regulation of gene expression in the mammalian eye and its relevance to eye disease todd scheetz et...
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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-week-old 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
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
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 non-contiguous-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
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