Heritability of the spatial distribution and peak density of macular pigment: a classical twin study.

PURPOSE: To elucidate the heritability of peak density and spatial width of macular pigment (MP) using a Classical Twin Study. METHODS: Fundus autofluorescence images were obtained at 488 nm from 86 subjects or 43 twin pairs (21 monozygotic (MZ) and 22 dizygotic (DZ)) (27 male, 59 female) aged from 55 to 76 years (mean 62.2 ± 5.3 years). The relative topographic distribution of MP was measured using a grey scale of intensity (0-255 units) in a 7° eccentricity around the fovea. Relative peak MP density (rPMPD) and relative spatial distribution of MP (rSDMP) were used as the main outcome measure in the statistical analysis. RESULTS: A significantly higher correlation was found within MZ pairs as compared with that within DZ pairs for rPMPD, (r=0.99, 95% confidence interval (95% CI) 0.93 to 1.00) and 0.22, 95% CI -0.34 to 0.71), respectively, suggesting strong heritability of this trait. When rSDMP was compared, there was no significant difference between the correlations within MZ pairs (r=0.48, 95% CI -0.02 to 0.83) and DZ pairs (r=0.63, 95% CI 0.32 to 0.83), thus rSDMP is unlikely to have a considerable heritable component. In addition, there was no difference between any MP parameter when normal maculae were compared with early age-related macular degeneration (AMD) (rPMPD 0.36 vs 0.34, t=1.18 P=0.243, rSDMP 1.75 vs 1.75, t=0.028 P=0.977). CONCLUSIONS: rPMPD is a strongly heritable trait whereas rSDMP has minimal genetic influence and a greater influence by environmental factors. The presence of macular changes associated with early AMD did not appear to influence any of these pigment parameters.

The hepatocyte growth factor receptor (MET) gene is not associated with refractive error and ocular biometrics in a Caucasian population.

PURPOSE: The purpose of this study was to determine if genetic variants in the hepatocyte growth factor receptor (MET) gene are associated with refractive error and ocular biometric measures in a Caucasian cohort. METHODS: A case-control association study using 818 Caucasian adults (37.2% male, 62.8% female; average age: 51.21+/-17.17 years) was undertaken. All individuals were genotyped for 16 tag single nucleotide polymorphisms (tSNPs) across the MET gene region. Myopia was defined as -0.5 DS or worse in both eyes and divided into high myopia (

The retinoic acid receptor alpha (RARA) gene is not associated with myopia, hypermetropia, and ocular biometric measures.

PURPOSE: The Retinoic Acid Receptor Alpha (RARA) gene is a potential candidate gene for myopia due to its differential expression in animal models during experimentally induced myopia. To test for whether RARA is associated with myopia we have undertaken a case-control study assessing for associations between RARA and myopia, hypermetropia, and ocular biometric measures. METHODS: A total of 802 Anglo-Celtic individuals were genotyped. Five tag single nucleotide polymorphisms (tSNPs) in RARA with an r(2) of 0.8 and a minor allele frequency greater than 5% were selected for genotyping. Genotype frequencies of these 5 tSNPs were compared between individuals with emmetropia and those with myopia or hypermetropia. A quantitative analysis was also performed to assess associations with ocular biometric measures including axial length, corneal curvature and anterior chamber depth. RESULTS: We did not identify any significant association between tSNPs in RARA with either myopia or hypermetropia as qualitative traits. Neither did we identify any significant associations of these tSNPs with the quantitative traits of axial length, corneal curvature and anterior chamber depth. CONCLUSIONS: This is the first study to assess for associations between RARA and myopia, hypermetropia, and ocular biometric measures. Our findings suggest that variations in the nucleotide sequence of RARA are not associated with myopia, hypermetropia, or ocular biometric measures in our population.

Role of genetic factors in lower- and higher-order aberrations--the genes in myopia twin study.

AIMS: We intended to investigate the relative genetic contribution in wavefront aberrations using a sub-group of twins recruited in the Genes in Myopia twin study, and subsequently provide direction for future studies into the aetiology of mono-chromatic aberrations. To our knowledge, the Genes in Myopia twin study is the first study to explore the role of genetic factors in both lower- and higher-order aberrations in a Caucasian population. METHODS: Each individual completed a general questionnaire and underwent a comprehensive eye examination. Higher-order wavefront aberrations were calculated with Zernike coefficients up to the fourth order. RESULTS: A total of 46 twin pairs with a mean age of 65.3 years were included in the analysis. Monozygotic intra-pair correlations were significantly higher compared to those in dizygotic twin pairs for defocus aberrations (p < 0.05). A trend for a genetic component was identified for higher-order aberrations. CONCLUSION: Genetic studies into refraction typically explore the genetic effects of lower-order aberrations such as myopia and hypermetropia; however, there is little to no research into the genetic basis of higher-order aberrations. The Genes in Myopia twin study indicates a potential genetic role for higher-order aberrations and provides useful insights into the aetiology of refractive error.

