Mitochondrial haplogroups H1, H2 and UK have previously been reported to be associated with proliferative diabetic retinopathy (PDR) in Caucasian patients with diabetes. We aimed to replicate this finding with a larger sample and expand the analysis to include different severities of DR, and diabetic macular edema (DME). Caucasian participants (n = 2935) with either type 1 or type 2 diabetes from the Australian Registry of Advanced Diabetic Retinopathy were enrolled in this study. Twenty-two mitochondrial single nucleotide polymorphisms were genotyped by MassArray and haplogroups reconstructed using Haplogrep. Chi square tests and logistic regressions were used to test associations between haplogroup and DR phenotypes including any DR, non-proliferative DR (NPDR), proliferative DR (PDR) and DME. After stratifying the samples in type 1 and type 2 diabetes groups, and adjusting for sex, age, diabetes duration, concurrent HbA1c and hypertension, neither haplogroups H1, H2, UK, K or JT were associated with any DR, NPDR, PDR or DME.
Diabetic retinopathy (DR) is a leading cause of vision loss from diabetes driven damage to the retina. It is becoming increasingly prevalent in spite of better risk factor control and screening1. Globally, from 1990 to 2010, visual impairment attributable to diabetes increased by 64% from 2.3 million to 3.7 million2. Vision loss occurs from proliferative diabetic retinopathy (PDR) and diabetic macula edema (DME). PDR is the most severe form of DR and is characterized by the growth of pathological vessels in the retina. DME can occur at any stage of DR and is characterized by oedema in the macula region of the retina.
DR has a complex genetic component3. While several studies have explored genes involved in inflammation and angiogenesis related pathways (such as vascular endothelial growth factor), little research has focused on the role of mitochondrial DNA (mtDNA) in the susceptibility of DR4,5. It is well established that oxidative stress plays a key role in the pathogenesis of diabetic complications, including DR6. A significant source of reactive oxygen species (ROS) is from the mitochondria. Mitochondrial overproduction of ROS is hypothesized to be the single upstream event that mediates multiple mechanisms of hyperglycemia induced damage to tissues including polyol pathway flux, increased formation of advanced glycation end products (AGEs), increased expression of AGE receptors and activating ligands, activation of protein kinase C isoforms and overactivity of the hexosamine pathway7. Furthermore, mtDNA is highly sensitive to oxidative damage and has a high mutation rate with implications for electron transport chain function and endothelial cell survival, even long after the initial hyperglycemic insult8,9,10.
A common classification system for mtDNA variation is mitochondrial haplogroup, which represents the major branch points on the mitochondrial phylogenetic tree of human evolution. Estopinal et al. reported that haplogroups H1, H2 and UK in a Caucasian sample (n = 392) were associated with PDR11. Haplogroup H1 and H2 were risk factors for the development of PDR from non-proliferative diabetic retinopathy (NPDR), while haplogroup UK was protective against PDR. Subsequently, Bregman et al. reported similar findings in a larger group from the same population (n = 637), and reported further that while mitochondrial haplogroup was associated with PDR, it was not associated with DR more generally12. A different case control study (149 with any type of DR and 78 with no DR) found a higher prevalence of haplogroup T in those with any DR (12.1% vs 5.1%; p = 0.046)13.
We sought to replicate these studies in a larger sample (n = 2935) with increased power to explore other DR phenotypes such as DME and to evaluate this association in participants with type 1 and type 2 diabetes mellitus.
This project has been approved by the human research ethics committees (HRECs) in Australia (Southern Adelaide Clinical HREC, Royal Adelaide Hospital HREC, The Queen Elizabeth Hospital HREC, Royal Melbourne Hospital HREC, Royal Victorian Eye and Ear Hospital HREC, St. Vincent’s Hospital (Melbourne) HREC, South Eastern Sydney Illawarra HREC, Tasmania Health and Medical HREC) and the NHS Health Research Authority in London. It adheres to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each participant before study enrolment.
Recruitment of patients and data collection
This study was carried out among Caucasian participants (identifying as of European descent) recruited in the Australian Registry of Advanced Diabetic Retinopathy (RADAR) and the Genetic Study of Diabetic Retinopathy based at Flinders University, South Australia. Multiple recruitment centres were involved and included the following Australian hospitals; Flinders Medical Centre, The Repatriation General Hospital, The Royal Adelaide Hospital, The Queen Elizabeth Hospital, The Royal Melbourne Hospital, Royal Victorian Eye and Ear Hospital, St. Vincent’s Hospital, Sydney Eye Hospital, Canberra Hospital, Royal Hobart Hospital, and from the United Kingdom; The National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom.
