Progressive Sutural Cataract Associated with a BFSP2 Mutation in a Chinese Family

Progressive sutural cataract associated with a BFSP2 mutation in a Chinese family

Lu Zhang,1 Linghan Gao,2 Zhijian Li,1 Wei Qin,2 Weiqi Gao,1 Xiaobo Cui,3 Guoyin Feng,2 Songbin Fu,3 Lin He,2 Ping Liu1

(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, Shang- hai Jiao Tong University, Shanghai, China; 3 Laboratory of Medical Genetics, Harbin Medical University, Harbin, China

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 To date, more than 20 loci for clinically diverse forms of of the eye, and account for one-tenth of the number of cases nonsyndromic Mendelian cataracts have been mapped on 12 of childhood blindness [1]. There have been few comprehen- human chromosomes. Genetic linkage methods have enabled sive studies of the incidence of congenital cataract, but two the discovery of seventeen loci in the human genome that are studies have estimated the incidence of this disorder to be be- associated with autosomal-dominant congenital cataract. Four- tween 2.2 and 2.49 per 10,000 live births [2,3]. The cataract teen distinct genes have been identified as causing may be isolated, may be associated with other developmental nonsyndromic autosomal dominant cataracts. These genes can abnormalities of the eye, or may form part of an inherited multi- be considered in four groups: (1) Genes coding for crystallines, system disorder. Approximately one-quarter to one-third of including seven genes; CRYAA [5], CRYAB [6,7], CRYBB1 [8], congenital cataracts is inherited and has been reported with CRYBB2 [9-11], CRYBA1 [12,13], CRYGC [14-16], CRYGD all three types of Mendelian inheritance, i.e., autosomal domi- [14,16-19]; (2) genes coding for membrane transport proteins, nant, autosomal recessive, and X-linked. Most inherited cata- including: one gene coding for major intrinsic protein (MIP racts manifest as an autosomal dominant trait in which pen- [20]), two genes coding for connexins (GJA8 [21,22], GJA3 etrance is almost complete but expressivity is highly variable [23,24]); (3) a gene coding for cytoskeletal proteins, includ- [4]. Despite congenital cataracts being a leading cause of blind- ing one gene coding for beaded filament structural protein 2 ness worldwide, the mechanisms of lens opacification have (BFSP2 [25,26]); and (4) genes coding for developmental regu- not been fully understood. Locating congenital cataract loci lators, such as PITX3 [27], MAF [28], and HSF4 [29]. and cloning candidate genes are important steps in identify- Sutural cataract is defined as an opacity affecting the ing the molecular lesions underlying congenital cataracts in whole or part of the anterior or posterior suture of one or both humans. eyes. Most sutural cataracts have been reported to be congenital without progression [30]. Sutural cataract is rarely inherited Correspondence to: Ping Liu, Eye Hospital, The First Affiliated Hos- without other forms of morphological change within the lens pital, Harbin Medical University, 23 Youzheng Road, Harbin 150001, and has been described in association with nuclear, pulveru- China; Phone: 0086-451-53643849-3958; FAX: 0086-451-53650320; lent, cerulean, and lamellar cataracts [31]. In this study, we email: [email protected] performed gene scan and linkage analysis on a four-genera- 1626 Molecular Vision 2006; 12:1626-31 ©2006 Molecular Vision tion Chinese family with progressive isolated sutural congenital Peripheral blood was collected from each of the four affected cataracts. We mapped the cataract locus to 3q21.1- q26.2, close and 11 unaffected individuals in the family, and genomic DNA to the BFSP2 gene. Subsequently, a delection mutation in exon was extracted from blood leukocytes using a QIAamp DNA 3 of the BFSP2 gene was observd in this pedigree. Blood Mini Kit (Qiagen, Hilden, Germany). Gene scan and linkage analysis: We conducted a gene METHODS scan based on a set of dinucleotide repeat microsatellite mark- Clinical evaluation and DNA specimens: A progressive su- ers spaced at approximately 10 cM intervals using an ABI tural congenital cataract was found in a Chinese family of Han PRISM Linkage Mapping Set, Version 2.5 (Perkin-Elmer, ethnicity living in Northern China. The four generation fam- Applied Biosystems, Foster City, CA). Ten additional ily was identified by the Eye Hospital of the First Affiliated microsatellite markers were synthesized using Invitrogen for Hospital, Harbin Medical University, Harbin, China. Informed precise targeting of candidate chromosomal regions. “Touch- consent in accordance with the Declaration of Helsinki and down” PCR was carried out in a 5 µl reaction volume contain- the Heilongjiang Institutional Review Board was obtained from ing 20 ng of genomic DNA, 1 µl of 10X PCR buffer, 7 mmol/ all participants. The family consisted of 30 members, includ- l of MgCl2, 0.2 mmol/l of dNTP, 0.3 U HotStar Taq DNA poly- ing seven affected and 23 unaffected individuals. Fifteen mem- merase, and 0.05 µmol/l of microsatellite markers. After an bers participated in the study, four affected and 11 unaffected initial denaturation of 12 min at 95 °C, the sample underwent (Figure 1). Affected status was determined by a history of cata- 14 cycles at 95 °C for 30 s, 63-56 °C for 30 s (each cycle -0.5 ract extraction or ophthalmologic examination, which included °C), and 72 °C for 1 min. After this, 30 cycles were performed visual acuity testing, slit lamp examination, intraocular pres- at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, followed sure measurement, and fundus examination with dilated pu- by an extension at 72 °C for 10 min and a final hold at 4 °C. pil. Phenotype was documented by slit lamp photography. 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 auto- mated 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). Mutation analysis: 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

