Biochem Genet (2013) 51:323–333 DOI 10.1007/s10528-012-9565-6 Contribution of TGFb1 Codon 10 Polymorphism to High Myopia in an Ethnic Kashmiri Population from India Shabhat Rasool • Ishfaq Ahmed • Rubiya Dar • Sheikh Gazalla Ayub • Sabia Rashid • Tariq Jan • Tahir Ahmed • Niyaz A. Naikoo • Khurshid I. Andrabi Received: 1 February 2012 / Accepted: 15 November 2012 / Published online: 17 January 2013 Ó Springer Science+Business Media New York 2013 Abstract This study looks at novel variants of the TGFb1 gene and their potential association with high myopia in an ethnic population from Kashmir, India. Allele frequencies of 247 Kashmiri subjects (from India) with high myopia and 176 ethnically matched healthy controls were tested for Hardy–Weinberg disequilibrium. S. Rasool  I. Ahmed  R. Dar  S. G. Ayub  K. I. Andrabi (&) Department of Biotechnology, Science Block, University of Kashmir, Hazratbal, Srinagar 190006, Jammu and Kashmir, India e-mail: andrabik@kashmiruniversity.net S. Rasool e-mail: shabhat_rasool@yahoo.com I. Ahmed e-mail: ishfi@rediffmail.com R. Dar e-mail: rubi07@rediffmail.com S. G. Ayub e-mail: gazallakhan@ymail.com S. Rashid Ophthalmology Unit, SMHS Hospital, Karan Nagar, Srinagar, Jammu and Kashmir, India e-mail: sabiarashid@gmail.com T. Jan Department of Statistics, University of Kashmir, Hazratbal, Srinagar, Jammu and Kashmir, India e-mail: trj527@gmail.com T. Ahmed Department of General Medicine, Sub-District Hospital, Kangan, Srinagar, Jammu and Kashmir, India e-mail: drtahir11@rediffmail.com N. A. Naikoo Immunology and Molecular Medicine SKIMS, Srinagar, Jammu and Kashmir, India e-mail: niyaznaik2005@gmail.com 123 324 Biochem Genet (2013) 51:323–333 The genotype and allele frequencies were evaluated using chi-square or Fisher’s exact tests. One of the three SNPs in codon 10 showed a significant difference between patients and control subjects (rs1982073: p genotype = 0.003, p allele = 0.001). There were no statistically significant differences between patients and control subjects for the other two SNPs, rs1800471 at codon 25 and a novel variant at codon 52. SNP rs1982073, substituting proline with leucine, appeared to be significantly associated with high myopia (p \ 0.05). In silico predictions show that substitutions are likely to have an impact on the structure and functional properties of the protein, making it imperative to understand their functional consequences in relation to high myopia. Keywords Myopia  Ethnic  Polymorphism  CSGE  TGFb1  Novel Introduction Development of myopia is a consequence of an incongruity between the power of the optical components and the axial length of the eye (Wensor et al. 1999). Lower grades of myopia (\ -6 diopters) are not associated with blinding conditions, but higher or pathological grades with a refractive error greater than -6 diopters are often associated with blinding conditions like macular degeneration, retinal detachment, and glaucoma (Lin et al. 2006). Ethnic diversity plays a great role in the progression of myopia, reaching as high as 70–90% in some parts of Asia, 30–40% in Americans and Europeans, and up to 20% in Africans (Chow et al. 1990; Lin et al. 2006). Comparative prevalence rates of high myopia from different parts of the world show considerable variability but still confirm that it affects a significant proportion of the population in different countries (Wang et al. 1994; Curtin 1970; Tokoro and Sato 1982; Lin et al. 1988; Wilson and Woo 1989; Fledelius 1988; Paluru et al. 2005). Ahmed et al. (2008) found the prevalence in India to be 19%, with 4% in Kashmir, and also reported the effects of age, gender, and socioeconomic conditions, showing an increase in myopia prevalence with increased age (3.76% for the group aged 6–10 years, 4.9% for 11–15 years, and 6.16% for 16–22 years). Additionally, girls on average were 1.52 times more likely to have myopia than boys (5.54% of girls and 3.6% of boys). Socioeconomic conditions had an impact on the prevalence of myopia. Only 3.23% of students from medium and high socioeconomic strata had myopia; it was about three times more prevalent in students from low socioeconomic strata (8.60%). Despite many decades of research, there is little knowledge about the precise molecular defects and abnormal biochemical pathways that result in myopia. It is a highly prevalent and complex phenotype involving both genetic and environmental factors (Ibay et al. 2004). Recent studies have mapped 14 genomic loci associated with myopia (MYP1 on Xq28, MYP2 on chromosome 18p, MYP3 on chromosome 12q, MYP4 on chromosome 7q, MYP5 on chromosome 17q, MYP6 on chromosome 22q12, MYP7 on chromosome 11p13, MYP8 on chromosome 3q26, MYP9 on chromosome 4q12, MYP10 on chromosome 8p23, MYP11 on chromosome 4q22–q27, MYP12 on 123 Biochem Genet (2013) 51:323–333 325 chromosome 2q37.1, MYP13 on Xq23–q25, and MYP14 on chromosome 1p36) (Paluru et al. 2003, 