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Genetics  |   March 2014
Mitochondrial Sequence Changes in Keratoconus Patients
Author Affiliations & Notes
  • Khaled K. Abu-Amero
    Ophthalmic Genetics Laboratory, Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
  • Taif Anwar Azad
    Ophthalmic Genetics Laboratory, Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
  • Hatem Kalantan
    Anterior Segment Unit, Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
  • Tahira Sultan
    Ophthalmic Genetics Laboratory, Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
  • Abdulrahman M. Al-Muammar
    Anterior Segment Unit, Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia
  • Correspondence: Khaled K. Abu-Amero, Ophthalmic Genetics Laboratory, Department of Ophthalmology, College of Medicine, King Saud University, P.O. Box 245, Riyadh 11411, Saudi Arabia; abuamero@gmail.com
Investigative Ophthalmology & Visual Science March 2014, Vol.55, 1706-1710. doi:10.1167/iovs.14-13938
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      Khaled K. Abu-Amero, Taif Anwar Azad, Hatem Kalantan, Tahira Sultan, Abdulrahman M. Al-Muammar; Mitochondrial Sequence Changes in Keratoconus Patients. Invest. Ophthalmol. Vis. Sci. 2014;55(3):1706-1710. doi: 10.1167/iovs.14-13938.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: We investigated whether a group of patients with keratoconus (KTCN) harbor mutations in the mitochondrial genome.

Methods.: We sequenced the full mitochondrial genome in a group of Saudi patients with KTCN (n = 26) and 100 ethnically matched controls who had no KTCN by examination.

Results.: A total of 10 KTCN patients (38.5%) had potentially pathogenic nonsynonymous mtDNA mutations. Of the nonsynonymous sequence changes detected, 4 (40%) were in Complex I, one was in the tRNAGlutamine, one was in tRNATryptophan, one was in tRNAAsparagine, one was in tRNAHistidine, and two were in the tRNALeucine2. One nonsynonymous sequence change was heteroplasmic, whereas all the remaining 9 were homoplasmic. These sequence changes were not detected in controls of similar ethnicity. Four sequence changes were novel (were not reported previously) and 5 were reported previously. Additionally, we detected 54 synonymous (does not result in an amino acid change) sequence changes with no pathologic significance.

Conclusions.: If our results are confirmed in a larger cohort and multiple ethnicities, then mtDNA mutation may be considered as a genetic risk factor contributing indirectly through the oxidative stress mechanism to the development and/or progression of KTCN.

