1. Regulation of gene expression in the mammalian eye and its relevance to eye disease Todd Scheetz et al. Presented by John MC Ma

2. Aims To determine loci related to genes expressed in rat eye by expression quantitative trait loci (eQTL) To verify certain undescribed genes by r 2 correlations on members of its gene groups

3. 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

4. Methods: Choosing rat strains Several criteria –Highly inbred –Readily available –Completely genotyped –Diverse origins between strains –Phenotypically normal SR/JrHsd and SHRSP were chosen

5. Methods: Rat breeding and Genotyping SR/JrHsd males and SHRSP females were mated to produce F 1 ; F 1 was interbred to produce 120 F 2. RNA was extracted from eye of 12-week- old F 2 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

6. 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

7. Results of eQTL 1,330 genes linked to at least one locus False discovery rate is 1.4%

8. 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

9. 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

10. 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

11. 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

12. Gene-linking locus (2) The locus having most of non- contiguous is D9Rat46, linked to 21 genes

13. 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

14. 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

15. 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

16. 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

17. 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

18. Correlating gene expressions Pearson’s r 2 statistics was used to calculate the correlation between different class of genes, compared to randomly selected groups Increased, but statistically insignificant increase in r 2 in functional groups

19. 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

20. 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

21. 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

22. 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 r 2 tests

23. 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

24. 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

25. End