Mitochondrial haplogroups are not associated with diabetic retinopathy in a large Australian and British Caucasian sample

Abstract

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.

Full text

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SCIENTIFIC REPORTS | (2019) 9:612 | DOI:10.1038/s41598-018-37388-8
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Mitochondrial haplogroups are
not associated with diabetic
retinopathy in a large Australian
and British Caucasian sample
Ebony Liu1, Georgia Kaidonis1, Mark C. Gillies2, Sotoodeh Abhary1, Rohan W. Essex
3,
John H. Chang4,5, Bishwanath Pal5, Mark Daniell6, Stewart Lake1, Jolly Gilhotra7,
Nikolai Petrovsky
8, Alex W. Hewitt
9, Alicia Jenkins10, Ecosse L. Lamoureux9,13,
Jonathan M. Gleadle
11, Kathryn P. Burdon
1,12 & Jamie E. Craig1
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 nding with a larger sample and expand the analysis to include dierent 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 becom-
ing 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 inammation
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 signicant 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
1Department of Ophthalmology, Flinders University, Flinders Medical Centre, Adelaide, South Australia, Australia.
2Save Sight Institute, Clinical Ophthalmology and Eye Health, the University of Sydney, Sydney, New South
Wales, Australia. 3Academic Unit of Ophthalmology, Australian National University, Canberra, Australia. 4School
of Medical Sciences, University of NSW, Sydney, New South Wales, Australia. 5Medical Retina Service, Moorelds
Eye Hospital, London, United Kingdom. 6Department of Ophthalmology, Royal Melbourne Hospital, Melbourne,
Victoria, Australia. 7Department of Ophthalmology, Royal Adelaide Hospital, Adelaide, South Australia, Australia.
8Department of Endocrinology, Flinders University, Flinders Medical Centre, Adelaide, South Australia, Australia.
9Centre for Eye Research Australia, University of Melbourne, Melbourne, Victoria, Australia. 10NHMRC Clinical Trials
Centre, University of Sydney, Camperdown, New South Wales and St Vincent’s Hospital, Fitzroy, Victoria, Australia.
11Department of Renal Medicine, College of Medicine and Public Health, Flinders University, Adelaide, South
Australia, Australia. 12Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia.
13Duke-NUS Medical School, Singapore, Singapore. Kathryn P. Burdon and Jamie E. Craig contributed equally.
Correspondence and requests for materials should be addressed to E.L. (email: ebony.liu@inders.edu.au)
Received: 11 June 2018
Accepted: 27 November 2018
Published: xx xx xxxx
OPEN
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SCIENTIFIC REPORTS | (2019) 9:612 | DOI:10.1038/s41598-018-37388-8
pathway ux, increased formation of advanced glycation end products (AGEs), increased expression of AGE recep-
tors 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 aer the initial hyperglycemic insult8–10.
A common classication 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 hap-
logroup UK was protective against PDR. Subsequently, Bregman et al. reported similar ndings 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 dierent 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.
Methods
Ethics statement. is project has been approved by the human research ethics committees (HRECs) in
Australia (Southern Adelaide Clinical HREC, Royal Adelaide Hospital HREC, e 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. is study was carried out among Caucasian partici-
pants (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, e
Repatriation General Hospital, e Royal Adelaide Hospital, e Queen Elizabeth Hospital, e 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; e 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). ose 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
(dened as the worst ever grading) and the presence of DME were determined from documented dilated fundus
exams performed by an ophthalmologist. DR grading was dened by the International Clinical DR Severity Scale14.
Clinically signicant macula edema (CSME) was dened 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 dened as either severe NPDR, PDR or CSME.
For each participant, approximately 8 mL of blood was collected in EDTA blood collection tubes and under-
went DNA extraction using the QIAamp Blood DNA Maxi Kits (Qiagen, Venlo, e 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 hap-
logroup (Supplementary Table1)11. Haplogrep soware was used to facilitate haplogroup identication17. Samples
identied as non-Caucasian aer haplogroup determination were removed from the analyses.
