Vision 2008; 14:1171-1175
Received 27 March 2008 | Accepted 9 June 2008 | Published 17 June 2008
1Centre for Genetic Disorders, Guru Nanak Dev University, Amritsar, India; 2Dr. Daljit Singh Eye Hospital, Amritsar, India; 3Institute of Human Genetics, Charitè, University Medicine of Berlin, Berlin, Germany
Correspondence to: Dr. Vanita Vanita, Centre for Genetic Disorders, Guru Nanak Dev University, Amritsar-143005, Punjab, India; Phone: + 0183-2258802-09 Ext. 3277, 3279; FAX: + 0183-2258863, +0183-2258820; email: email@example.com
Purpose: To detect the underlying genetic defect in a family with three members in two generations affected with bilateral congenital cataract.
Methods: Detailed family history and clinical data were recorded. Mutation screening in the candidate genes, αA-crystallin (CRYAA), βA1-crystallin (CRYBA1), βB2-crystallin (CRYBB2), γA–γD-crystallins (CRYGA, CRYGB, CRYGC, and CRYGD), connexin-46 (GJA3), and connexin-50 (GJA8), was performed by bidirectional sequencing of the amplified products.
Results: Affected individuals had “balloon-like” cataract with prominent Y-sutural opacities. Sequencing of the candidate genes showed a heterozygous c.262C>A change in the gene for connexin 50 (GJA8), which is localized at 1q21, that resulted in the replacement of a highly conserved proline by glutamine (p.P88Q). This sequence change was not observed in 96 ethnically matched controls.
Conclusions: We report a p.P88Q mutation in GJA8 associated with Y-sutural cataract in a family of Indian origin. Mutations of the same codon have previously been described in British families with pulverulent cataract, suggesting that modifying factors may determine the type of cataract.
Congenital cataract is one of the most significant causes of visual impairment and childhood blindness worldwide. The prevalence of non-syndromic congenital cataract is estimated to be 1–6 cases per 10,000 live births [1,2]. It is a clinically and genetically heterogeneous ocular lens disorder. Interfamilial and intrafamilial phenotypic variation is quite significant in congenital cataract, and various types and sub-types have been reported [3-5]. Approximately one-third of the cases show a positive family history . All three Mendelian modes of inheritance exist for congenital cataract. However, an autosomal dominant mode of transmission is reported to be the most common . At least 27 loci have been mapped. The identified genes encode proteins such as crystallins, the major structural lens proteins (CRYAA, CRYAB, CRYBA1/A3, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, and CRYGS), gap junctional proteins, the connexins (GJA3 and GJA8), major intrinsic protein (MIP/MIP26), lens integral membrane protein 2 (LIM2), beaded filament proteins (BFSP1, BFSP2), and heat shock protein (HSF4) as reviewed by Reddy et al.  and Hejtmancik .
Pulverulent cataract was the first autosomal disease locus mapped. In 1963, Renwick and Lawler described linkage to the Duffy blood group locus , which was found to cosegregate with an uncoiler element of chromosome 1q by Donahue in 1968 . Thirty years later, Shiels analyzed the same cataract family and reported a missense mutation in codon 88 of the connexin 50 (GJA8) gene at 1q21 , leading to the substitution of proline by serine (p.P88S). Here, we report a cataract family of Indian origin with three affected members in two generations with a proline to glutamine mutation of the same codon (p.P88Q) but with balloon-like cataract with prominent Y-sutural opacities. Now, more than a dozen different mutations in GJA8 associated with different cataract phenotypes have been identified (Table 1).
The proband, a seven-year-old male child, was diagnosed with bilateral cataract. The family history revealed three affected members in two generations (Figure 1). The ophthalmologic examination, including slit lamp examination, was performed on a total of four members of this family; the father (Figure 1; II:3), who had a history of cataract extraction in childhood, and two of his bilaterally affected children (Figure 1; III:1 and III:2). The proband’s mother (Figure 1; II:4) was diagnosed as unaffected.
