Received 14 June 2010 | Accepted 30 September 2010 | Published 12 October 2010
The first three authors contributed equally to the work
1Ruijin Hospital, State Key Laboratory of Medical Genomics, Molecular Medicine Center, Shanghai Institute of Endocrinology, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Department of Hematology/Oncology, Shanghai Children's Medical Center, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; 3School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, Zhejiang, China; 4Department of Ophthalmology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
Correspondence to: Huai-Dong Song, Ruijin Hospital, Shanghai Institute of Endocrinology, Ruijin road 2, Shanghai, 200025, China; Phone: 8621-64370045-610808; FAX: 8621-64743206; email: email@example.com
Purpose: Myopia and its extreme form, high myopia, are common vision disorders worldwide, especially in Asia. Identifying genetic markers is a useful step toward understanding the genetic basis of high myopia, particularly in the Chinese population, where it is highly prevalent. This study was conducted to provide evidence of linkage for autosomal dominant high myopia to a locus on chromosome 5p13.3-p15.1 in a large Chinese family.
Methods: After clinical evaluation, genomic DNA from 29 members of this family was genotyped. A genome-wide screen was then performed using 382 markers with an average inter-marker distance of 10 cM, and two-point linkage was analyzed using the MLINK program. Mutation analysis of the candidate genes was performed using direct sequencing.
Results: Linkage to the known autosomal dominant high myopia loci was excluded. The genome-wide screening identified a maximum two-point LOD score of 3.71 at θ=0.00 with the microsatellite marker D5S502. Fine mapping and haplotype analysis defined a critical region of 11.69 cM between D5S2096 and D5S1986 on chromosome 5p13.3-p15.1. Sequence analysis of the candidate genes inside the linked region did not identify any causative mutations.
Conclusions: A genetic locus was mapped to chromosome 5p13.3-p15.1 in a large Chinese family with autosomal dominant high myopia.
Myopia, the most common eye disease worldwide, is also the leading cause of visual impairment . The prevalence of myopia has been increasing in recent decades, especially in East Asian areas such as Japan, Singapore, and China [2-4]. The Chinese appear to be more susceptible to myopia than other populations. The prevalence of myopia in primary school children aged 5 to 16 years of Hong Kong is 36.71% . In adult persons more than 40 years old, Chinese residing in Singapore have a prevalence of myopia as high as 38.7% while the prevalence observed in European-derived populations in United States and Australia are 26.2% and 17% respectively [3,6,7]. High myopia, which is defined as a refractive error equal to or below −6.00 diopters (D), is also more prevalent in Chinese than Caucasian populations [3,8]. Individuals with high myopia have a greater chance of subsequently developing serious complications, including glaucoma, retinal detachment, and choroidal neovascularization, which if not treated early and appropriately, may lead to permanent visual impairment or blindness [9-11].
Genetic and environmental factors both contribute to the development of myopia. Environmental factors such as work at close range and prolonged reading are suggested to be involved in the progression of myopia [1,12,13] and a body of evidence supports the idea that heredity plays a central role in the etiology of myopia. Twin studies have reported a high degree of heritability for myopia, with monozygotic twins being more highly correlated than dizygotic twins [14,15]. In addition, the children of parents with myopia tend to have myopia more frequently than children of parents without myopia .
The inheritance of high myopia is equivocal. It may be inherited as an autosomal dominant, autosomal recessive, or X-linked recessive trait. Genetic mapping studies have identified at least 18 chromosomal regions suspected of harboring a myopia gene. X-linked recessive inheritance myopia has been mapped on Xq28 (MYP1) and Xq23–25 (MYP13) [17,18]. In addition, Yang et al.  found that the locus at 14q22.1-q24.2 (MYP18) was responsible for high myopia in a consanguineous Chinese family in an autosomal recessive pattern. Some research groups focusing on autosomal dominant high myopia have identified suggestive linkages on chromosome 18p11.31 (MYP2) , 12q21–23 (MYP3) , 7q36 (MYP4) , 17q21–22 (MYP5) , 4q22-q27 (MYP11) , 2q37.1 (MYP12) , 10q21.1 (MYP15) , and 5p15 (MYP16) . Furthermore, certain loci have also been implicated in common myopia: 22q12 (MYP6) , 11p13 (MYP7), 3q26 (MYP8), 4q12 (MYP9), 8p23 (MYP10) , 1p36 (MYP14) , and 7p15 (MYP17) . Identifying the genetic markers for myopia would be a useful step toward understanding the molecular defects that lead to the pathophysiology of myopia.
