Experimental Eye Research 132 (2015) 59e63
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Mitochondrial DNA copy number, but not haplogroup is associated with keratoconus in Han Chinese population Xiao-Dan Hao a, Peng Chen a, Ye Wang a, Su-Xia Li b, Li-Xin Xie a, * a
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China b Shandong Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Jinan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 31 December 2014 Accepted in revised form 18 January 2015 Available online 19 January 2015
Oxidative stress may play a role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is closely related to mitochondrion function, and variations may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. To test whether mtDNA background and copy number confer genetic susceptibility to KC in the Han Chinese population, we performed this association study. We analyzed mtDNA sequence variations in 210 KC patients and 309 matched individuals from China, and classified each subject by haplogroup. Mitochondrial DNA copy number was measured in a subset of these subjects (193 patients and 103 controls). Comparison of matrilineal components of the cases and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that KC patients had significantly lower mtDNA copy numbers than controls (P ¼ 0.0002), even when age, gender, and mtDNA background were considered. Our results suggest that mtDNA copy number, but not haplogroup, is associated with keratoconus, and may contribute to its pathogenesis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Keratoconus mtDNA Copy number Haplogroup Association study
1. Introduction Keratoconus (KC) is a degenerative disorder of the cornea that is characterized by corneal ectasia, thinning, and cone-shaped protrusion, resulting in reduced vision, irregular astigmatism, and cornea scarring (Krachmer et al., 1984). Keratoconus is mostly bilateral but can be unilateral. Onset is usually in puberty, and the progression slows after age 30, arresting around the fourth decade of life (Rabinowitz, 1998). It affects both genders and all ethnicities (Kok et al., 2012). The prevalence of this disease is approximately 1 per 2000 in the general population (Kok et al., 2012; Rabinowitz, 1998). Owing to the limited availability of medical treatments, it is a significant clinical problem worldwide and a leading indication for corneal transplantation.
Abbreviations: KC, keratoconus; mtDNA, mitochondrial DNA; ROS, reactive oxygen species; OS, oxidative stress; OXPHOS, oxidative phosphorylation; LHON, Leber's hereditary optic neuropathy; rCRS, revised Cambridge reference sequence; SE, standard error; ANOVA, analysis of variance. * Corresponding author. No. 5 Yanerdao Road, Qingdao 266071, China. E-mail addresses: [email protected] (X.-D. Hao), chenpeng599205@ 126.com (P. Chen), [email protected] (Y. Wang), [email protected] (S.-X. Li), [email protected] (L.-X. Xie). http://dx.doi.org/10.1016/j.exer.2015.01.016 0014-4835/© 2015 Elsevier Ltd. All rights reserved.
The molecular pathogenesis of KC is poorly understood. Despite extensive research, the exact cause of keratoconus remains unknown in the majority of patients. Studies have suggested that oxidative stress (OS) may play a role in the pathogenesis of KC (Behndig et al., 2001). In their study, Chwa et al. (2006) found that keratoconus corneal stromal fibroblasts had increased reactive oxygen species (ROS) and superoxides compared with levels in normal cultures. Oxidative phosphorylation (OXPHOS) occurs within mitochondria and is a significant endogenous source of ROS (Nohl, 1994). Mitochondria have their own genome, mitochondrial DNA (mtDNA), which encodes 13 subunits of respiratory complexes I, III, IV, and V (Anderson et al., 1981; Andrews et al., 1999). Mitochondrial DNA is closely related with mitochondrion function, and variations may affect the generation of ROS and be involved in pathogenesis of KC. Mitochondrial DNA is inherited maternally and is characterized by an absence of recombination, and high mutation rate. It can accumulate mutations during evolution, and a group of mtDNA that shares certain mtDNA ancestral variants can be traced to distinguishable haplogroups (Kong et al., 2006; Torroni et al., 2006). Certain mtDNA polymorphisms that distinguish haplogroups and subhaplogroups, or their combinations, may be predisposing