2005; Hammond et al. 2004; Naiglin et al. 2002; Schwartz et al. 1990; Stambolian et al. 2004; Wojciechowski et al. 2006; Young et al. 1998; Zhang et al. 2006; Inamori et al. 2007). A high heritability of myopia does not mean that environmental factors have no effect on its development. Close visual work in childhood has been hypothesized as an environmental risk factor for myopia progression (Saw et al. 2001). Initially one of the studies indicated a strong association between myopia and nightlight exposure (Quinn et al. 1999), but recent research has shown contradictory results (Saw et al. 2001; Zadnik et al. 2000; Gwiazda et al. 2000; Guggenheim et al. 2003). Myopia develops mainly because of excessive elongation in axial dimension rather than changes in corneal or lens power in human beings (Zadnik 1997). In animal models of myopia, active remodeling of sclera plays a crucial role in axial elongation (McBrien and Gentle 2003; Rada et al. 2006). Scleral remodeling involves reduced production of extracellular matrix, which results from reduced production of collagen and proteoglycans and from increased collagen degradation along with concomitant increased activity of matrix metalloproteinase 2 (MMP2) and a reduction in the activity of tissue inhibitors of MMP. Transforming growth factor b (TGFb) together with its receptor expressed in eye tissues (Saika 2006) also regulates the proliferation of fibroblasts and production of collagen, MMP2, and tissue inhibitors of MMP (Overall et al. 1989). TGFb is an obvious player in the regulation of scleral remodeling and accordingly has been implicated in the development of myopia (Zha et al. 2009). TGFb exists in three isomeric forms (TGFb1, TGFb2, and TGFb3), and during myopia development the expression of TGFb1 was found to be reduced in an isoform and time-specific manner in the sclera (Kusakari et al. 2001) and retina/choroid (Song et al. 2000) of chickens, whereas the TGFb2 level increased in both the retina/choroid and sclera of the chickens (Kusakari et al. 2001; Song et al. 2000). Cultured human retinal pigment epithelial cells have been shown to express at least TGFb1 and TGFb2 isoforms (Lam et al. 2003; Andrew et al. 2004; Tanihara et al. 1993; Seko et al. 1995). TGFb1 belongs to a family of polypeptides that display a broad range of multifunctional activities like transcriptional activation and increase in synthesis and secretion of matrix proteins (Guggenheim and McBrien 1996). The gene is encoded on chromosome l9ql3.1–ql3.3 and contains seven exons (Patel et al. 2005). Recent studies investigated the association of single-nucleotide polymorphisms (SNPs) of the TGFb1 gene and high myopia but produced conflicting results (Hayashi et al. 2007). Our study serves to clarify this relationship with a case– control design and ethnic purity of our population. Materials and Methods A total of 423 subjects (247 with high myopia of \ -6D and 176 healthy control subjects) were recruited from the local hospital (ophthalmology unit) as well as from our ophthalmologist’s clinic. Although we were interested in doing a familial study, it was very difficult to find ample numbers of families with high myopia. We therefore 123 326 Biochem Genet (2013) 51:323–333 designed a case control study. Informed consent was obtained from the study subjects after an explanation of the nature and possible consequences of the study. Criteria for selection included a history of onset of myopia in all affected subjects. Individuals were excluded if any ocular disease such as retinopathy or cataract was known or if they had a known genetic disease associated with myopia, such as stickler or Marfan syndrome. An ophthalmic examination of the participating subjects was performed by our ophthalmologist. Ophthalmic evaluation included measuring visual acquity, keratometry, retinoscopy, slit lamp examination of the anterior segment, fundus examination, and measurement of axial length. Autorefraction was taken and an A-scan was done on both eyes. Subjects were encouraged to narrate all the details relevant to this study. This included age of subject, history of onset of myopia, any associated ocular complications, and information regarding close work. The study was approved by the Research Ethics Committee. Polymerase Chain Reaction Genomic DNA was extracted from whole blood samples using standard protocols. The polymerase chain reaction (PCR) was carried out in a total volume of 50 ll, containing 50–100 ng genomic DNA, 2–6 pmol of each primer, 19 PCR buffer (Sigma Aldrich), and 0.5 U Taq DNA polymerase (Sigma Aldrich). The following primer sequences were used for amplification: 50 -GCCTCCCCACCACACCAG-30 (sense) and 50 -GCCGCAGCTTGGACAGGAT-30 (antisense) (Lin et al. 2006). The expected PCR product of 237 bp was generated successfully. The PCR cycling conditions involved one cycle of denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 