In this study, we recruited a four-generational Chinese family with autosomal dominant high myopia. Through genome-wide screening and linkage analysis, we mapped the disease to a locus on chromosome 5p13.3-p15.1.
A large family with autosomal dominant high myopia was identified in Zhejiang province, China. This family contained 11 affected individuals over four generations. Participating in this study were 29 individuals (aged 11 to 80 years): 10 affected and 19 unaffected (Figure 1). The study conformed to the guidelines involving human research as stated in the Declaration of Helsinki. Informed consent was obtained from every subject after an explanation of the nature, procedures and possible consequences of the study. This family was chosen based on the presence of numerous male and female family members and successful multiple generations with high myopia, suggesting an autosomal dominant mode of inheritance. Individuals with a spherical refractive error equal to or lower than −6.00 D, axial length longer than 26 mm in at least one eye and a history of myopia onset before 12 years of age were considered affected. Ophthalmology examination was performed for all of the members. No participant had any known ocular disease or insult that could predispose to myopia, such as a history of retinopathy of premature or neonatal problems, or a known genetic disease or connective tissue disorder associated with myopia, such as Stickler or Marfan syndrome. The results of the ophthalmic examinations are summarized in Table 1.
Genomic DNA was isolated from peripheral blood leucocytes by the standard proteinase K digestion and phenol-chloroform extraction. The genome-wide screen was conducted on ABI 3700 sequencer by using PRISM Linkage Mapping Set MD-10 (Applied Biosystems, Inc., Foster City, CA) that have 382 highly polymorphic fluorescent markers with an average spacing of 10 cM. The markers were ampliﬁed by polymerase chain reaction (PCR) under the following conditions: 50 ng genomic DNA, 2 pmol each primer, 0.2 μl dNTP (10 mM each), 1 μl 10× buffer, 0.6 μl MgCl2 (25 mM) and 0.4 U HotStar Taq DNA polymerase (Qiagen, Santa Clarita, CA) in a ﬁnal volume of 10 μl. Six to eight primer pairs were multiplexed in the ampliﬁcation reaction. Samples were incubated in a PTC-225 DNA Engine Tetrad (MJ Research, Waltham, MA) for 15 min at 95 °C to predenature, followed by 35 cycles of 30 s at 94 °C, 40 s at 55 °C, 40 s at 72 °C, and a ﬁnal extension at 72 °C for 10 min. Ampliﬁcation products were appropriately pooled into prescribed panels, diluted, and denatured for 5 min at 95 °C, then incubated on ice for 2 min. Subsequently, the products were run in an automated DNA sequencer (ABI Prism 3700; Applied Biosystems).
Data were analyzed using GeneScan 3.7NT and Genotyper 3.7NT software (Perkin Elmer, Foster City, CA). Two-point LOD scores were calculated using the MLINK program from the Linkage software package (version 5.2). For fine mapping, additional microsatellite markers spanning the chromosome 5p region were selected from the genetic map of the Marshfield Center for Medical Genetics (Marshfield, WI). The myopia in the family was analyzed as an autosomal dominant trait with 90% penetrance and with a disease-gene allele frequency of 0.01. Recombination frequencies were assumed to be equal between males and females. Haplotype analysis was performed with Cyrillic software (version 2.0) and confirmed by inspection.
The identiﬁed genes located in the linkage region were proposed as candidate genes on the basis of their functional information. Mutations of these genes were screened by direct sequencing. Using the soft Primer Express 2.0 (Perkin Elmer), primers were designed to amplify each exon including exon-intron boundaries regions of the candidate genes from genomic DNA (the sequences of all primers used in this study are summarized in Table 2). Screening for mutations was initially performed in two affected and two unaffected individuals. The PCRs were performed using Taq DNA polymerase and the products were sequenced directly with a dye-terminator cycle-sequencing system by ABI Prism 3700 DNA sequencer after puriﬁed by exonuclease I (Epicenter, Madison, WI) and shrimp alkaline phosphatase (USB, Cleveland, OH). The resulting sequences were compared with the corresponding wild-type sequences using Autoassembler software (version 2.0; Perkin Elmer). When a sequence variant was detected, the exon was ampliﬁed from the genomic DNA extracted from the other individuals to determine whether the base variant was speciﬁc to the patients. The NCBI SNP database was also referenced to determine whether the sequence variant was a polymorphism.
