|Molecular Vision 2006;
Received 11 October 2006 | Accepted 18 December 2006 | Published 20 December 2006
Progressive sutural cataract associated with a BFSP2 mutation in a Chinese family
(The first two authors contributed equally to this publication)
1Eye hospital, The First Affiliated Hospital, Harbin Medical University, Harbin, China; 2Bio-X life Science Research Center, Shanghai Jiao Tong University, Shanghai, China; 3 Laboratory of Medical Genetics, Harbin Medical University, Harbin, China
Correspondence to: Ping Liu, Eye Hospital, The First Affiliated Hospital, Harbin Medical University, 23 Youzheng Road, Harbin 150001, China; Phone: 0086-451-53643849-3958; FAX: 0086-451-53650320; email: Ping_Liu53@hotmail.com
Purpose: To identify the mutation underlying the segregation of progressive sutural congenital cataracts in a four-generation Chinese pedigree.
Methods: Genomic DNA was extracted from the peripheral blood samples of members of the pedigree. A genome-wide scan was performed using microsatellite markers spaced at about 10 cM intervals. Linkage analysis was carried out using a Linkage software package. Ten additional microsatellite markers for the positive region were selected for precise targeting, and haplotype data were processed using Cyrillic software to define the region of the disease gene. Mutation detection was carried out by sequencing candidate genes.
Results: Significant evidence of linkage was obtained at marker D3S1279 (LOD score [Z] =2.32, recombination fraction [θ]=0.0). Precise targeting and haplotype analysis traced the disease gene to a 38.6 cM region bounded by D3S1267 and D3S1614 at 3q21.1- q26.2 near BFSP2, which encodes a lens-specific beaded filament protein. Sequencing results revealed a 3-bp deletion of nucleotides 696-698 (GAA) in exon 3 of BFSP2, which is predicted to cause an in-frame deletion of glutamic acid residue 233 from the polypeptide encoded by the mutant gene. This deletion was seen neither in any unaffected member of the family nor in 50 unrelated control individuals.
Conclusions: We observed progressive isolated sutural cataract associated with a deletion mutation of the BFSP2 gene in a Chinese pedigree. It highlights the physiological importance of the beaded filament protein and supports the role of BFSP2 in human cataract formation.
Congenital cataracts are a common major abnormality of the eye, and account for one-tenth of the number of cases of childhood blindness . There have been few comprehensive studies of the incidence of congenital cataract, but two studies have estimated the incidence of this disorder to be between 2.2 and 2.49 per 10,000 live births [2,3]. The cataract may be isolated, may be associated with other developmental abnormalities of the eye, or may form part of an inherited multi-system disorder. Approximately one-quarter to one-third of congenital cataracts is inherited and has been reported with all three types of Mendelian inheritance, i.e., autosomal dominant, autosomal recessive, and X-linked. Most inherited cataracts manifest as an autosomal dominant trait in which penetrance is almost complete but expressivity is highly variable . Despite congenital cataracts being a leading cause of blindness worldwide, the mechanisms of lens opacification have not been fully understood. Locating congenital cataract loci and cloning candidate genes are important steps in identifying the molecular lesions underlying congenital cataracts in humans.
To date, more than 20 loci for clinically diverse forms of nonsyndromic Mendelian cataracts have been mapped on 12 human chromosomes. Genetic linkage methods have enabled the discovery of seventeen loci in the human genome that are associated with autosomal-dominant congenital cataract. Fourteen distinct genes have been identified as causing nonsyndromic autosomal dominant cataracts. These genes can be considered in four groups: (1) Genes coding for crystallines, including seven genes; CRYAA , CRYAB [6,7], CRYBB1 , CRYBB2 [9-11], CRYBA1 [12,13], CRYGC [14-16], CRYGD [14,16-19]; (2) genes coding for membrane transport proteins, including: one gene coding for major intrinsic protein (MIP ), two genes coding for connexins (GJA8 [21,22], GJA3 [23,24]); (3) a gene coding for cytoskeletal proteins, including one gene coding for beaded filament structural protein 2 (BFSP2 [25,26]); and (4) genes coding for developmental regulators, such as PITX3 , MAF , and HSF4 .
