Ocular Genetics

Myopia – genes in myopia (GEM) study

The Genes in Myopia (GEM) Study was established to investigate the genetic basis of myopia and to identify the genes involved in the development of both moderate as well as high to extreme myopia. Several approaches are being undertaken to achieve this goal.

Identification of myopia disease genes through family collection

Three thousand myopic patients who underwent corrective laser surgery at the Melbourne Excimer Laser Group Clinic (MELG) situated at the Centre for Eye Research Australia were eligible for this study. Of these, approximately 25% have vision of –6.00 or less (high myopia). All recruited individuals undertook a standard questionnaire, a comprehensive eye examination, including refractive and biometric measurements as well as exploration of potential risk factors including age, sex, level of education, ethnicity as well as general health history and use of medications. A blood sample was obtained and DNA was extracted from all individuals.

Heritability and modeling

Spherical equivalent and biometric trait measurements were obtained from members of 120 families collected through the GEM study. Heritability estimates and intra class correlation studies were undertaken and revealed that variance can be attributed to both genetic and environmental components for spherical equivalent whereas for axial length a substantial proportion of the variance is genetic. Model fitting for both traits indicated that an additive, common and unique environmental model best fitted the data.

Linkage analysis

Three of the larger families collected as part of the GEM study were genotyped using 400 markers at the Australian Genome Research Facility in Melbourne. The largest family containing 35 individuals had 18 individuals who were affected with myopia of –0.5D or less. Genotyping data was prepared and linkage analysis undertaken using the program MERLIN. A significant log of odds (LOD) score was detected on chromosome 2 for both non-parametric (model free) linkage analysis as well as for parametric linkage analysis. Further mapping is currently being undertaken to further refine the position of this locus.

Quantitative trait analysis (QTL)

Quantitative trait analysis is now underway to identify genome regions that are likely to contain loci for these traits. This will allow us to identify regions of the genome that code for spherical equivalent as well as biometric traits such as axial length.

The GEM Twin Study

Twins are an ideal sample population in which to undertake genetic analysis as they provide an opportunity to quantify the genetic and environmental components that influence a trait involved in disease. The twin methodology is based on the comparison of intra-pair correlations or similarities between genetically identical or monozygotic (MZ) twin pairs who share a 100% of their genes and non-identical or dizygotic (DZ) twin pairs who share up to 50% of their genes. A higher intra-pair correlation in MZ twin pairs compared to DZ twin pairs generally supports a genetic component to the trait. In addition, twins tend to share a common environment and thus in diseases where there are both genetic and environmental components, the effect of the common environment is minimized. These features make twin studies extremely useful in dissecting out components of complex diseases.

All twins (both MZ and DZ) recruited for this study were aged 18 years or older of either gender and lived in the state of Victoria. Twins were recruited through the Australian Twin Registry (ATR) in Melbourne. Following ATR invitations to participants, we were able to recruit over 620 twin pairs and this makes the GEM twin study, the world’s largest twin study in identifying factors involved in myopia. A large number of measurements and questionnaires on these twins (see GEM family study) have now been undertaken to better understand the genetic basis of myopia.

Results from this twin study indicate that correlation statistics and heritability estimates suggest a strong genetic component to spherical equivalent and axial length, providing further evidence of a genetic basis for these myopia traits

QTL mapping in twins

Four regions of the genome, previously suggestive of containing a QTL for spherical equivalence in a UK myopia twin study have now been genotyped for a series of markers using the AGRF. We are now in the process of statistical analysis of this data to ascertain if these regions can be replicated for spherical equivalence and also whether these regions contain QTLs for biometric traits.

SNP analysis in candidate loci regions using case control studies

A number of loci for high myopia have been previously described in the literature. We are undertaking a single nucleotide polymorphism (SNP) approach to genotyping a number of these regions. Comparisons will be made between different patient groups with different refractive properties to identify any SNPs in these regions that show significant association for individual traits.