A large, multigenerational, Chinese family with autosomal dominant high myopia was recruited and characterized (Figure 1). DNA was extracted from 29 blood samples of the family members (10 affected). The average age at diagnosis of myopia in the affected individuals was 6.9 years (range, 4 to 11 years). The average spherical component refractive error for the affected individuals was −11.59±5.26 D (range, −6.5 to −26 D). The mean axial lengths were 29.17±1.50 mm (range, 26.80 mm to 31.42 mm) and 23.59±1.04 mm (range, 22.03 mm to 25.72mm) for highly myopic and non-highly myopic subjects, respectively. Individual III:7 had the highest refractive error of −24.00 D for the right eye and −26.00 D for the left eye (Table 1).
Ophthalmological examination excluded known ocular diseases associated with myopia, including keratoconus, spherophakia, ectopia lentis, retinal dystrophy, and optic atrophy. Males and females in this family were equally affected.
All known syndromic myopia loci were excluded in this family. The LOD scores at θ=0.00 were as follows: D15S117 (Marfan syndrome), −2.43; D1S218 (juvenile glaucoma), −2.5; D12S85 (Stickler syndrome type 1), −6.44; D1S206 (Stickler syndrome type 2), −10.77; and D6S276 (Stickler syndrome type 3), −4.15. Linkage to all of the known loci for non-syndromic autosomal dominant high myopia showed no statistically significant or suggestive evidence of linkage in this family (data not shown). Through subsequent genome-wide screening, a two-point LOD score of 3.02 (θ=0.00) was initially obtained with the microsatellite marker D5S419, suggesting that the causative locus for the family with high myopia was mapped to a region adjacent to D5S419 on chromosome 5. For fine mapping, an additional 13 closely flanking microsatellite markers were tested, and the linkage analysis resulted in a significant LOD score at 5p13.3-p15.1. Seven microsatellite markers displayed positive LOD scores, with D5S502 having the highest LOD score, 3.71 at θ=0.00 (Table 3).
Haplotype analysis of the affected individuals revealed recombination events that narrowed the region containing the gene, as shown in Figure 1. Through haplotype analysis, it was discovered that in addition to the ten affected individuals, two unaffected siblings (III:15 and III:19) also inherited the putative disease allele. At the time of examination, III:15 and III:19 were 41 and 58 years of age, respectively, and it was not likely that they would develop high myopia. Interestingly, III:15 had only read for 5 years in primary school and III:19 had never attended school: both of them had spent less time reading.
The critical region was found to be between the markers D5S2096 and D5S1986. A telomeric recombinant event occurred between markers D5S2096 and D5S2074 in the affected individual III:17, which defined the distal limit of the region to marker D5S2096. Affected individual IV:9 displayed evidence of a centromeric recombinant event between markers D5S1986 and D5S1470. Another centromeric recombination event was observed between markers D5S819 and D5S1986 in two affected individuals, III:7 and III:11. These defined the proximal limit of the region to marker D5S1986. Ultimately, we mapped high myopia to a locus on chromosome 5p13.3-p15.1, covering an approximately 11.69 cM (14.14 Mb) region between D5S2096 and D5S1986.
Within the linkage region, the six genes cadherin 6, type II (CDH6), cadherin 10, type II (CDH10), cadherin 12, type II (CDH12), PDZ domain-containing protein 2 (PDZD2), Golgi phosphoprotein 3 (GOLPH3), zinc finger RNA binding protein (ZFR) were selected as candidate genes on the basis of their function: cell adhesion, intracellular signal transduction, protein trafficking, and DNA/RNA binding activities, which we thought were the functions most likely to be associated with myopia. A description of these genes was provided in Table 4. However, mutation analysis did not reveal any disease-causing mutation.
In this study, a locus for autosomal dominant high myopia in a large Chinese family was identified. Genome screening and linkage analysis located a critical region for high myopia on chromosome 5p13.3-p15.1 between D5S2096 and D5S1986, within an 11.69 cM interval. Linkage to the candidate gene regions for the Stickler syndromes, Marfan syndrome, and juvenile glaucoma was excluded, ensuring that this family did not exhibit a mild phenotypic expression of these conditions. Similarly, linkage was excluded from known autosomal myopia loci. This study has provided additional evidence for the genetic heterogeneity of autosomal dominant high myopia.
