Association of FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile Polymorphisms with Congenital Cataract and Microphthalmia

Abstract

Purpose:

To investigate the association of FOXE3-p.Ala170Ala (rs34082359) and PITX3-p.Ile95Ile (rs2281983) polymorphisms with congenital cataract and microphthalmia in a western Indian population.

Methods:

FOXE3-p.Ala170Ala (c.510C>T) and PITX3-p.Ile95Ile (c.285C>T) polymorphisms were genotyped in 561 subjects consisting of 242 cases with congenital cataract, 52 with microphthalmia, and 267 controls using polymerase chain reaction-restriction fragment length polymorphism. Approximately 10% of samples were randomly sequenced for each single nucleotide polymorphism to confirm the genotypes. The prediction of mRNA secondary structure for polymorphism FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile was performed.

Results:

A significantly high frequency of T allele and a borderline significance in the frequency of TT genotype of FOXE3-p.Ala170Ala was observed in microphthalmia cases, as compared to controls [T allele: OR: [CI] = 1.8 [1.15-2.72], P = 0.0115; TT: OR [CI] = 2.9 [1.14-7.16], P = 0.0291). The frequency of CC genotype was significantly low in microphthalmia cases when compared to controls (CC: OR [CI] = 0.5 [0.24-0.86, P = 0.0150). There was no significant difference in the allele and genotype frequencies of PITX3-p.Ile95Ile between cases and controls. A slight free energy change was observed in the secondary structure of mRNA between the FOXE3-p.Ala170Ala C-allele (-917.60 kcal/mol) and T-allele (-916.80 kcal/mol) and between PITX3-p.Ile95Ile C-allele (-659.80 kcal/mol) and T-allele (-658.40 kcal/mol).

Conclusion:

The present findings indicate that FOXE3-p.Ala170Ala ‘T’ allele and ‘TT’ genotype could be predisposing factors for microphthalmia while ‘CC’ genotype might play a protective role against it. A reduction in the free energy change associated with FOXE3-p.Ala170Ala ‘T’ allele could further contribute towards disease risk.

INTRODUCTION

Congenital cataract and microphthalmia are vision-threatening eye disorders that account for 10-11% of childhood blindness.[1,2] Congenital cataract represents lens opacity present at birth[3] and microphthalmia is characterized by the presence of small eye within the orbit.[1] Both disorders occur either in isolation or as part of a syndrome and are highly heterogeneous.[1,4] Several chromosomal, monogenic, and environmental factors have been identified as causes for the occurrence of congenital cataract and microphthalmia.[1,5,6] Nevertheless, genetic causes have been suggested to play a major role in these conditions.[1,7]

Several transcription factor genes are known to be indispensable for vertebrate eye development. Two such genes: forkhead box E3 (FOXE3) and paired-like domain transcription factor 3 (PITX3), play an important role in ocular development and map to chromosome 1p32 and 10q25 respectively. In a normal eye, FoxE3 controls lens epithelial proliferation and closure of lens vesicle,[8] and Pitx 3 controls lens epithelial maintenance and lens fiber differentiation.[9] In humans, mutations in FOXE3 and PITX3 are responsible for varying degree of eye phenotypes such as congenital cataract, anterior segment mesenchymal dysgenesis, and microphthalmia.[10,11,12,13,14,15] However, genetic association studies associating polymorphisms in FOXE3 and PITX3 genes and these eye disorders are sparse.

In our study on mutation screening in candidate genes for anophthalmia/microphthalmia, we observed a higher frequency of two polymorphisms: FOXE3-p.Ala170Ala (c.510C>T, rs34082359) and PITX3-p.Ile95Ile (c.285C>T, rs2281983), in isolated microphthalmia cases as compared to controls.[16] This prompted us to investigate the association of these polymorphisms in congenital cataract and isolated microphthalmia cases using a larger number of samples. Both these polymorphisms are synonymous and do not change the amino acid sequence. However, synonymous single nucleotide polymorphism (SNP) contributing towards the risk of several disorders are very well defined.[17,18,19] Genetic association studies of this kind have the potential to bring about major advancements in the field of congenital cataract and microphthalmia through diagnostics and risk prediction, as well as through modification in the therapeutic approaches, depending on the genetic profile of the patients. This is the first study to test for the association of polymorphisms in FOXE3 and PITX3 with congenital cataract and microphthalmia.