The role of birth weight in myopia--the genes in myopia twin study.

OBJECTIVE: It was the aim of this study to assess the role of birth weight in the development of myopia using a large cohort of Caucasian monozygotic (MZ) and dizygotic (DZ) twins that took part in the Genes in Myopia (GEM) twin study. METHODS: The recruitment of all twins in the GEM twin study was facilitated by the Australian Twin Registry. Each twin underwent a standard questionnaire and a comprehensive ocular examination, which included a dilated objective refraction through autorefraction. Myopia was defined as spherical equivalent equal to or worse than -0.50 diopters. Birth weight was determined through self-report as part of the standard questionnaire. RESULTS: A total of 1,224 twins (690 MZ and 534 DZ twins) aged between 18 and 86 years (mean age 52.36 years) were recruited into the GEM study. The mean birth weight was similar between MZ (2.34 kg) and DZ twins (2.46 kg; p > 0.05). Logistic regression showed no significant association with birth weight and myopia for all twins (p = 0.26), as well as for MZ (p = 0.18) and DZ twins (p = 0.70) separately, with no gender effect (p = 0.23). Moreover, there was no significant difference in mean birth weight between discordant (presence/absence) MZ (myopes = 2.33 kg, non-myopes = 2.39 kg) and DZ twin pairs (myopes = 2.39 kg, non-myopes = 2.43 kg; p = 0.91 and 0.95, respectively). CONCLUSION: Birth weight appears to have little to no role in the development of myopia. In addition, birth weight was not a predictor of the discordance of myopia in MZ and DZ twin pairs.

The dot-and-fleck retinopathy of X linked Alport syndrome is independent of complement factor H (CFH) gene polymorphisms.

BACKGROUND AND AIMS: X linked Alport syndrome is characterised by renal failure, hearing loss, lenticonus, and a central and peripheral dot-and-fleck retinopathy. complement factor H (CFH) gene variants are strongly associated with retinal drusen in macular degeneration and mesangiocapillary glomerulonephritis, and this study examines their role in the development of the Alport retinopathy. METHODS: Twenty-three males and 27 females from 27 unrelated families were examined and their DNA tested for the CFH risk allele (1277 T>C, h1, Y402H) and protective haplotypes (h2 and h4) using a MALDI-TOF-based method. RESULTS: The prevalence of the CFH risk allele was not increased in males with a central or peripheral retinopathy. Three of the nine (33%) with the central retinopathy had at least one copy of the risk allele, and five of the 14 (36%) without the retinopathy did (NS, OR 0.900, CI 0.154 to 5.259). Four of the 12 (33%) with either retinopathy had the risk allele, and two of the six (33%) with none did (NS OR 1.0, CI 0.125 to 7.996). CONCLUSION: The pathogenesis of the retinal dots and flecks in Alport syndrome is independent of CFH-dependent mechanisms and, like other clinical features, may depend on the nature of the underlying COL4A5 mutations.

Testing protocol and recruitment in the genes in myopia twin study.

PURPOSE: The genes in myopia twin study were established to assess the relative genetic contribution of spherical equivalent using a classical twin model. This manuscript will provide a detailed outline of the methodological design, twin recruitment, and the prevalence of myopia in the genes in myopia twin study. METHODS: All Victorian-based twins registered with the Australian Twin Registry aged 18 years or older were invited to participate genes in myopia twin study. Each subject underwent a general questionnaire, comprehensive eye examination, and a blood sample was collected. Myopia was defined as worse than or equal to -0.50 diopters sphere (in at least one eye). RESULTS: A total of 627 twin pairs out of 4,158 twin pairs consented to participate in the genes in myopia twin study. A total of 345 monozygotic and 267 dizygotic twin pairs aged between 18 and 86 years were examined. The response rate for monozygotic twins (19.8%) was almost double that of dizygotic twins (11.7%). The overall prevalence of myopia was 29.7% for all twins. CONCLUSIONS: The genes in myopia twin study is the first Australian-based twin study to assess refraction in an adult twin population and the largest of its kind in the world. The comprehensive testing protocol used in the in the genes in myopia twin study has provided an extensive twin database for genetic analysis. Participation rate was found to vary according to zygosity, gender, and age.

