Integration of genomics and transcriptomics predicts diabetic retinopathy susceptibility genes

  1. Andrew D Skol
  2. Segun C Jung
  3. Ana Marija Sokovic
  4. Siquan Chen
  5. Sarah Fazal
  6. Olukayode Sosina
  7. Poulami P Borkar
  8. Amy Lin
  9. Maria Sverdlov
  10. Dingcai Cao
  11. Anand Swaroop
  12. Ionut Bebu
  13. DCCT/EDIC Study group
  14. Barbara E Stranger
  15. Michael A Grassi

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Abstract

We determined differential gene expression in response to high glucose in lymphoblastoid cell lines derived from matched individuals with type 1 diabetes with and without retinopathy. Those genes exhibiting the largest difference in glucose response were assessed for association with diabetic retinopathy in a genome-wide association study meta-analysis. Expression quantitative trait loci (eQTLs) of the glucose response genes were tested for association with diabetic retinopathy. We detected an enrichment of the eQTLs from the glucose response genes among small association p-values and identified folliculin ( FLCN ) as a susceptibility gene for diabetic retinopathy. Expression of FLCN in response to glucose was greater in individuals with diabetic retinopathy. Independent cohorts of individuals with diabetes revealed an association of FLCN eQTLs with diabetic retinopathy. Mendelian randomization confirmed a direct positive effect of increased FLCN expression on retinopathy. Integrating genetic association with gene expression implicated FLCN as a disease gene for diabetic retinopathy.

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  1. Version published to 10.7554/elife.59980 on eLife
  2. eLife

    ###Reviewer #2:

    This study investigated gene expression profiles related to diabetic retinopathy by using several strategies. First, they tested differential gene expression associated with response to glucose by comparing lymphoblastoid cell lines (LCLs) between cases (with retinopathy) and controls (without retinopathy) with type 1 diabetes. Secondly, they identified significant eQTLs from gene expression analysis and public gene expression databases and then tested significant eSNPs by the meta-analysis GWAS using independent cohorts. Furthermore, they confirmed one gene expression, the FLCN gene, to be a mediator of diabetic retinopathy by the Mendelian Randomization method. The aims of the study are clear and the paper is well organized. However, the following points should be addressed.

    Comments:

    1. It is confusing that the authors used different selection criteria for gene identifications. In Results (Line 472), they identified 19 differential response genes (P <0.05) between retinopathy cases and controls. However, they have selected the top 103 genes with P<0.01 (Results, Line 494) for further investigation. The reason for this is unclear. I assume that the FLCN gene is in the top 103 gene set but not in the above 19 gene set. Explanations are needed for including specific genes for different analysis purposes.

    2. The authors selected LCLs from individuals of 3 groups, non-diabetes (nDM), type 1 diabetes without retinopathy (nDR) and type 1 diabetes with proliferative diabetic retinopathy (PDR). I didn't see much benefit of utilizing nDM samples in the analysis. Although both gene expression and GSEA methods were conducted, the results were not relevant to diabetic retinopathy. What is the purpose of including these samples?

    3. Similarly, it is not clear what the purpose of using the gene set enrichment analysis (GSEA) was. My understanding is that the authors performed most analyses to identify genetic components by gene-based or SNP-based methods in the manuscript.

    4. The authors tested gene expression profile and associations using data from type 1 diabetic retinopathy. However, for the confirmation with UK BioBank (UKBB) data, they included all samples with both type 1 and type 2 diabetes. Did you perform the analysis stratified by the type of diabetes? Do you have any explanations of possible differences?

  3. eLife

    ###Reviewer #1:

    This paper is based on the analysis of a blood cell line of 22 subjects from three different groups in relation to diabetic eye disease. It includes first a transcriptome analysis based on microarrays. Then the studies are mainly based on bioinformatics analyses with GWAS meta-analysis and GTEx data extraction. The in silico study is followed by a so-called validation in the UK biobank.

    The overall strategy is sound and the paper well written. It remains that the whole paper and it’s conclusions are based on a very small number of samples and not supported by strong experimental data about causality. This reviewer is surprised that the title only focused on "Mendelian randomization", which is an overstatement of this gene expression study. In addition stating that RM "identifies folliculin expression as a mediator of diabetic retinopathy" is also an overstatement for this reviewer (the mediator effect is not shown). Overall, the small group of studied subjects present huge differences in duration of diabetes and glucose control, the 2 main risk factors for retinopathy. How can you differentiate the biological effects of long term high glucose and their impact on retinopathy? In other words is it possible to change the title to "Mendelian randomization identifies folliculin expression as a mediator of long term uncontrolled diabetes"?

    Based on the transcriptome analysis this reviewer is afraid that the conclusion "This finding suggests that chronic glucose exposure depresses cellular immune responsiveness and may explain in part the increased risk of infection found in patients with diabetes" is not based on evidence as authors selected transcripts of their choice and also because causality is not shown. "Individuals with diabetic retinopathy exhibit a differential transcriptional response to glucose". Note that the level of association shown (especially for PDGF) is somewhat marginal. "Genes with differential response to glucose are implicated in the pathogenesis of diabetic retinopathy." This part is the most intriguing and original but it is based on expression in many tissues and thus the title is also overstated: it shows some kind of association but certainly not that these 103 genes "are implicated" in retinopathy.

    "Folliculin (FLCN) is a putative diabetic retinopathy disease gene" this part is also interesting (and includes some in vivo experiments) but this reviewer wants to stress that the original whole genome gene expression study did not detect FLNC as differentially expressed in the blood cells of the patients with retinopathy. Why?

    It is also noteworthy that to this reviewer's knowledge no GWAS found SNPs near FLCN associated with diabetes or complications. This is worrying.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    This study investigates gene expression profiling related to diabetic retinopathy using several strategies including differential gene expression associated with response to glucose by comparing lymphoblastoid cell lines (LCLs) between cases (with retinopathy) and controls (without retinopathy) with type 1 diabetes. The study identified significant eQTLs from gene expression analysis and public gene expression databases and then tested significant eSNPs by the meta-analysis GWAS using independent cohorts. The expression of one gene, FLCN, to be a mediator of diabetic retinopathy by the Mendelian Randomization method was confirmed.

  5. Version published to 10.1101/2020.06.09.143164v1 on bioRxiv