60
X.-D. Hao et al. / Experimental Eye Research 132 (2015) 59e63
factors for certain diseases (Torroni and Wallace, 1994). Previous studies have observed that mtDNA haplogroup affects genetic susceptibility to many diseases, including Leber's hereditary optic neuropathy (LHON) (Carelli et al., 2006), type 2 diabetes (Fuku et al., 2007) and Wolfram syndrome (Hofmann et al., 1997). A recently study established the mitochondrial haplogroups in Saudi patients with KC and healthy controls of same ethnicity, and found that individuals with mitochondrial haplogroups H and R are at increased risk to develop KC (Abu-Amero et al., 2014). However, the relationship between mtDNA haplogroup and KC in Chinese population has not yet been studied. Furthermore, each cell has hundreds to thousands of copies of mtDNA molecules, and the genetic background and factors in the physiological environment can affect mtDNA copy number alterations (Moraes, 2001). It has been reported that mtDNA copy number is involved in some diseases, such as renal cell carcinoma (Xing et al., 2008), non-Hodgkin lymphoma (Lan et al., 2008) and leprosy (Wang et al., 2012). Accumulating evidence shows that mtDNA copy number control is very important for mitochondrial biogenesis and normal cellular function (Clay Montier et al., 2009). In this study, we hypothesized that mitochondrial DNA may confer a genetic susceptibility to KC, and this may be reflected by mtDNA genetic background and mtDNA copy number. Here, mtDNA haplogroup composition and copy number in KC patients and healthy controls were studied to test this hypothesis. Our results showed that mtDNA haplogroup had no association with KC in a Han Chinese population, and decreased mtDNA copy number was a predisposing factor for KC and contributed to its pathogenesis. 2. Materials and methods 2.1. Ethics statement
(14.4%). The mean age of the first control cohort was 26.42 years, and the proportion of males was 74.7%, which are similar to the patients' group. The second control cohort included 126 Chinese individuals from the general population of Shandong province (Table 1). The data for mtDNA polymorphisms, variants and, haplogroups for the second control cohort were collected from the literature (Yao et al., 2002, 2003). 2.3. Classification of haplogroups by sequencing Total DNA was isolated from whole blood or cornea using the standard phenol/chloroform method. DNA concentration was measured by BioPhotometer (Eppendorf, Hamburg, Germany). We classified each subject by haplogroup no matter which tissue was available. The D-loop region of the mtDNA was amplified, as described in previous literature (Hao et al., 2013). The products were purified with Alkaline Phosphatase (Shrimp) (Takara, Dalian, China) and Exonuclease I (Takara, Dalian, China) and were subjected to direct DNA sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing kit and ABI PRISM 3730 sequencer (Applied Biosystems Inc., USA). The sequencing primers contained four internal primers (L15996, L16209, H16347, and L29) (Wang et al., 2008; Yao et al., 2003). Sequences were aligned and analyzed with the DNASTAR software package (DNASTAR Inc., USA). Sequence variations in each patient were scored relative to the revised Cambridge reference sequence (rCRS) (Andrews et al., 1999), and the haplogroup of every individual was defined using the online software Mitotool (http://www.mitotool.org/index. html). Further coding-region polymorphisms in specific lineages were typed as described in the previous studies (Yao et al., 2002, 2004) to confirm their predicted phylogenetic status. 2.4. Measurement of mtDNA copy number