Statistical 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 hap-
logroup 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 hyperten-
sion. Statistical signicance 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).
Results
Patient demographics (n = 2935) stratied by DR phenotype are presented in Table1. Chi square tests and
Mann-Whitney U tests were used to compare demographic variables between the dierent phenotype groups
(Table2). Diabetes duration, HbA1c and hypertension were associated with all subtypes of DR and DME.
Diabetes duration and HbA1c signicantly 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 signicant variable aecting
DR phenotypes PDR, any DME and CSME. Participants with PDR were younger than those with NPDR (median
59 versus 64 years, p < 0.0001).
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SCIENTIFIC REPORTS | (2019) 9:612 | DOI:10.1038/s41598-018-37388-8
Mitochondria haplogroup and DR phenotype. A total of 7 European mitochondrial haplogroups were
identied in our Caucasian sample. e 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 identied in our samples. As T2 is a subtype of J, we have therefore combined hap-
logroups 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 dis-
tributed similarly in each of the dierent phenotype groups, and that any dierences when compared with no DR
controls were not statistically signicant aer performing Chi Square association tests (Table3). We also found
no signicant associations when haplogroups were compared between NPDR and PDR. ere were no signicant
dierences 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) com-
pared 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 signicantly 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 aer adjustment for sex, age, diabetes duration, HbA1c and hypertension (Tables4 and 5).
Aer logistic regression, diabetes duration and HbA1c remain signicant risk factors for DR in both type 1 and
type 2 diabetes, while hypertension only remained signicant in type 1 diabetes.
e next most common haplogroups (JT and K separately from UK) were analysed separately (frequencies 12.4%
and 7.8% respectively). Signicant results were: haplogroup K was nominally associated with any DR (135 cases,
Demographic Tot a l No DR Any DR Any NPDR PDR Any DME CSME Sight threatening
n 2935 1124 1811 1161 650 936 643 1278
Female; n (%) 1309 (44.6) 521 (46.5) 788 (43.7) 514 (44.4) 274 (42.4) 420 (45.5) 289 (45.2) 558 (43.9)
Age in yrs; median (range) 61 (17–95) 65 (17–95) 63 (18–95) 65 (18–95) 59 (21–90) 65 (21–92) 65 (26–92) 63 (21–92)
Diabetes duration in yrs; median (range) 18 (5–70) 12 (5–67) 20 (5–70) 18 (5–65) 23 (5–70) 19 (5–64) 19 (5–59) 20 (5–70)
Type 1 diabetes; n (%) 670 (22.8) 239 (21.5) 431 (24.1) 208 (18.2) 223 (34.6) 148 (16.0) 100 (15.7) 297 (23.5)
HbA1c %; median (range) 8.07 (2–22) 7.40 (2–22) 8.10 (4–15) 7.90 (5–15) 8.50 (4–15) 8.20 (4–15) 8.10 (5–15) 8.25 (4–15)
Hypertension; n (%) 1935 (65.9) 695 (61.8) 1240 (68.5) 782 (67.4) 458 (70.5) 642 (68.6) 471 (73.3) 874 (68.4)
Table 1. Demographics of study population stratied by diabetic retinopathy phenotype.
No DR vs
any DR No DR vs
any NPDR No DR vs
PDR No DR vs
DME No DR vs
CSME No DR vs Sight
threatening
Sex 0.077 0.333 0.102 0.563 0.619 0.216
Age 0.103 0.190 <0.0001 0.761 0.848 0.041
Diabetes duration <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Diabetes type 0.103 0.051 <0.0001 1.79 × 10−33.79 × 10−30.258
HbA1c <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Hypertension 3.74 × 10−42.91 × 10−31.18 × 10−32.36 × 10−3<0.0001 1.37 × 10−3
Table 2. P values of demographic variables compared between the DR phenotype groups.
