Identification of First Potential Linkage against Myopia Locus

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Objectives: The genes responsible for myopia have not yet been identified although several chromosome loci have been suggested. Additional loci for the majority of high myopia, especially in Asian populations, await discovery. The purpose of this study was to search out the responsible locus of myopia by clinical and genetic investigation of myopic families. This is done by collecting various autosomal dominant families from the province Punjab of Pakistan. This is the first experimental study on myopia that is reported in Pakistan.

Method: Blood samples of various myopic families were collected from various areas of Punjab and their DNA was extracted with the standard protocol. The amplification of DNA was done with 3 primers of microsatellite markers spaced at intervals of about 6.0 cM, each belonging to the myopia loci MYP3, MYP4, MYP5, MYP6, MYP7 and MYP8. Genotyping was done for linkage analysis through PAGE (Polyacrylamide Gel Electrophoresis). This was then followed by haplotype analysis and LOD score calculation.

Results: The pedigrees of the families indicate that the inheritance pattern of myopia in these families is autosomal dominant. From the clinical data and analyzing cylindrical and spherical values it is manipulated that myopic patients included in the study were depicting simple to moderate myopia. The potential linkage was found between markers D11S904 and D11S935 in one of the families against the locus MYP7 i.e. on chromosome 11 with maximum LOD score of 0.0803. This is the 1st potential linkage that is observed in Pakistan.

Conclusion: Further mapping of this locus will be helpful in mapping out the genes responsible for this disease and will help to lay out the basis for research on genetic factors involved in developing this disease and its cure. It may also suggest new therapies and may lead to understand other molecular diseases. It is also found that both clinical and genetic factors are involved in the progression of myopia.

Key words: myopia, molecular investigation, genotyping, linkage analysis.

Introduction

Myopia is a major cause of impaired vision. Impaired vision basically means that the vision of human eye is below a certain defined standard and is a common cause of visual disability throughout the world (Edwards et al., 2004 and Fredrick, 2002). It is a refractive abnormality of the eye and is derived from a Greek word "muopia" that means "to close the eyes". It manifests itself as blurred distance vision, hence also called as "nearsightedsness." In this condition, the parallel light rays from an object at optical infinity are focused by the eye in front of the retina, rather than on the retina, resulting in an eye that is unable to focus at a distance (Jacobi et al., 2005). This disease has very serious pathological outcomes and is often found associated with potentially blinding conditions such as retinal detachment, macular degeneration and glaucoma (Fredrick, 2002).

Globally, the prevalence of myopia is estimated to be from 800 million to 2.3 billion (Dunaway and Berger 2006). Moreover, in Pakistan extrapolated prevalence of myopia is found to be approximately four billion (Shaheen et al., 2008). Both genetic and environment factors have been implicated in the etiology of myopia, but the precise molecular mechanisms responsible for myopia are still unknown.

It is a multi factorial problem whose development involves a long chain of processes that include environmental, genetic, biochemical and nutritional pathways etc. (Klaus, 2010). Environmental factors including near work are known to be involved in manipulating the progression of myopia but they justify only 12% of the observed phenotypic variation. It has been suggested that the remaining affects on myopia are due to inheritance of genes because a number of heritability studies provide evidence that between 50%-94% of population variance is affected by genetic factors (Dirani et al., 2008). Genetic factors predominate in familial occurrence of myopia with a Mendelian inheritance pattern. All kinds of Mendelian modes of inheritance of familial myopia have been studied. The most frequently being documented is autosomal dominant mode of inheritance (Jacobi et al., 2005). The studies on heritability factors and the correlation studies in families had shown that children having both myopic parents have a 33-60% prevalence rate of myopia. In children who have one parent with myopia, the prevalence rate is 23-40% and in case of non-myopic parents the chance of myopia in siblings is 6-15% (Daniel et al. 2007; Klaus, 2009).

In spite of many decades of research, the knowledge about the molecular defects and biochemical pathways that are responsible for causing myopia is very less. Animal models having abnormal eye growth and myopia (Mutti et al., 2007) propose that retina itself controls the eye growth to some extent, but its phenomenon is still unknown (Stambolian et al., 2004).

