Current Eye Research, Early Online, 1–7, 2014 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.997885
RESEARCH REPORT
Mutation Analysis in Chinese Families with Autosomal Dominant Hereditary Cataracts Zhenfei Yang1, Qian Li2,3, Xu Ma2,3,4 and Si Quan Zhu1
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1
Beijing Ophthalmology & Visual Sciences Key Lab, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China, 2Graduate School, Peking Union Medical College, Beijing, China, 3 National Research Institute for Family Planning, Beijing, China, and 4World Health Organization Collaborating Center for Research in Human Reproduction, Beijing, China
ABSTRACT Purpose: To identify the molecular basis and clinical phenotype in three Chinese families with hereditary cataracts. Methods: Detailed family history and clinical data were recorded. The phenotypes were documented using slitlamp photography. Candidate genes sequencing was performed to screen out the disease causing mutation. Bioinformatics analysis was performed to predict the function of mutant genes. Results: The phenotypes of the families were identified as nuclear cataract in Family 1, pulverulent cataract in Family 2, and nuclear cataract in Family 3. Direct sequencing revealed transversions of C4T at c.218 (p. S73F) in GJA8 in Family 1, A4C at c.125 (p. E42A) in GJA3 in Family 2, and C4T at c.268 (p. L90F) in GJA3 in Family 3. These mutations co-segregated with all affected individuals in the family and were not found in unaffected family members nor in the 100 unrelated controls. Bioinformatics analysis indicated that S73F in GJA8, E42A and L90F in GJA3 are highly conserved. S73F in GJA8, E42A and L90F in GJA3 could possibly be damaging predicted by PolyPhen-2, with score of 0.858, 1.000, 1.000, respectively. Conclusions: This study identified three mutations in three Chinese families with hereditary cataracts. Of the three mutations, two were novel (c.125 A4C in GJA3 and c.268 C4T in GJA3), one was previously reported (c.218 C4T in GJA8). Keywords: E42A, GJA8, GJA3, hereditary cataract, L90F, S73F
INTRODUCTION
to morphology, hereditary cataracts can be classified into several subtypes: sutural, pulverulent, whole lens, nuclear, lamellar, cortical, polar, cerulean, coralliform, and other minor subtypes.1,6 These subtypes of cataracts can result from mutations at different genetic loci and can have different inheritance patterns. Depending on the population studied, approximately half of the reported inherited cataract families have crystalin mutations,7 including crystalin alpha-A (CRYAA), crystalin alpha-B (CRYAB), crystalin beta A1 (CRYBA1/A3), crystalin beta B1 (CRYBB1), crystalin beta B2 (CRYBB2), crystalin gamma C (CRYGC), crystalin gamma D (CRYGD), crystalin gamma S
Hereditary cataract is the most common cause of treatable childhood blindness, which is characterized by opacification of all or part of the eye’s crystalin lens within the first year of life.1,2 The prevalence of hereditary cataracts is 1 to 6 per 10,000 live births.3 The cataract may be isolated, may be hereditary or secondary to an intra-uterine event. Approximately one-quarter to one-third of hereditary cataracts is inherited and has been reported with all three types of Mendelian inheritance, including autosomal dominant, autosomal recessive, and X-linked.4,5 According
Received 7 August 2014; revised 19 November 2014; accepted 7 December 2014; published online 30 December 2014 Correspondence: Professor Si Quan Zhu, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, 1 Dong Jiao Min Xiang, Beijing 100730, China. Tel: +8610-58269605. Fax: +8610-85110023. E-mail: [email protected]
