Polymorphisms in an intronic region of the myocilin gene associated with primary open-angle glaucoma--a possible role for alternate splicing

Abstract

Purpose: To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma.

Methods: Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed.

Results: A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the MYOC gene showed the presence of the predicted g.14072G>A polymorphism, g.1293C/T heterozygous polymorphism, instead of our predicted g.1293C/- polymorphism. Other than the prediction, two novel SNPs (g.1295G>T and g.1299T>G) and two reported SNPs (g.1284G>T and g.1286G>T) were also identified. Cluster analysis showed the g.14072G>A homozygous condition was more common in this cohort than the heterozygous condition.

Conclusions: We previously proposed that the disruption of dimer or oligomer formation by the C-term region allows greater chances of nucleation for aggregation. Here we suggest that polymorphisms in the myocilin genomic region that cause synonymous codon changes or those that occur in the intron regions can possibly lead to altered myocilin protein products through altered intron-exon splicing.

Figure 1

Full length myocilin is of 504 residues. The stop codon mutations Arg46Stop, Asp247Stop, Gln368Stop, Glu483Stop) in mutated myocilin contains 45 residues, 246 residues, and 367 residues, 482 residues, respectively and the rest of the region gets deleted. SNP121 results in shorter protein product containing 410 residues with amino acids 244 to 337 deleted from full length myocilin protein. SNP12 (rs2032555) consists of 248 residues with 243 to 248 residues altered from the full length myociln and the rest of the region from amino acid 249 to 504 deleted. SNP22 (rs10690049) has 218 residues with altered amino acids from 201 to 218 and 219 to 504 residues deleted. SNP67 (rs9600235) with 273 residues, 202 to 273 residues altered and 274 to 504 residues deleted. SNP68 (rs11295938) with 270 residues, 201 to 270 residues altered and 271 to 504 residues deleted. SNP88 (rs11366556) with 214 residues, 201 to 214 residues altered and 215 to 504 residues deleted. The maroon colored regions indicate sequence changes introduced because of the possible alternate splicing due to the Single nucleotide polymorphism indicated.

Figure 2

Space-filling model in four orientations for modified myocilin proteins. Full length myocilin, deletions due to presence of stop codon and deletions/modifications due to possible alternative splicing caused by the single nucleotide polymorphism shown in the model, as explained in Figure 1. Different regions in the model are colored as follows; NH₂-terminal region (Orange), coiled coil region (Pink), hinge region (Cyan), COOH-terminal region (Yellow), regions predicted to be deleted (White) due to stop codon mutation or possible alternative splicing, regions predicted to be modified due to possible alternative splicing.

Figure 3

Chromatogram of MYOC showing predicted splice site variation (A) homozygous g.14072G>A in patient-1 and (B) heterozygous g.14072G>A in patient-2. Top line: wild type sequence. Bottom line: observed sequence. The arrow indicates the position of sequence variation. Box represents the variation.

Figure 4

Eco72I restriction digestion to reconfirm the predicted g.14072G>A polymorphism. 1,4,5,7,8: POAG samples showing three distinct bands of 440bp, 233bp and 207 bp (reconfirms the heterozygous g.14072G>A polymorphism. 2,3,6,9,10,11,12: POAG samples showing a distinct band 440 bp (reconfirms the homozygous g.14072G>A polymorphism). C1: Control sample showing heterozygous g.14072 G>A polymorphism. C2: Control sample showing homozygous g.14072 G>A polymorphism. M: 100 bp DNA ladder. Arrow indicates the product size.

Figure 5

HinfI restriction digestion to screen the g.4453A>G polymorphism. 1 to 13 : Patient samples showing two distinct bands of 437 bp, 361 bp (absence of predicted g.4453A>G polymorphism). C: Control sample. M: 100bp DNA ladder. Arrow indicates the product size.

Figure 6

DNA sequence from Intron-1 of MYOC showing the g.1293C>T polymorphism. Top line: wild type sequence. Bottom line: observed sequence. The arrow indicates the position of sequence variation. Box represents the variation.

Figure 7

Chromatogram representing the g.1284G>T, g.1286G>T, and g.1299T>G polymorphisms from Intron-1 of MYOC. Top line: wild type sequence. Bottom line: observed sequence. The arrow indicates the position of sequence variation. Box represents the variation.

Figure 8

DNA sequence from Intron-1 of MYOC showing the g.1295G>T homozygous polymorphism. Top line: wild type sequence. Bottom line: observed sequence. The arrow indicates the position of sequence variation. Box represents the variation.