TABLE 1. PCR PRIMERS FOR MUTATION SCREENING OF BFSP2

Figure 1. Pedigree and haplotype of the progressive sutural cataract family. Pedigree and haplotype analysis of the cataract family showing segregation of ten microsatellite markers on chromosome 3q, listed in descending order from the centromere. Squares and circles symbolize males and females, respectively. Black and white sym- bols denote affected and unaffected individuals, respectively. The Primer pairs used for amplification and sequencing of the coding affected haplotype is indicated by the filled box. The slashed sym- exons for BFSP2 located on 3q. bols represent deceased indioviduals. 1627 Molecular Vision 2006; 12:1626-31 ©2006 Molecular Vision

BFSP2, we designed gene specific PCR primers that flanked RESULTS each exon and intron-exon junction (Table 1). Eight pairs of Clinical findings: We identified a four-generation Chi- primers were used to amplify the seven exons and the adja- nese family who had clear diagnosis of progressive sutural cent intron sequences of the gene. DNA samples from all avail- congenital cataracts. The affected individuals had bilateral iso- able affected and unaffected family members of the family lated Y-shaped sutural cataract present in both the whole ante- were screened for mutations in BFSP2 by direct cycle sequenc- rior Y-suture and the posterior inverted Y-suture (Figure 2), ing of the PCR products. The purified PCR products were se- which presented after birth and developed during infancy, pro- quenced from both directions using ABI 3100 sequencer gressing slowly with age. There was no family history of any (Perkin-Elmer, Applied Biosystems). Results were analyzed other ocular or systemic abnormalities. No affected individu- using the Sequence Scanner software (version 1.0). Two af- als had myopia. Most patients experienced decreased visual fected and two unaffected individuals were compared. After acuity around 7-8 years old. Some of those affected required identifying a mutation in exon 3, we screened all family mem- bilateral cataract surgery during childhood, usually in the early bers and 50 unrelated normal individuals. teens and occasionally in the 40s. The age at surgery ranged

Figure 2. Slit lamp photographs of affected individuals eyes. A: Individual III:5 at 12 years of age with corrected visual acuity of 60/200 in both eyes before surgery. B: Individual II:6 at 35 years of age with corrected visual acuity of 30/200 in both eyes before surgery. Lens opacity was only observed at the anterior Y and posterior inverted Y sutures, which show typical features of the Y-suture cataract.

TABLE 2. TWO-POINT LOD SCORES FOR LINKAGE BETWEEN CATARACT LOCUS AND CHROMOSOME 3 MARKERS

Two point LOD scores for linkage between the cataract locus and ten markers on chromosome 3q listed in genetic (sex-averaged) order using the Marshfield genetic database from p-tel, measured in centi-Morgans (cM).