1
2
Z. Yang et al.
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(CRYGS). About one quarter have mutations in gap junctional proteins,7 including gap junction protein, alpha 3 (GJA3), and gap junction protein, alpha 8 (GJA8), with the remainder divided among the genes for heat shock transcription factor-4 (HSF4), aquaporin-0 (MIP), and beaded filament structural protein-2 (BFSP2).2,8–10 In the present study, we sought to identify the genetic defect in three autosomal dominant hereditary cataract Han Chinese pedigrees. Functional candidate approach was used to test the known cataract-causing genes in Chinese family. We identified three transversions of the three mutations, two were novel (c.125 A4C in GJA3 and c.268 C4T in GJA3), one was previously- reported (c.218 C4T in GJA8).11
METHODS Clinical Examination and Isolation of Genomic DNA Three Chinese pedigrees with autosomal dominant hereditary cataract were collected from Beijing Tongren Hospital. We also recruited 100 unrelated subjects without eye diseases except mild myopia from the Ophthalmology Clinic of Beijing Tongren Hospital as normal controls in 2011.12 The ethics committee of Capital Medical University approved the research. All participants from the family gave their informed consent. The study protocol followed the principles of the Declaration of Helsinki. Histories of cataract extraction or ophthalmologic examination were used to determine those whose status was considered to be affected. All participating members underwent ophthalmic examination, including visual acuity, slit-lamp examination, intra-ocular pressure measurement, ultrasonography, and fundus examination of the dilated pupil. The phenotypes were documented by slit-lamp photography, and 5 ml of venous blood was collected in BD Vacutainers (BD, San Jose, CA) containing EDTA from participating family members and controls. Genomic DNA was extracted by QIAamp DNA Blood Mini Kits (Qiagen Science, Germantown, MD).
Mutation Detection All coding exons and intron–exon junction of the candidate genes known to be associated with hereditary cataract including CRYAA (GenBank NM_000394), CRYAB (GenBank NM_001885), CRYBA1 (GenBank NM_005208), CRYBB1 (GenBank NM_001887), CRYBB2 (GenBank NM_000496), CRYGC (GenBank NM_020989), CRYGD (GenBank NM_006891), CRYGS (GenBankNM_017541), GJA3 (GenBank NM_021954), GJA8 (GenBank NM_005267), MIP (GenBank
NM_012064.3), HSF4 (GenBank NM_001040667.2), and BFSP2 (GenBank NM_003571). Each exon and intron–exon junction of the genes were amplified by polymerase chain reaction (PCR) using previously published primer sequences.10 Each reaction mix (25 ml) contained 20 ng of genomic DNA, 1 PCR buffer, 1.5 mM MgCl2, 0.2 mMdNTPs, 0.5 mM each of forward and reverse primers and 2.5U of Taq DNA polymerase (TianGen, Beijing, China). A PCR program was performed for DNA amplifying: 95 C for 5 min; followed by 35 cycles at 95 C for 30 s, 57 C–63 C for 30 s (annealing temperature depending on different primer); 72 C for 30 s; and a final extension at 72 C for 10 min. The PCR products were sequenced using ABI 3730 Automated Sequencer (PE Biosystems, Foster City, CA). The sequencing results were analyzed using Chromas 2.33 (http://www.technelysium.com.au/ chromas.html) and compared to the reference sequence in the NCBI (http://www.ncbi.nlm.nih.gov/) database.
Bioinformatics Analysis The amino acid sequences of GJA8 and GJA3 from several different species were obtained from NCBI GenBank and conservation analysis was performed by CLC Main Workbench Software (Aarhus, Denmark). The function impact of the mutation was predicted by PolyPhen-2 (http:// genetics.bwh.harvard.edu/pph2/).