Analysis of the EFEMP1 gene in individuals and families with early onset drusen.

AIMS: Age-related macular degeneration (AMD) is considered a complex genetic disease, although the genetic influences are not yet fully understood. Genetic analysis is hampered by the late onset of disease and the difficulty in obtaining multigenerational families. To investigate this problem further we studied our population of early onset drusen cases. The Arg345Trp mutation on exon 10 of the EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) gene causes two clinical phenotypes of early onset drusen (Doyne honeycomb retinal dystrophy and Malattia Leventinese), yet does not appear to be involved in other early onset drusen phenotypes or typical AMD. We wished to ascertain the involvement of the EFEMP1 gene in our population of sporadic and familial subjects presenting with early onset drusen and their affected relatives. METHODS: Individuals presenting with drusen/end-stage maculopathy at 60 years or under were identified from retinal clinics in Melbourne. All available first- and second-degree relatives were also examined. In all, 116 ethnically matched controls were collected from the same community for comparison. RESULTS: Single stranded conformational polymorphism (SSCP) analysis and subsequent sequencing revealed four previously described and three novel sequence variations. Most occurred at similar frequencies in the case and control populations and were not thought to be disease associated. CONCLUSION: The term early onset drusen encompasses a wide range of phenotypes and our findings indicate that it is likely that more than one gene is involved in its causation. It is essential that these clinical phenotypes are well described and categorised to allow greater possibility of success in the search for other disease genes.

Generating mouse models of retinal disease using ENU mutagenesis.

We used the chemical mutagen, N-ethyl-N-nitrosourea, to induce random point mutations in the germline of the mouse strain C57BL/6 in order to generate models of retinal diseases. 1163 mutagenised first generation mice produced using this approach were examined for eye abnormalities. Approximately one-third (412) presented with some form of ocular abnormality. Most changes were unilateral and confined to the anterior segment of the eye. Less than 10% (44) of identified changes affected the posterior segment of the eye. 21 mice with varying ocular abnormalities, including 17 with retinal changes, were bred to produce second generation mice to confirm genetic inheritance. Genetic inheritance was confirmed in several of these lines including three with retinal changes.

Evidence for genetic heterogeneity within eight glaucoma families, with the GLC1A Gln368STOP mutation being an important phenotypic modifier.

OBJECTIVE: To investigate the phenotype and age-related penetrance of primary open-angle glaucoma (POAG) in Australian families with the most common Myocilin mutation (Gln368STOP). DESIGN: Cross-sectional genetic study. PARTICIPANTS: Eight pedigrees carrying the Gln368STOP mutation were ascertained from 1730 consecutive cases of POAG in the Glaucoma Inheritance Study in Tasmania. METHODS: Index cases and available family members were examined for signs of glaucoma, and the presence of the GLC1A Gln368STOP mutation was ascertained by single-strand conformation polymorphism analysis and subsequent direct sequencing. RESULTS: From the eight pedigrees, 29 Gln368STOP mutation-carrying individuals with either ocular hypertension (OHT) or POAG were found, with a mean age at diagnosis of 52.4 +/- 12.9 years and a mean peak intraocular pressure (IOP) of 28.4 +/- 4.7 mmHg. A further 11 mutation carriers older than 40 years have been studied, who as yet show no signs of OHT or POAG. Within the 8 pedigrees, a further 31 individuals with OHT or POAG were identified who did not carry the Gln368STOP mutation. For these individuals the mean age at diagnosis was higher (62.3 +/- 13.7 years, P < 0.01), and the mean peak IOP was lower (25.4 +/- 6.4 mmHg, P = 0.01). For Gln368STOP carriers, age-related penetrance for OHT or POAG was 72% at age 40 years and 82% at age 65 years. A positive family history of POAG was present in all index cases. Five of the eight pedigrees had a positive family history on both maternal and paternal sides. Seven of the eight pedigrees had one or more individuals with POAG who did not carry the mutation. Eight of the 29 Gln368STOP carriers with OHT or POAG had undergone trabeculectomy. CONCLUSIONS: The GLC1A Gln368STOP mutation is associated with POAG, which in the pedigrees studied is of a younger age of onset and higher peak IOP than non-mutation glaucoma cases. In addition, Gln368STOP mutation glaucoma cases were more likely to have undergone glaucoma drainage surgery. We have not observed simple autosomal dominant inheritance patterns for POAG in these pedigrees. Other factors, as yet uncharacterized, are involved in expression of the POAG phenotype in Gln368STOP pedigrees.

Variation of codons 1961 and 2177 of the Stargardt disease gene is not associated with age-related macular degeneration.