METHODS

Clinical Settings and Recruitment of Subjects

This study adhered to the tenets of the Declaration of Helsinki and was approved by the institutional review committee. All the subjects belonged to western India (Gujarat, Rajasthan, Maharashtra, and parts of Madhya Pradesh). All study subjects, or their guardians, were informed about the nature of the study and informed consent was obtained from them. Peripheral blood was obtained from 561 subjects consisting of 242 cases with congenital cataract (females, n = 138; males, n = 104), 52 with microphthalmia (females, n = 32; male, n = 20), and 267 age-and ethnicity-matched normal healthy controls (females, n = 144; males, n = 123). Diagnosis of congenital cataract was made using a slit lamp biomicroscope and classification of the type of cataract was based on zone and morphology of lens opacification. Congenital cataract cases consisted of nuclear (n = 42), lamellar (n = 59), posterior subcapsular (n = 46), membranous (n = 28), sutural (n = 15), and total (n = 52) cataracts. A globe with a total axial length at least two standard deviations less than the mean for age was diagnosed as microphthalmia.[20] The inclusion criteria comprised of isolated microphthalmia and congenital cataract diagnosed between the ages of 0.1-1 year. The controls had no family history of ocular malformations. Subjects with traumatic cataracts, eye disorders except for congenital cataract and microphthalmia, viral infections, chromosomal abnormalities, systemic diseases, neurodevelopmental disorders, and inborn errors of metabolism were excluded.

Single Nucleotide Polymorphism Genotyping

Genomic DNA was isolated from blood using the salting-out method.[21] The FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile polymorphisms were selected for genotyping based on observations from our previous study. Genotyping of FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile was carried out using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Primers for PCR-RFLP were designed using primer3.0 (http://primer3.ut.ee/) online program. FOXE3 was amplified using primers FOXE3-FW: 5’-CGCAAGTGGCAGAACAGCAT-3’ and FOXE3-RV: 3’-TAGCAGGAGTTTGAGTCCAG-5’ that gives an amplicon of length 769 base pairs (bp) while PITX3 was amplified using primers PITX3-FW: 5’-GGGAGCCAGCGAGTGGCTTAGGAG-3’, and PITX3-RV: 3’-GGGTGGAACCGCTGGCCTCCG-5’ that gives an amplicon of length 374 bp. The polymorphisms were analyzed using RestrictionMapper version 3.0 (www.restrictionmapper.org/) and were found to create a gain of DdeI restriction enzyme site for T allele of FOXE3-p.Ala170Ala and loss of MboI restriction enzyme site for T allele of PITX3-p.Ile95Ile. About 10% of samples were randomly selected and the genotypes for each SNP confirmed by Sanger's bi-directional sequencing using BigDye®Terminator V3.1 Cycle sequencing kit and run on the ABI 3130xl Genetic Analyzer.

Statistical Analysis

Allele and genotype frequencies were determined using (http://analysis.bio-x.cn/myanalysis.php).[22] Fisher's exact test was used to check the strength of association of SNPs between cases and controls. Odds ratio at 95% confidence interval (CI) was calculated using http://www.hutchon.net/confidor.htm. Correction for multiple testing was done by dividing 0.05 by the total number of SNPs evaluated. P values <0.05 were considered as statistically significant.

In silico Analysis

The functional effect of the two SNPs, FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile, were checked using tools such as PROVEAN,[23] SIFT,[24] and mutation taster[25] according to the standards and guidelines set by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.[26]

Analysis of RNA Secondary Structure

The in silico modeling was done by RNAfold web server http://rna. tbi. univie. ac. at/cgi-bin/RNAWebSuite/RNAfold. cgi to predict the secondary structure of mRNAs for FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile.

RESULTS

The study consisted of 242 congenital cataract cases (females: 57%, males: 43%; mean age: 6.11 ± 2.14 years), 52 microphthalmia cases (females: 61.5%, males: 38.5%; mean age: 5.19 ± 2.43 years), and 267 age and ethnicity matched controls (females: 53.9%, males: 46.1%; mean age: 5.83 ± 2.10 years). Congenital cataracts consisted of lamellar (24.4%), total (21.4%), posterior subcapsular (19.0%), nuclear (17.4%), membranous (11.6%), and sutural (6.2%) cataracts.

Genotyping by PCR-RFLP of FOXE3-p.Ala170Ala yielded four fragments (512, 189, 47, and 21 bp) for C allele and five fragments (339, 189, 173, 47, and 21 bp) for T allele when digested with DdeI (C↓TNAG) restriction enzyme, and PITX3- p.Ile95Ile yielded one fragment (374 bp) for T allele and two fragments (253 and 121 bp) for C allele when digested with MboI (^↓ GATC) restriction enzyme. The three possible genotypes of both polymorphisms were scored using agarose gel electrophoresis [Figure 1a and c]. The corresponding electropherograms are shown in Figure 1b and d.

Figure 1.

(a and c) Agarose gel electrophoresis showing PCR-RFLP patterns for FOXE3-p.Ala170Ala, and PITX3-p.Ile95Ile after digesting with DdeI and MboI restriction enzyme respectively. (b and d) Electropherograms confirming all the three possible genotypes of FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile respectively.