45 s, annealing at 59°C for 45 s, and extension at 72°C for 45 s, and one final 6 min elongation cycle at 72°C. PCR products were then purified using a purification kit or NaI. Conformation Sensitive Gel Electrophoresis (CSGE) Purified PCR products were subjected to denaturation and renaturation procedures for generation of potential heteroduplexes and analyzed by CSGE strictly as described by Ganguly et al. (1993). This mutation detection technique has many advantages over other techniques such as SSCP and PTT. Samples with unusual mobility during these assays (Fig. 1) were finally sequenced to confirm the presence of sequence variations along with controls (Macrogen, Korea). CSGE conditions described by Ganguly et al. (1993) for amplicons in the range 200–800 bp were able to detect 60 out of 63 single-base mismatches. Still, the migrating bands in this method are sometimes less clear and could lead to human error in reporting results. Therefore, sequencing of the samples screened with CSGE is relied on to provide accurate results (Blesa et al. 2004). Sequence Analysis Sequence results obtained in Fasta and PDF formats were analyzed using Clustal X version 2 software (Thompson et al. 1997; Larkin et al. 2007), and Chromas Pro 123 Biochem Genet (2013) 51:323–333 327 Fig. 1 Heteroduplex analysis of TGFb1 amplicons (237 bp) by conformation sensitive gel electrophoresis. Lanes 3, 5, and 6: samples with G?C variation. Lanes 8, 9, and 12: samples with C?T variation as confirmed by sequencing. Lane 4: separation pattern of 1 kb DNA marker; first band corresponds to 250 bp. Samples loaded in lanes 1, 2, 7, 10, 11, and 13 did not show any base variation on sequencing version 1.49 beta 2 software was used for the detailed inspection of individual chromatograms. Statistical Analysis Genotypes were obtained by direct counting with subsequent calculation of allele frequencies. Statistical analysis was undertaken using the chi-square test and significance value (p). A p value of less than 0.05 was considered significant. Adherence to the Hardy–Weinberg equilibrium constant was tested using the chisquare test with one degree of freedom. Odds ratio and confidence interval were also calculated. In Silico Analysis The amino acid sequence of the protein in Fasta format obtained from NCBI (www.ncbi.nlm.nih.gov) was submitted to the automated server I-TASSER (zhang.bioinformatics.ku.edu/I-TASSER) for three-dimensional (3D) structure prediction (Zhang 2007, 2008). The server furnishes predicted 3D structure in a PDB format. A Swiss PDB Viewer was used for viewing files and computing the free energy of the predicted 3D structures (Camacho et al. 2000; Camacho and Gatchell 2003; Comeau et al. 2004). Results Two missense variants, C/T (rs1982073) at codon 10 and G/C (rs1800471) at codon 25, corresponded to previously reported SNPs in public databases. A silent variation (G/A at codon 52) observed in the study population appeared to be novel (Table 1). Genotype analysis of individual variants revealed the presence of both heterozygous 123 328 Biochem Genet (2013) 51:323–333 Table 1 Polymorphism detected in exon 1 of TGFb1 gene in ethnic Kashmiri population Codon rs Wild-type nucleotide Observed base pair change Amino acid change 10 1982073 C T Pro to Leu 25 1800471 G C Arg to Pro 52 Novel G A Silent Chromatogram and homozygous genotypes. A subtle and statistically significant difference in the genotypic frequency for the codon 10 variant (p allele = 0.001, p genotype = 0.003; v2 allele = 10.36, v2 genotype = 11.451; OR 1.59, CI 95%: 1.9–2.11) was indicative of its possible association with high myopia (Table 2). The relative frequency of occurrence of variants at codon 25 (Table 3) and codon 52 (Table 4) for high myopes was found to be statistically insignificant, compared with their occurrence in healthy controls. In Silico Prediction Results TGFb1 was modeled by I-TASSER to obtain its PDB structure. Analysis (energy calculations) was done using the PDB Viewer. Discussion Diverse populations have presented inconsistent profiles of association data owing largely to the heterogeneous nature of the subject populations, but the TGFb1 codon 123 Biochem Genet (2013) 51:323–333 329 Table 2 Distribution of alleles and genotypes of TGFb1 gene polymorphism at codon 10 in healthy and high myopic subjects Allele or genotype Control (n = 176) Affected (n = 247) n % n % C 116 33 217 44 T 236 67 277 56 CC 15 8.52 41 16.6 CT 86 48.86 135 54.66 TT 75 42.62 71 28.74 v2 p Value OR CI (95%) 10.36 0.001* 1.59 1.9–2.11 11.451 0.003* * Statistically significant at p \ 0.05 Table 3 Distribution of alleles and genotypes of TGFb1 gene polymorphism at codon 25 in healthy and high myopic subjects Allele or genotype Control (n = 176) Affected (n = 247) n % n % G 237 67 306 62 C 115 33 188 38 GG 77 43.75 96 38.87 GC 83 47.16 114 46.15 CC 16 9.09 37 14.98 v2 p Value OR CI (95%) 2.59 