OBJECTIVES: To investigate the role of 2 specific alleles of the Stargardt disease gene (ABCA4) in the pathogenesis of age-related macular degeneration (AMD). Secondary objectives were to investigate differences in frequency of the G1961E allele in selected ethnic groups as well as to examine the segregation of both G1961E and D2177N alleles in 5 multiplex families with AMD. METHODS: Five hundred forty-four patients with AMD and 689 controls were ascertained from 3 continents. Blood samples from 62 normal individuals of Somalian ancestry were also obtained. Participants were screened for the presence of these ABCA4 alleles with a combination of restriction digestion and single-strand conformation polymorphism analysis of polymerase chain reaction amplification products. Detected alleles were confirmed by DNA sequencing. The number of subjects exhibiting the G1961E or D2177N variants were compared between AMD and control groups using a 2-tailed Fisher exact test. RESULTS: There was no significant difference (P >.1) in the frequency of the G1961E and D2177N alleles in patients with AMD (2.2%) vs controls (1.0%). In contrast, there was a significant difference (P< .001) in the frequency of the G1961E alleles between normal individuals of Somali ancestry (11.3%) and normal individuals from other populations (0.4%). There was no evidence of cosegregation of these alleles and the AMD phenotype in the 5 multiplex families with AMD examined. These two ABCA4 alleles were slightly more frequent in patients with AMD with choroidal neovascularization (2.7%) than those without this complication (2.5%). CONCLUSIONS: Somali ancestry is more than 100 times more strongly associated with presence of the G1961E allele than the AMD phenotype. This study did not find any statistically significant evidence for involvement of the G1961E or D2177N alleles of the ABCA4 gene in AMD. CLINICAL RELEVANCE: The ABCA4 gene is definitively involved in the pathogenesis of Stargardt disease and some cases of photoreceptor degeneration. However, it does not seem to be involved in a statistically significant fraction of AMD cases.

The Taa1 restriction enzyme provides a simple means to identify the Q368STOP mutation of the myocilin gene in primary open angle glaucoma.

PURPOSE: To identify a rapid and reliable method to detect the Glutamine 368 STOP (Q368STOP) disease-predisposing allele of the myocilin gene associated with adult onset, primary, open-angle glaucoma. METHODS: Individuals with the Q368STOP mutation of the myocilin gene were identified from a cohort of primary open-angle glaucoma patients from Tasmania and subjected to Taa1 restriction digestion. RESULTS: In the Tasmanian family presented, screening with the Taa1 restriction enzyme successfully confirmed identification of all individuals with the Q368STOP mutation. CONCLUSIONS: The use of the Taa1 restriction enzyme offers a relatively simple, rapid, and reproducible technique that could be applied to detect the Q368STOP mutation of the myocilin gene.

Expression of the Wilms' tumor gene (WT1) in normal hemopoiesis.

The Wilms tumor suppressor gene (WT1) is mutated in a number of cases of Wilms' tumor as well as in mesothelioma and leukemia. It encodes a transcription factor derived from any one of four alternate transcripts. WT1 has a restricted pattern of expression within the body and within the hemopoietic system its expression is limited to primitive leukemias and a number of leukemic cell lines. Given the overexpression of WT1 in leukemias, we have addressed the question of whether this gene is expressed within the normal hemopoietic system. Mononuclear bone marrow (BM) cells obtained from normal donors were separated by fluorescence-activated cell sorting (FACS) into "primitive" (CD34+) and "mature" (CD34-) cell populations. Total RNA extracted from these cells was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using primers based on the WT1 sequence, to examine the expression of this gene within the hemopoietic system. Phenotypic purity of cells was guaranteed by performing single-cell sorting followed by RT-PCR to define the precise cellular phenotypes that express WT1. Expression of WT1 was detected in cells bearing the CD34+ phenotype but not in those cells lacking expression of CD34. In addition, single-cell analysis revealed that expression of WT1 occurred in the candidate stem cell-containing population of hemopoietic cells which have the phenotype CD34+ CD38-. Moreover, the single-cell RT-PCR analysis also demonstrated that differential expression of alternate transcripts of WT1 occurs between hemopoietic progenitor cells with the same phenotype. In conclusion, expression of WT1 is limited to early progenitors of the blood system, which suggests that this gene plays a critical role in hemopoietic development.

Cytokine receptor genes: structure, chromosomal location, and involvement in human disease.