Introduction
Keratoconus (KTCN) is a complex condition of multifactorial etiology. Genetic and environmental factors are associated with KTCN. Evidence of genetic etiology includes the condition's familial inheritance, discordance between dizygotic twins, and its association with other known genetic disorders. Several chromosomal loci and genes were reported to be associated with KTCN. 1,2 However, some eventually were excluded, 1,3 while in other studies, no confirmed association with the disease have been established. 4,5 This, of course, is not the case for the visual system homebox 1 (VSX1) gene, where mutations associated with KTCN cases have been found in different studies. 69 Having said that, there also are various studies, including our own study, that did not report VSX1 mutations in cohorts of KTCN patients from various populations. 912 This indicates that KTCN is a complex condition of multifactorial etiology and that mutations in the VSX1 gene cannot be responsible for all cases of KTCN. Genome-wide association study (GWAS) allows the interrogation of the whole genome in one experiment. A few candidate KTCN genes were identified in GWAS, including IL1B, 13 CDH11, NUB1, COL27A1, and HGF. A recent GWAS study suggested that SNP rs4954218, located near the RAB3GAP1 gene, reported previously to be associated with corneal malformation, is a potential susceptibility locus for KTCN. 14 As these findings were relatively recent, it awaits conformations in larger cohorts and in multiethnicities. 
It was reported previously that mitochondrial oxidative stress in Tet-mev-1 mice causes excessive apoptosis in several tissues leading to precocious age-dependent corneal physiologic changes, delayed corneal epithelialization, decreased corneal endothelial cells, thickened Descemet's membrane, and thinning of parenchyma with corneal pathologic dysfunctions, such as keratitis, Fuchs' corneal dystrophy (FCD), and probably KTCN. 15 Under transmission electron microscopy (TEM), swelling of the mitochondria were observed in KTCN corneal tissues. 16 The KTCN corneas exhibited more mtDNA damage than do normal corneas. 17 The KTCN fibroblasts had increased basal generation of reactive oxygen species and were more susceptible to stressful challenges (low pH and/or H2O2 conditions) than were normal fibroblasts.18 Additionally, cultured KTCN fibroblasts have an inherent, hypersensitive response to oxidative stress that involves mitochondrial dysfunction and mtDNA damage. As a result, it was suggested that KTCN fibroblast hypersensitivity may have a role in the development and progression of KTCN. 19 To the best of our knowledge, no studies have investigated whether KTCN patients harbor mtDNA mutations. Here, we sequenced the full mitochondrial genome in a group of Saudi patients with KTCN. 
Materials and Methods
Study Population
The study adheres to the tenets of the Declaration of Helsinki, and all participants signed an informed consent. The study was approved by the College of Medicine ethical committee (proposal number 09-659). All study subjects were self-identified as Saudi Arabian ethnicity. Family names all were present in the database of Arab families of Saudi Arabian origin. Additionally, these names indicated that all five major Saudi Arabian provinces were represented in the study population. Patients (n = 26) were selected from the anterior segment clinic at King Abdulaziz University Hospital after examination by two of the authors (AMA-M and HK). Patients were diagnosed with KTCN if the Schimpff-flow–based elevation map showed posterior corneal elevation within the central 5 to ≥+20 μm, inferior-superior dioptric asymmetry (I-S value) > 1.2 diopters (D), and the steepest keratometry > 47D. Patients were labeled as sporadic after examining the immediate family members and identifying the patient as isolated case of KTCN. Exclusion criteria were refusal to participate or post-LASIK ectasia. 
The controls (n = 100) were recruited from the general ophthalmology clinic that had no ocular disease(s) or previous ophthalmic surgeries. Their slit-lamp exam showed clear cornea and their Schimpff-flow–based elevation map was within normal limit. 
All KTCN cases secondary to causes, like trauma, surgery, Ehlers-Danlos syndrome, osteogenesis imperfecta, and pellucid marginal degeneration, were excluded from the study. 
Sample Collection and DNA Extractions
Ficoll-Paque-PLUS (Pharmacia Biotech AB, Uppsala, Sweden) was used for lymphocyte separation and isolation from peripheral blood as detailed previously. 20 DNA was extracted from whole blood samples of all KTCN patients and controls using the PUREGENE DNA isolation kit from Gentra Systems (Minneapolis, MN). 
DNA Amplification and Sequencing
The entire coding region of the mitochondrial genome was amplified in 24 separate PCRs using single set cycling conditions as detailed previously 21 for all patients and controls. Each successfully amplified fragment was sequenced directly using the BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA), and samples were run on the ABI prism 3100 sequencer (Applied Biosystems). 
Sequence Analysis of the Mitochondrial DNA Coding Region
Sequencing results were compared to the corrected Cambridge reference sequence. 22 All fragments were sequenced in forward and reverse directions at least twice for confirmation of a detected variant. Patient mtDNA sequences were compared to those from local controls, and all sequence variants were compared to the MITOMAP database, 22 the Human Mitochondrial Genome Database (available in the public domain at http://www.genpat.uu.se/mtDB), GenBank (available in the public domain at http://www.ncbi.nlm.nih.gov/Genbank/index.html), and Medline-listed publications. 
Prediction of Pathogenicity
Pathogenic characteristics of a previously undescribed (novel) nonsynonymous mtDNA sequence changes were determined according to a combination of standard criteria, 23 an evaluation of interspecies conservation using the Polymorphism phenotyping v2 (PolyPhen-2 database, available in the public domain at http://genetics.bwh.harvard.edu/pph2/) and, when necessary, the Mamit-tRNA website (available in the public domain at http://mamit-trna.u-strasbg.fr/index.html), the human mitochondrial genome database (MitoMap website, available in the public domain at http://www.mitomap.org/MITOMAP), and available English literature. Therefore, a nonsynonymous nucleotide change was considered potentially pathogenic if it was not reported in mitochondrial databases or Medline-listed literature as a confirmed polymorphism, it was not present in local controls, it changed a moderately or highly conserved amino acid, it occurred in a region of high interspecies conservation, and it was assessed as possibly or probably pathogenic by PolyPhen-2. 
For previously reported nonsynonymous nucleotide changes, consideration was given to pathogenic status determined by others and by mitochondrial databases in addition to the criteria described above. 
Results
Table 1 displays the 10 nonsynonymous (resulting in an amino acid change) mtDNA sequence variants detected in 10 unique patients (each patient had one unique mutation) after sequencing the full mitochondrial genome in our KTCN patient group (n = 26). A total of 16 KTCN patients had no known or those we believe are potentially pathologic sequence changes. The 10 nonsynonymous variants listed in Table 1, are those that we believe were potentially pathologic based on the stringent criteria described in the Methods. 
Table 1
 
