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Heritability of myopia and its relation with GDJ2 and RASGRF1 genes in Lithuania

Abstract

Background

This study aimed to assess heritability of myopia in Lithuania and evaluate both genes GJD2 (Gap Junction Protein, Delta 2) and RASGRF1 (RAS protein-specific guanine nucleotide-releasing factor 1) relation with myopia.

Methods

In this study Lithuanian twin population aged between 18 and 40 (n = 460) were examined. Single-nucleotide polymorphisms of the RASGRF1 (rs8027411) and GJD2 (rs634990) genes were assessed by real-time polymerase chain reaction method.

Results

Intrapair correlations for spherical equivalent in all twin pairs were significantly higher in MZ twin pairs r = 0.539 (p < 0.001, 95% CI 0.353–0.684) than in DZ twin pairs r = 0.203 (p < 0.01, 95% CI 0.0633–0.442) in myopia group. Correlations for spherical equivalent in emmetropia group were not significant in MZ twin pairs r = 0.091 (p > 0.05, 95% CI -0.215-0.381) and in DZ twin pairs r = − 0.220 (p > 0.05, 95% CI -0.587-0.222). The odds ratio (95% CI) were 2.7 (1.018–7.460) for combinations of genotypes of rs634990 CC and rs8027411 GT (p = 0.046).

Conclusions

Our studies have shown that the heritability of myopia makes 67.2% in Lithuania. Persons with combinations of genotypes rs634990 CC and rs8027411 GT have 2.7 times higher odds to have myopia.

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Background

Refractive error is one of the priority targets of World Health Organization Vision 2020 [1]. More than 150 million people in the world are estimated to be visually impaired because of uncorrected refractive error, 8 million of them are functionally blind [2]. Myopia affects approximately one-third of adults older than 20 years in the United States, and in areas with high prevalence, specifically in urban East Asia, more than 80% of students graduating from school are myopic [3]. It is estimated that by the year 2020, 2.5 billion people - one third of the world’s population will have been affected by myopia alone [4]. Worldwide, myopia affects approximately one in four individuals in western population and is the most common visual disorder [4, 5].

In Lithuania, the data on refractive errors are systematically collected, and the official statistics are published by the Lithuanian Department of Statistics. In the population of Lithuania from 2007 to 2014, the prevalence of myopia increased from 44.3 to 63.9 per 1000 population (www.sic.hi.lt).

Both genetic and environmental factors can affect development of myopia, but the exact causes are not fully understood [6]. The genetic contribution to a trait often is assessed through heritability. Heritability is the proportion of phenotypic variability in a population trait that is due to genetic differences [7]. Up to now, the heritability of myopia has not been studied in Lithuania.

Twin studies allow us to estimate the overall gene influence, and the results can show the heritability of myopia. The twin method assumes that monozygotic (MZ) twins are influenced by largely similar environmental differences as dizygotic (DZ) twins, but MZ twins share the same genes whereas DZ twins on average share only half their genes [8]. Twin method is excellent for the estimation of heritability of myopia, but it does not show the specific genes that may possibly be involved in the heritability of myopia. Thus, we chose genetic markers, in order to establish whether any of them are significantly associated with myopia.

Genome-wide association studies (GWAS) for refractive error showed that single nucleotide polymorphisms (SNPs) in 15q25 and 15q14 were associated with refractive error and myopia [9]. RASGFR1 is a gene made of 28 exons. This gene has a significant influence on development of myopia. RASGFR1 gene encodes Ras protein-specific guanine nucleotide-releasing factor-1, which is highly expressed in the retina and neurons. Then RASGFR1 gene proceeds to activate Ras [10, 11]. Also, RASGRF1 is a nuclear exchange factor that promotes GDP/GTP exchange on the Ras family GTPases and is related to synaptic transmission of the photoreceptor responses [12]. Muscarinic receptors and retinoic acid can regulate RASGRF1 expression as well [11]. Some animal and human studies showed that muscarinic inhibitors prevented the development of myopia [13]. In animal models of myopia there was detected reduced synthesis of choroidal retinoic acid [14]. To date, studies of SNP rs8027411 of RASGRF1 gene associations with high myopia in different populations have provided controversial results [6, 15,16,17].