0.107 0.78 0.59–1.05 3.46 0.17 Table 4 Distribution of alleles and genotypes of TGFb1 gene polymorphism at codon 52 in healthy and high myopic subjects Allele or genotype Control (n = 176) Affected (n = 247) n % n % G 246 70 361 73 A 106 30 133 27 GG 90 51.14 136 55.06 GA 66 37.5 91 36.03 AA 20 11.36 22 8.91 v2 p Value OR CI (95%) 1.032 0.310 1.16 0.86–1.58 0.928 0.629 10 (rs 1800470) polymorphism has been found to be associated with high myopia in Taiwanese Chinese, showing a strong association of the CC genotype with high myopia (Lin et al. 2006). A later study by Hayashi et al. (2007) on TGFb1 gene polymorphism in high myopia revealed no significant association with high myopia, excluding TGFb1 as a candidate gene for myopia in the Japanese population. A recent study of TGFb1 polymorphism in high myopia affecting Chinese subjects of Hong Kong revealed the association of four SNPs in the 50 half of the TGFb1 locus with high myopia. This study could successfully replicate the positive finding of Lin et al. (2006), supporting the association of TGFb1 with myopia susceptibility 123 330 Biochem Genet (2013) 51:323–333 (Zha et al. 2009; Sandhya et al. 2011). The Kashmiri population representing a homogeneous cohort of common ethnicity provided an opportunity to revalidate the significance of TGFb1 sequence variants (if any) for defining their relevance in the pathogenesis of the disease. Genetic polymorphisms have been widely used to test the association of a gene with a commonly seen and multifactorial disease instead of a single gene disease. Since nucleotide polymorphism is not strong enough to result in a lethal phenotype, this allele will not eventually disappear or reach frequency equilibrium without any selective disadvantage for individuals. Since ethnic differences do exist, it is imperative to substantiate or dispute the relevance of such polymorphism in genetically purer cohorts. To date, variations in several genes have been reported to associate with high myopia but only a few studies have been replicated successfully. Our study is a kind of replication study (Lin et al. 2006; Zha et al. 2009) associating TGFb1 codon 10 polymorphism with high myopia in a population wherein heterogeneity effects seen in other populations are neutralized to a large extent. Investigating the genetics of common and complex disorders such as myopia remains one of the great challenges in human genetics. Myopia is considered to be a complex and multigenic condition involving several overlapping signaling pathways, each mediated by a group of distinct genes. Therefore, studying the genetic polymorphisms of myopia-related genes can further clarify the relationship between genetics and myopia. This association has helped increase our knowledge of prevention and treatment of myopia. The relationship between TGFb and sclera remodeling during the development of myopia is well established (Honda et al. 1996; Kusakari et al. 2001). TGFb1 has been analyzed as a candidate gene because of its differential expression in experimental chicken myopia (Jobling et al. 2004) and its functional relation with TGIF (Chen et al. 2003). In the earlier study (Lin et al. 2006), only one (rs1982073 at codon 10, 29T/C, Leu10Pro) was analyzed and reported to be associated with high myopia in a Chinese population living in Taiwan (p = 0.001). At the same time, 10 SNPs (rs1982073 not included) and related haplotypes in TGFb1 were analyzed in 330 Japanese patients with high myopia and 330 control subjects, but none was associated with high myopia (Hayashi et al. 2007), and a further study on TGFb1 was suggested (Wang et al. 2009). Our study adds support to the idea that the codon 10 polymorphism of the TGFb1 gene contributes to the pathogenesis of myopia. Further investigation is needed to establish the precise role played by TGFb1 in the development of high myopia, especially in the context of codon 10 polymorphism. In silico predictions show higher energy states for both codon 10 (-8,931.029 kJ/mol) and codon 25 (-8,102.402 kJ/mol) variants than for wild-type protein (-9,573.964 kJ/mol) and protein that has both variations together (-9,501.950 kJ/mol), which may affect the stability of the protein. Since these SNPs change the energy state of the protein, an interference with its functional properties and stability may be possible. Genes farther up- and downstream of TGFb1 also need to be investigated, as it is likely that a number of genes will form the genetic background in individuals with myopia, on which environmental factors will act. In conclusion, we observed that the frequency of the C allele at codon 10 of TGFb1 was higher in the high myopia group than in the control group. People who 123 Biochem Genet (2013) 51:323–333 331 have the CC/CT genotypes at codon 10 may be at greater risk for developing high myopia (Table 2). 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