Haplogroup
Proportion (%) P value (Chi Square tests)
comparison to controls (No DR)H UK JT I, R,W,X
No DR 50.7 22.5 12.2 14.6 NA
Any DR 50.4 22.9 12.9 13.8 0.885
NPDR 50.9 22.9 12.4 13.8 0.954
PDR 49.5 22.8 13.8 13.8 0.760
DME 51.0 23.3 12.5 13.2 0.830
CSME 49.6 23.3 13.2 13.8 0.867
Sight
threatening DR 50.8 23.2 12.9 13.1 0.721
NPDR 50.4 22.9 12.8 13.9 0.793 (compared with PDR)
Table 3. Haplogroup distribution (H, UK, JT, Other) according to DR phenotype.
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SCIENTIFIC REPORTS | (2019) 9:612 | DOI:10.1038/s41598-018-37388-8
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) ese results should be treated with cau-
tion as the numbers are small and the association does not survive correction for multiple hypothesis testing.
Discussion
In our larger Caucasian sample, unlike earlier smaller studies, we found no signicant associations between mito-
chondrial haplogroup and the presence of any DR, DME, nor more severe phenotypes such as PDR, CSME or
sight-threatening DR (severe NPDR, CSME or PDR). is was true for analysis as a group or when stratied for
type of diabetes, in spite of following the same methods of previous studies which found positive associations.
Estopinal et al. rst demonstrated that haplogroups H1 and H2 (analysed collectively) and UK were associated
with PDR when compared with NPDR in an American Caucasian sample (n = 197 NPDR, 195 PDR)11. Having
either haplogroup H1 or H2 was a risk factor, while haplogroup UK was protective. Bregman et al. expanded
from this initial study with 513 additional diabetic controls in the same databases (Vanderbilt Eye Institute and
Vanderbilt University)12. ey found that haplogroup H1 and H2, and UK were not associated with any incident
DR compared with no DR. Using the same cohort, Mitchell et al. found duration of diabetes and HbA1c was
signicantly associated with PDR in haplogroups H1 and H2, but not UK, suggesting that mitochondrial hap-
logroups modify these clinical risk factors for the development of PDR in type 2 diabetes18. In a dierent study,
Koer et al. reported haplogroup T was signicantly associated with any DR compared with no DR (12.1% vs
5.1%; p = 0.046)13.
Inconsistent results are common in all areas of haplogroup association studies. For example Crispim et al.
reported haplogroup cluster J/T was signicantly associated with insulin resistance in a Caucasian Brazilian pop-
ulation19, but this was refuted by two other studies of Caucasian samples20,21. Challenges in interpreting mito-
chondrial association studies include dierences in study design, case and control denitions, statistical analysis,
population stratication, inadequate power and lack of replication22.
Dierent results could be due to dierent populations and study design, however in examining the demo-
graphics and distribution of the haplogroups, our group appears to be similar to the group from the Vanderbilt
Eye Institute and Vanderbilt University. Both groups consist of Caucasian patients of European descent. We used
the same criteria for selection of retinopathy cases and controls and the same statistical analyses. e most com-
mon haplogroups were H1 and H2, and UK; 73.3% in our study, compared with 68% in Bregman et al.'s study.
As expected in both studies, age, diabetes duration, type of diabetes and HbA1c were strongly associated with
increasing severity of DR.