Haemopoietic cytokines regulate haemopoietic cell function via specific cell surface receptors. These receptors are members of a large superfamily of transmembrane proteins and are characterised by a 200 amino acid extracellular sequence encoding the ligand binding domain. Several of the genes for members of this superfamily have now been characterised at the molecular level revealing a highly conserved organisation and a number of these genes have been localised cytogenetically. The recent finding that genes for the IL-3 and GM-CSF receptor alpha chain subunits colocalise to a small region of the pseudoautosomal region and the observation that the LIF receptor locus is present in a cluster of receptor genes on chromosome 5 suggest the possibility that subsets of cytokine receptor genes may be organised into clusters. This possibility is discussed and the potential significance of cytokine receptor gene clusters is assessed. Several of the receptor genes are known to be involved in inherited disorders and there is evidence to suggest lesions in cytokine receptor genes could have a role in leukaemia. We review the gene organisation, localisation and involvement in disease for the known cytokine receptor loci. This large family of receptors is expanding with the steady discovery of new members--all of which have the potential to be involved in human disorders.

Molecular genetic analysis of chromosome 11p in familial Wilms tumour.

In the family reported here, a mother and both of her children developed a Wilms tumour, and all three tumours were of the relatively rare monomorphous epithelial histopathological subtype. Using restriction fragment length polymorphism analysis, both sibs were shown to inherit the same maternal allele from the 11p13 region but different maternal alleles from the 11p15 region. Using a combination of single-strand conformation polymorphism (SSCP) and polymerase chain reaction (PCR) sequencing techniques, no mutations were identified in the WT1 tumour-suppressor gene from the 11p13 region, but a novel polymorphism was identified in exon 1. mRNA expression studies using the insulin-like growth factor II (IGF-II) gene, located in 11p15, showed that there was no relaxation of imprinting at this locus. There was also no evidence of loss of heterozygosity on the long arm of chromosome 16. These findings indicate that the WT1 and IGF-II genes, together with the long arm of chromosome 16, are not directly implicated in tumorigenesis in this Wilms family, but that a recombination event has occurred on the short arm of chromosome 11.

Insertional inactivation of the WT1 gene in tumour cells from a patient with WAGR syndrome.

The WT1 gene was analysed using DNA from a Wilms' tumour derived from a patient with the WAGR syndrome using single strand conformation polymorphism analysis and polymerase chain reaction sequencing. A 14-bp insertion was found in the intron part of the splice donor site of exon 7 and was a tandem duplication of an upstream exon sequence. This mutation would be expected to disrupt the correct processing of the WT1 mRNA and is predicted to result in a non-functional protein. This observation further supports the role of WT1 in Wilms' tumorigenesis in patients with constitutional 11p13 deletions.

A single base pair polymorphism in the WT1 gene detected by single-strand conformation polymorphism analysis.

The Wilms' tumor predisposition gene, WT1, was analysed exon-by-exon in a variety of tumours using the single-strand conformation polymorphism (SSCP) technique. A consistent variation in the usual band pattern for exon 7 was detected in this survey. On sequencing, a silent mutation was noted in codon 313 resulting in an A-->G transition in an arginine codon. The A-->G transition destroys an AflIII restriction enzyme recognition site, which provides a rapid means of identifying heterozygotes at this locus. Analysis of the segregation of this polymorphism in families demonstrated a co-dominant inheritance pattern. In an analysis of 21 randomly selected individuals 25% were heterozygous at this locus, which makes this polymorphism useful in a variety of genetic analyses.

Identification of mutations in the WT1 gene in tumours from patients with the WAGR syndrome.

Individuals with constitutional, heterozygous deletions of chromosome region 11p13 are predisposed to the development of Wilms' tumour, indicating the site of the tumour predisposition gene. The WT1 gene is a candidate for this cancer predisposition gene. If this gene is truly involved in tumorigenesis it would be expected to be mutant in tumour tissue from patients with 11p13 deletions. We have used single-stranded conformation polymorphism (SSCP) and polymerase chain reaction sequencing to test this hypothesis in an exon-by-exon analysis of the gene. Four tumours were analysed, two of which were from unilaterally affected individuals and two from a bilaterally affected patient. SSCP analysis identified mutations in the two unilateral tumours which, on sequencing, were shown to involve a 10-bp insertion in exon 7 and a single base pair change in exon 8. Both mutations result in the generation of premature stop codons and the predicted proteins would lack part of the zinc finger motif. Despite complete sequencing of the WT1 gene in both of the bilateral tumours, no mutations were identified. These results possibly suggest that WT1 may not be involved in tumorigenesis in all tumours. All four tumours retained heterozygosity in the 11p15 region, making it unlikely that a second recessive oncogene in this region was involved in tumorigenicity.