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Regulation of gene expression in
the mammalian eye and its
relevance to eye disease
Todd Scheetz et al.
Presented by John MC Ma
Aims
• To determine loci related to genes
expressed in rat eye by expression
quantitative trait loci (eQTL)
• To verify certain undescribed genes by r2
correlations on members of its gene
groups
Background: QTL
• Quantitative trait locus
• Statistically links a certain genetic locus to
a quantitative trait and seeks association
– Phenotype QTL: The trait is a phenotype, eg
blood pressure, blood sucrose
– Expression QTL: The trait is the expression of
a gene.
• Expression QTL is used in this paper
Methods: Choosing rat strains
• Several criteria
– Highly inbred
– Readily available
– Completely genotyped
– Diverse origins between strains
– Phenotypically normal
• SR/JrHsd and SHRSP were chosen
Methods: Rat breeding and
Genotyping
• SR/JrHsd males and SHRSP females
were mated to produce F1; F1 was
interbred to produce 120 F2.
• RNA was extracted from eye of 12-weekold F2 and is analyzed on Affymetrix Rat
230 Rat version 2 microarray
• Rats were genotyped using 399 sets of
short tandem-repeat polymorphisms with
PCR and gel electrophoresis
Methods: eQTL Analysis
• 18,976 eye-expressed probes out of the
over 31,000 probe sets of the microarray
was selected
• Expression of probe sets analyzed:
– ANOVA for linkage;
– Permutation test and q-value for multiple
testing correction;
– Bonferroni correction for gene-level analysis
Results of eQTL
• 1,330 genes
linked to at least
one locus
• False discovery
rate is 1.4%
Reading the dot plot (1)
• X-axis refers to the genetic marker by location,
where Y-axis refers to the genes by location.
• A: original data for genes in chr 4
• B: A, corrected for adjacent assuming all
adjacent hits is one hit
Reading the dot plot (2)
• Right: genome-wide
dot plot
• Diagonal:
Neighbourhood
effects
• Vertical: One locus
affects multiple genes
• Horizontal: One gene
affects multiple loci
Neighbourhood effects
• Most of the significant
linkages falls within
50cM of the gene
concerned
• Although it is usually
due to cis-acting
regulatory regions,
some are due to
trans-action
Gene-linking locus
• 278 markers shown
significant linkage to
2+ genes
• Largest amount of
linkage is marker
D20Rat2: 33 genes,
but within 10mb
– This area has a high
gene density and the
genes are in the same
family related to
immune response
Gene-linking locus (2)
• The locus having
most of noncontiguous is
D9Rat46, linked to 21
genes
Locus-linking genes
• Statistical limitations
limit how many genes
can be statistically
linked to a single locus
– Single expression value
is used to calculate all
the influences to its
expression
• Only 2 genes are seen
with larger level of
regulation
Linkages to retinal disease
genes
• 114 retinal disease
genes from RetNet
were investigated; 62
are adequately
expressed in the rats
• 7 has contiguous-only
linkages, 11 are noncontiguous-only, and
7 has both kinds
Case of Abca4
• Example of contiguous-linkage-only gene
• Associated to Stargardt disease, an
inheritable, juvenile kind of macular
dystrophy
• Log odds score highest at marker closest
to gene
• Sequencing shown the insertion of cREL
promotion factor 6.6k upstream
Case of Opn1sw
• Example of non-contiguous-linkage-only
gene
• Codes an opsin most sensitive to blue light
• Linked to markers D10RAT27 and D15MIT2
D10Rat27
D15Mit2
Case of Opn1sw (Cont’d)
• D15MIT2 contains Thrb, known to
determine fate of optic cones
• Sequencing found a mutation
(Ser56Asn) that decreases expression
of this gene by 30% if homozygous
Correlating gene expressions
• Pearson’s r2 statistics
was used to calculate
the correlation
between different class
of genes, compared to
randomly selected
groups
• Increased, but
statistically
insignificant increase
in r2 in functional
groups
Correlating Bardet-Biedl
Syndrome - Background
• Rare genetic disease that cause multiple
syndromes like:
– Retinitis pigmentosa
– Obesity
– Polydactyly
• Autosomal recessive
• Several genes have seen linkage, but they
only account for about half of cases
Correlating Bardet-Biedl
Syndrome
• Hypothesis:
Expression of known
Bardet-Biedl
syndrome-related
genes are highly
correlated
• Result: Rat orthologs
of BBS genes are
strongly positively
correlated
Bardet-Biedl Syndrome: the
Case of BBS9
• The investigators
previously identified
BBS9 through
comparative
genomics
• Hypothesis: BBS9
correlates well with
other BBS genes
• Result: hypothesis
confirmed
Bardet-Biedl Syndrome: the
Case of BBS11
• Investigators also
previously identified
BBS11 through SNP
genotyping
• BBS11 is also found
to be corerelated to
other BBS genes
through pairwise r2
tests
Discussion
• Previous eQTL analyses has shown that
gene regulation is more complex than
expected
• In this investigation:
– Changes in expression are can be traced to
inheritable factors
– Multiple loci controls one gene
• Experience on Abca4 and Opn1sw shown
eQTL is effective method for determining role
of gene regulation in diseases
Discussion (Cont’d)
• Regulatory relativity: functionally related
gene are expressionally related; genes
that response to a change can be inferred
to be functionally related
• In this paper, recombination between the
genomes became the permutation
• RG is found plausible in research and
useful in finding undescribed disease
genes
End