Linkage analysis is a powerful method not only for mapping new locations but also refining intervals where myopia causing loci have been previously mapped (Van Camp and Smith, 2000). Linkage analysis is usually performed to predict transmission of a disease gene and to detect new genes as well. It helps in finding out the position of the gene under analysis by using genetic markers especially microsatellite markers that are also called as short tandem repeats (STRs) (Anna et al., 2002). These markers prove to be an efficient and successful tool to investigate linkage analysis in humans. There are multiple reasons to use them as genetic detectors including, high level of polymorphism, rapid mutation rate and range of variety of microsatellite markers available.

Susceptible genes for myopia can be studied through candidate gene analysis in which the genes that are expressed in the eyes and are located within or near the chromosome regions associated with myopia are selected and then studied. Identifying the exact loci and genes for myopia may become difficult due to two major problems. One is discrimination between inherited myopia and acquired myopia and second is the permeability of any myopia-related gene into a family such as by marriage can create confusion in identifying loci and genes. Therefore, a myopia family having well defined phenotypes and least environmental impact should be considered best for finding the locus or gene responsible for the disease (Schäche et al., 2009).

Clinically, the mechanism of myopia development involves the cillary muscles to remain in a contracted state in order to keep the light on fovea (main region of retina) in comparison to the normal condition in which the ciliary muscles must be relaxed to focus the image perfectly. This constant contraction to focus near image changes the shape of lens and elongates the eye ball thus results the eye to be myopic (Hughes, 2004). It is also observed that females are more myopic in comparison to males but the reason behind this is still un known. The most common unit for measuring the degree of severity of myopia is Diopter (D). Myopia is classified according to various aspects and parameters and according to this classification, if the value is between -1D to -3D, it is simple myopia, if the value is between -3D to -6 D, it is moderate myopia and if the value is between -6 D to -8 D, it is high myopia. Spherical value of myopia greater than -8 and up till -20 is said to be extreme myopia (David et al., 2006) Moreover, it has also been observed that about 150 genetic syndromes that are associated with various levels of myopia (Jacobi et al., 2005).

Myopia may be inherited as an autosomal dominant, autosomal recessive, or as X-linked recessive trait. In the past two decades, genetic mapping studies have recognized 20 chromosomal regions assumed to be harboring a myopia gene. Of these, 13 are concerned with high myopia (MYP1, MYP2, MYP3, MYP4, MYP5, MYP11, MYP12, MYP13, MYP15, MYP16, MYP17, MYP18) and seven with common myopia (MYP6, MYP7, MYP8, MYP9, MYP10, MYP14 and locus 2q37). Nine of the loci (MYP2, MYP3, MYP6, MYP7, MYP8, MYP9, MYP10, MYP12 and MYP13) have been verified through replication studies in independent family studies. Some of these still remain to be confirmed. Identification of these loci is the first step towards understanding the molecular basis of myopia and subsequently towards the prevention and treatment of this sight threatening problem (Yang et al., 2009).

In this study, we collected five autosomal dominant families from province Punjab and performed linkage analysis against six loci of myopia i.e. MYP3, MYP4, MYP5, MYP6, MYP7 and MYP8. In Pakistan, the molecular investigation and characterization of myopia has not yet been done, so this would be the first molecular genetic study on it. This work will provide the genetic data that will be helpful in finding out the responsible locus for myopia. According to Jacobi, F. K. and C. M. Pusch, 2010, the future of genetic research in this area will likely depend increasingly on microchip array technology.

Material and Methods:

Enrollment of families:

Five myopic families, MYO-1, MYO-2, MYO-3, MYO-4 and MYO-5, with autosomal dominant mode of inheritance were identified from various areas of Punjab (Fig.1). All these families were identified and selected on the basis of their eye-sight and the spherical power of their eyes. Individuals having eye-sight and spherical power equal to or greater than -1.0 D were considered myopic in this study.

Clinical Evaluation:

The myopic individuals from the selected families were clinically assessed and evaluated as well. The clinical details were collected through the clinical perfoma filled in by the participating individuals. The optical cards given by their ophthalmologists were also obtained from them and their spherical power (Sph), cyclic power (Cyc) of right and left eye and axis of both eyes were noted. Their age of onset and age at examination was also noted.

