Keratoconus in 18 pairs of twins
Petra Liskova, MD, PhD
Institute of Ophthalmology
UCL, 11-43 Bath Street
Tel: + 44 20 7608 6945
Fax: + 44 20 7608 6863
Purpose: To describe the concordance of keratoconus in 18 sets of twins.
Methods: Thirteen monozygotic (MZ) and five dizygotic (DZ) pairs of twins were identified during an investigation of familial keratoconus. We used 16 forensic microsatellite markers to confirm the zygosity of same sex twins. Patients and available relatives were examined for signs of keratoconus using corneal topography. For each pair of twins, the severity of keratoconus in each eye was graded according to the steepest keratometry value and the average difference in score between the MZ and DZ twins compared.
Results: All of the MZ twins and four of the five DZ twins were concordant for keratoconus but with differences in age of onset and severity of disease. The subjective age of onset of keratoconus tended to be earlier in the MZ twins (16.4 years, SD 4.66) than in the DZ twins (20.3 years, SD 7.55) (p = 0.086). Additional relatives with keratoconus were identified in two (16%) of the families with MZ twins and in three (60%) of the families of DZ twins. The mean difference in severity scores was 1.4 (SD 1.73) for the MZ twins and 3.0 (SD 1.00) for the DZ twins (p = 0.035).
Conclusion: This data provide evidence that the severity of keratoconus is more concordant in MZ than in DZ twins. The results support the currently accepted hypothesis of an important genetic contribution towards the pathogenesis of keratoconus, but suggest that there is also an environmental effect on the expression of disease.
Keratoconus is characterized by thinning and ectasia of the cornea that causes irregular astigmatism and visual loss. The prevalence of keratoconus is estimated to be between 29 and 229 per 100 000 depending on ethnicity (Ihalainen 1986; Kennedy et al. 1986; Pearson et al. 2000; Nielsen et al. 2007). Although the disease is almost always bilateral, the involvement of the two eyes can be markedly asymmetric (Wilson et al. 1991). The pathogenesis of keratoconus is still unknown, but genetic factors are thought to play a major role in the development of disease (Wang et al. 2000; Edwards et al. 2001). Familial disease occurs and all modes of Mendelian inheritance have been described, but autosomal dominant transmission with variable phenotypic expression is the most common (Edwards et al. 2001). Several loci and sequence variants that might contribute to the disease phenotype have been identified in non-syndromic keratoconus (Fullerton et al. 2002; Heon et al. 2002; Tyynismaa et al. 2002; Brancati et al. 2004; Hutchings et al. 2005; Tang et al. 2005; Li et al. 2006; Burdon et al. 2008; Bisceglia et al. 2009; Liskova et al. 2010).
Twin studies provide an estimate of the relative contribution of the genotype and environment toward the phenotype of a disease (Boomsma et al. 2002). To further define the role of genetic factors in the aetiology of keratoconus, we compared the concordance of disease in 13 monozygotic (MZ) and five dizygotic (DZ) pairs of twins with keratoconus. In same sex pairs, the zygosity was confirmed by genotyping using established forensic markers.
Patients and Methods
The study was approved by the local research ethics committees and conformed to the tenets of the Declaration of Helsinki, and informed consent was obtained for all participating subjects. For this study, we recruited twins identified during an investigation of the genetic basis of keratoconus. For each twin pair, we recorded the details of the interval from birth that they had shared the same home, the age of diagnosis and family history for keratoconus, and whether there was a history of asthma, eczema or hay fever. Ethnic background was classified as white, Pakistani or Indian, or black. Where possible, the clinical examination included an objective assessment of the corneal topography using computer-assisted videokeratography (Orbscan II; Bausch & Lomb, Rochester, NY, USA).