The GJD2 gene at 15q14 encodes a neuron-specific protein connexin 36 (CX36), a 36 kDa protein, which is a neuron-specific protein of a family of integral membrane proteins [18]. CX36 forms gap junction channels between adjacent membranes of neuronal cells. It is present in photoreceptors, bipolar and amacrine cells, and, by enabling intercellular transport of small molecules and ions, plays an essential role in the transmission process of the retinal electric circuitry [18, 19].

The aim of our research was to find associations between the GJD2, RASGRF1 genes and myopia development and to assess the heritability of myopia in Lithuania.

Methods

Ethics statement

Permission (Number P1–52/2005) to undertake the study was obtained from the Kaunas Regional Biomedical Research Ethics Committee. Before the study, the procedure and purpose of the study was explained, and an informed consent was obtained from all participants.

Study samples

The twins participating in this study were from the Twin Centre of Lithuanian University of Health Sciences. The Twin Centre has registered more than 600 twin pairs who agreed to participate in various medical and genetic studies. The study was conducted in the Institute of Biological Systems and Genetics Research, Lithuanian University of Health Sciences.

Refractive error measurement

Refractive error was measured with Sol. Cyclopentolate 1% using an autorefractor (Accuref-K9001, Shin-Nippon, Japan) and calculated by the mean spherical equivalent for each of the two eyes of every individual. The mean spherical equivalent was calculated using the standard formula: spherical equivalent = sphere+(cylinder/2).

MZ and DZ twins with spherical equivalent of at least one eye > = − 0.5 D were assigned to the myopia group. Twins whose spherical equivalent was between 0.49 and − 0.49 D were included in the emmetropia group. The myopia degree was determined by the strength or optical power of a corrective lens that focuses distant images on the retina: from − 0.5 D to − 3 D mild-degree myopia; from − 3 D to − 6.0 D medium-degree myopia; and − 6.0 D and over high-degree myopia [20, 21].

The exclusion criteria were as follows: 1) cataract, refractive surgery or other previous interventions that might have affected refractions; 2) other refractive errors; 3) refusal to participate in the research.

The inclusion criteria were as follows: 1) no ophthalmological eye disorders were found on detailed ophthalmological evaluation; 2) participation consent.

Lenses were evaluated by a slim-lamp biomicroscopy with the illumination source at a 45 degree angle and the light beam set being set to 2 mm width.

Verification of zygosity

Zygosity was determined using a DNA test. The polymerase chain reaction set (AmpFlSTR® Identifiler®, Applied Biosystems, Foster City, CA, USA) was used to amplify short tandem repeats. 15 specific DNA markers were used for comparison of genetic profiles: D8S1179, D21S11, D7S820, CSF180, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TROX, D18S51, D5S818, and Amelogenin. The sample’s gender, age, zygosity characteristics and spherical equivalents are shown in Table 1.

Table 1 Characteristics of twin pairs, defined by zygosity

DNA extraction

Peripheral blood samples were collected from each individual in ethylenediaminetetraacetic (EDTA) tubes for DNA extraction. DNA was extracted from leukocytes using a reagent kit (NucleoSpin Blood L Kit; Macherey & Nagel, Düren, Germany). DNA samples from one member of each MZ pair were used for genotyping.

Genotyping

SNP of the GJD2 gene (rs634990) were assessed using a commercial genotyping kit C_2088259_10. SNP of the RASGRF1 gene (rs8027411) was assessed using a commercial genotyping kit C_185318_10 (Applied Biosystems, Foster City, CA, USA). The Applied Biosystems 7900HT Real-Time Polymerase Chain Reaction System was used for detecting the SNPs. The cycling program started with heating for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Allelic discrimination was carried out using the software of Applied Biosystems. Both SNPs (rs8027411, rs634990) were present in two previous GWASs.

Statistical analysis

The data was analysed with the statistical software package SPSS version 19.0 for Windows. Odds ratios and 95% confidence intervals were computed to assess the association between two SNPs and myopia multivariate logistic regression (Tables 3). Statistical significance was determined at a two-tailed p = 0.05 level. Estimate of heritability (h2) was obtained Pearson’s correlations (r) for MZ and DZ twin pairs: h2 = 2×(rMZ-rDZ) [22].

Results

230 pairs of twins (135 MZ and 95 DZ) aged between 18 and 40 participated in the study, their mean age being 25.08 years (SE 0.7 years) (Table 1). The mean spherical equivalent was − 1.324 ± 0.150, with a range from 0.49 D to − 7.375 D. There were no significant differences between MZ and DZ twins in age and spherical equivalent of the left and right eyes.