Ethical approval for the study was granted by the Ethics Committee of the Shandong Eye Institute, Qingdao, China. Written consent was obtained from each subject or the subject's guardian (for subjects under 18 years of age). 2.2. Samples A total of 210 unrelated Chinese KC patients and 309 unrelated controls were included in the present study. Patients affected with keratoconus, most of them from Shandong province, were recruited from the cornea clinics of Qingdao Eye Hospital and Shandong Eye Hospital. The cases were diagnosed on the basis of clinical examination (corneal stromal thinning, Vogt's striae, Fleischer's ring, Munson's sign, signs of videokeratography, and refractive errors). Cornea or blood samples from each patient were collected according to availability. The healthy controls contained two cohorts. The first control cohort included 183 individuals recruited at Qingdao Eye Hospital and Shandong Eye Hospital. These individuals had accidental injuries but no eye disease. A blood sample was collected from each individual. Controls were confirmed to have no sign of KC using the same examination protocol as used for the cases. The mean age of the KC patients was 21.01 years, and there were more males (85.6%) than females
The mtDNA content is inversely associated with age (He et al., 2014; Mengel-From et al., 2014), and different tissues have different mitochondrial content (Benard et al., 2006). To exclude these effects, we measured the mtDNA copy number in 193 blood samples of patients (mean age ± SE, 20.58 ± 0.431) and 103 controls of matched age (mean age ± SE, 21.18 ± 0.213) from the first control cohort. There was no health cornea as a control, so KC corneal mtDNA copy numbers were not measured. Relative mtDNA copy number was measured by fluorescence-based quantitative realtime PCR and the 2DDCT method, as described in previous studies (Bi et al., 2010; Wang et al., 2012). In brief, primer pair L394/H475 was used for measuring the mtDNA, and amplification of the singlecopy nuclear b-globin gene was used for normalization (Wang et al., 2012). The amplification assays were performed with FastStart Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany) on the Rotor-Gene Q Real-Time PCR system (QIAGEN, Germany), and results were obtained and analyzed using the RotorGene Q Series Software (version 2.1.0). The relative mtDNA copy number of each sample was calibrated by one sample of the controls (n025, Supplementary Table S1). To ensure data quality, samples with a standard deviation greater than 0.25 across replicates were measured again. 2.5. Statistical analysis
Table 1 Second control cohort's information. Geographic location and population
Abbreviation No. of subjects
References
Qingdao Shandong Taian Shandong
SD-QD SD-TA
Yao et al., 2002 Yao et al., 2003
50 76
Statistical analysis was performed using SPSS 17.0 (SPSS Inc., USA). Statistical significance was defined at a ¼ 0.05. A chi-squared (c2) test or Fisher's exact test, as appropriate, was used to assess differences in the distribution of each haplogroup between cases and controls. Each haplogroup was compared with all other haplogroups pooled into one group. Power calculations were
X.-D. Hao et al. / Experimental Eye Research 132 (2015) 59e63
performed using Quanto version 1.2.4 in our population. Allele frequency and optional inheritance modes are not applicable for mtDNA haplogroup, so we just focused on the final susceptibility frequency in the power calculation. We have 80% power to detect a risk of 2.07e1.67 or a resistance of 0.34e0.60 within a haplogroup with a frequency in the range of 10%e50%. Relative mtDNA copy number was analyzed as a continuous variable. Analysis of Variance (ANOVA) was used to determine the differences in mtDNA copy number in different groups. 3. Results 3.1. MtDNA haplogroup distribution Fifteen main haplogroups, including A, B, C, D, F, G, M7, M8a, M9, M10, M11, N9a'b, R11, Y, and Z, were detected in the cases and controls. The most frequent haplogroups were D (29.5%), F (12.4%), A (9.5%), B (9.5%), and G (7.6%) for KC patients; and D (24.6%), B (13.9%), F (13.6%), A (7.8%) and G (7.1%) for the combined control group. The distribution of mtDNA haplogroups in 210 unrelated Chinese KC patients and 309 Chinese controls is presented in Table 2. Detailed information about the sequence variations in each patient and the first control cohort is shown in Supplementary Table S2. 