Eligible participants were actively recruited from ophthalmology, diabetes and renal clinics, with the following inclusion criteria: 1) type 1 (T1DM) or type 2 diabetes mellitus (T2DM). Those with T2DM must have received at least 5 years of medical treatment for diabetes (oral hypoglycemic agents or insulin) prior to enrolment, and must have been over 18 years of age. All participants underwent a questionnaire and venous blood sample collection for DNA analysis. Clinical information was collected from case notes and electronic records, including the average of three most recent, available HbA1c measurements (or three measurements immediately prior to a diagnosis of PDR), renal and lipid measures, medications and the presence of non-ocular diabetic complications. DR grading (defined as the worst ever grading) and the presence of DME were determined from documented dilated fundus exams performed by an ophthalmologist. DR grading was defined by the International Clinical DR Severity Scale14. Clinically significant macula edema (CSME) was defined according to the Early Treatment Diabetic Retinopathy Study protocol: 1) retinal thickening within 500 μm of the center of the macula, 2) hard exudates at or within 500 μm of the centre of the macular if associated with thickening of the adjacent retina or 3) retinal thickening 1 disc area in size, within 1 disc diameter of the centre15. Sight threatening DR was defined as either severe NPDR, PDR or CSME.
For each participant, approximately 8 mL of blood was collected in EDTA blood collection tubes and underwent DNA extraction using the QIAamp Blood DNA Maxi Kits (Qiagen, Venlo, The Netherlands). More detail regarding the data collection method has been described previously16.
Genotyping and mitochondrial haplogroup determination
Genotyping was performed through the Australian Genome Research Facility (AGRF), using the Agena Bioscience MassARRAY platform. We utilized the same panel of 22 mtDNA SNPs designed by Estopinal et al. in previous studies to determine mitochondrial haplogroup (Supplementary Table 1)11. Haplogrep software was used to facilitate haplogroup identification17. Samples identified as non-Caucasian after haplogroup determination were removed from the analyses.
Statistical analysis was performed with Statistical Package for Social Sciences versions 23.0 (For Windows; IBM Corp, Armonk, NY). Chi Square tests were performed to analyse the association of haplogroup type with various DR phenotypes such as any DR, any NPDR, PDR, DME and CSME. Logistic regression was used to adjust for covariates age, sex, type of diabetes, duration of diabetes, HbA1c and presence of hypertension. Statistical significance was taken at p < 0.05. Further analysis was performed by stratifying the analysis into T1DM and T2DM cohorts, and the major European haplogroups (H1 and H2, UK).
Patient demographics (n = 2935) stratified by DR phenotype are presented in Table 1. Chi square tests and Mann-Whitney U tests were used to compare demographic variables between the different phenotype groups (Table 2). Diabetes duration, HbA1c and hypertension were associated with all subtypes of DR and DME. Diabetes duration and HbA1c significantly increased from no DR to NPDR, PDR, DME or CSME(p < 0.0001, Mann-Whitney U test) and similarly from NPDR to PDR. Type of diabetes was also a significant variable affecting DR phenotypes PDR, any DME and CSME. Participants with PDR were younger than those with NPDR (median 59 versus 64 years, p < 0.0001).
Mitochondria haplogroup and DR phenotype
A total of 7 European mitochondrial haplogroups were identified in our Caucasian sample. The most common ones were haplogroup H1 and H2 (analysed collectively) and UK at 50.8% and 22.5% respectively. Other types included JT (12.4%), R (7.1%), I (4.2%), W (2.0%) and X (1.0%) (Supplementary Table 2). One SNP (rs3088053, rCRS position 11812) failed genotyping and therefore Haplogroup T2 could not be identified in our samples. As T2 is a subtype of J, we have therefore combined haplogroups J, T1 (and T2) in our analyses.
We found the percentages of the three most common haplotype groups (H1 and H2, UK and JT) were distributed similarly in each of the different phenotype groups, and that any differences when compared with no DR controls were not statistically significant after performing Chi Square association tests (Table 3). We also found no significant associations when haplogroups were compared between NPDR and PDR. There were no significant differences when all 7 haplogroups were analysed separately instead of grouping less common haplogroups into one category.
After separating the samples per diabetes type, the majority were T2DM participants (n = 2265) compared with T1DM participants (n = 670). The demographics of the T1DM and T2DM groups are given in Supplementary Tables 3 and 5 respectively. P values comparing the demographic variables between cases and controls in the T1DM and T2DM groups are given in Supplementary Table 5 and 6 respectively. Duration of diabetes, HbA1c and the presence of hypertension were significantly increased from no DR to NPDR, PDR, DME or CSME (p < 0.01, Mann-Whitney U test) and similarly from NPDR to PDR in both types of diabetes. Binary logistic regression show that haplogroups H1 and H2, and UK were not associated with any DR phenotypes in either T1DM or T2DM after adjustment for sex, age, diabetes duration, HbA1c and hypertension (Tables 4 and 5). After logistic regression, diabetes duration and HbA1c remain significant risk factors for DR in both type 1 and type 2 diabetes, while hypertension only remained significant in type 1 diabetes.
The next most common haplogroups (JT and K separately from UK) were analysed separately (frequencies 12.4% and 7.8% respectively). Significant results were: haplogroup K was nominally associated with any DR (135 cases, 98 controls, OR 0.49, 96% CI 0.24–1.00, p = 0.05) and NPDR (85 cases, 98 controls, OR 0.31, 95% CI 0.13–0.78, p = 0.012). JT was nominally associated with NPDR (144 cases, 137 controls, OR 2.20, 95%CI 1.09–4.43, p = 0.027) and CSME (85 cases, 137 controls, OR 2.06, 95% CI 1.16–8.08, p = 0.024) These results should be treated with caution as the numbers are small and the association does not survive correction for multiple hypothesis testing.