DNA Extraction:

DNA was extracted from blood samples by inorganic method (Lerner and Lerner, 2006; Sambrook and Russel, 2001). After DNA quantification, concentration of all the DNA samples was brought to same level i.e. 50ng/mL.

Microsatellite markers selection:

D12S1684, D12S1716 and D12S1605 belonging to MYP3, D7S1815, D7S2447 and D7S2423 of MYP4, D17S787, D17S1160 and D17S808 of MYP5, D22S689, D22S685 and D22S683 of MYP6, D11S904, D11S2014 and D11S935 of MYP7 and D3S1614, D3S3725 and D3S1565 of MYP8 loci were selected for this study. Their primers were designed using software "Primer 3".

Amplification Reaction:

Primers of these microsatellite markers were optimized for their amplification conditions. Total PCR reaction volume was 25 μL containing DNA (50ng/μL), MgCl2 (2.0 mM), dNTPs (2.0 mM), forward and reverse primers (10 pM each), DNA Taq polymerase (0.1U / primer) and double distilled water. Thermocycler carried out the reaction in four steps. First step was initial denaturion at 950C for 4 minutes. Second step comprised of 35 cycles. Each cycle had denaturation at 940 C for 45 sec, primer annealing at 540C (as optimized) for 45 seconds and extension for 1 min at 720 C. Third step was final extension for 10 min at 720 C and fourth step was maintaining the final temperature at 40C.

Genotyping:

PCR products were subjected to non denaturing Polyacrylamide Gel Electrophoresis for genotyping (Wang et al., 2003). Gel with 8.0 % final concentration was used having Acrylamide and Bisacrylamide solution 30% (W/V), TAE buffer 50X, Ammonium per sulfate 10% (W/V), Tetra methyl ethylene diamine (TEMED) 0.08% (W/V) and double distilled water. 10 μL PCR product along with 2 μL of loading dye was loaded in the gel and were run at 200 volts for 2 hours in PAGE unit of Major Science, model no. MV-20DSYS.

Haplotype Analysis:

After visualizing the alleles of all the individuals of a family on the gel, they were arranged for various markers and their haplotypes were created that showed the inheritance pattern of the disease. The genotyping data was further analyzed by performing multipoint linkage analysis through "Easy Linkage software". Through this software the probability of linkage among the genotyped families was evaluated.

MYO-1 MYO-2

MYO-3 MYO-4

MYO-5

Fig: 1. Pedigrees of the families showing autosomal dominant mode of inheritance.

Results:

All the five families included in the study were collected from different areas of Lahore. It was evaluated from the ophthalmic examination that two of the affected individuals had early childhood/ school myopia. While seven of the individuals had early onset myopia and five had youth onset myopia. Only one myopic individual had late adult onset myopia. One subject had cataract in right eye. One affected individual had a surgery of radial keratotomy and another one had recent surgery of macular atrophy.

After clinical evaluation, it was found that the spherical values range from -1.00 D to -4.75 D and cylindrical values were low which showed that the affected individuals participating in the study had simple to moderate myopia. Also from the calculated axial length, ranging from 24.1 mm to 25.9 mm showed that the individuals in this study depicted moderate myopia. The focal length of the myopic patients involved in this study range from -21 cm to -100 cm. It was also analyzed that in elderly people, increase in spherical component increased the positive value of lens that means hyperopia (far sightedness). All the five families reported no other high ocular disorder except that three of the five families reported of hypertension, cardiac problem and diabetes running in the family. No systematic abnormalities were noted in affected individuals.

The results of the genotyping showed that family MYO-3 showed potential linkage against the locus MYP7 with maximum LOD score of 0.0803. This family belongs to the caste "Khawaja" and was enrolled from PCSIR Phase II Lahore, Punjab. This family had three generations and contained nine myopic individuals in three loops. Only three myopic and one normal individual from one of the loops were included in the study. After pedigree analysis, the family was found to have autosomal dominant mode of inheritance. After genotyping and haplotype analysis of the family, potential linkage was observed with chromosome 11p13 markers. The clinical data of this family showed that the range of the spherical power in myopic patients was from -2.00 D to -3.75 D and that of cylindrical values was from -0.5 to -1.5 (Table.1). According to a study by Llorente et al. (2004), the increase in spherical and cylindrical power with age has been found to be directly proportional to increase in myopia pathogenesis and also its progression.

