|Molecular Vision 2005;
Received 12 May 2005 | Accepted 18 August 2005 | Published 16 September 2005
Progressive polymorphic congenital cataract caused by a CRYBB2 mutation in a Chinese family
Ke Yao,1 Xiajing Tang,1
Xingchao Shentu,1 Kaijun Wang,1 Huiying Rao,1 Kun
1Eye Center, Affiliated Second Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 2National Laboratory of Medical Genetics of China, Changsha, China
Correspondence to: Ke Yao, Eye Center, Affiliated Second Hospital, College of Medicine and Institute of Ophthalmology, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China; Phone: 0086-571-87783897; FAX: 0086-571-87783908; email: firstname.lastname@example.org
Purpose: To report and identify the genetic defect that causes progressive polymorphic congenital cataracts affecting a large five generation Chinese family.
Methods: Family history and phenotypic data were recorded, and the phenotypes were documented by slit lamp photography. Genetic linkage analysis was performed on the known genetic loci for autosomal dominant congenital cataract (ADCC) with 41 short tandem repeat polymorphic markers. Mutations were screened by DNA sequencing and restriction fragment length analysis (RFLP).
Results: A significant two point LOD score was generated at marker D22S420, D22S539 and D22S315 for 22q11.2. The highest observed LOD score was 6.26 (θ=0.00) with marker D22S315. Mutation screening of the CRYBB2 gene in this family revealed an C->T transition at position 475 (Q155X) of the cDNA sequence, creating a novel SpeI restriction site that cosegregated with affected members of the pedigree, but was not present in unaffected members or any of the 100 unrelated individuals tested.
Conclusions: Our finding expands the spectrum of cataract phenotypes caused by the Q155X mutation of CRYBB2, confirms the phenotypic heterogeneity of this mutation and suggests the mechanism that influences the congenital cataract formation in different ethnic backgrounds.
Although surgical techniques and visual prognosis have been improved recently, congenital cataracts remain the leading cause of visual disability in children worldwide. The incidence of congenital cataracts has been estimated to be 2.22-2.49 per 10,000 births [1,2], and genetic mutation is the most common cause. In fact, about one third of all congenital cataracts are inherited, with the most common being the nonsyndromic autosomal dominant form . The frequent autosomal dominant inheritance represents a tool to identify the genes involved in lens development and cataract formation. To date, more than 18 candidate loci have been identified and 13 cataract-related genes characterized. These genes can be considered in four groups. (1) Crystallin genes encode greater than 90% of the structural proteins in the lens. Mutations in 7 crystallin genes have been identified as the cause of autosomal dominant congenital cataract (ADCC) including: CRYAA [4,5], CRYAB , CRYBA1 [7,8], CRYBB1 , CRYBB2 [10-12], CRYGC [13,14], and CRYGD [14-18]. (2) Genes encoding membrane transport proteins including: MIP , GJA3 [20,21], and GJA8 [22,23]. (3) Genes encoding cytoskeletal proteins such as BFSP2 [24,25]. (4) Genes encoding transcription factors such as PITX3  and HSF4 .
An ever growing number of genes implicated in cataractogenesis indicate the genetic heterogeneity of congenital cataract, which in turn causes phenotypic heterogeneity. Depending on the position and the morphology of the lens opacity, the phenotypes of isolated inherited cataracts have been categorized as anterior polar, posterior polar, nuclear, lamellar, pulverulent, aceuliform, cerulean, total, cortical, polymorphic, and sutural cataracts . However, the relationship between the genotype and the phenotype of inherited congenital cataracts is still undetermined.
Polymorphic congenital cataract refer to lens opacities of variable morphology even within the same family. This type of cataract has been characterized in ADCC families with mutations in the CRYG and MIP genes [29,30]. In this study, a five generation Chinese family with progressive polymorphic congenital cataracts was studied in an attempt to identify the genetic defect associated with this special phenotype. Using linkage analysis, we mapped an associated locus to 22q22.1, close to the CRYBB2 gene. Finally we identified a mutation in exon 6 of this gene, and this mutation is present in all affected family members.
Clinical evaluation and DNA specimens
The five generation family was ascertained through the Eye Center of Affiliated Second Hospital, College of Medicine, Zhejiang University, Hangzhou, China. Informed consent in accordance with the Zhejiang Institutional Review Board approval was obtained from all participants. Forty-one individuals participated in the study, 17 affected individuals and 24 unaffected individuals among whom 12 were spouses (Figure 1). Affected status was determined by a history of cataract extraction or ophthalmologic examination, which included visual function, slit lamp, and fundus examination with the dilated pupil. Phenotype was documented by slit lamp photography. Blood specimens (5 ml) were collected in EDTA and leukocyte genomic DNA was extracted use the 3-Spin Blood DNA Isolation Kit (Biocolors, Shanghai, China).
