Kumar Manoj, Tanwar Mukesh, Faiq Muneeb Ahmad, Pani Jhumur, Shamsi Monis Bilal, Dada Tanuj, Dada Rima
Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India.
Mol Vis. 2013;19:220-30. Epub 2013 Feb 1.
Primary congenital glaucoma (PCG) is the second most common cause of blindness, accounting for 0.01%-0.04% of total blindness worldwide. Most congenital glaucoma cases are mapped to the GLC3A locus, and many aspects of PCG are still unknown. Recent studies have reported an increased frequency of mitochondrial DNA (mtDNA) sequence changes in primary open-angle glaucoma, primary angle-closure glaucoma, and pseudoexfoliation glaucoma compared to controls. Thus, this study was planned with the aim of detecting mitochondrial DNA variations in PCG cases.
Twenty primary congenital glaucoma cases were selected from Dr. R. P. Centre for Ophthalmic Sciences of All India Institute of Medical Sciences (AIIMS), New Delhi, India. DNA was isolated from whole blood samples. The entire coding region of the mitochondrial genome was amplified by PCR in 20 patients and 20 controls. The full mtDNA genome was sequenced and analyzed against mitochondrial reference sequence NC_012920.
MtDNA sequencing revealed a total of 195 nucleotide variations in PCG patients and 58 in controls. Of the 195 changes, 43 (22.05%) were nonsynonymous, 82 (42.05%) were synonymous, and 30 were in RNA genes. A total of 39/195 (20.00%) variations were observed in the D-loop (hypervariable region), 19/195 (9.74%) in different ribosomal RNA (rRNAs), 11/195 (5.64%) in transfer RNA (tRNAs), 66/195 (33.84%) in complex I, 17/195 (8.71%) in complex III, 27/195 (13.84%) in complex IV, and 15/195 (7.69%) in complex V. Of 58 variations in the controls, 14 were nonsynonymous changes. The Sorting Intolerant from Tolerant and Polymorphism Phenotyping analyses of all nonsynonymous changes from patients revealed two pathogenic changes in NADH-ubiquinone oxidoreductase chain 2 (ND2) and cytochrome oxidase subunit III (COXIII) subunits. In one of the patients, the insertion of cytosine introduced a frame shift change (p.Ile104AsnfsX26) in the cytochrome b (CYB) subunit of the electron transport chain. In another patient, a variation (G8572A) in ATP synthase 8 (ATpase8) led to the introduction of a stop codon or termination at amino acid position 69. Haplogroup/phylogenetic analysis of mtDNA showed that primary congenital glaucoma patients belong to three macrohaplogroups: M (4), N (15), and L (1). Fifty percent of the patients belonged to the H2a2a lineage of the N-derived haplogroup.
Although several mutations were found at a higher frequency among our population, there is a need to complement this study with functional studies and to analyze a large number of samples in different populations of different haplogroups, as penetrance varies among haplogroups.
原发性先天性青光眼（PCG）是导致失明的第二大常见原因，占全球失明总数的0.01%-0.04%。大多数先天性青光眼病例定位于GLC3A基因座，PCG的许多方面仍不清楚。最近的研究报告称，与对照组相比，原发性开角型青光眼、原发性闭角型青光眼和剥脱性青光眼患者中线粒体DNA（mtDNA）序列变化的频率增加。因此，本研究旨在检测PCG病例中的线粒体DNA变异。
从印度新德里全印度医学科学研究所（AIIMS）的R.P.眼科科学中心选取20例原发性先天性青光眼病例。从全血样本中分离DNA。通过聚合酶链反应（PCR）扩增20例患者和20例对照的线粒体基因组的整个编码区。对完整的mtDNA基因组进行测序，并与线粒体参考序列NC_012920进行比对分析。
mtDNA测序显示，PCG患者共有195个核苷酸变异，对照组有58个。在195个变化中，43个（22.05%）为非同义突变，82个（42.05%）为同义突变，30个位于RNA基因中。在D环（高变区）共观察到39/195（20.00%）个变异，在不同的核糖体RNA（rRNA）中有19/195（9.74%）个，在转运RNA（tRNA）中有11/195（5.64%）个，在复合体I中有66/195（33.84%）个，在复合体III中有17/195（8.71%）个，在复合体IV中有27/195（13.84%）个，在复合体V中有15/195（7.69%）个。对照组的58个变异中，14个为非同义变化。对患者所有非同义变化进行的耐受与不耐受排序及多态性表型分析显示，烟酰胺腺嘌呤二核苷酸-泛醌氧化还原酶链2（ND2）和细胞色素氧化酶亚基III（COXIII）亚基中有两个致病变化。在其中一名患者中，胞嘧啶的插入在电子传递链的细胞色素b（CYB）亚基中引入了移码变化（p.Ile104AsnfsX26）。在另一名患者中，ATP合酶8（ATpase8）中的一个变异（G8572A）导致在氨基酸位置69处引入了一个终止密码子或终止。mtDNA的单倍群/系统发育分析表明，原发性先天性青光眼患者属于三个大的单倍群：M（4例）、N（15例）和L（1例）。50%的患者属于N衍生单倍群的H2a2a谱系。
尽管在我们的研究人群中发现了几种突变的频率较高，但由于不同单倍群的外显率不同，需要通过功能研究对本研究进行补充，并分析不同单倍群的不同人群中的大量样本。
