacacacatgctt
ggctgcacacacacacacacacacacatgctt
ggctgcacacacacacacacacacatgctt
ggctgcacacacacacacacacatgctt
ggctgcacacacacacacacatgctt
Can follow “segregation” of ancestral
“haplotypes” of linked marker alleles in
families
Recombination events prune marker
haplotypes, defining “genetic interval” that
must contain the disease gene
•Only true for
Mendelian traits
•For polygenic traits,
linkage can only
localize disease genes
within rough
probability distribution
of location
Genetic Linkage Analysis
• Statistical measure is LOD (log of odds)
score
LOD = Log10
Likelihood of data if loci linked at 
Likelihood of data if loci unlinked
• Significance level: LOD >3.0 for Mendelian trait
LOD >3.3 for Polygenic trait
Single-Nucleotide Polymorphisms (SNPs)
[bi-allelic; ~1/50-300 bp; mostly used for
association analysis]
SNP1 Allele 1 CCGAGATCCAGAAATCCTGAACATAA
SNP1 Allele 2 CTGAGATCCAGAAATCCTGAACATAA
SNP2 Allele 1 CCGAGATCCAGAAATCCTGAACATAA
SNP2 Allele 2 CCGAGATCCAGAAAGCCTGAACATAA
• Occurrence/allele frequencies differ in different ethic
groups/populations
• Can be in genes (~4,000,000) on not (~8,000,000), can result in
amino acid substitutions or not
• Each occurs in local context (haplotype) of surrounding SNPs
(in example above, SNP2 is on background of SNP1 C allele)
Haplotype Map of Human Genome
International HapMAP Project
•Recombination breaks macro-patterns of polymorphic
genotypes on the same chromosome into haplotypes
•Recombination is not truly random, so very close
polymorphism genotypes carried on the same
chromosome cluster into ~10-50 kb haplotype blocks in
which SNP alleles are in linkage disequilibrium (marker
alleles within blocks tend to be co-inherited on same
piece of DNA, because recombination within blocks is
uncommon); blocks smaller in African than Caucasian
or Asian pops. because African pop. is more ancient
•HapMap genotyped SNPs in different populations to
characterize haplotype block distributions
Copy-Number Variants (CNVs)
[bi-allelic]
Basically are common genomic deletions, hundreds to
tens of thousands of nucleotides in size
May be detected by LD with local SNP patterns:
Allele
--1---1---1----1---2----1----2----1-----1----2----1----1----1----1--Allele
--2---2---2----1---1----2----2----1-----1----2----2----2----1----2--CNV Allele --1---1—[
]--1----2---
• Tens of thousands known
• Like SNPS, occurrence/allele frequencies differ in different
ethic groups/populations
• Individually most are rare (< 1%), collectively common
• Can be in genes or not, can include genes
• NOT commonly definitively causal for human disease
1000 Genomes Project, UK10K Project
International projects to sequence 1000/10000
genomes from different ethnic groups
• Will catalog human genetic variations (particularly SNPs)
– Essential for sequence-based analysis of rare variants that
may be causal for common diseases