Genotyping and linkage analysis
The initial strategy consisted of screening 18 known loci related to ADCC formation and 41 fluorescent short tandem repeat polymorphic markers (ABI PRISM Linkage Mapping Set, Version 2.0, Foster City, CA) were used (Table 1). Multiplex PCR was carried out in a 5 μl reaction mixture containing 30 ng of genomic DNA, 200 μM of each dNTP, 80 pmol each of forward and reverse primers, 0.2 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Branchburg, NJ), 3.0 mM MgCl2, and primary PCR buffer. Samples were incubated in a thermocycler for 12 min at 94 °C and 30 s at 94 °C; the annealing temperature was programmed to initiate from 63 °C at 1 min and decrease 0.5 °C every cycle; 72 °C for 110 s, for 15 cycles; followed by 94 °C for 30 s, 56 °C for 1 min, 72 °C for 1 min, 110 s for 25 cycles; a final extension at 72 °C for 15 min was performed. The PCR products were appropriately pooled and an aliquot was loaded onto a 5% standard denaturing polyacrylamide gel and run in an Applied Biosystems 377XL DNA sequencer. The size of each allele was determined on the basis of an internal size standard (Genescan-400HD ROX, Perkin Elmer, Foster City, CA) in each lane, and results were analyzed by Genescan 3.0 and Genotyper 2.1 software (Perkin Elmer). Two point LOD scores between the disease locus and markers were calculated using the MLINK routine of the LINKAGE software package, version 5.1. The disease locus was specified to be an autosomal dominant trait with a disease allele frequency of 0.0001. The allele frequencies for each marker were assumed to be equal as were the recombination frequencies in males and females. Genetic penetrance was assigned to be full.
PCR and DNA sequencing
A strong candidate gene, the βB2-crystallin gene (CRYBB2; NM_000496), is comprised of six exons. To screen the coding regions of CRYBB2, gene-specific PCR primers were designed flanking each exon and intron-exon junction. Five pairs of primers were the same as those used by Santhiya et al.  except for the exon 3 primers (5'-TGA GGG TCT GAG TCT CGC-3' and 5'-GGT GGA ACC TGG ATT TGA-3') were used. A 10 μl PCR reaction mixture contain 30 ng DNA template, 200 μM of each dNTP, 0.1 U Taq DNA polymerase (TAKARA, Dalian, China), 10 pmol each of forward and reverse primers, and primary PCR buffer was prepared. The cycling conditions for PCR included a 95 °C preactivation of the enzyme for 5 min, 10 cycles of touchdown PCR with a 1 °C decrement of the annealing temperature per cycle from 68 °C to 58 °C, followed by 25 cycles with annealing at 62 °C for 35 s with denaturation at 94 °C for 45 s and extension at 72 °C for 50 s. PCR products were purified from 8% polyacrylamide gels by ethanol precipitation and were TA subcloned by means of the pGEM-T vector system II (Promega, Madison, WI). Plasmid DNA was purified by means of the QIAprep spin miniprep kit (Qiagen, Hilden, Germany), and insert DNA was sequenced commercially. T7 and SP6 primers were used to sequence in both directions. Two affected and two unaffected individuals were compared.
Restriction fragment length polymorphism analysis
After identifying a mutation in exon 6 of the CRYBB2 gene, all family members and 100 unrelated normal individuals were examined by restriction fragment length polymorphism analysis. The mutation created a novel SpeI site. PCR products of exon 6 of the CRYBB2 gene were digested for 1 h at 37 °C with SpeI (TAKARA) and electrophoresized in 6% polyacrylamide gels with silver staining.
We have identified a five generation Chinese family with clear diagnosis of congenital cataracts. Opacification of the lens was bilateral in all affected cases, but the appearance of white opacities distributed in the nucleus and cortex were highly variable, which included pulverulent, dot, strip, star-like and sheet shapes (Figure 2). In addition, the opacities became denser as age increased. Visual acuity in the unoperated eyes of those affected individuals ranged from 1.0 to 0.1. Most affected individuals noticed their visual impairments before the age of ten, and then their visual acuity decreased gradually until surgery was required to improve their visual function after the age of 40. Also, there was no family history of other ocular or systemic abnormalities aside from age related disorders. Based on the presence of affected individuals in each of the five generations, and male to male transmission, autosomal dominant inheritance of the cataract was demonstrated.
