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

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 non-contiguous 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 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

End