Informed consent was obtained for each individual studied. This study was approved by the ethics review board of the Guru Nanak Dev University (Amritsar, Punjab, India), consistent with the provisions of the Declaration of Helsinki. Blood was drawn and DNA isolated using method of Adeli and Ogbonna . Mutation screening was performed in the exonic regions of the following candidate genes: CRYAA (21q22.3; GenBank NM_000394), CRYBA1 (17q11-q12; GenBank NM_005208), CRYBB2 (22q11.2; GenBank NM_000496), CRYGA (2q33-q35; GenBank NM_014617), CRYGB (2q33-q35; GenBank NM_005210), CRYGC (2q33-q35; GenBank NM_020989), CRYGD (2q33-q35; GenBank NM_006891), GJA3 (13q11-q13; GenBank NM_021954), and GJA8 (1q21-q25; GenBank NM_005267). The coding regions and exon-intron boundaries of the candidate genes were amplified using previously published primer sequences [10,12-16]. Genomic DNA from all three affected and one unaffected individual was amplified. Purified polymerase chain reaction (PCR) products were sequenced bidirectionally with ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit version 3.1 (Applied Biosystems, Foster City, CA) for a 10 µl final volume containing 5.0 µl of purified PCR product, 4.0 µl of BigDye Terminator ready reaction mix, and 3.2 pmol of primer. Cycling conditions were: 95 °C for 2 min, 25 cycles at 95 °C for 30 s, 52 °C for 15 s, and 60 °C for 4 min. The sequencing reaction products were purified by the isopropanol precipitation method (ABI protocol; Applied Biosystems), resuspended in 10 µl of loading buffer (5:1 ratio of deionized formamide and 25 mM EDTA with blue dextran [50 mg/ml]), denatured at 95 °C for 5 min, and electrophoresed on 4% denaturing polyacrylamide gels on the DNA sequencer (ABI-Prism 377; Applied Biosystems). Sequencing results were assembled and analyzed using the SeqMan II program of the Lasergene package (DNA STAR Inc., Madison, WI).
Slit lamp examination of the lenses in affected individuals (III:1, III:2) showed that the cataract affected the fetal nucleus with additional changes on its surface. The fetal nucleus appeared semi-opaque. The Y-sutures on the anterior surface seem sharp and narrow (Figure 2A). In between the Y-sutures, there appeared feathery opacities extending up to three-fourths of the length of the Y-sutures. Along the perimeter of the fetal nucleus, three prominent riders were present. The posterior Y-sutures were not clearly seen through the anterior central feathery opacification, but its presence is in no doubt. Inside the semi-opaque fetal nucleus, there appeared no opacities.
Bidirectional sequencing of the coding regions of the candidate genes showed a heterozygous change, C>A (Figure 3), at position 262 (c.262 C>A) in GJA8 in all three affected individuals. This substitution was not seen in the unaffected mother or in 96 unrelated control subjects (192 chromosomes) from the same North Indian population as tested by bidirectional sequence analysis (data not shown). The substitution replaces an evolutionarily highly conserved proline by glutamine at amino acid position 88 (p.P88Q) in the second α-helical transmembrane domain 2 (M2) of connexin 50.
Connexins are integral membrane proteins with four transmembrane domains, two extracellular loops, and an intracellular loop with both NH2- and COOH-termini localized in the cytoplasm. In humans, at least 20 connexins classified into three families are known [17,18]. Connexin 46 and connexin 50 are responsible for joining the lens cells into a functional syncytium. In addition, connexin 50 is also important for lens growth . The observed p.P88Q substitution is centrally located within the second α-helical transmembrane domain (M2) of connexin 50 and replaces the highly conserved hydrophobic proline by the non-polar glutamine at position 88, in association with the congenital cataract in the present family.
The same mutation was seen in a British family. However, this mutation was associated with lamellar pulverulent cataract . In another family of English descent, a mutation of the same codon lead to the substitution of proline by serine (p.P88S), which is associated with nuclear pulverulent cataract, characterized by innumerable powdery opacities located in the nuclear (central) and perinuclear (lamellar) zones of the lens [8,10]. Further, functional analyses revealed that mutants p.P88S and p.P88Q act as dominant negative inhibitors and significantly decreases the activity of co-expressed wild type connexin 50 [20,21].
Mutations in connexin 46 and connexin 50 have been reported to be linked with cataractogenesis in humans as well as in mice. In humans, over a dozen mutations in each connexin 46 and connexin 50 gene have so far been detected in association with congenital cataract [2,7] and significant interfamilial phenotypic variability. The phenotype in most cases with mutations in connexin 46 and connexin 50 has been described as zonular/nuclear pulverulent cataract [3,5,7]. The cataract phenotype in the present family differs from these as no “pulverized” dust-like opacities are seen in the lens (Table 1). It also differs from the British family with the identical mutation , which showed linear dense vertical opacities inside the fetal nucleus with the embryonic nucleus remaining clear and without sutural opacities (Figure 2B). In the present family, Y-sutural opacities are very prominent, comparable with the p.V79L mutation in the second transmembrane domain (M2) of connexin 50, which is linked with “full moon” like cataract with Y-sutural opacities (Table 1) in another Indian family having 15 affected members in three generations reported previously by us .
In summary, we describe a heterozygous p.P88Q mutation in connexin 50 that showed marked phenotypic differences to previously reported cases affecting the same codon. Thus, variants in other genes might act as modifiers of the cataract phenotype.
We wish to thank the patients and their relatives for their cooperation. We are grateful to Dr. P.N. Robinson for critically reading the manuscript. This work was in part supported by grant number DBT/BT/IC/71/89/Pt from the Department of Biotechnology, Ministry of Science and Technology, Government of India sanctioned to J.R.S. and INI 331 from Bundesministerium für Bildung und Forschung, Federal Ministry of Education and Research, Bonn, Germany to K.S.