An important reason why our results are dierent is because our study consisted of a much larger popula-
tion; 1124 diabetic retinopathy controls (no DR), 1161 NPDR cases and 650 PDR cases. erefore our study
has increased statistical power to identify any true associations. We were unable to replicate previously reported
OR (95% CI)
P-value No DR vs Any DR No DR vs
NPDR No DR vs PDR No DR vs DME No DR vs CSME No DR vs sight
threatening NPDR vs PDR
Haplogroup H – Type 1 Diabetes
Haplogroup H 0.90 (0.58–1.40)
P = 0.652 0.92 (0.57–1.48)
P = 0.718 0.95 (0.53–1.69)
P = 0.848 0.95 (0.52–1.77)
P = 0.881 0.79 (0.38–1.63)
P = 0.524 0.97 (0.58–1.63)
P = 0.918 0.98 (0.62–1.55)
P = 0.924
Sex (female) 1.34 (0.86–2.09)
P = 0.199 1.30 (0.80–2.10)
P = 0.294 1.44 (0.79–2.62)
P = 0.231 1.43 (0.77–2.64)
P = 0.255 1.54 (0.78–3.17)
P = 0.243 1.43 (0.85–2.41)
P = 0.182 0.84 (0.53–1.33)
P = 0.453
Age 1.02 (1.0–1.03)
P = 0.063 1.02 (1.0–1.04)
P = 0.011 0.99 (0.96–1.01)
P = 0.347 1.04 (1.02–1.06)
P = 3.81 × 10−31.03 (1.01–1.06)
P = 0.007 1.02(1.0–1.04)
P = 0.104 0.97 (0.95–0.99)
P = 0.001
Diabetes duration 1.13 (1.10–1.16)
P < 0.0001 1.10 (1.07–1.13)
P < 0.0001 1.14 (1.11–1.19)
P < 0.0001 1.08 (1.04–1.11)
P < 0.0001 1.08 (1.04–1.12)
P < 0.0001 1.13 (1.10–1.17)
P < 0.0001 1.08 (1.06–1.11)
P < 0.0001
HbA1c 1.46 (1.26–1.70)
P < 0.0001 1.38 (1.18–1.63)
P < 0.0001 1.54 (1.28–1.86)
P < 0.0001 1.57 (1.30–1.91)
P < 0.0001 1.49 (1.19–1.87)
P < 0.0001 1.52 (1.28–1.79)
P < 0.0001 1.25 (1.07–1.46)
P = 0.004
Hypertension 2.19 (1.30–3.68)
P < 0.0001 1.61 (0.90–2.87)
P = 0.108 4.34 (2.24–8.41)
P < 0.0001 2.60 (1.34–5.07)
P = 4.85 × 10−32.61 (1.19–5.73)
P = 0.017 2.82 (1.57–5.05)
P = 0.001 2.21 (1.31–3.73)
P = 0.003
Haplogroup UK – Type 1 Diabetes
Haplogroup UK 0.80 (0.49–1.32)
P = 0.391 0.68 (0.39–1.21)
P = 0.191 (0.53–1.91)
P = 0.989 0.65 (0.31–1.36)
P = 0.255 0.50 (0.21–1.24)
P = 0.136 0.88 (0.49–1.58)
P = 0.674 1.60 (0.93–2.75)
P = 0.087
Sex (female) 1.34 (0.86–2.09)
P = 0.201 1.28 (0.79–2.08)
P = 0.311 1.44 (0.79–2.62)
P = 0.231 1.46 (0.79–2.70)
P = 0.231 1.53 (0.74–3.16)
P = 0.251 1.43 (0.85–2.42)
P = 0.177 0.84 (0.53–1.34)
P = 0.457
Age 1.02 (1.0–1.03)
P = 0.064 1.02 (1.00–1.04)
P = 0.011 0.99 (0.96–1.01)
P = 0.351 1.04 (1.02–1.06)
P = 4.17 × 10−41.04 (1.00–1.06)
P = 0.007 1.02 (1.00–1.04)
P = 0.108 0.97 (0.95–0.99)
P = 0.001
Diabetes duration 1.13 (1.09–1.16)
P < 0.0001 1.10 (1.06–1.13)
P < 0.0001 1.14 (1.11–1.19)
P < 0.0001 1.08 (1.04–1.11)
P < 0.0001 1.08 (1.04–1.12)
P < 0.0001 1.13 (1.10–1.17)
P < 0.0001 1.08 (1.06–1.11)
P < 0.0001
HbA1c 1.46 (1.26–1.70)
P < 0.0001 1.39 (1.18–1.63)
P < 0.0001 1.54 (1.28–1.86)
P < 0.0001 1.56 (1.28–1.89)
P < 0.0001 1.47 (1.17–1.85)
P = 0.001 1.51 (1.28–1.79)
P < 0.0001 1.25 (1.07–1.46)
P = 0.004
Hypertension 2.22 (1.32–3.75)
P = 0.003 1.67 (0.93–2.98)
P = 0.086 4.34 (2.23–8.42)
P < 0.0001 2.75 (1.40–5.41)
P = 0.003 2.86 (1.28–6.40)
P = 0.011 2.85 (1.58–5.13)
P = 4.80 × 10−42.27 (1.35–3.84)
P = 0.002
Table 4. Association of haplogroup H (H1 and H2) and UK with DR in type 1 diabetes: binary logistic
regression adjusting for gender, age, diabetes duration and HbA1c.