Eyes were graded on a severity scale adapted from the Collaborative Longitudinal Evaluation of Keratoconus study (Zadnik et al. 1998). Normal eyes were graded 0, keratoconus suspect was graded 1, eyes with definite keratoconus and a steep keratometry reading of <45 dioptre (D) were considered as two, those with a keratometry reading between 45 and 52 D were graded 3, and eyes with a steep keratometry reading >52 D were graded 4. Eyes with extensive scarring that precluded keratometry and eyes that had been grafted were also included as grade 4. A keratoconus suspect was defined as an eye without characteristic slit-lamp signs (e.g. Fleischer ring, Vogt striae) and with spectacle-corrected visual acuity of 20/20 or better. In addition, two of the following topographic abnormalities were required: (i) irregular astigmatism (asymmetric bow tie or skewed radial axes); (ii) posterior corneal elevation >45 μm; (iii) inferior and/or nasal–temporal decentration of the maximum point of anterior and/or posterior corneal elevation; (iv) central corneal pachymetry <473 μm (two standard deviations bellow normal) (Doughty & Jonuscheit 2007; Shirayama-Suzuki et al. 2009; Guell et al. 2010). For each twin, a severity grade was calculated as the sum of the scores for both eyes. The arithmetic difference between the severity scores for each twin pair was then determined and the average severity difference summarized. We then used a t-test (with unequal variances) to test the hypothesis that the average score was the same in the two types of twins.
To confirm zygosity, sixteen forensic markers were used; D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA and amelogenin (Perez-Lezaun et al. 2000; Cerda-Flores et al. 2002; Lee et al. 2002; Kubat et al. 2004). Genotyping was performed in a masked fashion by an accredited laboratory (DNA Bioscience, London, UK).
We identified 18 pairs of twins, of which 13 were MZ twins (eight male and five female pairs) (Table 1) and five DZ twins (four male pairs and one opposite sex) (Table 2). All of the twin pairs shared the same home environments until at least the age of 18 years. The subjective age of onset of keratoconus tended to be earlier in the MZ twins (16.4 years, SD 4.66) than in the DZ twins (20.3 years, SD 7.55) (p = 0.086). Additional relatives with keratoconus were identified in 2 (16%) of the families with MZ twins and in 3 (60%) of the families of DZ twins. The mean difference in keratoconus severity scores was 1.4 (SD 1.73) for the MZ twins and 3.0 (SD 1.00) for the DZ twins (p = 0.035). The only twin pair discordant for keratoconus was the DZ siblings from family 3, although the unaffected twin had thin corneas (central corneal thickness 474 μm in the right eye and 472 μm in the left eye) despite regular topography. Systemic and other ocular disorders were uncommon except for atopy, which was reported in nine (35%) of the 26 MZ twins and in four (40%) of the 10 DZ twins. Both MZ twins from family 10 had developmental delay and both MZ twins from family nine had insulin-dependent diabetes of identical onset at the age 30 years. One DZ twin (a) from family 1 also suffered from non-insulin-dependent diabetes, and one DZ twin (a) from family 4 was colour blind, whereas his brother had normal colour vision.
Table 1. Data collected on monozygotic twin pairs.
|b||20||N||2||4|| || || || || || |
|b||16||Y||UA||4|| || || || || || |
|3||a||M||55||Son of twin A||UA||N||3||4||0||1|
|b||UA||N||3||3|| || || || || || |
|b||13||N||4||4|| || || || || || |
|b||13||N||3||3|| || || || || || |
|b||17||N||4||4|| || || || || || |
|b||17||N||4||4|| || || || || || |
|b||20||Y||3||4|| || || || || || |
|b||UA||N||1||1|| || || || || || |
|b||13||N||4||3|| || || || || || |
|b||10||N||3||3|| || || || || || |
|b||13||Y||2||3|| || || || || || |
|b||23||Y||4||3|| || || || || || |
Table 2. Data collected on dizygotic twin pairs.
|b||M||35||N||3||1|| || || || || |
|b||M||N||Y||1||1|| || || || || |
|3||a||M||31||Sister and brother||UA||N||1||3||1||3|
|b||F||N||N||0||0|| || || || || |
|4||a||M||37||Second degree cousin||28||N||3||2||0||2|
|b||M||21||Y||3||4|| || || || || |
|b||M||16||N||3||2|| || || || || |
DNA analysis revealed that three twin pairs self-reporting themselves as DZ were MZ. The probability of monozygosity for twins was ≥99.99997% and likelihood of being MZ ≥4494075 to 1.
In this study, we report on the phenotype of 18 keratoconus twin pairs with zygosity confirmed by genetic markers, of which five were found to be DZ. Although there are published clinical descriptions of at least 19 MZ twins with keratoconus (Weed et al. 2006; Aknin et al. 2007), no reports of DZ twins exist, which is attributed to a lower rate of concordance (Edwards et al. 2001). Until this study, only two sets of twins with keratoconus have been reported from UK centres (Weed et al. 2006), and zygosity status has only been confirmed by genotyping in two sets (Mc Mahon et al. 1999). The usefulness of DNA analysis for establishing zygosity is highlighted by the fact that MZ twins can be designated in error as DZ based on differences in their appearance (Machin 2009).
