Molecular Vision 2005; 11:758-763 <>
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 Xia2

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:


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 [3]. 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 [6], CRYBA1 [7,8], CRYBB1 [9], CRYBB2 [10-12], CRYGC [13,14], and CRYGD [14-18]. (2) Genes encoding membrane transport proteins including: MIP [19], 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 [26] and HSF4 [27].

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 [28]. 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. [31] 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.


Clinical evaluation

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.

Linkage analysis

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.

Mutation analysis

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 [10]. 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 [11]. 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 [32].

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 [33]. 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 [34]. 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 [35]. 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 [36]. 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).


1. Rahi JS, Dezateaux C, British Congenital Cataract Interest Group. Measuring and interpreting the incidence of congenital ocular anomalies: lessons from a national study of congenital cataract in the UK. Invest Ophthalmol Vis Sci 2001; 42:1444-8.

2. Wirth MG, Russell-Eggitt IM, Craig JE, Elder JE, Mackey DA. Aetiology of congenital and paediatric cataract in an Australian population. Br J Ophthalmol 2002; 86:782-6.

3. Rahi JS, Dezateux C. Congenital and infantile cataract in the United Kingdom: underlying or associated factors. British Congenital Cataract Interest Group. Invest Ophthalmol Vis Sci 2000; 41:2108-14.

4. Litt M, Kramer P, LaMorticella DM, Murphey W, Lovrien EW, Weleber RG. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet 1998; 7:471-4.

5. Mackay DS, Andley UP, Shiels A. Cell death triggered by a novel mutation in the alphaA-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q. Eur J Hum Genet 2003; 11:784-93.

6. Berry V, Francis P, Reddy MA, Collyer D, Vithana E, MacKay I, Dawson G, Carey AH, Moore A, Bhattacharya SS, Quinlan RA. Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans. Am J Hum Genet 2001; 69:1141-5.

7. Padma T, Ayyagari R, Murty JS, Basti S, Fletcher T, Rao GN, Kaiser-Kupfer M, Hejtmancik JF. Autosomal dominant zonular cataract with sutural opacities localized to chromosome 17q11-12. Am J Hum Genet 1995; 57:840-5.

8. Qi Y, Jia H, Huang S, Lin H, Gu J, Su H, Zhang T, Gao Y, Qu L, Li D, Li Y. A deletion mutation in the betaA1/A3 crystallin gene (CRYBA1/A3) is associated with autosomal dominant congenital nuclear cataract in a Chinese family. Hum Genet 2004; 114:192-7.

9. Mackay DS, Boskovska OB, Knopf HL, Lampi KJ, Shiels A. A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q. Am J Hum Genet 2002; 71:1216-21.

10. Litt M, Carrero-Valenzuela R, LaMorticella DM, Schultz DW, Mitchell TN, Kramer P, Maumenee IH. Autosomal dominant cerulean cataract is associated with a chain termination mutation in the human beta-crystallin gene CRYBB2. Hum Mol Genet 1997; 6:665-8.

11. Gill D, Klose R, Munier FL, McFadden M, Priston M, Billingsley G, Ducrey N, Schorderet DF, Heon E. Genetic heterogeneity of the Coppock-like cataract: a mutation in CRYBB2 on chromosome 22q11.2. Invest Ophthalmol Vis Sci 2000; 41:159-65.

12. Vanita, Sarhadi V, Reis A, Jung M, Singh D, Sperling K, Singh JR, Burger J. A unique form of autosomal dominant cataract explained by gene conversion between beta-crystallin B2 and its pseudogene. J Med Genet 2001; 38:392-6.

13. Ren Z, Li A, Shastry BS, Padma T, Ayyagari R, Scott MH, Parks MM, Kaiser-Kupfer MI, Hejtmancik JF. A 5-base insertion in the gammaC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract. Hum Genet 2000; 106:531-7.

14. Heon E, Priston M, Schorderet DF, Billingsley GD, Girard PO, Lubsen N, Munier FL. The gamma-crystallins and human cataracts: a puzzle made clearer. Am J Hum Genet 1999; 65:1261-7.

15. Stephan DA, Gillanders E, Vanderveen D, Freas-Lutz D, Wistow G, Baxevanis AD, Robbins CM, VanAuken A, Quesenberry MI, Bailey-Wilson J, Juo SH, Trent JM, Smith L, Brownstein MJ. Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene. Proc Natl Acad Sci U S A 1999; 96:1008-12.

16. Kmoch S, Brynda J, Asfaw B, Bezouska K, Novak P, Rezacova P, Ondrova L, Filipec M, Sedlacek J, Elleder M. Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography. Hum Mol Genet 2000; 9:1779-86.

17. Shentu X, Yao K, Xu W, Zheng S, Hu S, Gong X. Special fasciculiform cataract caused by a mutation in the gammaD-crystallin gene. Mol Vis 2004; 10:233-9 <>.