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associations, suggesting that these previous association may be false. Smaller studies and sub analyses of pheno-
type groups lead to a higher risk of type 1 errors23. Our larger study size allowed us to analyse other phenotypes
such as DME and CSME, as well as to separately analyse less common haplogroups such as JT, and K separate
from UK. e only statistically signicant results we found were haplogroup K was nominally associated with
any DR, and haplogroup JT was nominally associated with NPDR and CSME. As the numbers were small in
these comparisons and the result does not survive multiple hypothesis testing, this is likely to be type 1 error.
Haplogroup K was not a common haplogroup in the previous two studies and is not implicated in diabetes and
other associated diseases. Koer et al. reported haplogroup T was signicantly associated with any DR but this
study also had a much smaller sample size (149 with any DR and 78 with no DR)13. As noted in our results, we were
unable to separately analyse haplogroup T due to genotyping failure, and so direct comparison to Koer et al.’s
study could not be made.
In addition to study size, strengths of this study include the inclusion of both T1DM and T2DM subjects from
multiple sites, rigour of retinopathy status characterisation, wide range of levels of DR and use of the same haplo-
typing methods and statistical analyses as previous studies so comparisons could be made.
e haplotyping method we utilized from previous studies has an important limitation. SNPs chosen to rep-
resent the H haplogroup (rCRS position 3010 and 1438) only identify haplogroups H1 and H2. erefore 7 other
major subtypes of haplogroup H were not analysed. In our study, one SNP completely failed genotyping (rCRS
11812, determination of haplogroup T2) and therefore we could not analyse haplogroup T separately. Another
4 SNPs had a 2% failure rate, and this could have contributed to the percentage of samples with haplogroup R (a
major clade consisting of H, J, T, and UK). We chose our Caucasian sample based on participants self-identifying
as Caucasian, but a small number had non-Caucasian haplogroups (for example haplogroup A, B, C, L, M, N and
Q). Some of the 22 SNPs chosen for haplogroup determination are also found in other ethnic populations (for
example rCRS position 3197 determines U5 but also L3e3 which is found in Asian populations). erefore, all
samples with a non-Caucasian haplogroup were removed to minimize any confounding eect and reduce popu-
lation stratication.
We recognise that even larger studies and studies in dierent ethnic groups, particularly those at high risk of
diabetic retinopathy, are desirable. We only studied 7 haplogroups, while the human mitochondrial phylogenetic
tree consists of hundreds of haplogroups. e hypothesis that variations in mitochondrial genetics contribute
to DR risk is logical given the role that the mitochondria play in oxidative stress7 and their presence in the ret-
ina24. Mitochondrial DNA are inherited completely from the maternal line, unlike nuclear DNA which has equal
maternal and paternal contributions25. Risk of T1DM in the ospring varies by parental status; being two-fold
lower if the mother has T1DM rather than the father26. Epidemiology studies show that certain ethnicities are
at greater risk of DR such as people of Asian, African and Indigenous ethnic groups27. Complex biological and
environmental factors explain this observation, and mitochondrial genetics could also play a role, but this has
not been studied.
