|Molecular Vision 2006;
Received 10 May 2006 | Accepted 19 July 2006 | Published 20 July 2006
A novel mutation in the connexin 46 gene (GJA3) causes autosomal dominant zonular pulverulent cataract in a Hispanic family
P. K. F. Addison,1 V.
Berry,1 K. R. Holden,2 D. Espinal,3 B. Rivera,3
H. Su,3 A. K. Srivastava,2 S. S.
1Department of Molecular Genetics, Institute of Ophthalmology, University College London, London, UK; 2J. C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC; 3Hospital General San Felipe, Tegucigalpa, Honduras
Correspondence to: Professor S. S. Bhattacharya, Department of Molecular Genetics, Institute of Ophthalmology, London EC1V 9EL, UK; Phone: 020 7608 6826; FAX: 020 7608 6863; email: firstname.lastname@example.org
Purpose: A five-generation Hispanic pedigree with autosomal dominant zonular pulverulent cataract was studied to identify the causative mutation in connexin 46 (Cx46), a gap junction protein responsible for maintaining lens homeostasis.
Methods: Twenty-six individuals from the family were comprehensively clinically examined. DNA was extracted from their peripheral blood samples. The DNA was used for automated genotyping with fluorescently labeled microsatellite markers and for mutation detection by automated sequencing.
Results: A novel D3Y missense mutation in GJA3 segregated with autosomal dominant (AD) zonular pulverulent cataract throughout the family. The mutation was absent in the unaffected individuals in the family and in 230 control chromosomes.
Conclusions: A novel mutation causing AD zonular pulverulent cataract has been identified in a Hispanic Central American family. This is the first report of a mutation in GJA3 causing autosomal dominant congenital cataract (ADCC) in this ethnic group. It is also the first reported cataract-causing mutation in the NH2-terminal region of the Cx46 protein.
Cataract, opacification of the crystalline lens of the eye, is the most common cause of blindness in the world . The World Health Organization estimates that 45 million people in the world are blind, 19 million of them as a result of cataract . Cataracts may be broadly divided into adult onset and childhood onset (either congenital or infantile). Congenital cataract is defined as cataract which is present from birth and is responsible for approximately one-tenth of worldwide childhood blindness . The incidence of congenital cataract is between 2.2 and 2.49 per 10,000 live births [3,4]. About one third of isolated congenital cataracts are familial , the most common mode of inheritance being autosomal dominant [4,5]. Fourteen genes and at least six additional loci have been implicated in autosomal dominant congenital cataract (ADCC) . The genes that have been identified comprise seven crystallins [7-18], three transcription factors [19-21], one cytoskeletal protein , and three transmembrane proteins [23-34]. The transmembrane proteins consist of connexin 46 (Cx46) [23,26-30,32], connexin 50 (Cx50) [24,31,33,34], and major intrinsic protein of the lens (MIP) .
Cx46 is a member of the connexin family of proteins. The connexins comprise proteins important for the formation of gap junction channels. Connexin proteins form hexamers known as connexons in cell membranes. Connexons in neighboring cells dock to form gap junctions which allow the transport of small metabolites between cells . In humans, at least 20 connexin genes have been associated with several different diseases including genetic deafness, skin disease, peripheral neuropathies, heart defects and cataracts . The lens expresses three distinct connexins, connexin 43 (Cx43), Cx46, and Cx50, all of which appear to have different functions in maintaining lens homeostasis . The lens is an avascular structure and lens fibers lose all intracellular organelles during development. The lens has therefore developed an extensive intercellular communication system using gap junctions to maintain tissue homeostasis and hence transparency . Cx43 is expressed mainly in the lens epithelial cells, while Cx46 and Cx50 are expressed in lens fiber cells [38-40]. Hence, mutations in Cx46 and Cx50 may lead to congenital cataracts.
Pulverulent cataracts have a pulverized (powdery) appearance to opacification . "Zonular pulverulent cataract" is a term which has been used to describe pulverulent cataracts which involve the nucleus minimally but markedly affect lamellar regions beyond it. AD zonular pulverulent cataracts have been described in association with mutations in GJA3 [30,32], GJA8 [24,31,33], and CRYGC  genes.