Refractive errors for twin 1 versus twin 2 for mean of spherical equivalent are shown in figs. 1 and 2. Intrapair correlations for spherical equivalent in twin pairs were significantly higher in MZ twin pairs r = 0.539 (p < 0.001, 95% CI 0.353–0.684) than in DZ twin pairs r = 0.203 (p < 0.01, 95% CI 0.0633–0.442) in myopia group. Correlations for spherical equivalent in emmetropia group were not significant: r = 0.091 (p > 0.05,995% CI -0.215-0.381) in MZ twin pairs and r = − 0.220 (p > 0.05, 95% CI -0.587-0.222) in DZ twin pairs. The correlations of MZ were clearly higher compared to DZ pairs, indicating genetic effects on myopia.

Fig. 1
figure1

Scatter plots of refractive error as spherical equivalent for each twin‘s right eye, twin 1 versus twin 2 in 73 MZ and 56 DZ twin pairs in myopia group

Fig. 2
figure2

Scatter plots of refractive error as spherical equivalent for each twin‘s right eye, for twin 1 versus twin 2 in 65 MZ and 43 DZ twin pairs in emmetropia group

We genotyped two SNPs (rs634990 and rs8027411) in 189 myopia (83 MZ and 106 DZ) and 96 emmetropia (25 MZ and 71 DZ) subjects. The results are shown in Table 2. The distribution of the two SNP genotypes matched the Hardy-Weinberg equilibrium (p ≥ 0.05). High-degree myopia was present in 11 cases, 33 twins had medium-degree 10 and 145 twins mild-degree myopia. But we didn’t find significant correlations between myopia degree and genotypes.

Table 2 Frequency of GJD2 and RASGRF1 genotypes and allele (%)

5 models were used to calculate the odds ratios to have myopia separately with each gene (GJD2 or RASGRF1), results were not significant (Table 3, Table 4).

Table 3 Pooled results of the associations between GJD2 and myopia
Table 4 Pooled results of the associations between RASGRF1 and myopia

But we found significant association between the combinations of GJD2 CC and RASGRF1 GT and myopia (Table 5). The odds ratio of myopia compared to emmetropia (95% confidence intervals [CIs]) was 2.7 (1.018–7.460) for GJD2 CC and RASGRF1 GT genotypes.

Table 5 Odds ratio of myopia according to the combinations of GJD2 and RASGRF1

The number with combinations of genotypes rs634990 CC and rs8027411 GT and myopia degrees are shown in Table 6.

Table 6 Distribution of SNP rs634990 and SNP rs8027411 genotypes according to degrees of myopia

Discussion

We estimated heritability of myopia according to correlations for MZ and DZ twin pairs and our study showed 67.2% heritability of myopia. Three published twin studies of refractive error have found high heritability from 84 to 86% [23], 89 to 94% [24] and 75 to 88% [25]. It is indicate that heritability in Lithuania is lower than in other Europian populations. Dirani et al. have reported that different populations have shown a wide range of heritability estimates ranging from 50 to 90% [25]. Results shown that the samples of the population and different methods may affect the estimates of heritability [25]. In our study 77% twins had mild-degree myopia, 17% - medium-degree and 6% - high-degree myopia. Meanwhile, the medium-degree myopia accounted for the largest portion in the mentioned studies of heritability in Europe.

Study showed that the gene GJD2, located nearest to the locus 15q14, and RASGRF1 15q25 are important for the transmission and processing of visual signals [23, 26]. The studies of genetic associations in some European and Japanese populations showed that common genetic variations located in GJD2 and RASGRF1 were associated with common myopia and refractive error [5, 11, 15].

A study of genome-wide associations (GWASs) showed associations of SNP with refractive error in 5328 individuals of the Dutch population which were not related. They found that carriers of the C allele of rs634990 have a higher risk of myopia [5]. Qiang et al. found that RASGRF1 gene was significantly associated with high-degree myopia (risk allele T) but GJD2 gene was not [15]. Also, results of meta-analysis, which included 2529 individuals with high-degree myopia and 3127 controls, showed that RASGRF1 was significantly associated with high-degree myopia in Chinese and Japanese populations. However, carriers of the RASGRF1 G allele had a lower risk of high-degree myopia compared to carriers of the T allele (G versus T) [27]. Also, Hysi et al. found that individuals carrying TT alleles on the RASGRF1 were significantly more likely to have myopia than those homozygous for the non-susceptibility GG alleles. We found a significant association between combinations of GJD2 and RASGRF1 genotypes and myopia. Our study showed that individuals with combinations of GJD2 CC and RASGRF1 GT genotypes were 2.7 times more likely to have myopia (p = 0.046). This indicates that some of our results are consistent with the previous reports. Individuals carrying CC alleles on the GJD2 were significantly more likely to have myopia than carriers of TT alleles. But carriers of GT allele on the RASGRF1 gene had more risk to have myopia than carriers of wild type alleles.