OR, 95% CI, P
value No DR vs Any
DR No DR vs NPDR No DR vs PDR No DR vs DME No DR vs
CSME No DR vs sight
threatening NPDR vs PDR
Haplogroup H – Type 2 Diabetes
Haplogroup H 1.03 (0.83–1.28)
P = 0.777 1.07 (0.85–1.34)
P = 0.571 0.92 (0.66–1.27)
P = 0.612 1.04 (0.80–1.34)
P = 0.780 0.91 (0.69–1.21)
P = 0.521 1.04 (0.81–1.32)
P = 0.763 0.93 (0.69–1.23)
P = 0.598
Sex (female) 0.75 (0.60–0.93)
P = 0.008 0.76 (0.60–0.96)
P = 0.019 0.70 (0.50–0.97)
P = 0.033 0.87 (0.67–1.12)
P = 0.270 0.87 (0.65–1.15)
P = 0.328 0.79 (0.63–1.01)
P = 0.064 0.95 (0.70–1.27)
P = 0.711
Age 0.98 (0.97–0.99)
P < 0.0001 0.99 (0.98–1.0)
P = 0.016 0.96 (0.94–0.97)
P < 0.0001 0.98 (0.97–0.99)
P = 4.81 × 10−40.98 (0.97–0.99)
P = 0.002 0.97 (0.96–0.99)
P < 0.0001 0.97 (0.96–0.98)
P < 0.0001
Diabetes duration 1.09 (1.08–1.11)
P < 0.0001 1.08 (1.07–1.10)
P < 0.0001 1.12 (1.10–1.15)
P < 0.0001 1.10 (1.08–1.12)
P < 0.0001 1.10 (1.08–1.12)
P < 0.0001 1.11 (1.09–1.12)
P < 0.0001 1.04 (1.02–1.06)
P < 0.0001
HbA1c 1.37 (1.27–1.48)
P < 0.0001 1.33 (1.22–1.44)
P < 0.0001 1.50 (1.34–1.67)
P < 0.0001 1.45 (1.32–1.58)
P < 0.0001 1.44 (1.30–1.60)
P < 0.0001 1.45 (1.33–1.58)
P < 0.0001 1.15 (1.06–1.26)
P = 0.001
Hypertension 0.96 (0.74–1.24)
P = 0.742 0.94 (0.71–1.25)
P = 0.681 0.96 (0.65–1.43)
P = 0.846 0.77 (0.57–1.04)
P = 0.092 1.06 (0.74–1.51)
P = 0.758 0.83 (0.62–1.11)
P = 0.208 1.01 (0.71–1.44)
P = 0.943
Haplogroup UK – Type 2 Diabetes
Haplogroup UK (0.77–1.29)
P = 0.981 0.98 (0.74–1.29)
P = 0.863 1.13 (0.76–1.67)
P = 0.541 1.08 (0.80–1.46)
P = 0.630 1.13 (0.81–1.59)
P = 0.464 1.03 (0.77–1.38)
P = 0.849 1.07 (0.75–1.51)
P = 0.714
Sex (female) 0.75 (0.60–0.93)
P = 0.008 0.76 (0.60–0.95)
P = 0.018 0.70 (0.50–0.97)
P = 0.034 0.87 (0.67–1.12)
P = 0.267 0.87 (0.66–1.16)
P = 0.348 0.79 (0.62–1.01)
P = 0.063 0.95 (0.70–1.27)
P = 0.712
Age 0.98 (0.97–0.99)
P < 0.0001 0.99 (0.98–1.0)
P = 0.016 0.96 (0.94–0.97)
P < 0.0001 0.98 (0.97–0.99)
P = 4.73 × 10−40.98 (0.97–0.99)
P = 0.002 0.97 (0.96–0.99)
P < 0.0001 0.97 (0.96–0.98)
P < 0.0001
Diabetes duration 1.09 (1.08–1.11)
P < 0.0001 1.08 (1.07–1.10)
P < 0.0001 1.12 (1.10–1.15)
P < 0.0001 1.10 (1.08–1.12)
P < 0.0001 1.10 (1.08–1.12)
P < 0.0001 1.11 (1.09–1.12)
P < 0.0001 1.04 (1.02–1.06)
P < 0.0001
HbA1c 1.37 (1.27–1.48)
P < 0.0001 1.33 (1.22–1.44)
P < 0.0001 1.50 (1.34–1.67)
P < 0.0001 1.45 (1.32–1.58)
P < 0.0001 1.44 (1.30–1.60)
P < 0.0001 1.45 (1.33–1.58)
P < 0.0001 1.15 (1.06–1.26)
P = 0.001
Hypertension 0.96 (0.74–1.25)
P = 0.746 0.95 (0.72–1.25)
P = 0.695 0.96 (0.64–1.43)
P = 0.839 0.77 (0.57–1.04)
P = 0.091 1.05 (0.74–1.50)
P = 0.772 0.83 (0.62–1.11)
P = 0.207 1.01 (0.71–1.44)
P = 0.953
Table 5. Association of haplogroup H and UK with DR in type 2 diabetes: binary logistic regression adjusting
for gender, age, diabetes duration and HbA1c.
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SCIENTIFIC REPORTS | (2019) 9:612 | DOI:10.1038/s41598-018-37388-8