18. Santhiya ST, Shyam Manohar M, Rawlley D, Vijayalakshmi P, Namperumalsamy P, Gopinath PM, Loster J, Graw J. Novel mutations in the gamma-crystallin genes cause autosomal dominant congenital cataracts. J Med Genet 2002; 39:352-8.

19. Berry V, Francis P, Kaushal S, Moore A, Bhattacharya S. Missense mutations in MIP underlie autosomal dominant 'polymorphic' and lamellar cataracts linked to 12q. Nat Genet 2000; 25:15-7.

20. Mackay D, Ionides A, Kibar Z, Rouleau G, Berry V, Moore A, Shiels A, Bhattacharya S. Connexin46 mutations in autosomal dominant congenital cataract. Am J Hum Genet 1999; 64:1357-64.

21. Rees MI, Watts P, Fenton I, Clarke A, Snell RG, Owen MJ, Gray J. Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA3). Hum Genet 2000; 106:206-9.

22. Shiels A, Mackay D, Ionides A, Berry V, Moore A, Bhattacharya S. A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q. Am J Hum Genet 1998; 62:526-32.

23. Berry V, Mackay D, Khaliq S, Francis PJ, Hameed A, Anwar K, Mehdi SQ, Newbold RJ, Ionides A, Shiels A, Moore T, Bhattacharya SS. Connexin 50 mutation in a family with congenital "zonular nuclear" pulverulent cataract of Pakistani origin. Hum Genet 1999; 105:168-70.

24. Conley YP, Erturk D, Keverline A, Mah TS, Keravala A, Barnes LR, Bruchis A, Hess JF, FitzGerald PG, Weeks DE, Ferrell RE, Gorin MB. A juvenile-onset, progressive cataract locus on chromosome 3q21-q22 is associated with a missense mutation in the beaded filament structural protein-2. Am J Hum Genet 2000; 66:1426-31.

25. Jakobs PM, Hess JF, FitzGerald PG, Kramer P, Weleber RG, Litt M. Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2. Am J Hum Genet 2000; 66:1432-6.

26. Semina EV, Ferrell RE, Mintz-Hittner HA, Bitoun P, Alward WL, Reiter RS, Funkhauser C, Daack-Hirsch S, Murray JC. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD. Nat Genet 1998; 19:167-70.

27. Bu L, Jin Y, Shi Y, Chu R, Ban A, Eiberg H, Andres L, Jiang H, Zheng G, Qian M, Cui B, Xia Y, Liu J, Hu L, Zhao G, Hayden MR, Kong X. Mutant DNA-binding domain of HSF4 is associated with autosomal dominant lamellar and Marner cataract. Nat Genet 2002; 31:276-8.

28. Reddy MA, Francis PJ, Berry V, Bhattacharya SS, Moore AT. Molecular genetic basis of inherited cataract and associated phenotypes. Surv Ophthalmol 2004; 49:300-15.

29. Rogaev EI, Rogaeva EA, Korovaitseva GI, Farrer LA, Petrin AN, Keryanov SA, Turaeva S, Chumakov I, St George-Hyslop P, Ginter EK. Linkage of polymorphic congenital cataract to the gamma-crystallin gene locus on human chromosome 2q33-35. Hum Mol Genet 1996; 5:699-703.

30. Francis P, Berry V, Bhattacharya S, Moore A. Congenital progressive polymorphic cataract caused by a mutation in the major intrinsic protein of the lens, MIP (AQP0). Br J Ophthalmol 2000; 84:1376-9.

31. Santhiya ST, Manisastry SM, Rawlley D, Malathi R, Anishetty S, Gopinath PM, Vijayalakshmi P, Namperumalsamy P, Adamski J, Graw J. Mutation analysis of congenital cataracts in Indian families: identification of SNPS and a new causative allele in CRYBB2 gene. Invest Ophthalmol Vis Sci 2004; 45:3599-607.

32. Van Leen RW, Breuer ML, Lubsen NH, Schoenmakers JG. Developmental expression of crystallin genes: in situ hybridization reveals a differential localization of specific mRNAs. Dev Biol 1987; 123:338-45.

33. Blundell T, Lindley P, Miller L, Moss D, Slingsby C, Tickle I, Turnell B, Wistow G. The molecular structure and stability of the eye lens: x-ray analysis of gamma-crystallin II. Nature 1981; 289:771-7.

34. Inana G, Piatigorsky J, Norman B, Slingsby C, Blundell T. Gene and protein structure of a beta-crystallin polypeptide in murine lens: relationship of exons and structural motifs. Nature 1983; 302:310-5.

35. Crabbe MJ, Goode D. Protein folds and functional similarity; the Greek key/immunoglobulin fold. Comput Chem 1995; 19:343-9.

36. Graw J, Loster J, Soewarto D, Fuchs H, Reis A, Wolf E, Balling R, Hrabe de Angelis M. Aey2, a new mutation in the betaB2-crystallin-encoding gene of the mouse. Invest Ophthalmol Vis Sci 2001; 42:1574-80.

Yao, Mol Vis 2005; 11:758-763 <>
©2005 Molecular Vision <>
ISSN 1090-0535