Personality profiles and myopia

In collaboration with A/Prof Nick Haslam, Dept Psychology, University of Melbourne, personality has been implicated in myopia and we wish to explore the role that this may have in disease. The short version of the “Big 5” personality questionnaire deriving information on personality has been undertaken on 600 twin pairs in the GEM study. Following some interesting associations we are following up on these observations with a larger questionnaire (the International personality item pool or IPIP) in twins. This will allow us to explore aspects of personality and other psychometric indicators that may impact on myopia.

Age related macular degeneration (AMD)

In collaboration with A/Prof Robyn Guymer of the Macular Research Unit, CERA

In order to undertake genetic studies on AMD, we have ascertained an extensive and unique patient resource over the past five years. This resource comprises 957 AMD cases and 621 controls (including 114 over the age of 70 years), 320 individuals from 93 sib / multiplex families with between 2-7 effected individuals, 350 twin pairs (over 55 years), and 254 individuals with AMD who have been examined over an average of seven years (range six to nine years) in 2 AMD progression studies. DNA and sera, autofluoresence, psychophysical and optical coherence tomography measurements have been obtained together with extensive risk factor information and medical histories on these individuals. Colour fundus photographs including both 35˚ stereo macular photographs and optic nerve head are available for all individuals (TOPCON retinal camera TRC 50X). We have already reported on several genes including the apolipoprotein E gene, paraoxonase gene, EFEMP1, PKD2 and ABCA4 genes and their involvement in AMD.

The complement factor H (CFH) gene

Analysis of the recently described Y402H change in the complement factor H (CFH) gene of the alternative complement pathway has been undertaken using the Mass Array System of the AGRF in our collection of AMD patients. A significantly increased association of this variant with disease was identified. Further analysis of how this gene is involved in AMD is ongoing.

Analysis of other genes in AMD

Several other genes recently reported as involved in AMD are also under analysis. These include the LOC387715 locus, APOE, VEGF and, BF and C2 genes. Analysis will involve SNP association studies of cases and controls from our AMD patient repository.

Linkage analysis in AMD families

A large number of families where there is evidence of AMD at an early age have allowed us to recruit multigenerational families. Three of our largest AMD families, each with a minimum of eight effected individuals have undergone whole genome scan analysis. Genotyping of an age matched control cohort followed by statistical analysis is currently taking place. This will allow us to identify whether these families link to previously identified loci or novel loci.

Dissection of gene-environmental interactions in twins

A total of 340 twin pairs over the age of 50 years have been recruited through the Australian Twin Registry in Melbourne, Victoria. A questionnaire, eye exam, fundus photograph, as well as other retinal measures such as optical coherence tomography and DNA sample have been taken from each twin. Features of disease as well as genetic analysis are being undertaken in these twins.

Protein expression studies in AMD

Donor Eyes

In collaboration with A/Prof Robyn Guymer, Macular Research Unit, CERA, eyes from volunteer donors with AMD or with no disease, are collected either through the Lions Eye Bank at the Royal Victorian Eye and Ear Hospital, Melbourne, St Vincent’s Hospital, Melbourne or through the Flinders Medical Centre, South Australia. Macular trephines are taken and stored at –80 0C to allow RNA and protein expression studies to be undertaken. Tissue sections from eye blocks are analysed for the presence of proteins using immunohistochemistry and Western blotting. A range of antibodies against proteins typically associated with other neurodegenerative disorders including Alzheimer’s disease have so far been used on these sections.

AMD biomarker discovery using proteomics

In collaboration with Dr Matt Perugini at BIO21, Melbourne

Plasma samples from our patient repository are collected and analysed using a proteomic approach with the aim of identifying differences in protein expression based on their apolipoprotein profile. Both disease and non-disease individuals are compared to assess proteome differences by the use of 2D gels and Mass spectrometry. These studies are being undertaken with the aim of identifying candidate biomarkers in AMD.