Mitochondrial haplogroup is not a specic marker for mitochondrial genetic variability. A haplogroup con-
sists of many genetic variants that are inherited together. erefore, if there are specic mitochondrial variants
that contribute to DR, these cannot be studied eectively. Single nucleotide polymorphisms in mitochondria
cause diseases such as Leber hereditary optic neuropathy, and in complex diseases such as diabetes and cancer,
it is increasingly recognised that small mitochondrial defects could lead to subtle bioenergetics alterations with
major clinical implications28. Specic mitochondrial variants that have been studied for DR include mutations in
UCP2 and Mn-SOD genes4.
Few studies have demonstrated whether mitochondrial haplogroup directly aects mitochondrial function.
Fang et al. recently reported lower respiratory chain complex activity in haplogroup N9a compared with D4j,
G4a2 and Y1, using transmitochondrial technology29. Mueller et al. reported mitochondrial haplogroup T cell
cybrids had a higher survival rate than haplogroup H cybrids under oxidative stress conditions such as when
challenged with hydrogen peroxide30. Haplogroup K cybrids showed dierent gene expression levels compared
with H cybrids aer amyloid-beta toxicity31. Untreated retinal cell cybrids of H and J haplogroups also showed
dierent gene expression and methylation status32. Future studies and techniques designed to explore the mito-
chondria genome in better detail than currently available can help us understand whether mitochondrial genetics
contribute to DR risk33.
Conclusion
In contrast to previous studies, our much larger study found no association between the major European mito-
chondrial haplogroup H1, H2, UK, and DR phenotypes in either type 1 or type 2 diabetes. No signicant asso-
ciations were found for dierent severities of DR and DME, or other subsets of mitochondrial haplogroups that
were analysed by this study.
Data Availability
e dataset generated for analysis in the current study is available under a CC BY-NC-ND license at: https://doi.
org/10.25957/5c060cbdb9162.
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Acknowledgements
is work was funded by the National Health and Medical Research Council (NHMRC) of Australia (project
grant #595918), the Ophthalmic Research Institute of Australia, the National Institute for Health Research
(NIHR) Biomedical Research Centre at Moorelds Eye Hospital NHS Foundation Trust and UCL Institute of
Ophthalmology. e funding organization had no role in the design or conduct on of this study.
Author Contributions
E.L. wrote the main manuscript text and prepared the tables. All authors contributed to the recruitment of
participants from various sites for the study. All authors reviewed the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-37388-8.
Competing Interests: e authors declare no competing interests.
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