|Molecular Vision 2006;
Received 20 June 2006 | Accepted 28 November 2006 | Published 5 December 2006
An autosomal dominant progressive congenital zonular nuclear cataract linked to chromosome 20p12.2-p11.23
Ningdong Li,1 Yongjia Yang,1,2 Juan Bu,1 Chen
Zhao,1 Shasha Lu,1 Jun Zhao,1 Li Yan,1 Lihong
Cui,1 Rongchang Zheng,1 Jianjun Li,3 Jinsheng
Tang,4 Kanxing Zhao1
(The first two authors contributed equally to this publication)
1Tianjin Eye Hospital, Tianjin Medical University, Tianjin; 2School of Biosciences and Technologies of Central South University, Changsha; 3The People Hospital of Lintao county, Gansu province; 4The Second Affiliated Hospital, Central South University, Changsha, People's Republic of China
Correspondence to: Kanxing Zhao, Tianjin Eye Hospital, Tianjin Medical University, Tianjin, 300040, People's Republic of China; Phone: +86-22-23542766; FAX: +86-22-23542524; email: firstname.lastname@example.org
Purpose: To map and to identify the causal gene for autosomal dominant congenital cataract (ADCC) in a Chinese family.
Methods: A four-generation family with a history of progressive congenital cataracts was investigated. Twenty-three members of the family were examined ophthalmologically. Blood samples were collected from twenty-nine family members for genetic linkage analysis. Two-point LOD scores were calculated. Multi-point linkage analysis and haplotype construction were performed to define the optimal cosegregating interval. Direct sequence analysis of the candidate gene, beaded filament structural protein 1, filensin (BFSP1) in the critical region was carried out.
Results: Fifteen family members were affected with autosomal dominant progressive congenital zonular nuclear cataract (ADPCZNC). The maximum two-point LOD Score of 6.02 was obtained for marker D20S904 (θ=0). The cataract locus in this family was mapped to chromosome 20p12.2-p11.23, a 9.34 Mb (16.37 cM) interval between markers D20S186 and D20S912. Although BFSP1 was in this critical region, we found no evidence that the condition in the family was caused by a BFSP1 mutation.
Conclusions: We have mapped the genetic locus of ADPCZNC to chromosome 20p12.2-p11.23 in an ADCC family. This is the first time ADPCZNC was linked to this region.
Congenital cataract is one of the major causes of blindness in human beings. Although the visual acuity of the patients can be improved dramatically by surgical intervention, the congenital cataract patients are usually accompanied with intractable complications after operation. In some cases, congenital cataracts are hereditary. To date, various inheritance patterns for congenital cataract have been reported , including autosomal dominant, autosomal recessive, and X-linked . Among these types, autosomal dominant inheritance appears the most common . Currently, a number of loci on different chromosomes associated with ADCC have been documented , suggesting that ADCC is a highly heterogeneous condition. In non-consanguineous families, clinically identical cataracts have been mapped to inconsistent loci . Conversely, an identical defective gene or a same mutation has resulted in different phenotypes [5-7]. In this report, we studied a Chinese family with autosomal dominant progressive congenital zonular nuclear cataract (ADPCZNC) and we have presented evidence of linkage to chromosome 20p. Haplotype construction and multiple linkage analysis assigned the cataract locus to a 9.34 Mb (16.37 cM) interval between the markers D20S186 and D20S912.
A large four-generation family with ADCC from Gansu province of China was investigated (Figure 1). All adult-individuals and the parents of the minors in this family gave informed consent to the study protocol, which was approved by the Ethics committee of Tianjin Eye Hospital (Tianjin, China). Twenty-three members of the family were invited for a detailed clinical examination including direct-flashlight test, distance visual acuity, examination with slit-lamp microscope and examination of the fundus. Detailed medical and family histories were obtained from each available family member.
Blood samples were collected from 29 family members. Genomic DNA of all 29 members was isolated by standard procedures. All specimens were quantified by spectrophotometry and diluted to 30 ng/μl for polymerase chain reaction (PCR) amplification. Genome-wide screening was performed with 382 markers spaced an average of 10 cM apart (ABI PRISM Linkage Mapping Set, Version 2.5, Applied Bio-systems, Foster City, CA). Fine mapping was accomplished using fluorescently labeled primers from the Decode linkage map . Multiplex PCR was carried out in a 5 μl reaction mixture containing 50 ng of genomic DNA, 1X PCR buffer, 100 μM of each dNTP, 3.0 mM MgCl2, 60 pmol each of forward and reverse primers, and 0.2 U of Ampli Taq Gold DNA polymerase. The mixture including reaction products (1 μl), Liz Size Standard-500 (0.2 μl), and Hi-Di Formanmide (9 μl) were electrophoresed and visualized on a 3130 Genetic Analyzer (Applied Biosystems). Alleles were analyzed by Genescan analysis software V3.7 and Genotyper V3.7 (Applied Biosystems).