Conclusion

Our studies have shown that the heritability of myopia makes 66.4% in Lithuania. We detected significant associations between the combinations of GJD2 CC and RASGRF1 GT and odds ratio of developing myopia.

Abbreviations

D:

Diopters

DZ:

Dizygotic twins

GJD2:

Gap Junction Protein Delta 2

MZ:

Monozygotic twins

RASGRF1:

RAS protein-specific guanine nucleotide-releasing factor 1

SE:

Standard error

References

  1. 1.

    Dandona L, Dandona R. What is the global burden of visual impairment? BMC Med. 2006;4:6.

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Holden BA, Fricke TR, Ho SM, et al. Global vision impairment due to uncorrected presbyopia. Arch Ophthalmol. 2008;126:1731–9.

    Article  PubMed  Google Scholar 

  3. 3.

    Lin LL, Shih YF, Hsiao CK, Chen CJ. Prevalence of myopia in Taiwanese schoolchildren: 1983 to 2000. Ann Acad Med Singap. 2004;33:27–33.

    PubMed  CAS  Google Scholar 

  4. 4.

    Kempen JH, Mitchell P, Lee KE, et al. The prevalence of refractive errors among adults in the United States, Western Europe, and Australia. Arch Ophthalmol. 2004;122:495–505.

    Article  PubMed  Google Scholar 

  5. 5.

    Solouki AM, Verhoeven VJ, Duijn CM, et al. A genome-wide association study identifies a susceptibility locus for refractive errors and myopia at 15q14. Nat Genet. 2010;42:897–901.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. 6.

    Hayashi H, Yamashiro K, Nakanishi H, et al. Association of 15q14 and 15q25 with high myopia in Japanese. Invest Ophthalmol Vis Sci. 2011;52:4853–8.

    Article  PubMed  CAS  Google Scholar 

  7. 7.

    Liew SH, Elsner H, Spector TD, Hammond CJ. The first “classical” twin study? Analysis of refractive error using monozygotic and dizygotic twins published in 1922. Twin Res Hum Genet. 2005;8:198–200.

    PubMed  Google Scholar 

  8. 8.

    Luft FC. Twins in cardiovascular genetic research. Hypertension. 2001;37(2):350–6.

  9. 9.

    Nakanishi H, Yamada R, Gotoh N, et al. A genome-wide association analysis identified a novel susceptible locus for pathological myopia at 11q24.1. PLoS Genet; 2009. https://doi.org/10.137/journal.pgen.1000660.

  10. 10.

    Zippel R, Gnesutta N, Matus-Leibovitch N, et al. Ras-grf, the activator of ras, is expressed preferentially in mature neurons of the central nervous system. Brain Res Mol Brain Res. 1997;48:140–4.

    Article  PubMed  CAS  Google Scholar 

  11. 11.

    Hysi PG, Young TL, Mackey DA. A genome-wide association study for myopia and refractive error identifies a susceptibility locus at 15q25. Nat Genet. 2010;42:902–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 12.

    Shi Y, Gong B, Chen L, et al. A genome-wide meta-analysis identifies two novel loci associated with high myopia in the Han Chinese population. Hum Mol Genet. 2013;22:2325–33.

    Article  PubMed  CAS  Google Scholar 

  13. 13.

    Tong L, Huang XL, Koh AL, Zhang X, Tan DT, Chua WH. Atropine for the treatment of childhood myopia: effect on myopia progression after cessation of atropine. Ophthalmology. 2009;116:572–9.

    Article  PubMed  Google Scholar 

  14. 14.

    Mertz JR, Wallman J. Choroidal retinoic acid synthesis. A possible mediator between refractive error and compensatory eye growth. Exp Eye Res. 2000;70:519–27.