Environmental and genetic factors both play a role in the development of keratoconus, but their relative contribution is debated. Comparison of the phenotype of twins can provide an insight into this question. DZ twins are no more genetically similar than they are to other siblings; however, they are matched for age as well as the environment they shared during development. If there was a strong genetic effect for the development of keratoconus, one would expect greater differences in concordance in DZ twins compared to MZ twins. Conversely, if the risks for developing keratoconus were mainly environmental, there would be a similar concordance in MZ and DZ twins. To address this question, the accurate diagnosis of keratoconus should be supported by computer-assisted videokeratography, and zygosity should be confirmed by genetic markers. Using this approach, our study provides evidence that the severity of keratoconus is more concordant in MZ than in DZ twins, supporting the currently accepted hypothesis that genetic contribution plays an important role in the pathogenesis of keratoconus (Wang et al. 2000; Li et al. 2012).
Since the introduction of computer-assisted videokeratography, two pairs of MZ twins discordant for keratoconus have been described (McMahon et al. 1999). However, in this report, the unaffected twins had an asymmetric bow tie pattern and asymmetric steepening in one eye on computer-assisted corneal videokeratography, but other relevant values such as pachymetry or anterior and posterior corneal elevation were not provided. It is, therefore, possible that a diagnosis of keratoconus suspect would have been made if these additional diagnostic criteria were applied to the unaffected twin, establishing concordance for the presence of keratoconus but with variation in disease severity. When the protocol for this study was developed, neither Scheimpflug imaging nor the measurement of corneal hysteresis was available at our hospital. We used a uniform protocol for the duration of the study, although we appreciate that the use of these addition techniques might have added to the sensitivity of the measurements.
In this study, there was not a marked difference in concordance between the MZ and the DZ twins, with only one pair of DZ twins discordant for the disease. However, there were significant differences in the phenotype between MZ and DZ twin pairs when assessed using a stratified classification of disease severity. This suggests that there is a strong environmental influence on the severity of keratoconus in addition to a genetic effect, although preferential recruitment of patients with a family history of keratoconus may have introduced a selection bias.
It is likely that keratoconus is a complex trait determined by multiple genes with an associated environmental component. This conclusion is supported by several observations; a digenic inheritance was identified in an Australian pedigree (Burdon et al. 2008), familial occurrence of the disease occurs in only a small proportion of the cases (Edwards et al. 2001), the relatively infrequent identification of large pedigrees and the finding of numerous susceptibility loci without the identification of unequivocally disease causing changes in candidate genes (Brancati et al. 2004; Hutchings et al. 2005; Li et al. 2006; Burdon et al. 2008; Bisceglia et al. 2009; Gajecka et al. 2009; Liskova et al. 2010). Finally, several environmental factors that contribute to the development of the disease have been reported (Edwards et al. 2001; Zadnik et al. 2002). Multiple alleles that act additively could explain the distinct subset of patients with apparently non-progressive keratoconus, as well as abnormalities of corneal thickness and shape found in clinically unaffected relatives of patients with keratoconus (Levy et al. 2004; Steele et al. 2008). Recent research into the genetics of keratoconus has included genome-wide association studies to detect genetic loci contributing to the susceptibility to disease (Li et al. 2012).
To the best of our knowledge, this is the first study describing both MZ and DZ twins with keratoconus. We conclude that the greater concordance of keratoconus in MZ twins than DZ twins, as well as the greater similarity of phenotype in MZ twins, supports a genetic effect on the development of keratoconus. Phenotype variability between MZ twins could be explained by possible differences in environmental influences, epigenetic mechanisms as well as other factors (Machin 2009; Bell & Spector 2011).
This material was presented in part at the meeting of Association for Research in Vision & Ophthalmology, Fort Lauderdale May 1-May 4 2011. Statistical consultation was provided by Catey Bunce. The research was funded by the Department of Health through the award made by the National Institute for Health Research (NIHR) to Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology for a Specialist Biomedical Research Centre for Ophthalmology (BMRC 089) and by Moorfields Eye Hospital Special Trustees. PL was supported by the Research Project of the Ministry of Education, Youth and Sports of the Czech Republic (MSM0021620806). None of the authors has declared any conflict of interest related to the study presented in this article.