The allele and genotype frequencies of both polymorphisms were in Hardy–Weinberg equilibrium in both cases and controls. The frequency of FOXE3-p.Ala170Ala T allele was found to be significantly increased in microphthalmia cases when compared to controls (OR [CI] = 1.8 [1.15-2.72], P = 0.0115). The frequency of CT and TT genotype of FOXE3-p.Ala170Ala was found to be significantly high in microphthalmia cases when compared to controls (CT: OR [CI] = 2.0 [1.04-3.98], P = 0.0459; TT: OR [CI] = 2.9 [1.14-7.16], P = 0.0291) [Table 1]. The genotyping of PITX3-p.Ile95Ile did not show any significant difference in the allele and genotype frequencies between cases and controls [Table 2]. A significant difference in the frequency of CC genotype of FOXE3-p.Ala170Ala was observed between microphthalmia cases and controls using dominant model (OR [CI] = 0.5 [0.24-0.86, P = 0.0150) [Table 3]. After correcting the P value for multiple testing (P = 0.025), the frequency of FOXE3-p.Ala170Ala T allele was found to be significantly associated with microphthalmia and there was a borderline significance in the association of TT genotype with microphthalmia. The FOXE3-p.Ala170Ala CC genotype was found to confer protective effect towards the development of microphthalmia. There was no significant difference in the allele and genotype frequencies of FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile among the different cataract types and the controls (data not shown). In silico analysis by Provean, SIFT, and mutation taster classified both FOXE3-p.Ala170A and PITX3-p.Ile95Ile as ‘neutral’, ‘tolerated’, and ‘polymorphisms’ respectively. A slight change in free energy was observed between C allele (-917.60 kcal/mol) and T allele (-916.80 kcal/mol) of FOXE3-p.Ala170A [Figure 2a and b] and C allele (-659.80 kcal/mol) and T allele (-658.40 kcal/mol) of PITX3-p.Ile95Ile [Figure 2c and d].

Table 1.

Distribution of FOXE3-p.Ala170Ala in the study groups

Table 2.

Distribution of PITX3-p.Ile95Ile in the study groups

Table 3.

Dominant and recessive models for FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile

Figure 2.

Predicted RNA secondary structure and energies for FOXE3-p.Ala170Ala (a) C allele and (b) T allele and PITX3-p.Ile95Ile (c) C allele and (d) T allele measured using RNA fold. The alteration in secondary structure has been shown by an arrow. The stability of the RNA structure is determined using the free energy.

DISCUSSION

Normal eye development requires sequential activation and silencing of thousands of genes in a spatial and temporal manner. Several tissue-specific transcription factors control these developmental processes. An alteration in any of these transcription factors can have serious consequences for normal ocular development and could lead to partial or total blindness. In humans, mutations in several transcription factor genes have been reported to cause disruption of vertebrate eye development or maintenance.[10,11,12,13,14,15,27,28,29,30,31,32] This led to the hypothesis that polymorphisms in these genes may also predispose towards the risk of congenital eye disorders. In our previous studies on mutation screening in candidate genes for congenital cataract and microphthalmia, we observed that the frequency of two polymorphisms FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile was higher in congenital cataract and microphthalmia cases as compared to controls. Hence in the present study, we evaluated the association of polymorphisms in FOXE3 and PITX3 transcription factors with the risk of developing congenital cataract and microphthalmia.

The polymorphisms FOXE3-p.Ala170Ala and PITX3-p.Ile95Ile tested in the present study are synonymous variations that do not change the protein sequence. However, synonymous SNPs are gaining much attention for their ability to cause changes in protein expression, conformation, and function, and also for their contribution towards human disease risk and other complex traits.[17,18,19] In our study, the FOXE3-p.Ala170Ala T allele and TT genotype (borderline) was found to be associated with the risk of microphthalmia while the CC genotype was found to have a protective effect towards its development.

During the translation process, the stability of mRNA has a profound impact on the expression level of protein. Any alteration in the free energy change of the secondary structure of mRNA can reduce the translation efficiency up to four-fold.[33] In the RNA secondary structure, a decrease in predicted free energy change (ΔG) has been suggested to reduce the relative expression of the protein.[33] In our study, we observed a minor free energy change in the secondary structure of FOXE3 mRNA between the C allele (-917.60 kcal/mol) and T allele (-916.80 kcal/mol) and also of PITX3 mRNA between the C allele (-659.80 kcal/mol) and T allele (-658.40 kcal/mol). Hence, we believe that the T allele of FOXE3-p.Ala170Ala might contribute to decreased FOXE3 mRNA expression when compared to C allele carriers which might further increase disease susceptibility. However, this idea needs to be functionally validated by in vitro studies.

Our study did not find any significant association of FOXE3-p.Ala170Ala with congenital cataract and PITX3-p.Ile95Ile with both congenital cataract and microphthalmia. This suggests that in addition to mutations in candidate genes and an individual's genotype, there are several other modifying factors such as environmental factors, gene-gene interactions etc. that could pose a serious risk towards the onset and maturation of the disease. This study has the limitation of not including the normal, unaffected blood relatives of the study population and also the moderately small sample size (especially microphthalmia) and hence to substantiate the present findings, similar studies using the blood relatives and large cohorts in different populations is essential.

Financial Support and Sponsorship

This study was partially supported by grants from the Indian Council of Medical Research, Government of India (File No. 5/4/6/10/Oph./11-NCD II and File No. 5/4/6/2012-RMC).

Conflicts of Interest

There are no conflicts of interest.