Sutural cataract is defined as an opacity affecting the whole or part of the anterior or posterior suture of one or both eyes. Most sutural cataracts have been reported to be congenital without progression . Sutural cataract is rarely inherited without other forms of morphological change within the lens and has been described in association with nuclear, pulverulent, cerulean, and lamellar cataracts . In this study, we performed gene scan and linkage analysis on a four-generation Chinese family with progressive isolated sutural congenital cataracts. We mapped the cataract locus to 3q21.1- q26.2, close to the BFSP2 gene. Subsequently, a delection mutation in exon 3 of the BFSP2 gene was observd in this pedigree.
Clinical evaluation and DNA specimens
A progressive sutural congenital cataract was found in a Chinese family of Han ethnicity living in Northern China. The four generation family was identified by the Eye Hospital of the First Affiliated Hospital, Harbin Medical University, Harbin, China. Informed consent in accordance with the Declaration of Helsinki and the Heilongjiang Institutional Review Board was obtained from all participants. The family consisted of 30 members, including seven affected and 23 unaffected individuals. Fifteen members participated in the study, four affected and 11 unaffected (Figure 1). Affected status was determined by a history of cataract extraction or ophthalmologic examination, which included visual acuity testing, slit lamp examination, intraocular pressure measurement, and fundus examination with dilated pupil. Phenotype was documented by slit lamp photography. Peripheral blood was collected from each of the four affected and 11 unaffected individuals in the family, and genomic DNA was extracted from blood leukocytes using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany).
Gene scan and linkage analysis
We conducted a gene scan based on a set of dinucleotide repeat microsatellite markers spaced at approximately 10 cM intervals using an ABI PRISM Linkage Mapping Set, Version 2.5 (Perkin-Elmer, Applied Biosystems, Foster City, CA). Ten additional microsatellite markers were synthesized using Invitrogen for precise targeting of candidate chromosomal regions. "Touchdown" PCR was carried out in a 5 μl reaction volume containing 20 ng of genomic DNA, 1 μl of 10X PCR buffer, 7 mmol/l of MgCl2, 0.2 mmol/l of dNTP, 0.3 U HotStar Taq DNA polymerase, and 0.05 μmol/l of microsatellite markers. After an initial denaturation of 12 min at 95 °C, the sample underwent 14 cycles at 95 °C for 30 s, 63-56 °C for 30 s (each cycle -0.5 °C), and 72 °C for 1 min. After this, 30 cycles were performed at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, followed by an extension at 72 °C for 10 min and a final hold at 4 °C. The PCR products were pooled on the basis of size (Genescan-400HD ROX, Perkin Elmer, Foster City, CA) and denatured at 95 °C for 3 min and electrophoresed on a 96-capillary automated DNA sequencer (MegaBACE 1000, Amersham, Freiburg, Germany). The results were analyzed by Genetic Profiler (version 1.5). Two-point linkage analysis was performed on the MLINK sub-program of the LINKAGE software package (version 5.1). Autosomal dominant inheritance, with a full penetrance and a disease-gene frequency of 0.0001 was assumed. Marker allele frequencies were assumed to be equal. LOD scores were calculated at recombination fractions (θ) of 0.00, 0.1, 0.2, 0.3, 0.4, and 0.5. The pedigree and haplotype was constructed using Cyrillic software (version 2.1).
A strong candidate gene, the beaded filament structural protein 2 gene (BFSP2; NM_003571), is comprised of seven exons. To screen the coding regions of BFSP2, we designed gene specific PCR primers that flanked each exon and intron-exon junction (Table 1). Eight pairs of primers were used to amplify the seven exons and the adjacent intron sequences of the gene. DNA samples from all available affected and unaffected family members of the family were screened for mutations in BFSP2 by direct cycle sequencing of the PCR products. The purified PCR products were sequenced from both directions using ABI 3100 sequencer (Perkin-Elmer, Applied Biosystems). Results were analyzed using the Sequence Scanner software (version 1.0). Two affected and two unaffected individuals were compared. After identifying a mutation in exon 3, we screened all family members and 50 unrelated normal individuals.