A linkage approach was used to investigate the known cataract genes and loci in a large Hispanic pedigree from Honduras with zonular pulverulent cataract with the aim of identifying the causative mutation. A novel causative mutation in the connexin 46 gene (GJA3) was identified.
Patient ascertainment and collection of genetic material
A five-generation pedigree from Honduras with zonular pulverulent cataract and without gross chromosomal abnormalities was identified. Written informed consent for molecular studies, ethically approved by the Institutional Review Board of Self Regional Healthcare IRC, Greenwood, SC, was obtained from all individuals involved in the study. Both affected and unaffected individuals underwent full ophthalmic and clinical examination. Peripheral blood samples were collected from which DNA was extracted for subsequent molecular genetic analysis.
Individuals were genotyped using fluorescently labeled microsatellite markers at known cataract loci. Alleles were assigned after the analysis of the PCR products on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Warrington, UK). Two point LOD scores were calculated using the FASTLINK package . The pedigree was constructed using Cyrillic version 2.1.3 software (FamilyGenetix Ltd., Oxford, UK).
The entire coding region of GJA3 was sequenced using the same pairs of overlapping primers as used by Jiang et al. . PCR products, amplified using ReddyMix PCR master mix (Abgene, Epsom, UK), were cycle sequenced with Big Dye Terminator Ready Reaction Mix (Applied Biosystems) and analyzed on an ABI PRISM 3100 DNA sequencer (Applied Biosystems).
The relevant part of the pedigree is shown in Figure 1. AD inheritance is supported by the presence of affected individuals in each of the five generations, equal numbers of affected males and females, and male-to-male transmission. Zonular pulverulent cataract (Figure 2) was fully penetrant and exhibited some variable expressivity. There were no other ocular or systemic abnormalities. Bilateral zonular pulverulent cataracts typically presented in the first few months of life and progressed to total opacity over time. Visual acuities of affected individuals at diagnosis ranged from 6/18 to counting fingers. In many cases, there was amblyopia. To date, almost all of the affected individuals have had cataract surgery.
Twenty-six members of the family, including 16 affected individuals and 10 unaffected individuals, were genotyped. The initial approach was to exclude all known cataract loci. Candidate loci implicated in ADCC (1p36, GJA8, CRYGC, CRYGD, BFSP2, and PITX3) were excluded by linkage analysis (LOD scores less than 1 at θ=0). A LOD score of 2.53 was obtained at θ=0 for D13S175, a marker genetically close to GJA3. The LOD score was not above 3 since this marker was relatively uninformative in the family. Haplotyping refined the region of possible linkage (Figure 1) as being between the tip of chromosome 13q above and the marker D13S1275 (the lower crossover) below. It was decided that the LOD score of 2.53 for marker D13S175 together with correlation between the family's phenotype and the phenotype typical of cataracts associated with GJA3 mutations warranted screening of this gene.
GJA3 (GenBank NM_021954) was sequenced in both affected and unaffected individuals. The variant 7G->T, causing a novel heterozygous missense mutation D3Y, was identified in all 16 affected individuals but in neither 10 unaffected individuals nor 230 control chromosomes from an ethnically mixed panel with a high proportion of Hispanic individuals. The aspartate residue is conserved across species represented in GenBank (Figure 3). Its substitution by a tyrosine residue is therefore consistent with a significant change in the protein.
The investigation of this large Hispanic cataract pedigree has revealed a novel mutation, D3Y, in GJA3. The D3Y mutation is likely to be causative since it segregates with affected status throughout the pedigree and is absent both in unaffected individuals within the pedigree and in unaffected, unrelated controls. The mutation changes a negatively-charged amino acid aspartate (D) to an uncharged amino acid tyrosine (Y).
Nine different cataract-causing mutations in GJA3 [23,26-30,32] and four different cataract-causing mutations in the connexin 50 gene (GJA8) have so far been reported [24,31,33,34] (Table 1). These are the only two connexin genes expressed in lens fibers. For both of them, there is a clear genotype-phenotype relationship. There is a strong association with pulverulent congenital cataracts, either predominantly in the nuclear or lamellar regions of the lens. The phenotype in the Hispanic pedigree resembles those previously reported.
