Ng Jun B, Poh Rozaida Y Y, Lee Kenneth R, Subrayan Visvaraja, Deva Jenny P, Lau Amy Y L, Tan Jin A M A
Clin Lab. 2016 Sep 1;62(9):1731-1737. doi: 10.7754/Clin.Lab.2016.160144.
Keratoconus is an ocular degeneration characterized by the thinning of corneal stroma that may lead to varying degrees of myopia and visual impairment. Genetic factors have been reported in the pathology of keratoconus where Asians have a higher incidence, earlier onset, and undergo earlier corneal grafts compared to Caucasians. The visual system homeobox 1 (VSX1) gene forms part of a paired-like homeodomain transcription factor which is responsible for ocular development. The gene was marked as a candidate in genetic studies of keratoconus in various populations. Single nucleotide polymorphisms (SNPs) in the VSX1 gene have been reported to be associated with keratoconus. The detection of the SNPs involves DNA amplification of the VSX1 gene followed by genomic sequencing. Thus, the objective of this study aims to establish sensitive and accurate screening protocols for the molecular characterization of VSX1 polymorphisms.
Keratoconic (n = 74) and control subjects (n = 96) were recruited based on clinical diagnostic tests and selection criteria. DNA extracted from the blood samples was used to genotype VSX1 polymorphisms. In-house designed primers and optimization of PCR conditions were carried out to amplify exons 1 and 3 of the VSX1 gene. PCR conditions including percentage GC content, melting temperatures, and differences in melting temperatures of primers were evaluated to produce sensitive and specific DNA amplifications.
Genotyping was successfully carried out in 4 exons of the VSX1 gene. Primer annealing temperatures were observed to be crucial in enhancing PCR sensitivity and specificity. Annealing temperatures were carefully evaluated to produce increased specificity, yet not allowing sensitivity to be compromised. In addition, exon 1 of the VSX1 gene was amplified using 2 different sets of primers to produce 2 smaller amplified products with absence of non-specific bands. DNA amplification of exons 1 and 3 consistently showed single band products which were successfully sequenced to yield reproducible data.
The use of in-house designed primers and optimized PCR conditions allowed sensitive and specific DNA amplifications that produced distinct single bands. The in-house designed primers and DNA amplification protocols established in this study provide an addition to the current repertoire of primers for accurate molecular characterization of VSX1 gene polymorphisms in keratoconus research.
圆锥角膜是一种以角膜基质变薄为特征的眼部退行性病变，可导致不同程度的近视和视力损害。已有报道称圆锥角膜的病理学存在遗传因素，与白种人相比，亚洲人发病率更高、发病更早且更早接受角膜移植。视觉系统同源框1（VSX1）基因是配对样同源结构域转录因子的一部分，负责眼部发育。该基因在不同人群圆锥角膜的遗传研究中被标记为候选基因。据报道，VSX1基因中的单核苷酸多态性（SNP）与圆锥角膜有关。SNP的检测包括VSX1基因的DNA扩增，随后进行基因组测序。因此，本研究的目的是建立敏感且准确的筛选方案，用于VSX1多态性的分子特征分析。
根据临床诊断测试和选择标准招募圆锥角膜患者（n = 74）和对照受试者（n = 96）。从血样中提取的DNA用于VSX1多态性基因分型。进行内部设计的引物和PCR条件优化，以扩增VSX1基因的外显子1和3。评估PCR条件，包括GC含量百分比、解链温度以及引物解链温度的差异，以产生敏感且特异的DNA扩增。
成功对VSX1基因的4个外显子进行了基因分型。观察到引物退火温度对于提高PCR敏感性和特异性至关重要。仔细评估退火温度以提高特异性，同时不损害敏感性。此外，使用2组不同的引物扩增VSX1基因的外显子1，产生2个较小的扩增产物，且无非特异性条带。外显子1和3的DNA扩增始终显示单一条带产物，成功测序以产生可重复的数据。
使用内部设计的引物和优化的PCR条件可实现敏感且特异的DNA扩增，产生清晰的单一条带。本研究建立的内部设计引物和DNA扩增方案为圆锥角膜研究中准确进行VSX1基因多态性分子特征分析的现有引物库增添了内容。
