Role of MYOC and OPTN sequence variations in Spanish patients with primary open-angle glaucoma

Abstract

Purpose: To retrospectively investigate the contribution of myocilin (MYOC) and optineurin (OPTN) sequence variations to adult-onset ocular hypertension (OHT) and primary open-angle glaucoma (POAG) in Spanish patients.

Methods: The promoter region and the three exons of MYOC were analyzed by direct PCR DNA sequencing in 40 OHT and 110 POAG unrelated patients. We used 98 subjects in whom OHT or glaucoma had been ruled out as controls. We also screened the complete coding region of the OPTN gene (exons 4-16) in all subjects by single-stranded conformational polymorphisms (SSCPs).

Results: We identified six common single nucleotide polymorphisms (SNPs) in the promoter region of MYOC (-1000C>G, -387C>T, -306G>A, -224T>C, -126T>C and -83G>A) and a polymorphic GT microsatellite (-339(GT)11-19). In addition, we detected four novel, rare DNA polymorphisms. None of these DNA sequence variations were associated with either OHT or POAG. We also found three (2.7%) POAG patients with MYOC pathogenic mutations. Two of these pathogenic mutations (Gln368Stop and Ala445Val) were previously described whereas the third (Tyr479His) was novel. Transient expression of the novel mutation in 293T cells supported its pathogenicity. Only two OPTN polymorphisms, which are not associated with the disease, were detected.

Conclusions: Overall, our data show that in Spain a minority of adult-onset high-pressure POAG patients carry heterozygous disease-causing mutations in the MYOC gene and that OPTN is not involved in either OHT or POAG.

Figure 1

Genomic structure of the human myocilin gene and location of identified DNA sequence variations. The promoter and the three exons are represented by boxes. Consensus regulatory sequences in the promoter region are depicted in the inset. Pathogenic mutations and polymorphisms in the coding region are indicated by solid and dashed arrows, respectively. Sequence variations in the promoter region are shown by arrowheads. Novel mutations are shown by asterisks. All mutations are defined in terms of the one-letter code. Designated features include AP-1-like and AP-2-like sequences, putative TATA and SAC boxes, glucocorticoid response element (GRE), negative glucocorticoid response element (nGRE), thyroid response element (TRE) and a MIR repeat [26,63]. -700_699ins: -700_699insCAGACACACATATACATGCACATACACA.

Figure 2

Pairwise linkage disequilibrium pattern of myocilin single nucleotide polymorphisms measured by D'. The location of each tested SNP along the MYOC gene is indicated at the top. The strength of LD is depicted by grey intensity, which moves from light grey to black as D' progresses from 0 to 1.

Figure 3

Multiple amino acid sequence alignment of myocilin from different species. Sequence alignment was generated by ClustalW. Residues affected by mutations are indicated by arrowheads. Asterisks indicate amino acid positions at which all query sequences are identical. Amino acid positions at which all analyzed sequences have amino acids that are chemically similar are denoted by two dots (:). One dot denotes amino acid positions with weak chemical similarity (.). Arrows indicated regions of the polypeptide chain which are predicted to fold into a beta-sheet conformation.

Figure 4

Western immunoblot of two novel myocilin mutations found in this study and expressed in transiently transfected 293T cells. Two hundred nanograms of DNA constructs encoding myc epitope-tagged versions of mutant myocilin forms (Arg346Thr, Tyr479His, Gln368Stop and Pro370Leu) were transfected into 293T cells. Separation of culture medium, soluble cellular fractions, and insoluble cellular fractions were carried out as indicated in the Materials and Methods. Detection was performed with an anti-myc monoclonal antibody. Myc-tagged wild-type myocilin was used as a control of normal expression and the myocilin mutation Pro370Leu was employed as a control of disease-causing mutation. The arrow and arrowhead indicate the position of the 55 kDa and 35 kDa myocilin bands, respectively. c.m.: culture medium; s.c.f.: soluble cellular fraction; i.c.f.: insoluble cellular fraction.

Figure 5

Subcellular distribution in transiently transfected 293T cells of human wild-type myocilin-GFP and two novel myocilin mutations found in this study. Two hundred nanograms of DNA constructs encoding wild-type myocilin (A), mutant myocilin forms, Arg346Thr (B) and Tyr479His (C), and the control, Pro370Leu (a disease causing mutation; D) were transfected into 293T cells. Wild-type myocilin was mainly detected in structures compatible with the Golgi apparatus and secretory vesicles. Note that the three mutant versions accumulated in the ER. The asterisk indicates the location of the Golgi apparatus. Arrows indicate the position of intracellular myocilin aggregates. Original magnification: X1600.