Potentially Pathogenic Nonsynonymous mtDNA Sequence Changes Detected in KTCN Patients
Table 1
 
Potentially Pathogenic Nonsynonymous mtDNA Sequence Changes Detected in KTCN Patients
Nucleotide Substitution Codon Location % in Patients % in Controls Heteroplasmy Novel Interspecies Conservation
m.4218 T>A Y304X ND1 3.85 0 Yes No High
m.4381 A>G t–RNA Gln 3.85 0 No Yes Moderate
m.5567 T>C t–RNA Trp 3.85 0 No No High
m.5664 A>G t–RNA Asn 3.85 0 No Yes High
m.11393 C>T L212F ND4 3.85 0 No Yes High
m.12178 C>T t–RNA His 3.85 0 No No High
m.12308 A>G t–RNA Leu2 3.85 0 No No High
m.12310 insA t–RNA Leu2 3.85 0 No No High
m.12504 G>A C56W ND5 3.85 0 No Yes Moderate
m.14000 T>A L555Q ND5 3.85 0 No No High
Of the nonsynonymous sequence changes listed in Table 1, 4 (40%) were in complex I, one was in the tRNAGlutamine, one was in tRNATryptophan, one was in tRNAAsparagine, one was in tRNAHistidine, and two were in the tRNALeucine2. One nonsynonymous sequence change was heteroplasmic, whereas all the remaining 9 were homoplasmic. These sequence changes were not detected in controls of similar ethnicity. Four sequence changes were novel (were not reported previously) and 5 were reported previously. 
As for the synonymous (does not result in amino acid change) sequence changes, we detected 54. Of those, 50 (92.6%) were in complex I (NADH dehydrogenase, also called NADH:ubiquinone oxidoreductase), 3 (5.5%) were in complex IV (cytochrome c oxidase), and 1 (1.9%) in complex III (cytochrome bc1 complex, Table 2). All synonymous mtDNA sequence changes listed in Table 2 also were detected in controls with similar frequencies. 
Table 2
 
