Alhaddad Exit Seminar

Linkage Disequilibrium and
Recombination in the Domestic Cat:
Applications to Genome-wide
Association and Linkage Studies
Hasan Alhaddad
Advisor: Dr. Leslie Lyons

Presentation Overview
• Introduction
• Linkage disequilibrium in cats
• Population recombination rate and
recombination hotspots
• Genome-wide analyses of progressive retinal
atrophy in Persian cats
• Conclusion
• Acknowledgments

Introduction Linkage Disequilibrium Recombination hotspots Persian PRA Conclusion
A bit of history Cat domestication Cat breeds Phenotype genetics Disease genetics Genetic resources
“An Angora cat and a regular male cat (first
generation) have produced only regular cats
(second generation). If you looked at them,
you would give the father a high significance.
The young cats however had a lot of Angora
blood despite their regular look. That is
because after mating two of the same, there
was in the third generation besides regular
also a female Angora cat (unchanged). The
chance for Angora blood may be even better
in the fourth generation. We would have not
reached that conclusion if we only relied on
the visible traits and hence we should not
trust them, because how would two regular
cats produce an Angora cat?”
C. Nägeli, 1884

Personal synthesis
a. Ecological domestication
b. Selection from standing variation in RB
c. Selection from standing variation in breed
d. Breeds from de novo mutation
e. Hybridizing two (more) breeds
f. Interspecies hybridization

• What is a cat breed?
• Breeders vs. Genetics?
• Cat Fancy Association (CFA) recognizes 40 breeds.
• The International Cat Association (TICA) recognizes 55 breeds.
• Cat breed designation is interesting and challenging.

Variation within FGF5 (long hair) differentiate between Persian
Filler et al., J Hered, (2012)
Gandolfi et al., Scientific Reports, (2013)
and Exotic shorthair cats

CFA registry record of cat breeds
for the period 1958-2011
(1,901,585 cats)

Burmese
Hypokalemia
Head defect
Oralfacial pain
Persian family
Polycystic Kidney
Retinal degeneration
Osteochondroplasia
Diabetes
Hypertrophic
cardiomyopathy

• Somatic cell hybrid panels
O’Brien and Nash, Science 1982
• Intra-inter species linkage map
Menotti-Raymond et al., Genomics 1999
Menotti-Raymond et al., Journal of Heredity 2003
Menotti-Raymond et al., Genomics 2009
• RH panel
Murphy et al., Genome Research 2000
Bach et al., Cytogenet Genome Res 2012
• Genome sequencing
Pontius et al., Genome Research 2007 – 2X ~ 60% genome
Mullikin et al., BMC Genomics 2010 – 3X ~ 80% genome and 1,000,000
SNPs
• 63K Illumina Array chip

Objectives Hypotheses
O1: Estimate the extent of LD in cats.
O2: Provide insights for design of GWA studies.
H1: Linkage disequilibrium varies across populations and
genomic regions.
H2: Variation in linkage disequilibrium among populations
reflects population histories.

Linkage Disequilibrium Markers Samples LD in cats Haplotypes Cat LD & GWAS LD in mammals
• Linkage disequilibrium is the non-random association of alleles at different
loci in a gamete.

Markers Samples LD in cats Haplotypes Cat LD & GWAS LD in mammals
• LD is measured as the difference between the frequency of a haplotype
and the frequencies of the alleles.
D` =
DAB , where Dmax = {
min (PA PB, Pa Pb), when DAB < 0
Dmax min (PA Pb, Pa PB), when DAB > 0
• Squared correlation coefficient
r2 =
D2
PA Pa PB Pb
• Normalized D` (D prime)
DAB = PAB – PAPB
Linkage Disequilibrium

LD decay and comparison across populations
Hill and Weir, Theor Popul Biol 33, 54 (1988)

• A custom Illumina GoldenGate array (1536 SNPs).
• Ten (1 Mb) regions from various locations relative to centromere.
• ~ 150 SNPs/region with higher density of marker in one end of the region.