3.2. Lack of association of mtDNA haplogroup with keratoconus We determined the distribution of fifteen main haplogroups in both patients and controls in order to investigate the difference between them. First, we compared the haplogroup frequency between KC patients and the first control cohort (Table 2). Our results showed that there was no significant difference in mtDNA haplogroup frequency between the patients and the first control cohort. Haplogroups H and R were reported to be associated with keratoconus in Saudis, so we analyzed these haplogroups in our population too. Haplogroup R is a macrohaplogroup, including haplogroups B, F, and R11 for Han Chinese. The frequency of haplogroup R in patients was 23.8%, which was similar to that in the first control cohort (26.8%). There was no significant difference in the frequency of haplogroup R between the cases and the first control cohort (P ¼ 0.499). Haplogroup H, which was associated with KC in Saudi Arabians, was not observed in our patients (0/210) (Supplementary Table S2). To confirm these results, we assessed differences in the distribution of haplogroups between the cases
Table 2 MtDNA haplogroup distribution in patients and controls. Haplogroup KC patients 1st control
A B C D F G M7 M8a M9 M10 M11 N9a'b R11 Y Z Others Total
2nd control
Controls
N (%)
N (%)
P
N (%)
P
N (%)
P
20 (9.5) 20 (9.5) 8 (3.8) 62 (29.5) 26 (12.4) 16 (7.6) 12 (5.7) 7 (3.3) 3 (1.4) 5 (2.4) 2 (1.0) 11 (5.2) 4 (1.9) 1 (0.5) 8 (3.8) 5 (2.4) 210
16 (8.7) 23 (12.6) 6 (3.3) 43 (23.5) 25 (13.7) 15 (8.2) 9 (4.9) 1 (0.5) 3 (1.6) 3 (1.6) 3 (1.6) 11 (6.0) 1 (0.5) 4 (2.2) 9 (4.9) 11 (6.0) 183
0.789 0.335 0.777 0.178 0.706 0.832 0.726 0.073 1.000 0.729 0.667 0.740 0.378 0.189 0.590
8 (6.3) 20 (15.9) 3 (2.4) 33 (26.2) 17 (13.5) 7 (5.6) 6 (4.8) 7 (5.6) 5 (4.0) 2 (1.6) 3 (2.4) 5 (4.0) 2 (1.6) 4 (3.2) 3 (2.4) 1 (0.8) 126
0.308 0.082 0.546 0.511 0.768 0.468 0.707 0.324 0.157 0.715 0.368 0.792 1.000 0.068 0.546
24 (7.8) 43 (13.9) 9 (2.9) 76 (24.6) 42 (13.6) 22 (7.1) 15 (4.9) 8 (2.6) 8 (2.6) 5 (1.6) 6 (1.9) 16 (5.2) 3 (1.0) 8 (2.6) 12 (3.9) 12 (3.9) 309
0.481 0.133 0.573 0.212 0.688 0.830 0.665 0.619 0.538 0.535 0.483 0.976 0.449 0.091 0.966
61
and the second control cohort, and between the cases and the combined controls. The results were consistent across all comparisons (Table 2). These results further support the absence of any association of mtDNA haplogroup with keratoconus. 3.3. Decreased mtDNA copy number in KC patients To test whether variations of mtDNA copy number affect KC, we measured the mtDNA copy number in 193 blood samples of KC patients (mean age ± SE, 20.58 ± 0.431) and 103 matched controls (mean age ± SE, 21.18 ± 0.213). As shown in Table 3, the mean relative mtDNA copy number for patients and controls was 0.978 (SE ¼ 0.039) and 1.344 (SE ¼ 0.112) respectively. There was a significant decrease of mtDNA copy number in KC patients compared to control subjects (P ¼ 0.0002). The medians and the 25th and 75th percentiles of relative mtDNA copy number of patients and controls are shown in Fig. 1. Detailed information about mtDNA copy number in each patient and controls from the first cohort is shown in Supplementary Table S1. To test whether other parameters, such as age, gender, and mtDNA background, affect the relationship between KC and mtDNA copy number, we compared the mtDNA copy number between different groups. When we grouped the KC patients and controls according to their sex, age, or mtDNA background, there was also a significant difference of mtDNA copy number between cases and controls (Table 3). The male KC patients had significantly lower mtDNA copy numbers compared to the male control samples. The females showed the same trend, with an insignificant P value, which may be due to the small sample size. Regardless of whether the patients were older (>mean age of patients) or younger (