Candidate loci related to autosomal dominant congenital cataract were initially screened with 41 markers. After the other loci related to ADCC were excluded, significant linkage was found with markers of the CRYBB2 locus in the 22q11.2 region. Two point maximum likelihood data for markers of this region was summarized (Table 2). Significant two point LOD scores were generated with markers D22S420, D22S539, and D22S315, and the highest observed LOD score was 6.26 (θ=0.00) with marker D22S315.
By sequencing of exon 6 of the CRYBB2 gene, we found a base change (C->T) at position 475 of the CRYBB2 cDNA. This mutation creates a premature stop codon, and this nonsense mutation creates a novel SpeI restriction site that segregated with all affected members in this Chinese family, but was not detected in the 100 unrelated normal controls and unaffected pedigree members (Figure 3).
We have described a novel progressive polymorphic congenital cataract phenotype caused by the Q155X mutation of the CRYBB2 gene. The cataract phenotype of this Chinese family was highly variable. White opacities varied from pulverulent, dot, strip, star-like, and sheet shapes distributed in the nucleus and cortex of the lens. The cataract appeared after birth and progressed in the early years of life. In addition, this phenotype is phenotypically distinct from the cerulean cataracts and nuclear cataracts in other families with an identical gene mutation. The cerulean cataract is characterized by coarser, punctate lens opacities in the nuclear and cortex of the lens have a distinct blue hue . As for the Coppock-like cataract linked to the same mutation, the cataract was characterized by a pulverulent opacification of the embryonic nucleus, giving a gray disc appearance associated with zonular opacities of variable degree. The lens opacity is present in the more internal layers of the nucleus in this Swiss family . However, the morphology of lens opacities is similar in each member of the above two cataractous families. In contrast, the five generation Chinese family in our study showed highly variable lens opacities between the family members. Diverse cataract phenotypes caused by exactly the same mutation of the CRYBB2 gene in different ethic backgrounds suggest that ethic background including environmental factors or, more likely, other genetic modifiers may influence the expression and function of this gene in lens development and cataract formation.
However, there are some similarities in these different families influenced by the Q155X mutation. The cataracts were all dominant and progressed in early life. This unique clinical manifestation is in accordance with the function of CRYBB2, which encodes the most abundant crystallin of the adult lens.The increasing severity of the phenotype is temporally correlated with the increased expression of the CRYBB2 gene throughout life .
β-Crystallins are recognized as a member of the β/γ-crystallin superfamily. Both of β-crystallin and γ-crystallin contain four Greek key motifs. In the β-crystallins, individual Greek key motifs are encoded by separate exons . The CRYBB2 gene consists of six exons; the first exon is not translated, the second exon encodes the NH2-terminal extension, and the subsequent four exons are responsible for one Greek key motif each . The Q155X mutation results in an in-frame stop codon at nucleotide 14 of exon 6 that causes 51 amino acids to be truncated from the COOH-terminal end of βB2-crystallin. The corresponding alteration affects not only the length of the COOH-terminal arms but also the formation the fourth Greek key motif formation in βB2-crystallin. Although the function of the Greek key motifs has not been elaborated in detail, computer-based analysis suggests that it may be responsible for particular protein-protein interactions within the lens and is postulated to be critical in the maintenance of lens transparency . This mutation affects the Greek key motifs, and it is predicted to change the folding properties of βB2-crystallin and change its steric coordinations with other proteins in lens. Animal experiments indicate that the altered form of βB2-crystallin is present primarily in the heavy molecular weight fraction . Whether the interactions of the altered βB2-crystallin with other lens proteins cause a rapid aggregation of the cellular proteins, which leads to the formation of the heavy molecular weight material and resulting finally in a cataract, is still to be determined.
In conclusion, the present article described a novel progressive polymorphic congenital cataract caused by the Q155X mutation of the CRYBB2 gene, expanding the spectrum of phenotypes caused by this mutation. Further studies of this cataract-related genetic defect and the factors that modify their variable phenotypes will improve our understanding of the mechanism of cataract formation and illuminate the developmental biology and biochemistry of the lens.
This work was supported by Nature Science Key Fund of Zhejiang Province, China (491020-N20205).
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