    Article  PubMed  CAS  Google Scholar 

  15. 15.

    Qiang Y, Li W, Wang Q, et al. Association study of 15q14 and 15q25 with high myopia in the Han Chinese population. BMC Genet. 2014;15:51.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. 16.

    Zhu JY, Rong WN, Jia Q, et al. Associations of single nucleotide polymorphisms of chromosomes 15q14, 15q25 and 13q12.12 regions with high myopic eyes in hui and Han population of Chinese Ningxia area. Chin J Exp Ophthalmol. 2014;32:354–8.

    Google Scholar 

  17. 17.

    Jiao X, Wang P, Li S, et al. Association of markers at chromosome 15q14 in Chinese patients with moderate to high myopia. Mol Vis. 2012;18:2633–46.

    PubMed  PubMed Central  CAS  Google Scholar 

  18. 18.

    Kihara AH, Paschon V, Cardoso CM, et al. Connexin36, an essential element in the rod pathway, is highly expressed in the essentially rodless retina of Gallus gallus. J Comp Neurol. 2009;512:651–63.

    Article  PubMed  CAS  Google Scholar 

  19. 19.

    Striedinger K, Petrasch-Parwez E, Zoidl G, et al. Loss of connexin36 increases retinal cell vulnerability to secondary cell loss. Eur J Neurosci. 2005;22:605–16.

    Article  PubMed  Google Scholar 

  20. 20.

    Young TL, Metlapally R, Shay AE. Complex trait genetics of refractive error. Arch Ophthalmol. 2007;125:38–48.

    Article  PubMed  CAS  Google Scholar 

  21. 21.

    Fernández-Medarde A, et al. RasGRF1 disruption causes retinal photoreception defects and associated transcriptomic alterations. J Neurochem. 2009;110:641–52.

    Article  PubMed  CAS  Google Scholar 

  22. 22.

    Neale MC, Cardon LR. Methodology for genetic studies of twins and families. NATO ASI series. Dordrech: Kluwer; 1992.

    Book  Google Scholar 

  23. 23.

    Hammond CJ, Snieder H, Gilbert CE, Spector TD. Genes and environment in refractive error: the twin eye study. Invest Ophthalmol Vis Sci. 2001;42:1232–6.

    PubMed  CAS  Google Scholar 

  24. 24.

    Lyhne N, Sjolie AK, Kyvik KO, Green A. The importance of genes and environment for ocular refraction and its determiners: a population based study among 20–45 year old twins. Br J Ophthalmol. 2001;85:1470–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. 25.

    Dirani M, Chamberlain M, Shekar SN, et al. Heritability of refractive error and ocular biometrics: the genes in myopia (GEM) twin study. Invest Ophthalmol Vis Sci. 2006;47:4756–61.

    Article  PubMed  Google Scholar 

  26. 26.

    Deans MR, Volgyi B, Goodenough DA, Bloomfield SA, Paul DL. Connexin36 is essential for transmission of rod-mediated visual signals in the mammalian retina. Neuron. 2002;36(4):703–12. https://doi.org/10.1016/S0896-6273(02)01046-2.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. 27.

    Chen T, Shan G, Ma J, Zhong Y. Polymorphism in the RASGRF1 gene with high myopia: a meta-analysis. Mol Vis. 2015;21:1272–80.

    PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We wish to thanks all the twins who agreed to participate in this study.

Funding

Funding for the research was obtained from the Lithuanian University of Health Sciences.

Availability of data and materials

The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

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Authors

Contributions

EK, RL, MS analyzed the data. EK, RL wrote the manuscript. EK, AS developed the structure for the paper. AS, RL, MS made critical revisions. EK, AS, ITM approved final version. All authors approved of the final manuscript.

Corresponding author

Correspondence to Edita Kunceviciene.

Ethics declarations

Ethics approval and consent to participate

Permission (Number P1–52/2005) to undertake the study was obtained from the Kaunas Regional Biomedical Research Ethics Committee. Participation in the study form is completed.

Competing interests

The authors declare that they have no competing interests.

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Kunceviciene, E., Sriubiene, M., Liutkeviciene, R. et al. Heritability of myopia and its relation with GDJ2 and RASGRF1 genes in Lithuania. BMC Ophthalmol 18, 124 (2018). https://doi.org/10.1186/s12886-018-0787-1

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Keywords

  • RASGRF1
  • GJD2
  • Twin
  • Myopia