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Mitochondrial DNA copy number, but not haplogroup is associated with keratoconus in Han Chinese population Xiao-Dan Hao a, Peng Chen a, Ye Wang a, Su-Xia Li b, Li-Xin Xie a, * a
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China b Shandong Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Jinan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 31 December 2014 Accepted in revised form 18 January 2015 Available online 19 January 2015
Oxidative stress may play a role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is closely related to mitochondrion function, and variations may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. To test whether mtDNA background and copy number confer genetic susceptibility to KC in the Han Chinese population, we performed this association study. We analyzed mtDNA sequence variations in 210 KC patients and 309 matched individuals from China, and classified each subject by haplogroup. Mitochondrial DNA copy number was measured in a subset of these subjects (193 patients and 103 controls). Comparison of matrilineal components of the cases and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that KC patients had significantly lower mtDNA copy numbers than controls (P ¼ 0.0002), even when age, gender, and mtDNA background were considered. Our results suggest that mtDNA copy number, but not haplogroup, is associated with keratoconus, and may contribute to its pathogenesis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Keratoconus mtDNA Copy number Haplogroup Association study
1. Introduction Keratoconus (KC) is a degenerative disorder of the cornea that is characterized by corneal ectasia, thinning, and cone-shaped protrusion, resulting in reduced vision, irregular astigmatism, and cornea scarring (Krachmer et al., 1984). Keratoconus is mostly bilateral but can be unilateral. Onset is usually in puberty, and the progression slows after age 30, arresting around the fourth decade of life (Rabinowitz, 1998). It affects both genders and all ethnicities (Kok et al., 2012). The prevalence of this disease is approximately 1 per 2000 in the general population (Kok et al., 2012; Rabinowitz, 1998). Owing to the limited availability of medical treatments, it is a significant clinical problem worldwide and a leading indication for corneal transplantation.
Abbreviations: KC, keratoconus; mtDNA, mitochondrial DNA; ROS, reactive oxygen species; OS, oxidative stress; OXPHOS, oxidative phosphorylation; LHON, Leber's hereditary optic neuropathy; rCRS, revised Cambridge reference sequence; SE, standard error; ANOVA, analysis of variance. * Corresponding author. No. 5 Yanerdao Road, Qingdao 266071, China. E-mail addresses: [email protected] (X.-D. Hao), chenpeng599205@ 126.com (P. Chen), [email protected] (Y. Wang), [email protected] (S.-X. Li), [email protected] (L.-X. Xie). http://dx.doi.org/10.1016/j.exer.2015.01.016 0014-4835/© 2015 Elsevier Ltd. All rights reserved.
The molecular pathogenesis of KC is poorly understood. Despite extensive research, the exact cause of keratoconus remains unknown in the majority of patients. Studies have suggested that oxidative stress (OS) may play a role in the pathogenesis of KC (Behndig et al., 2001). In their study, Chwa et al. (2006) found that keratoconus corneal stromal fibroblasts had increased reactive oxygen species (ROS) and superoxides compared with levels in normal cultures. Oxidative phosphorylation (OXPHOS) occurs within mitochondria and is a significant endogenous source of ROS (Nohl, 1994). Mitochondria have their own genome, mitochondrial DNA (mtDNA), which encodes 13 subunits of respiratory complexes I, III, IV, and V (Anderson et al., 1981; Andrews et al., 1999). Mitochondrial DNA is closely related with mitochondrion function, and variations may affect the generation of ROS and be involved in pathogenesis of KC. Mitochondrial DNA is inherited maternally and is characterized by an absence of recombination, and high mutation rate. It can accumulate mutations during evolution, and a group of mtDNA that shares certain mtDNA ancestral variants can be traced to distinguishable haplogroups (Kong et al., 2006; Torroni et al., 2006). Certain mtDNA polymorphisms that distinguish haplogroups and subhaplogroups, or their combinations, may be predisposing