GJA3 mutations have previously been reported in pedigrees of Caucasian, Chinese, and Indian ancestry. Here, a novel GJA3 mutation is reported in a family of Hispanic Central American origin. This increases the diversity of ethnic groups in which these mutations cause cataract, adding to the evidence that mutations in GJA3 are an important cause of cataract in widely different ethnic groups on a worldwide scale.
All connexins have four transmembrane domains and two extracellular loops with cytoplasmic NH2- and COOH-termini. The previously reported mutations associated with congenital cataracts are summarized in Table 1.
GJA3 encodes a 435 amino acid protein in humans and is predominantly expressed in lens fiber cells . The D3 residue of GJA3 is phylogenetically conserved from zebrafish to man (Figure 3), indicating that the aspartate is likely to be functionally important and that the mutation may therefore have a detrimental physiological effect. The 7G->T change results in an aspartate to tyrosine amino acid substitution within the NH2-terminal cytoplasmic tail. This is the first reported mutation within this region of GJA3 associated with congenital cataract. The mutation leads to replacement of a negatively charged amino acid with an uncharged amino acid at position 3 in the amino terminus. Substitutions in the amino acid residues of the NH2-terminus may interfere with the conformation and flexibility of the amino terminus and also with voltage gating [43,44]. Cx46 protein functions in gap junction communication between elongated fiber cells , which constitute the bulk of the lens mass and represent the target cells for cataract formation. Lens fiber cells are dependent on intercellular communication for their survival . Given that the mutation affects the NH2-terminal domain, it is likely that it affects intercellular communication through the gap junction channel by affecting voltage gating. Functional work is required to confirm that this is the mechanism by which the mutation affects the protein.
In summary, a novel mutation of the human GJA3 gene has been found to segregate with zonular pulverulent cataract. This expands the spectrum of GJA3 mutations causing AD pulverulent cataract both in terms of ethnicity and in terms of location of the mutation in the NH2-terminal region of the protein.
We would like to thank the family members for taking part in these studies. We thank Mr. Sailesh D. Menon, Dr. Patricia C. Holden and Dr. Ramon H. Alvarenga for their assistance. We gratefully acknowledge the assistance provided by The Franciscan Institute for Rehabilitation of the Non-Sighted (INFRACNOVI). This work was supported by The Wellcome Trust (project grant number 063969/Z/01) and by The Special Trustees of Moorfields Eye Hospital. The authors have no competing interests.
1. Life in the 21st century: a vision for all. Geneva: World Health Organization; 1998.
2. Gilbert CE, Canovas R, Hagan M, Rao S, Foster A. Causes of childhood blindness: results from west Africa, south India and Chile. Eye 1993; 7:184-8.
3. Rahi JS, Dezateux 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.
4. 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.
5. 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.
6. 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.
7. Bateman JB, Geyer DD, Flodman P, Johannes M, Sikela J, Walter N, Moreira AT, Clancy K, Spence MA. A new betaA1-crystallin splice junction mutation in autosomal dominant cataract. Invest Ophthalmol Vis Sci 2000; 41:3278-85.
8. 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.
9. 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.
10. Kannabiran C, Rogan PK, Olmos L, Basti S, Rao GN, Kaiser-Kupfer M, Hejtmancik JF. Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the betaA3/A1-crystallin gene. Mol Vis 1998; 4:21 <http://www.molvis.org/molvis/v4/a21/>.
11. 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.
12. 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.
13. 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.
14. 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.
15. Nandrot E, Slingsby C, Basak A, Cherif-Chefchaouni M, Benazzouz B, Hajaji Y, Boutayeb S, Gribouval O, Arbogast L, Berraho A, Abitbol M, Hilal L. Gamma-D crystallin gene (CRYGD) mutation causes autosomal dominant congenital cerulean cataracts. J Med Genet 2003; 40:262-7.
16. 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.
17. 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.
18. Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D, Fardeau M. A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet 1998; 20:92-5.
19. Berry V, Yang Z, Addison PK, Francis PJ, Ionides A, Karan G, Jiang L, Lin W, Hu J, Yang R, Moore A, Zhang K, Bhattacharya SS. Recurrent 17 bp duplication in PITX3 is primarily associated with posterior polar cataract (CPP4). J Med Genet 2004; 41:e109.