We identified a four-generation Chinese family who had clear diagnosis of progressive sutural congenital cataracts. The affected individuals had bilateral isolated Y-shaped sutural cataract present in both the whole anterior Y-suture and the posterior inverted Y-suture (Figure 2), which presented after birth and developed during infancy, progressing slowly with age. There was no family history of any other ocular or systemic abnormalities. No affected individuals had myopia. Most patients experienced decreased visual acuity around 7-8 years old. Some of those affected required bilateral cataract surgery during childhood, usually in the early teens and occasionally in the 40s. The age at surgery ranged from 12 to 44 years. One family member had never had surgery, even in the presence of moderate sutural opacities, and the unoperated visual acuity was as good as 60/200. Autosomal dominant inheritance of the cataract was supported by the presence of affected individuals in each of the four generations, and male to male transmission.
Linkage and haplotype analysis
After excluding large regions of the genome, we obtained a positive LOD score for marker D3S1569. Further analysis of markers showed a positive LOD score at the recombination fraction of 0.00 and strongly supported this region as a candidate. The maximum two-point LOD score (Zmax) of 2.32 was obtained at marker D3S1279 with recombination θ=0.00. Ten additional markers flanking D3S1279 were analyzed in this pedigree. The order and genetic distances of the markers were derived from the Marshfield database. The adjacent markers (D3S3637, D3S1309, D3S1569, and D3S1593) also showed a LOD score above 2.0. The result of the two-point LOD scores is summarized (Table 2).
We constructed haplotypes of the family using the computer program Cyrillic version 2.1. The markers used are listed in Table 2. The haplotype were checked by visual inspection. Haplotype data are given in Figure 1. A crossover between D3S1278 and D3S1267 in individual III:5 and III:6, and one between D3S1267 and D3S3606 in individual III:1 defines the proximal border of the region, and one between D3S1279 and D3S1614 in individual II:5, III:2, and III:5 define the distal border. All affected individuals had an affected parent, and there no unaffected individuals carried the disease haplotype. Thus, penetrance appears to be virtually complete in this family. The disease-associated haplotype shared by all affected members was identified. The results of both linkage and haplotype analyses situated the disease gene in a 38.6 cM region bounded by D3S1267 and D3S1614 at 3q21.1-q26.2.
Based on former linkage and mutation studies [25,26], we predicted the BFSP2 gene as the candidate gene and sequenced all 7 exons of BFSP2 in all members of the family. Sequencing results revealed a deletion in all affected members in this family, but not in normal relatives, (Figure 3)namely a 3 bp deletion of nucleotides 696-698 (GAA) in exon 3 of BFSP2, which is predicted to cause an in-frame deletion of glutamic acid residue 233 (E233del) from the polypeptide encoded by the mutant gene. Furthermore, this deletion did not exist in 50 unrelated individuals, thus excluding the possibility that it is a rare polymorphism.
We have identified a progressive isolated sutural congenital cataract associated with a 3 bp deletion mutation of the BFSP2 gene. The nucleotides 696-698 (GAA) deletion is predicted to cause an in-frame deletion of glutamic acid residue 233 (E233del) from the polypeptide encoded by the mutant gene.