Synonymous Sequence Changes Detected in KTCN Patients and Controls
Table 2
 
Synonymous Sequence Changes Detected in KTCN Patients and Controls
Nucleotide Substitution Codon Location % in Patients % in Controls Heteroplasmy Novel
m.3357 G>A M17M ND1 3.85 4 No No
m.3594 C>T V96V ND1 11.5 15 No No
m.3603 C>T N99N ND1 3.85 4 No Yes
m.3666 G>A G120G ND1 3.85 5 No No
m.3768 A>G L154L ND1 3.85 5 No No
m.3847 T>C L181L ND1 14.3 17 No No
m.4059 C>T S251S ND1 3.85 6 No No
m.4092 G>A K262K ND1 3.85 5 No No
m.4104 A>G L266L ND1 11.5 15 No No
m.4529 A>T T20T ND2 3.85 5 No No
m.4769 A>G M100M ND2 11.5 16 No No
m.4991 G>A Q174Q ND2 8.69 9 No No
m.5237 G>A P256P ND2 3.85 4 No No
m.9965 T>C Y253Y CO III 3.85 4 No Yes
m.10115 T>C I19I ND3 11.5 15 No No
m.10238 T>C I60I ND3 11.5 19 No No
m.11009 T>C L84L ND4 3.85 8 No No
m.11056 A>G L99L ND4 3.85 6 No Yes
m.11251 A>G L164L ND4 8.7 9 No No
m.11344 A>G M195M ND4 3.85 9 No Yes
m.11437 T>C A226A ND4 11.53 17 No No
m.11467 A>G L236L ND4 3.85 5 No No
m.11485 T>C G242G ND4 3.85 6 No No
m.11569 T>C I270I ND4 3.85 5 No Yes
m.11620 A>G A287A ND4 3.85 5 No No
m.11653 A>G V298V ND4 3.85 5 No No
m.11719 G>A G320G ND4 61.53 71 No No
m.11761 C>T Y334Y ND4 3.85 5 No No
m.11944 T>C L395L ND4 11.53 13 No No
m.11983 C>T L408L ND4 3.85 5 No Yes
m.12372 G>A L12L ND5 8.69 9 No No
m.12501 G>A M55M ND5 11.53 14 No No
m.12612 A>G V92V ND5 8.69 10 No No
m.12693 A>G K119K ND5 11.53 14 No No
m.12696 T>C Y120Y ND5 3.85 4 No No
m.12705 C>T I123I ND5 46.15 51 No No
m.12816 C>T A160A ND5 3.85 4 No No
m.12843 T>C I169I ND5 3.84 5 No Yes
m.12879 T>C G181G ND5 3.84 5 No No
m.13111 T>C L259L ND5 3.84 6 No No
m.13174 T>C L280L ND5 3.84 6 No No
m.13188 C>T T284T ND5 23.07 28 No No
m.13422 A>G L362L ND5 3.84 6 No No
m.13590 G>A L418L ND5 11.53 13 No No
m.13650 C>T P438P ND5 8.69 10 No No
m.13803 A>G T389T ND5 11.53 12 No No
m.14070 A>G S578S ND5 3.84 5 No No
m.14364 G>A L104L ND6 3.84 5 No No
m.14470 T>C G68G ND6 8.69 10 No No
m.14544 G>A L44L ND6 8.69 12 No No
m.14566 A>G G36G ND6 11.53 12 No No
m.14783 T>C L13L CYTB 8.69 10 No No
m.15043 G>A G99G CYTB 11.42 13 No No
m.15148 G>A P134P CYTB 3.84 7 No No
As for mitochondrial haplogrouping, we detected 12 different mtDNA haplogroups among our patient group (n = 26). We detected mtDNA haplogroup R0a in 3 patients, L3c in 3, N1a3 in 3, and J1b8 in 3, while 2 patients had the H63a, 2 had the L4A, 3 had the HV, 3 had the K1b1a, and one each had the mtDNA haplogroups U8, N2, M12G, and J1a. 
Discussion
We enrolled 26 KTCN patients into this unique study, which was conducted to investigate whether KTCN patients possessed a pathogenic mtDNA sequence change. The 26 KTCN patients were found to lack mutations in the VSX1 gene 12 and also lack any chromosomal abnormalities, 24 and, thus, the genetic causes, if any, for these KTCN cases still are not known. Previous studies had indicated that oxidative stress, mitochondrial dysfunction, and mtDNA damage may have a role in the development and progression of KTCN. 15,1719 Therefore, we investigated whether our KTCN patients possessed a pathogenic mtDNA mutation(s) in their mitochondrial genome. To our knowledge, this the first study that investigated an mtDNA mutation possible link to KTCN. We previously carried out similar studies in other ophthalmic diseases, such as Leber's hereditary optic neuropathy (LHON), 25 LHON-plus, 26 primary open angle glaucoma, 27 primary angle closure glaucoma, 28 and pseudoexofoliation glaucoma. 29  