60
X.-D. Hao et al. / Experimental Eye Research 132 (2015) 59e63
factors for certain diseases (Torroni and Wallace, 1994). Previous studies have observed that mtDNA haplogroup affects genetic susceptibility to many diseases, including Leber's hereditary optic neuropathy (LHON) (Carelli et al., 2006), type 2 diabetes (Fuku et al., 2007) and Wolfram syndrome (Hofmann et al., 1997). A recently study established the mitochondrial haplogroups in Saudi patients with KC and healthy controls of same ethnicity, and found that individuals with mitochondrial haplogroups H and R are at increased risk to develop KC (Abu-Amero et al., 2014). However, the relationship between mtDNA haplogroup and KC in Chinese population has not yet been studied. Furthermore, each cell has hundreds to thousands of copies of mtDNA molecules, and the genetic background and factors in the physiological environment can affect mtDNA copy number alterations (Moraes, 2001). It has been reported that mtDNA copy number is involved in some diseases, such as renal cell carcinoma (Xing et al., 2008), non-Hodgkin lymphoma (Lan et al., 2008) and leprosy (Wang et al., 2012). Accumulating evidence shows that mtDNA copy number control is very important for mitochondrial biogenesis and normal cellular function (Clay Montier et al., 2009). In this study, we hypothesized that mitochondrial DNA may confer a genetic susceptibility to KC, and this may be reflected by mtDNA genetic background and mtDNA copy number. Here, mtDNA haplogroup composition and copy number in KC patients and healthy controls were studied to test this hypothesis. Our results showed that mtDNA haplogroup had no association with KC in a Han Chinese population, and decreased mtDNA copy number was a predisposing factor for KC and contributed to its pathogenesis. 2. Materials and methods 2.1. Ethics statement
(14.4%). The mean age of the first control cohort was 26.42 years, and the proportion of males was 74.7%, which are similar to the patients' group. The second control cohort included 126 Chinese individuals from the general population of Shandong province (Table 1). The data for mtDNA polymorphisms, variants and, haplogroups for the second control cohort were collected from the literature (Yao et al., 2002, 2003). 2.3. Classification of haplogroups by sequencing Total DNA was isolated from whole blood or cornea using the standard phenol/chloroform method. DNA concentration was measured by BioPhotometer (Eppendorf, Hamburg, Germany). We classified each subject by haplogroup no matter which tissue was available. The D-loop region of the mtDNA was amplified, as described in previous literature (Hao et al., 2013). The products were purified with Alkaline Phosphatase (Shrimp) (Takara, Dalian, China) and Exonuclease I (Takara, Dalian, China) and were subjected to direct DNA sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing kit and ABI PRISM 3730 sequencer (Applied Biosystems Inc., USA). The sequencing primers contained four internal primers (L15996, L16209, H16347, and L29) (Wang et al., 2008; Yao et al., 2003). Sequences were aligned and analyzed with the DNASTAR software package (DNASTAR Inc., USA). Sequence variations in each patient were scored relative to the revised Cambridge reference sequence (rCRS) (Andrews et al., 1999), and the haplogroup of every individual was defined using the online software Mitotool (http://www.mitotool.org/index. html). Further coding-region polymorphisms in specific lineages were typed as described in the previous studies (Yao et al., 2002, 2004) to confirm their predicted phylogenetic status. 2.4. Measurement of mtDNA copy number