1628 Molecular Vision 2006; 12:1626-31 ©2006 Molecular Vision from 12 to 44 years. One family member had never had sur- Mutation analysis: Based on former linkage and muta- gery, even in the presence of moderate sutural opacities, and tion studies [25,26], we predicted the BFSP2 gene as the can- the unoperated visual acuity was as good as 60/200. Autoso- didate gene and sequenced all 7 exons of BFSP2 in all mem- mal dominant inheritance of the cataract was supported by the bers of the family. Sequencing results revealed a deletion in presence of affected individuals in each of the four genera- all affected members in this family, but not in normal rela- tions, and male to male transmission. tives, (Figure 3)namely a 3 bp deletion of nucleotides 696- Linkage and haplotype analysis: After excluding large 698 (GAA) in exon 3 of BFSP2, which is predicted to cause regions of the genome, we obtained a positive LOD score for an in-frame deletion of glutamic acid residue 233 (E233del) marker D3S1569. Further analysis of markers showed a posi- from the polypeptide encoded by the mutant gene. Further- tive LOD score at the recombination fraction of 0.00 and more, this deletion did not exist in 50 unrelated individuals, strongly supported this region as a candidate. The maximum thus excluding the possibility that it is a rare polymorphism. two-point LOD score (Zmax) of 2.32 was obtained at marker D3S1279 with recombination θ=0.00. Ten additional markers DISCUSSION flanking D3S1279 were analyzed in this pedigree. The order We have identified a progressive isolated sutural congenital and genetic distances of the markers were derived from the cataract associated with a 3 bp deletion mutation of the BFSP2 Marshfield database. The adjacent markers (D3S3637, gene. The nucleotides 696-698 (GAA) deletion is predicted to D3S1309, D3S1569, and D3S1593) also showed a LOD score cause an in-frame deletion of glutamic acid residue 233 above 2.0. The result of the two-point LOD scores is summa- (E233del) from the polypeptide encoded by the mutant gene. rized (Table 2). Mutations in BFSP2 have been identified in other three We constructed haplotypes of the family using the com- cataract families. However, progressive isolated sutural cata- puter program Cyrillic version 2.1. The markers used are listed ract phenotype had not been identified in previous reports. in Table 2. The haplotype were checked by visual inspection. Conley et al. [25] identified an R287W mutation in a juve- Haplotype data are given in Figure 1. A crossover between nile-onset cataract family. The lens was clear at birth but gradu- D3S1278 and D3S1267 in individual III:5 and III:6, and one ally developed opacities in the second and third decades of between D3S1267 and D3S3606 in individual III:1 defines life. Jakobs et al. [26] identified a E233del mutation associ- the proximal border of the region, and one between D3S1279 ated with cogenital cataract. Affected members had congeni- and D3S1614 in individual II:5, III:2, and III:5 define the dis- tal nuclear, sutural, and stellate or spoke-like cortical cata- tal border. All affected individuals had an affected parent, and racts that varied in severity among different individuals. Zhang there no unaffected individuals carried the disease haplotype. et al. [30] observed a E233del mutation in a Chinese family Thus, penetrance appears to be virtually complete in this fam- with sutural cataracts and myopia. Affected members had bi- ily. The disease-associated haplotype shared by all affected lateral lens opacities, involving the sutures, showing a feather- members was identified. The results of both linkage and hap- duster-like appearance. In contrast, the morphology of the lotype analyses situated the disease gene in a 38.6 cM region cataracts in our study is different from that in the three previ- bounded by D3S1267 and D3S1614 at 3q21.1-q26.2. ously 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. Inter- estingly, 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. Sec- ondly, 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. [30] comes from southern China. So the possibility of the migration of the same ancestor is very little. Figure 3. Sequence analysis of BFSP2 at exon 3. A: Sequence of Although the family of Zhang et al. [30] and our family have unaffected individual (individual II:5 in Figure 1). B: Sequence of the same mutation and both of them have strutral opacity in affected (individual II:6 in Figure 1). A 3 bp deletion (GAA) is evi- lens, but the clinical features of the cataract we observed were dent at nucleotides 696-698. not the same. The former has strural cataract associated with 1629 Molecular Vision 2006; 12:1626-31 ©2006 Molecular Vision myopia as well, and the latter is an isolated progressive strural 195) and the Fund of Harbin Medical University. cataract and neither of the family members has myopia. The opacity of their family shows a kind of feather-duster-like REFERENCES appearance, and the opacity of ours has not that kind of 1. Gilbert CE, Canovas R, Hagan M, Rao S, Foster A. Causes of appenrence. childhood blindness: results from west Africa, south India and The affected gene product, CP49 (phakinin), and its as- Chile. 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The print version of this article was created on 20 Dec 2006. This reflects all typographical corrections and errata to the article through that date. Details of any changes may be found in the online version of the article. α 1631