Stringent criteria were used to assess the potential pathogenic status of the nonsynonymous mtDNA sequence changes detected here. Usually, the mitochondrial genome is highly mutated and one must be extremely careful in assigning pathogenic status to various sequence changes. In our cohort, only 38.5% (10/26) had mtDNA sequence changes that were potentially pathogenic. Most of the mutations detected were in complex I, a situation somehow resembling those for LHON. None of our KTCN patients had any of the three primary LHON mutations described previously, 30 although one mutation (m.4381 A>G) in the t-RNAGln was reported previously in LHON patients 25 who lack any of the three primary LHON mutations. This sequence change is located in pairing chain position 53 of the t-RNAGln, just before the TψC loop. PolyPhen could not predict the effect of this sequence change due to lack of data; however, phylogenetic data for 32 different species (Mamit-tRNA website; available in the public domain at http://mamit-trna.u-strasbg.fr/index.html) showed the sequence to be moderately conserved. This sequence alteration was not reported previously as a polymorphism and was absent in controls, and, therefore, was considered potentially pathogenic. 
As for the m.4218 T>A, which is the only heteroplasmic mutation detected, it results in the introduction of a premature stop-codon in the ND1 gene. The high level of heteroplasmy (74%), the fact it results in premature stop-codon, and the fact that it was not detected in controls indicate that this sequence change is potentially pathogenic. As for m.5567 T>C, this mutation was reported previously in cases of mitochondrial encephalomyopathy 31 and was not found in our controls. Its location and its high interspecies conservation made us rank this as potentially pathogenic. As for the m.12308 A>G, this mutation was reported previously in the patients with chronic progressive external ophthalmoplegia (CPEO). 32 This mutation was not found in our controls and, similar to other tRNA mutations, was highly conserved, and, therefore, it is potentially pathogenic. The rest of the mutations in Table 1 (5664, 11393, 12178, 12310, 12504, and 14000) were not reported previously in association with diseases. We applied the same pathogenic criteria and we concluded that they are potentially pathogenic. 
As for mtDNA haplogroups, we detected 12 different mtDNA haplogroups in our relatively small cohort. There was no particular mtDNA haplogroup that could confer susceptibility to KTCN, at least in our small cohort. We believe that a larger study will determine whether mtDNA haplogroups may confer susceptibility to KTCN or not. 
Detecting potentially pathogenic mtDNA sequence changes in 38.5% (10/26) of our KTCN cohort certainly is a sign that mtDNA mutations may have a role in KTCN pathogenesis. We reported a fairly small group of patients from a restricted ethnic population, and this type of evaluation must be repeated in other centers. If these results are confirmed, then mtDNA mutation may be considered as a genetic risk factor contributing indirectly through the oxidative stress mechanism to the development and/or progression of KTCN. 
Acknowledgments
Supported by the Glaucoma Research Chair at King Saud University, Riyadh, Saudi Arabia. 
Disclosure: K.K. Abu-Amero, None; T.A. Azad, None; H. Kalantan, None; T. Sultan, None; A.M. Al-Muammar, None 
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Table 1
 