Ethical approval for the study was granted by the Ethics Committee of the Shandong Eye Institute, Qingdao, China. Written consent was obtained from each subject or the subject's guardian (for subjects under 18 years of age). 2.2. Samples A total of 210 unrelated Chinese KC patients and 309 unrelated controls were included in the present study. Patients affected with keratoconus, most of them from Shandong province, were recruited from the cornea clinics of Qingdao Eye Hospital and Shandong Eye Hospital. The cases were diagnosed on the basis of clinical examination (corneal stromal thinning, Vogt's striae, Fleischer's ring, Munson's sign, signs of videokeratography, and refractive errors). Cornea or blood samples from each patient were collected according to availability. The healthy controls contained two cohorts. The first control cohort included 183 individuals recruited at Qingdao Eye Hospital and Shandong Eye Hospital. These individuals had accidental injuries but no eye disease. A blood sample was collected from each individual. Controls were confirmed to have no sign of KC using the same examination protocol as used for the cases. The mean age of the KC patients was 21.01 years, and there were more males (85.6%) than females
The mtDNA content is inversely associated with age (He et al., 2014; Mengel-From et al., 2014), and different tissues have different mitochondrial content (Benard et al., 2006). To exclude these effects, we measured the mtDNA copy number in 193 blood samples of patients (mean age ± SE, 20.58 ± 0.431) and 103 controls of matched age (mean age ± SE, 21.18 ± 0.213) from the first control cohort. There was no health cornea as a control, so KC corneal mtDNA copy numbers were not measured. Relative mtDNA copy number was measured by fluorescence-based quantitative realtime PCR and the 2DDCT method, as described in previous studies (Bi et al., 2010; Wang et al., 2012). In brief, primer pair L394/H475 was used for measuring the mtDNA, and amplification of the singlecopy nuclear b-globin gene was used for normalization (Wang et al., 2012). The amplification assays were performed with FastStart Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany) on the Rotor-Gene Q Real-Time PCR system (QIAGEN, Germany), and results were obtained and analyzed using the RotorGene Q Series Software (version 2.1.0). The relative mtDNA copy number of each sample was calibrated by one sample of the controls (n025, Supplementary Table S1). To ensure data quality, samples with a standard deviation greater than 0.25 across replicates were measured again. 2.5. Statistical analysis
Table 1 Second control cohort's information. Geographic location and population
Abbreviation No. of subjects
References
Qingdao Shandong Taian Shandong
SD-QD SD-TA
Yao et al., 2002 Yao et al., 2003
50 76
Statistical analysis was performed using SPSS 17.0 (SPSS Inc., USA). Statistical significance was defined at a ¼ 0.05. A chi-squared (c2) test or Fisher's exact test, as appropriate, was used to assess differences in the distribution of each haplogroup between cases and controls. Each haplogroup was compared with all other haplogroups pooled into one group. Power calculations were
X.-D. Hao et al. / Experimental Eye Research 132 (2015) 59e63
performed using Quanto version 1.2.4 in our population. Allele frequency and optional inheritance modes are not applicable for mtDNA haplogroup, so we just focused on the final susceptibility frequency in the power calculation. We have 80% power to detect a risk of 2.07e1.67 or a resistance of 0.34e0.60 within a haplogroup with a frequency in the range of 10%e50%. Relative mtDNA copy number was analyzed as a continuous variable. Analysis of Variance (ANOVA) was used to determine the differences in mtDNA copy number in different groups. 3. Results 3.1. MtDNA haplogroup distribution Fifteen main haplogroups, including A, B, C, D, F, G, M7, M8a, M9, M10, M11, N9a'b, R11, Y, and Z, were detected in the cases and controls. The most frequent haplogroups were D (29.5%), F (12.4%), A (9.5%), B (9.5%), and G (7.6%) for KC patients; and D (24.6%), B (13.9%), F (13.6%), A (7.8%) and G (7.1%) for the combined control group. The distribution of mtDNA haplogroups in 210 unrelated Chinese KC patients and 309 Chinese controls is presented in Table 2. Detailed information about the sequence variations in each patient and the first control cohort is shown in Supplementary Table S2. 