20. 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.
21. Jamieson RV, Munier F, Balmer A, Farrar N, Perveen R, Black GC. Pulverulent cataract with variably associated microcornea and iris coloboma in a MAF mutation family. Br J Ophthalmol 2003; 87:411-2.
22. 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.
23. Bennett TM, Mackay DS, Knopf HL, Shiels A. A novel missense mutation in the gene for gap-junction protein alpha3 (GJA3) associated with autosomal dominant "nuclear punctate" cataracts linked to chromosome 13q. Mol Vis 2004; 10:376-82 <http://www.molvis.org/molvis/v10/a47/>.
24. 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.
25. 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.
26. Burdon KP, Wirth MG, Mackey DA, Russell-Eggitt IM, Craig JE, Elder JE, Dickinson JL, Sale MM. A novel mutation in the Connexin 46 gene causes autosomal dominant congenital cataract with incomplete penetrance. J Med Genet 2004; 41:e106. Erratum in: J Med Genet 2005; 42:288.
27. Devi RR, Reena C, Vijayalakshmi P. Novel mutations in GJA3 associated with autosomal dominant congenital cataract in the Indian population. Mol Vis 2005; 11:846-52 <http://www.molvis.org/molvis/v11/a100/>.
28. Jiang H, Jin Y, Bu L, Zhang W, Liu J, Cui B, Kong X, Hu L. A novel mutation in GJA3 (connexin46) for autosomal dominant congenital nuclear pulverulent cataract. Mol Vis 2003; 9:579-83 <http://www.molvis.org/molvis/v9/a70/>.
29. Li Y, Wang J, Dong B, Man H. A novel connexin46 (GJA3) mutation in autosomal dominant congenital nuclear pulverulent cataract. Mol Vis 2004; 10:668-71 <http://www.molvis.org/molvis/v10/a80/>.
30. 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.
31. Polyakov AV, Shagina IA, Khlebnikova OV, Evgrafov OV. Mutation in the connexin 50 gene (GJA8) in a Russian family with zonular pulverulent cataract. Clin Genet 2001; 60:476-8.
32. 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.
33. 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.
34. Willoughby CE, Arab S, Gandhi R, Zeinali S, Arab S, Luk D, Billingsley G, Munier FL, Heon E. A novel GJA8 mutation in an Iranian family with progressive autosomal dominant congenital nuclear cataract. J Med Genet 2003; 40:e124.
35. Kumar NM, Gilula NB. The gap junction communication channel. Cell 1996; 84:381-8.
36. Gerido DA, White TW. Connexin disorders of the ear, skin, and lens. Biochim Biophys Acta 2004; 1662:159-70.
37. White TW. Unique and redundant connexin contributions to lens development. Science 2002; 295:319-20.
38. Paul DL, Ebihara L, Takemoto LJ, Swenson KI, Goodenough DA. Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol 1991; 115:1077-89.
39. White TW, Bruzzone R, Goodenough DA, Paul DL. Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol Biol Cell 1992; 3:711-20.
40. Goodenough DA. The crystalline lens. A system networked by gap junctional intercellular communication. Semin Cell Biol 1992; 3:49-58.
41. Ionides A, Francis P, Berry V, Mackay D, Bhattacharya S, Shiels A, Moore A. Clinical and genetic heterogeneity in autosomal dominant cataract. Br J Ophthalmol 1999; 83:802-8.
42. Cottingham RW Jr, Idury RM, Schaffer AA. Faster sequential genetic linkage computations. Am J Hum Genet 1993; 53:252-63.
43. Purnick PE, Benjamin DC, Verselis VK, Bargiello TA, Dowd TL. Structure of the amino terminus of a gap junction protein. Arch Biochem Biophys 2000; 381:181-90.
44. Rouan F, White TW, Brown N, Taylor AM, Lucke TW, Paul DL, Munro CS, Uitto J, Hodgins MB, Richard G. trans-dominant inhibition of connexin-43 by mutant connexin-26: implications for dominant connexin disorders affecting epidermal differentiation. J Cell Sci 2001; 114:2105-13.