Potentially Pathogenic Nonsynonymous mtDNA Sequence Changes Detected in KTCN Patients
Table 1
 
Potentially Pathogenic Nonsynonymous mtDNA Sequence Changes Detected in KTCN Patients
Nucleotide Substitution Codon Location % in Patients % in Controls Heteroplasmy Novel Interspecies Conservation
m.4218 T>A Y304X ND1 3.85 0 Yes No High
m.4381 A>G t–RNA Gln 3.85 0 No Yes Moderate
m.5567 T>C t–RNA Trp 3.85 0 No No High
m.5664 A>G t–RNA Asn 3.85 0 No Yes High
m.11393 C>T L212F ND4 3.85 0 No Yes High
m.12178 C>T t–RNA His 3.85 0 No No High
m.12308 A>G t–RNA Leu2 3.85 0 No No High
m.12310 insA t–RNA Leu2 3.85 0 No No High
m.12504 G>A C56W ND5 3.85 0 No Yes Moderate
m.14000 T>A L555Q ND5 3.85 0 No No High
Table 2
 
Synonymous Sequence Changes Detected in KTCN Patients and Controls
Table 2
 
Synonymous Sequence Changes Detected in KTCN Patients and Controls
Nucleotide Substitution Codon Location % in Patients % in Controls Heteroplasmy Novel
m.3357 G>A M17M ND1 3.85 4 No No
m.3594 C>T V96V ND1 11.5 15 No No
m.3603 C>T N99N ND1 3.85 4 No Yes
m.3666 G>A G120G ND1 3.85 5 No No
m.3768 A>G L154L ND1 3.85 5 No No
m.3847 T>C L181L ND1 14.3 17 No No
m.4059 C>T S251S ND1 3.85 6 No No
m.4092 G>A K262K ND1 3.85 5 No No
m.4104 A>G L266L ND1 11.5 15 No No
m.4529 A>T T20T ND2 3.85 5 No No
m.4769 A>G M100M ND2 11.5 16 No No
m.4991 G>A Q174Q ND2 8.69 9 No No
m.5237 G>A P256P ND2 3.85 4 No No
m.9965 T>C Y253Y CO III 3.85 4 No Yes
m.10115 T>C I19I ND3 11.5 15 No No
m.10238 T>C I60I ND3 11.5 19 No No
m.11009 T>C L84L ND4 3.85 8 No No
m.11056 A>G L99L ND4 3.85 6 No Yes
m.11251 A>G L164L ND4 8.7 9 No No
m.11344 A>G M195M ND4 3.85 9 No Yes
m.11437 T>C A226A ND4 11.53 17 No No
m.11467 A>G L236L ND4 3.85 5 No No
m.11485 T>C G242G ND4 3.85 6 No No
m.11569 T>C I270I ND4 3.85 5 No Yes
m.11620 A>G A287A ND4 3.85 5 No No
m.11653 A>G V298V ND4 3.85 5 No No
m.11719 G>A G320G ND4 61.53 71 No No
m.11761 C>T Y334Y ND4 3.85 5 No No
m.11944 T>C L395L ND4 11.53 13 No No
m.11983 C>T L408L ND4 3.85 5 No Yes
m.12372 G>A L12L ND5 8.69 9 No No
m.12501 G>A M55M ND5 11.53 14 No No
m.12612 A>G V92V ND5 8.69 10 No No
m.12693 A>G K119K ND5 11.53 14 No No
m.12696 T>C Y120Y ND5 3.85 4 No No
m.12705 C>T I123I ND5 46.15 51 No No
m.12816 C>T A160A ND5 3.85 4 No No
m.12843 T>C I169I ND5 3.84 5 No Yes
m.12879 T>C G181G ND5 3.84 5 No No
m.13111 T>C L259L ND5 3.84 6 No No
m.13174 T>C L280L ND5 3.84 6 No No
m.13188 C>T T284T ND5 23.07 28 No No
m.13422 A>G L362L ND5 3.84 6 No No
m.13590 G>A L418L ND5 11.53 13 No No
m.13650 C>T P438P ND5 8.69 10 No No
m.13803 A>G T389T ND5 11.53 12 No No
m.14070 A>G S578S ND5 3.84 5 No No
m.14364 G>A L104L ND6 3.84 5 No No
m.14470 T>C G68G ND6 8.69 10 No No
m.14544 G>A L44L ND6 8.69 12 No No
m.14566 A>G G36G ND6 11.53 12 No No
m.14783 T>C L13L CYTB 8.69 10 No No
m.15043 G>A G99G CYTB 11.42 13 No No
m.15148 G>A P134P CYTB 3.84 7 No No
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