18 breeds
2 random bred populations
Total of 408 samples
~ 18 cats/population
Breed cats pedigreed verified
to be unrelated to the
grandparent level

Extent of LD in cats on each chromosomal region

• Lowest LD among breeds found in Siberian and Manx breed (~ 20Kb). These breeds
largely resemble a random bred population.
• Highest LD (> 200 Kb) found in Eastern breeds (Birman, Burmese, and Siamese).
These breeds are known to have restricted phenotypes and under selection for very
defined phenotypes.
• Persian cats exhibit moderate LD (~ 75 Kb) compared to other breeds and is
consistent with the large population size of the breed.

• Eastern Breeds have higher fractions of SNPs useful for GWA
studies.
• Association studies for phenotypic vs. disease traits.
• Successes so far.

• LD calculated using
the same measure.
• Extent of LD
compared at the same
value.
• Each data point
corresponds to the LD
of a single population.

LD Markers Samples LD in cats Haplotypes Cat LD & GWAS LD in mammals
Conclusion
• Extent of LD varies across chromosomal localities and among
breeds.
• LD of cat breeds reflects the demographic and breeding histories.
• Eastern breeds exhibit larger extent of LD. Association studies in
involving such breeds are likely to be successful.
• Breeds such as Manx and Siberian (may be Persian) require a
higher density SNP array or alternative approaches.
Any questions?

A glance at recombination hotspots in the
domestic cat
Alhaddad, H., Zhang C., Rannala B., and Lyons L.A.

O: Use coalescent based methods to infer population
parameters.
O: Understand recombination hotspots in cats.
H1: Population recombination rates vary at different genomic
localities.
H2: Variation in population recombination rate at different
regions is due to the presence of specific genomic features
(i.e. recombination hotspots)

Recombination Coalescent Markers samples program Estimates&interpretation Hot&warm-spots G. features
Parent
Recombination
• Recombination: shuffling the genome during meiosis
and formation of gametes.
• Recombination hotspots: regions of elevated
recombination rate.
• Correlation with hotspots in humans:
• GC content
• Repeat elements
• Motif
• PRDM9-zinc finger domain
• Dog hotspots different !

• Coalescent + recombination = Ancestral
Recombination Graph (ARG).
• Allows estimation of population
parameter (rho) = scaled recombination
rate.
• Identify recombination hotspots.
• Overcome the need for large pedigrees
and sperm typing.
Rosenberg & Nordborg, Nat Rev Genet 3, 380 (2002)

• 22 Eastern random bred (feral) cats.
• 701 SNPs distributed over ten regions.
• inferRho: coalescent-based Bayesian method.
• Bayesian hypothesis testing and Bayes factor:
BF
• Spot designation:
(1 )
p p
i i
q q
(1 )
i i
• Bayes factor ≥ 100 “hot spot”
• Bayes factor 10 – 100 “warm-spot”
• Bayes factor < 10 “neutral spot”
Wang and Rannala, Philos Trans R Soc Lond B Biol Sci 363, 3921 (2008)
Wang and Rannala, Proc Natl Acad Sci U S A 106, 6215 (2009)

• Hotspot: Analysis of chromosome E2 region
• Local recombination rate.
• Posterior probability.
• Bayes Factor.
• Four recombination hotspots
in three regions.
• hotspot size: 1.8 – 4.6 Kb.
• 52 warm-spots found in all
regions.
• Warm-spot size: 0.4 – 5.6 Kb.

Lack of significant correlation between GC content and
hot/warm-spot locality

• L2 LINE elements were present in three of the four hotspot regions.
• tRNA-Lys family SINE elements are present in three of the four hotspots.
• MIR family SINE elements were present in all hotspot regions.
• Other repeat elements inconsistently present across the hotspot regions.
Hotspots Warm-spots Neutral-spots

Conclusion
• Population recombination rate varies across regions examined.
• Regions with higher recombination rates contain hotspots.
• Four recombination hotspots were identified.
• No correlation between GC content and hotspot locality (need
more data).
• L2, MIR, tRNA-Lys likely to be a signature of hotspots in cats
(need more data).
Any questions?