3.2. Lack of association of mtDNA haplogroup with keratoconus We determined the distribution of fifteen main haplogroups in both patients and controls in order to investigate the difference between them. First, we compared the haplogroup frequency between KC patients and the first control cohort (Table 2). Our results showed that there was no significant difference in mtDNA haplogroup frequency between the patients and the first control cohort. Haplogroups H and R were reported to be associated with keratoconus in Saudis, so we analyzed these haplogroups in our population too. Haplogroup R is a macrohaplogroup, including haplogroups B, F, and R11 for Han Chinese. The frequency of haplogroup R in patients was 23.8%, which was similar to that in the first control cohort (26.8%). There was no significant difference in the frequency of haplogroup R between the cases and the first control cohort (P ¼ 0.499). Haplogroup H, which was associated with KC in Saudi Arabians, was not observed in our patients (0/210) (Supplementary Table S2). To confirm these results, we assessed differences in the distribution of haplogroups between the cases
Table 2 MtDNA haplogroup distribution in patients and controls. Haplogroup KC patients 1st control
A B C D F G M7 M8a M9 M10 M11 N9a'b R11 Y Z Others Total
2nd control
Controls
N (%)
N (%)
P
N (%)
P
N (%)
P
20 (9.5) 20 (9.5) 8 (3.8) 62 (29.5) 26 (12.4) 16 (7.6) 12 (5.7) 7 (3.3) 3 (1.4) 5 (2.4) 2 (1.0) 11 (5.2) 4 (1.9) 1 (0.5) 8 (3.8) 5 (2.4) 210
16 (8.7) 23 (12.6) 6 (3.3) 43 (23.5) 25 (13.7) 15 (8.2) 9 (4.9) 1 (0.5) 3 (1.6) 3 (1.6) 3 (1.6) 11 (6.0) 1 (0.5) 4 (2.2) 9 (4.9) 11 (6.0) 183
0.789 0.335 0.777 0.178 0.706 0.832 0.726 0.073 1.000 0.729 0.667 0.740 0.378 0.189 0.590
8 (6.3) 20 (15.9) 3 (2.4) 33 (26.2) 17 (13.5) 7 (5.6) 6 (4.8) 7 (5.6) 5 (4.0) 2 (1.6) 3 (2.4) 5 (4.0) 2 (1.6) 4 (3.2) 3 (2.4) 1 (0.8) 126
0.308 0.082 0.546 0.511 0.768 0.468 0.707 0.324 0.157 0.715 0.368 0.792 1.000 0.068 0.546
24 (7.8) 43 (13.9) 9 (2.9) 76 (24.6) 42 (13.6) 22 (7.1) 15 (4.9) 8 (2.6) 8 (2.6) 5 (1.6) 6 (1.9) 16 (5.2) 3 (1.0) 8 (2.6) 12 (3.9) 12 (3.9) 309
0.481 0.133 0.573 0.212 0.688 0.830 0.665 0.619 0.538 0.535 0.483 0.976 0.449 0.091 0.966
61
and the second control cohort, and between the cases and the combined controls. The results were consistent across all comparisons (Table 2). These results further support the absence of any association of mtDNA haplogroup with keratoconus. 3.3. Decreased mtDNA copy number in KC patients To test whether variations of mtDNA copy number affect KC, we measured the mtDNA copy number in 193 blood samples of KC patients (mean age ± SE, 20.58 ± 0.431) and 103 matched controls (mean age ± SE, 21.18 ± 0.213). As shown in Table 3, the mean relative mtDNA copy number for patients and controls was 0.978 (SE ¼ 0.039) and 1.344 (SE ¼ 0.112) respectively. There was a significant decrease of mtDNA copy number in KC patients compared to control subjects (P ¼ 0.0002). The medians and the 25th and 75th percentiles of relative mtDNA copy number of patients and controls are shown in Fig. 1. Detailed information about mtDNA copy number in each patient and controls from the first cohort is shown in Supplementary Table S1. To test whether other parameters, such as age, gender, and mtDNA background, affect the relationship between KC and mtDNA copy number, we compared the mtDNA copy number between different groups. When we grouped the KC patients and controls according to their sex, age, or mtDNA background, there was also a significant difference of mtDNA copy number between cases and controls (Table 3). The male KC patients had significantly lower mtDNA copy numbers compared to the male control samples. The females showed the same trend, with an insignificant P value, which may be due to the small sample size. Regardless of whether the patients were older (>mean age of patients) or younger (
