Polymorphisms in COL4A3 and COL4A4 genes associated with keratoconus - PubMed (original) (raw)

. 2009 Dec 20:15:2848-60.

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Polymorphisms in COL4A3 and COL4A4 genes associated with keratoconus

Mirna Stabuc-Silih et al. Mol Vis. 2009.

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1

Figure 1

A: Three different PCR–single stranded conformational analysis patterns on one gel, representing COL4A3 exons 17, 48, and 49. PCR fragments were loaded in succession from shortest to longest PCR fragment at 30-min intervals. Exons 48 and 49 did not show any differences in elution shifts; in exon 17, the different patterns were subsequently sequenced. B: Partial sequence of exon 17 with heterozygous substitution D326Y (976GT). C: Partial sequence of exon 17 with homozygous substitution 326Y (976TT). K1 to K4 marked patterns are patterns of three exons (17, 48, and 49) of COL4A3 from keratoconus patients, multiplied by PCR and analysed with SSCA. C1 to C3 marked patterns are patterns of three exons (17, 48, and 49) of COL4A3 from controls multiplied by PCR and analysed with SSCA. '1/1', '2/2' denote different genotypes at position 976 (1/2 being GT and 2/2 being TT) in COL4A3. GG genotypes, being '1/1’ genotypes, are unmarked. Single stranded DNA (ssDNA) and double stranded DNA (dsDNA) patterns are marked on the side of the SSCA gel.

Figure 2

Figure 2

A: Three different PCR–single stranded conformational analysis patterns on one gel, representing COL4A4 exons 9, 10, and 42. PCR fragments were loaded in succession from shortest to longest PCR fragment at 30-min intervals. Exons 9 and 10 did not show any differences in elution shifts; in exon 42, different patterns were subsequently sequenced. B: Partial sequence of exon 42, made with reverse primer, showing homozygous substitution 1327M (3797AA). The position marked on reverse sequence is 3797TT. C: Partial sequence of exon 42, made with reverse primer, showing homozygous substitution 1327V (3797GG). The position marked on reverse sequence is 3797CC. '1/1',’1/2’ and '2/2' denote different genotypes at position 1327 in COL4A4. Single stranded DNA (ssDNA) and double stranded DNA (dsDNA) patterns are marked on the side of the SSCA gel.

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