Localization of progressive retinal atrophy
of Persian cats using genome-wide
analyses
Alhaddad, H., Gandolfi B., Grahn R.A., Rah H., Peterson C.B., Maggs
D.J., Pedersen N.C., and Lyons L.A.

O: Evaluate genome-wide methods for cat pedigree data.
O: Identify the causative mutation of PRA in Persian cats.
H: Progressive Retinal Atrophy (PRA) of Persian cats can
be localized using a dense genotype data of a family.
H: The causative mutation can be identified using
candidate gene approach.

PRA Samples Linkage TDT sib-TDT Case-Control Haplotype Candidate genes
• Two identified mutations cause retinal
degeneration in Abyssinian cats.
• CEP290
• CRX
• Progressive Retinal Atrophy in Persian cat.
• Gradual degeneration of photoreceptor.
• Early onset: starts 2-3 weeks of age.
• Rapid disease progression.
• Complete loss of photoreceptors at 16-17
weeks of age.
• Autosomal recessive mode of inheritance.
Rah et al., Invest Ophthalmol Vis Sci 46, 1742 (2005)

• Pedigree composed of 202 cats.
• 126 cats (blue and red) were genotyped using 63K SNP array.
• After QC 106 included (37 affected and 69 unaffected).

• Two variation of
nonparametric linkage
analysis – not significant
• Recessive mode of
inheritance – parametric
analysis.
• Parametric analysis: 35
markers LOD score ~ 14.
• Linkage found on cat
chromosome E1 (~1.75 Mb).

TDT
sib-TDT
Spielman et al., Am J Hum Genet 52, 506 (1993)
Spielman, and Ewens, Am J Hum Genet 62, 450 (1998)
• Family based
methods tests
linkage given the
presence of
association between
a marker locus and
a disease locus.
• TDT: 33 trios.
• sibTDT: 85 sib
pairs

• Case-control using pedigree
data is inappropriate.
• Population substructure within
the pedigree.
• Reduction of genomic inflation
(3.18 to 1.3).
• Association consistent with
TDT and sibTDT.

• Association and linkage markers
points to a single haplotype among
affected cats.
• Three control cats share the same
haplotype of the affected.
• After examination two founder cats
confirmed affected.
• One cat mislabeled as unaffected.

• Haplotype region contains 22 eye related genes.
• Mutation within PITPNM3, AIPL1, and ARRB2 known to cause retinal
degeneration in humans.

Conclusion
• Parametric linkage and various association analyses consistently
points to the same region.
• Progressive Retinal Atrophy of Persian cats was localized to ~
1.36Mb region on cat chromosome E1.
• Three genes are likely candidates.
Any questions?

Future Direction
• Genome-wide estimation of LD in cats using 63K array.
• Investigating recombination across the genome and
studying genomic signatures of recombination hotspots.
• Comparing recombination hotspots across different cat
populations.
• Investigating PRDM9 in cats and analyzing the motif
recognition portion.
• Studying PRA localized region via candidate gene
sequencing, targeted sequencing, or whole genome
sequencing.
• Continue on the development of cat genetic resources.

Acknowledgments
Dissertation Committee
• Dr. Leslie A. Lyons (Chair)
• Dr. Bruce Rannala
• Dr. Jeffrey Ross-Ibarra
Lyons Laboratory
• Dr. Barbara Gandolfi
• Dr Robert A. Grahn
• Razib Khan
• Many grad students
• Many Many cool undergrads
CCAH
• Dr. Niels Pederson and lab
• Dr. Ben Sacks and lab
• Dr. Holly Ernest and lab
Collaborator
• Dr. Chi Zhang
GGG
• Carolyn Yrigollen
• Gavin Rice
•Lattha Souvannaseng
• Rebecca Nitcher
• My cohort (Fall 2009)
• All GGG students
• Demian Sainz
• Ellen Picht
Vet genetics lab
• All are cool

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