Myopia is thought to be multifactorial, caused by a variety of environmental and genetic factors as well as their interactions. Compared to the remarkable progress in identifying the genes for retinal degeneration, genes causing non-syndromic myopia (common or high) have proven difficult to identify. The likely explanation for this difficulty is that the gene-environment interplay affects even Mendelian patterns of myopia [1,12,13]. The underlying pattern of genetic and/or environmental factors in myopic subjects is highly variable and incomplete penetrance is common in high myopia, as reported in other high myopia families [26,32]. It was noted that all patients with high myopia in this family carried the putative disease haplotype; but two individuals, III:15 and III:19, both of whom inherited the putative disease allele from their mother, did not have high myopia. At the time of examination, III:15 and III:19 were 41 and 58 years of age, respectively, and it was not likely that they would develop high myopia. Interestingly, III:15 had only read for 5 years in primary school and III:19 had never entered school, so these two siblings had spent less time reading. These suggested that the variability in the phenotype might be mostly attributable to the interplay of genetic and environmental factors, leading to incomplete penetrance of the disease.
Six candidate genes CDH6, CDH10, CDH12, PDZD2, GOLPH3, and ZFR at this high myopia locus were selected on the basis of their function to screen for gene mutations by re-sequencing. The classical cadherins mediate homophilic cell–cell adhesion and are key regulators of many morphogenetic processes .
Loss-of-function studies demonstrate that the classical cadherins play a crucial role in vertebrate retinogenesis. They have multiple morphoregulatory functions in retinal proliferation, migration, differentiation, and layer formation, as well as axonal outgrowth, pathfinding, target recognition, and synaptogenesis [34,35]. CDH6 regulates the differentiation of retinal ganglion cells, amacrine cells, and photoreceptors in Zebrafish . CDH10 and CDH12 were detected in the mouse eye during the first postnatal week when several developmental processes, such as cell migration and formation of synaptic connections, occur simultaneously . PDZD2 is a ubiquitously expressed multi-PDZ-domain protein . PDZ domain scaffolds have been shown by genetic, electrophysiological, and morphological studies to be essential for controlling the structure, strength, and plasticity of synapses, which may play a role in the process of vision formation . GOLPH3 is a peripheral membrane protein of the Golgi stack. It is required for trafficking from the Golgi to the plasma membrane and for the normal extended Golgi ribbon. Depletion of GOLPH3 alters the Golgi ribbon, changing its normal appearance of extending partially around the nucleus, to condensing at one end of the nucleus . ZFR contains three widely spaced zinc finger domains. Zinc finger proteins with a similar pattern of zinc finger motifs are known to bind RNA, DNA, and DNA/RNA hybrids . ZFR can be involved in DNA repair and chromosome organization . Analyses of ZFR knockout mice indicate that ZFR is essential for at least some developmental pathways, as embryonic death occurs at 8–9 days gestation in these mice. In homozygotes, genetic ablation of ZFR causes increased embryonic cell death and/or decreased cell proliferation rates . In the current study, PCR and sequencing primers were synthesized for the exons and peripheral intron regions of these candidate genes, and direct sequencing analysis was performed. However, no disease mutation was identified.
A previous study revealed an autosomal dominant high myopia locus mapped to chromosome 5p15.33-p15.2 with an interval of 17.45 cM between D5S1970 and D5S1987 in three Chinese pedigrees originating from Hong Kong (HK) . In our study, the locus for high myopia of the pedigree was mapped to the critical region between D5S2096 and D5S1986 on chromosome 5p13.3-p15.1. The physical distance of the two markers yielding the peak two-point LOD score (D5S2505 in HK families and D5S502 in Zhejiang family) is approximately 19.7 Mb. The physical distance of the nearest two markers displaying a strong linkage to high myopia in these two studies (D5S1987 and D5S2074) was 9.71 Mb. However, the linkage regions with high myopia on the short arm of chromosome 5 identified in these two studies did not show any overlap (Figure 2). The fact that the two causative loci identified in these Chinese families with inherited high myopia did not overlap, but were adjacent, suggested that there may be disease gene(s) for high myopia on the short arm of chromosome 5 in the Chinese population. Although no mutation has yet been identified for the putative candidate genes, a more refined mutation screen is needed to identify the causative gene(s).
In summary, we have mapped a genetic locus for autosomal dominant high myopia in a large Chinese family. Myopia is the most common eye disease. Identification of the mutant gene(s) for myopia potentially would advance the understanding of the causes of this common eye disorder, and may thus lead to methods for preventing or slowing its progression.
We are extremely grateful to all members of the family who participated in this study. This work was supported in part by grants from the High Tech Program (863) (2006AA02Z175), the National Natural Science Foundation of China (30771017, 30971595, and 30971383), the Commission for Education of Shanghai, the Zhejiang Provincial Natural Science Foundation of China (Y205322), and the Zhejiang Provincial Medical Science Foundation (2005A085).