Stabuc-Silih Mirna, Ravnik-Glavac Metka, Glavac Damjan, Hawlina Marko, Strazisar Mojca
Eye Hospital, University Medical Centre, Ljubljana, Slovenia.
Mol Vis. 2009 Dec 20;15:2848-60.
Alterations in collagen type IV, alpha-3 (COL4A3) and collagen type IV, alpha-4 (COL4A4) genes may be responsible for a decrease in collagen types I and III, a feature often detected in keratoconus (KC). To evaluate the significance of alterations in COL4A3 and COL4A4 genes in KC patients, we screened both genes and estimated the significance of polymorphisms in Slovenian patients with KC.
The study included 104 unrelated patients with KC and 157 healthy blood donors. Diagnosis was established by clinical examination, electronic refractometry, and keratometry. DNA was extracted from blood, and gene exons were amplified by PCR. Non-isotopic high-resolution single-stranded conformation analysis (SSCA) was used to screen COL4A3 and COL4A4 genes, and migration shifts detected by SSCA were subsequently sequenced. For statistical evaluation, control blood donors were chosen according to age, sex, and not having blood relationship. Neither patients nor control blood donors chosen for statistical analysis were in blood relationship. We used Fisher's exact test for statistical evaluation, with p<0.05 considered significant.
We detected eight polymorphisms in the COL4A3 gene and six in the COL4A4 gene. Allele differences in D326Y in COL4A3 and M1237V and F1644F in COL4A4 are significantly distinctive of KC patients (Fisher's exact test, p<0.05). When analyzing different genotypes under three models (dominant, recessive, and additive), we established that P141L, D326Y, and G895G in COL4A3 and P482S, M1327V, V1516V, and F1644F in COL4A4 have significant differences in genotype distribution between KC patients and the control group.
This is the first mutational screening of COL4A3 and COL4A4 genes in KC patients to establish the status of these genes and compare them to a control population. Analysis of COL4A3 and COL4A4 revealed no mutations related to KC patients, but specific genotypes of seven previously described polymorphisms are significantly associated with KC under dominant, recessive, or additive models. Differences in the expression of type IV collagen in previously published data about chromosomal instabilities in the regions in which the analyzed genes were mapped and our data indicate a probability that some of the polymorphisms we detected could be related to KC.
IV型胶原α-3（COL4A3）基因和IV型胶原α-4（COL4A4）基因的改变可能是I型和III型胶原减少的原因，这一特征在圆锥角膜（KC）中经常被检测到。为了评估COL4A3和COL4A4基因改变在KC患者中的意义，我们对这两个基因进行了筛查，并评估了斯洛文尼亚KC患者中多态性的意义。
该研究纳入了104例无亲缘关系的KC患者和157名健康献血者。通过临床检查、电子验光和角膜曲率测量来确诊。从血液中提取DNA，通过PCR扩增基因外显子。采用非同位素高分辨率单链构象分析（SSCA）对COL4A3和COL4A4基因进行筛查，随后对SSCA检测到的迁移位移进行测序。为了进行统计学评估，根据年龄、性别和无血缘关系选择对照献血者。用于统计分析的患者和对照献血者均无血缘关系。我们使用Fisher精确检验进行统计学评估，p<0.05被认为具有统计学意义。
我们在COL4A3基因中检测到8个多态性，在COL4A4基因中检测到6个多态性。COL4A3基因中D326Y的等位基因差异以及COL4A4基因中M1237V和F1644F的等位基因差异在KC患者中具有显著差异（Fisher精确检验，p<0.05）。在三种模型（显性、隐性和加性）下分析不同基因型时，我们发现COL4A3基因中的P141L、D326Y和G895G以及COL4A4基因中的P482S、M1327V、V1516V和F1644F在KC患者和对照组之间的基因型分布存在显著差异。
这是首次对KC患者的COL4A3和COL4A4基因进行突变筛查，以确定这些基因的状态并与对照人群进行比较。对COL4A3和COL4A4的分析未发现与KC患者相关的突变，但在显性、隐性或加性模型下，7种先前描述的多态性的特定基因型与KC显著相关。在先前发表的关于所分析基因定位区域染色体不稳定性的数据中，IV型胶原表达的差异以及我们的数据表明，我们检测到的一些多态性可能与KC有关。