Table: 1. Clinical data of the family MYO-3

The loop of this family involved in the study contained an affected father, a normal mother and two affected sons. Father had the heterozygous alleles F1= 1, 2, 1 and F2= 2, 2, 1. Mother carried the alleles M1= 1, 1, 2 and M2= 2, 1, 1. Both affected children carried the F1 allele from father and M2 allele from mother. Hence, the F1 allele (1, 2, 1) was considered as the affected allele that was inherited in all the affected members of the family without any recombination. This allele was considered to be potentially linked with the chromosome number11 in the family (See fig: 1).

Fig: 2. Pedigree and haplotype diagram of the family MYO-3 showing potential linkage against MYP7 locus. Filled squares (male) or circles (female) represent individuals affected with myopia.

The three microsatellite markers examined for this family and the haplotype construction of these markers supported that this locus is potentially linked with the family. The values of LOD scores for all the markers were below 1.0. Maximum LOD score calculated came out to be 0.0803 for the marker D11S904 that shows very low percentage of linkage. This low value of LOD score is may be due to less number of affected and normal individuals in the family. So further confirmation is required by extending the loop members (Fig: 3, table: 3).

Fig: 3. Graphical representation of LOD Scores for markers D11S904, D11S2014 and D11S935 for pedigree no. MYO-3

Table: 3. Maximum LOD Score obtained for marker D11S904

Marker

CHR

cM

pLOD

D11S904

11

33.57

0.0803

Discussion:

In this study, a locus is identified that is potentially considered to be responsible for causing myopia. Microsatellite markers have been proved as an efficient and powerful tool for discovering any linked locus. In every individual a pair of alleles of each microsatellite marker is present, one on each homologous chromosome. Combined profile of different microsatellite marker alleles gives a specific identity or haplotype of every individual.

Although many experiments have been conducted for the genetic analysis of myopia loci in many parts of the world, but up till now, no study has been carried out for this purpose in Pakistan. So, in the present study, a panel of eighteen microsatellite markers belonging to MYP3, MYP4, MYP5, MYP6, MYP7 and MYP8 loci of myopia has been developed to carry out linkage analysis for these loci. To fulfill this purpose, five myopic families, MYO-1, MYO-2, MYO-3, MYO-4 and MYO-5 from province Punjab, were collected to find out that which locus is responsible for making them myopic.

All these families were identified and selected on the basis of their eye-sight and the spherical power of their eyes. Individuals having eye-sight equal to or greater than -1.0D were considered myopic in this study. According to a study by Llorente et al. (2004), the increase in spherical and cylindrical power with age has been found to be directly proportional to increase in myopia pathogenesis and also its progression. Only those families having a minimum of three myopic individuals in them were involved in the study. The total individuals included in the study were 25, of which, 16 were myopic and the remaining were normal. After pedigree analysis, all families showed autosomal dominant mode of inheritance.

After genotyping all the families, it was found that families MYO-1, MYO-2, MYO-3, MYO-4 &MYO-5 did not show any linkage against the locus MYP3 (12q21-23). While according to the past study by Young et al., (1998) on German/Italian family, showed significant linkage against this locus on 12q arm of chromosome. The evidence of significant linkage against the locus MYP3 was further verified by Farbrother et al., (2004). He studied UK families and found the evidence of significant linkage against this locus.

These five families also did not show any significant linkage against locus MYP4 (7q36). Whereas, Naiglin et al., (2002) provided the evidence of presence of significant linkage against this locus in French and Algerian families.

Against the locus MYP5, these five families again did not show any significant linkage. However, Paluru et al., (2003) identified significant linkage against this locus MYP5 (17q21-22) in multi-generation English/ Canadian family.