Mutations in BFSP2 have been identified in other three cataract families. However, progressive isolated sutural cataract phenotype had not been identified in previous reports. Conley et al.  identified an R287W mutation in a juvenile-onset cataract family. The lens was clear at birth but gradually developed opacities in the second and third decades of life. Jakobs et al.  identified a E233del mutation associated with cogenital cataract. Affected members had congenital nuclear, sutural, and stellate or spoke-like cortical cataracts that varied in severity among different individuals. Zhang et al.  observed a E233del mutation in a Chinese family with sutural cataracts and myopia. Affected members had bilateral lens opacities, involving the sutures, showing a feather-duster-like appearance. In contrast, the morphology of the cataracts in our study is different from that in the three previously reported families. In our family, affected members had bilateral isolated Y shaped sutural cataract present in both the whole anterior Y-suture and the posterior inverted Y-suture. The cataract appeared after birth and progressed slowly in the early years of life. There is no nuclear or cortex opacity. Interestingly, three of these four families have the same E233del mutation, which indicates that this deletion position is a hot spot for congenital cataracts or that the deletion nucleotides are very important for the gene function.
Although the same mutation has been observed in two Chinese families, we are inclined to believe that our mutation is an independent reoccurrence mutation, instead of a founder mutation. The first reason is that we did not observe this mutation in the 50 normal controls from northern China. Secondly, the penetrance of congenital cataract is high. Thirdly, our family comes from a remote small village in Heilongjiang province of China, the most northern province in China. The village is isolated and the population flow is very small. The family of Zhang et al.  comes from southern China. So the possibility of the migration of the same ancestor is very little. Although the family of Zhang et al.  and our family have the same mutation and both of them have strutral opacity in lens, but the clinical features of the cataract we observed were not the same. The former has strural cataract associated with myopia as well, and the latter is an isolated progressive strural cataract and neither of the family members has myopia. The opacity of their family shows a kind of feather-duster-like appearance, and the opacity of ours has not that kind of appenrence.
The affected gene product, CP49 (phakinin), and its assembly partner, filensin, are both members of the intermediate filament (IF) family of proteins. Both proteins localize to a cytoskeletal element referred to as the beaded filament . CP49 shows most of the secondary structural features that are conserved among IF proteins. Among these is a central rod domain dominated by runs of alpha helix. These runs of alpha helix exhibit a heptad repeat pattern in which the 1 and 4 positions of the heptad are dominated by hydrophobic residues. Deletion of residue E233 would result in a phase shift in the 1,4 periodicity and would perturb an otherwise highly conserved property among IF proteins . The role of the beaded filament in lens biology has not been defined, but two lines of evidence suggest that the beaded filament is a requirement for sustained optical clarity: (1) mutations in human CP49 have been implicated in two separate families who have autosomal dominant inherited cataract. These individuals are born with clear lenses, but opacities develop when they are children or young adults [25,26]; and, (2) targeted deletion of CP49 expression in mice results in a subtle opacification that worsens over time . It is significant that in both cases the opacification is not evident at birth but progresses in severity with age.
The actual mechanism whereby a mutation in BFSP2 gives rise to cataracts is unknown. Some reports state that CP49 and filensin, together with α-crystallins, have been shown to immunolocalize to unique cytoskeletal structures within the lens fiber cells known as beaded filaments. They are considered to be important in facilitating the chaperone activity of α-crystallin assemblies [31,34]. On the other hand, analysis of a targeted mouse CP49 knock-out revealed dramatic alterations in lens fiber cell surface, thus identifying a functional link between transparency, the cytoskeleton, and membrane organization . In another murine knock-out model, targeted deletion of CP49 was found to dysregulate the protein levels of its assembly partner filensin, suggesting a mechanism for the regulation of beaded filament protein stoichiometry . The nature of the complex mechanism is still to be determined.
In conclusion, we have identified a progressive isolated sutural congenital cataract associated with a 3 bp deletion mutation of the BFSP2 gene in a northern Chinese family. This mutation supports the previously proposed role of BFSP2 in the lens opacification process and may help delineate the functional domains of BFSP2. It highlights the physiological importance of the beaded filament protein and supports the role of BFSP2 in human cataracts formation.
We thank the patients and their families for participation in this study. We also acknowledge the financial support of the Fund of Heilongjiang Provincial Health Bureau (Grant 2005-195) and the Fund of Harbin Medical University.
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