The MLINK program of the LINKAGE package (version 5.1) was used for calculating two-point LOD scores. The disease was assumed to be an autosomal dominant trait with 99% penetrance. Equal male and female recombination rates were assumed. Marker allele frequencies were set at 1/n (where n is the number of alleles observed). We assumed gene frequencies of 0.0001. Multi-point linkage analysis was used to estimate the optimal position. For multi-point linkage calculation, the genetic distances between loci were obtained from the Decode linkage map . The haplotype was constructed using the Cyrillic program to define the borders of the cosegregating region. Direct cycle sequencing of beaded filament structural protein 1, filensin (BFSP1) was carried out on three family members (two affected and one unaffected).
A four-generation Chinese family from Ganshu province was identified (Figure 1). After informed consent, 23 individuals with a family history of cataract and six spouses were included in this study. Fifteen of these 23 family members were affected with ADPCZNC. None of the spouses showed signs of the disease. Some parents of the affected individuals recalled that the cataracts presented at birth and a progressive visual loss appeared during the first decade of life (most of them were 2-6 years old). There was no history of other ocular or systemic abnormalities in the family. Autosomal dominant inheritance was supported by the results of pedigree analysis, which presented affected individuals in each of four generations, about equal number of affected males and females and male to male transmission. Penetrance in this family appeared complete.
In this family, 15 affected members had bilateral zonular nuclear cataracts (typical features shown in Figure 2). The opacification was symmetrical, homogeneous, and of the same density in both eyes. In some cases, cataracts occurred with the anterior Y suture and were central pulverant. The extent of opacification was variable among different individuals in this family, so some of the affected members retained mild visual acuity, whereas other family member's visual acuity had been damaged severely and required surgical intervention.
Linkage analysis and sequence analysis
Initially, 71 microsatellite markers (commonly associated with ADCC) from candidate regions on chromosomes 1, 2, 3, 11, 12, 13, 16, 17, 21, and 22 were genotyped. No significant positive LOD score was found at these candidate loci (data not shown). Subsequently, a genome-wide scanning was performed. Linkage was found to marker D20S112 on chromosome 20p. Further analysis with refining markers showed a maximum two-point LOD score of 6.02 at marker D20S904 (θ=0; Table 1). Haplotypes were constructed for the analyzed markers on 20p (Figure 3). On individuals II:3 and IV:7, recombination events were found. Individual II:3 showed recombination proximal to marker D20S912, whereas individual IV:7 showed recombination distal to marker D20S186. Such critical recombination events defined a disease gene interval of 9.34 Mb (16.37 cM) between markers D20S186 and D20S912. Multi-point linkage analysis also supported the same interval (Figure 3). Direct cycle sequencing of the amplified fragments of exons and exon/intron boundaries of BFSP1 on three family individuals (two affected and one unaffected) was performed and no mutation was detected.
We have mapped the genetic cataract locus to chromosome 20p12.2-p11.23 in an ADPCZNC family. This is the first time ADPCZNC was linked to chromosome 20p12.2-p11.23. Due to the high heterogeneity of congenital cataracts, different phenotypes can be caused by an identical gene defect . It is possible that the ADPCZNC locus is allelic to the CPP3 locus, which was assigned to chromosome 20p12-20q12 in a Japanese autosomal dominant posterior polar cataract family . In case of that, our results confirmed the ADCC locus on chromosome 20 and refined it to 9.34 cM. The critical interval in this report is less than half of the size of the CPP3 cataract locus (Figure 4). It is worthy to note the cataract phenotypes are different between the CPP3 and ADPCZNC families. Perhaps there are two distinct genes involved in the pathogenesis of ADCC on chromosome 20.
The upper boundary of the co-segregating interval in this report was defined by the recombination event occurring on individual IV:7. Individual IV:7 was a 19 year old unaffected family member. His unaffected status was ascertained by three independent ophthalmologists. Since the youngest affected member was 3 years old and the penetrance was complete in this family, it is credible that the locus can be assigned by such a recombination event. The gene BFSP1 (and BFSP2), was considered functioning as the major cytoskeletal element of the eye lens and it was essential to the optical properties of the eye lens . Secondly, mutations in the CP49-encoding gene BFSP2 (as a partner of BFSP1) were shown to be responsible for congenital cataracts in human beings [11-13]. Furthermore, BFSP1 has been mapped to chromosome 20p11.23-p12.1 . Such a critical region has a high overlap with the ADPCZNC locus. We believe that BFSP1 is a potential candidate gene in this family. In our study, we performed direct cycle-sequencing at exons and exon-intron-boundaries of BFSP1 on two affected family members (and a control). However, no mutation was found, suggesting that there may be a new cataract gene in this interval. It was also possible that the mutation occurred in introns or the promoter area of BFSP1.
In conclusion, our study assigned a locus for ADPCZNC to a 9.34 cM interval on chromosome 20p12.2-p11.23 in a Chinese family.
The authors are grateful for the participation of the family members.
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