Regarding the locus MYP6, MYP7 & MYP8, it was found that four of five families, MYO-1, MYO-2, MYO-4 and MYO-5, did not show any linkage against the locus MYP6 (22q12). While the past studies on American families of Ashkenazi Jewish descent by Stambolian et al., (2004) showed significant linkage against this locus. Similarly Stambolian et al. (2006) studied some additional families of Jewish descent and found significant linkage against locus MYP6 on chromosome 22. The same locus was studied by Klein et al., 2007 in Americans of Northern European or German ancestry. He also found linkage to the 22q12 region. The same families i.e.; MYO-1, MYO-2, MYO-4 and MYO-5 also did not show any linkage against the locus MYP8. However, Hammond et al. (2004) studied UK population and got significant linkage against the locus MYP8 on chromosome 3.

The family Myo-3 showed potential linkage against the locus MYP7. The maximum LOD score calculated came out to be 0.0803 at the marker D11S904. But further confirmation is still required by extending the loop members. In 2004, Hammond et al. discovered this locus MYP7 to be linked with UK population. In his study the LOD score was 6.10 that showed significant linkage.

In regard with the clinical parameters of myopia in past studies it has been reported by many scientists and ophthalmologists that the more the cylindrical power value is, myopia is classified as high myopia and condition becomes worse. Helen et al., (1999) in their book on refractive surgery report that patients having spherical power -6.00D and cylindrical component -3.00D and above are having the worse myopic conditions (Jane et al., 2004). Llorente et al., (2004) investigated the properties of spherical component of eye and reported that the increase in the sphere power with age has been attributed to a shift of the crystalline lens towards positive value (Glasser and Campbell, 1998). It was also observed that increase in the sphere refractive power increases the myopia pathogenesis and also increase its progression.

Comparing these findings with the present study suggest the cylindrical values of the myopic individuals analyzed were having low cylindrical values. The range of cylindrical values of the myopic individuals is from -0.25 to -3.5. Only two of the affected individuals showed rather high cylindrical value between -2 to -3.5, also possessing spherical component ranging from -2.5 to -2.75. It is likely from the cylindrical component and also spherical component that the form of myopia in these two individuals may depict a pattern of high myopia in later time. While the findings of rest of affected individuals interpret that the individuals in this study do not posses high or severe form of myopia rather they have simple to moderate myopia.

Similarly, regarding the spherical component, findings in this study were in accordance with the findings of Llorente et al., (2004). The Sphere refractive power of individuals in this study ranges from -1.00 to - 4.75 and this increase in Sphere power was observed in 90 % of individuals studied. This increases the progression of myopia in selected individuals. Also increase in spherical component with age shifts the lens more towards positive value (hyperopia) was also observed in elderly affected individuals. In this study the axial length of myopic individuals ranges from 24.1mm to 25.9mm with 24.1 the minimum and 25.9 the maximum. It was also observed from the calculated value of axial length that a direct proportional relationship was found between axial length and sphere power. The increase in sphere power increases the axial length. The focal length of the myopic patients involved in this study range from - 21 to -100 centimeter with -21 the maximum focal length and -100 the minimum. The relationship between focal length and sphere power was also found to be directly proportional. The more the sphere power value is, the value of focal length increases. Other ocular problems and surgery were also reported.

One of the major problem that contribute to the difficulty in identifying linkage for the selected families is that whether the status of affected and unaffected individual is determined correctly or not? Secondly, as high myopia is found to be very much prevalent in Pakistani population, through cousin marriage introduction of another myopia related gene can also confound the identification of linkage. Therefore, a high myopia family with less environmental impact and clear phenotypes would be ideal for identification of loci as well as gene in Lahore population.

This was a pioneering study never held in Pakistan before. As a result of this study, a locus MYP7 was identified in a myopic family Myo-3 of Punjab, Pakistan. Now, by further enhancing the study by extending the pedigree for confirmation of this locus, it would become helpful in mapping out the genes in this locus that are responsible for this disease and to find out its treatments methods.

This study will be helpful in mapping out the genes in this locus that are responsible for this disease and will help to lay out the basis for research on genetic factors involved in developing this disease and its cure. Afterwards this study can be extended by working on other loci of myopia. The future cloning and mutational characterization of the genes for myopia will clarify the molecular mechanisms underlying increased eye growth and lead to a better understanding of the clinical consequences of the mutations of these loci. It would also be helpful to develop pharmacogenomics, may also lead to allow development of DNA based diagnosis including pre-symptomatic and post-natal diagnosis. It may suggest new therapies and may lead to understand other molecular diseases.

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