RESULTS AND DISCUSSION Clinical Evaluation Family 1 Seven family members (I:2,II:3,II:5,II:6,III:2,III:3,III:4) of a three-generation Chinese family with a history of cataracts participated in the study, including four affected and three unaffected individuals (Figure 1A). All patients in this family had bilateral cataracts. The proband, who was a 14-year-old girl, experienced vision decrease at 2 years old and had been diagnosed with bilateral cataracts at age 3. Slit-lamp examination revealed dense nuclear cataract in the center (Figure 1B). The girl did not have nystagmus, her best corrected visual acuity (no mydriasis) was 0.2/0.3. Family 2 Nine family members (I:1,I:2,II:1,II:3,II:5,II:6,III:2, III:3,III:4) of a three-generation Chinese family with a history of cataracts participated in the study, including three affected and six unaffected individuals (Figure 2A). Most patients experienced decreased visual acuity at 5 years old, and then Current Eye Research
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Mutation Analysis in Hereditary Cataracts
FIGURE 1 The results of Family 1. (A) Pedigree of Family 1 with autosomal dominant cataract. The arrow indicates the proband. The family members diagnosed with hereditary cataract by slit-lamp are marked with black. Individuals marked with a star indicate family members participated in this study. (B) The photograph of the proband. Slit-lamp biomicroscopy photographs showed dense nuclear cataract of proband in the left eye. (C) The mutation C4T at c.218 (p. S73F) in GJA8 was identified in all the affected participants, but was not found in unaffected family members nor in the 100 unrelated control subjects. (D) A multiple-sequence alignment of the amino acid sequence in GJA8 from different species. The alignment data indicate that the Ser at the 73th amino acid position is highly conserved among many species (indicated by an arrow).
their visual acuity decreased gradually until surgery was required. The proband, who was a 5-year-old girl experienced vision decrease and had been diagnosed with bilateral cataracts. Slit-lamp !
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examination revealed pulverulent nuclear cataract (Figure 2B). The girl did not have nystagmus, her best corrected visual acuity was 0.3/0.3 (no mydriasis).
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FIGURE 2 The results of Family 2. (A) Pedigree of Family 2 with autosomal dominant cataract. The arrow indicates the proband. The family members diagnosed with hereditary cataract by slit-lamp are marked with black. Individuals marked with a star indicate family members participated in this study. (B) The photograph of the proband. Diffuse slit-lamp biomicroscopy photographs showed nuclear pulverulent cataract of proband in the right eye. (C) The mutation A4C at c.125 (p. E42A) in GJA3 was identified in all the affected participants, but was not found in unaffected family members nor in the 100 unrelated control subjects. (D) A multiplesequence alignment of the amino acid sequence in GJA3 from different species. The alignment data indicate that the Glu at the 42nd amino acid position is highly conserved among many species (indicated by an arrow).
Family 3 Seven family members (I:1,I:2,II:1,II:3,II:5,III:1,III:2) of a three-generation Chinese family with a history of cataracts participated in the study, including five affected and two unaffected individuals (Figure 3A). All patients in this family had bilateral
cataracts. The proband, who was a 38-year-old man, did not have nystagmus and experienced vision decrease at 6 years old. Slit-lamp examination revealed nuclear cataract with lamellar opacity (Figure 3B). His best corrected visual acuity was 0.3/0.4 (no mydriasis). Current Eye Research
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Mutation Analysis in Hereditary Cataracts
FIGURE 3 The results of Family 3. (A) Pedigree of Family 3 with autosomal dominant cataract. The arrow indicates the proband. The family members diagnosed with hereditary cataract by slit-lamp are marked with black. Individuals marked with a star indicate family members participated in this study. (B) The photograph of the proband. Diffuse slit-lamp biomicroscopy photographs showed nuclear cataract with lamellar opacity of proband in the right eye. (C) The mutation C4T at c.268 (p. L90F) in GJA3 was identified in all the affected participants, but was not found in unaffected family members nor in the 100 unrelated control subjects. (D) A multiplesequence alignment of the amino acid sequence in GJA3 from different species. The alignment data indicate that the Leu at the 90th amino acid position is highly conserved among many species (indicated by an arrow).
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FIGURE 4 Schematic diagrams of GJA8 and GJA3 reported mutations. (A) Schematic diagram of GJA8 protein containing all reported human mutations associated with cataracts. The identified mutation (p. S73F) in this study is marked with rectangle. (B) Schematic diagram of GJA3 protein containing all reported human mutations associated with cataracts. The identified two mutations (p. E42A and p.L90F) in this study are marked with rectangle.
Mutation Analysis Seven family members (I:2,II:3,II:5,II:6,III:2,III:3,III:4) in Family 1, nine family members (I:1,I:2,II:1, II:3,II:5,II:6,III:2,III:3,III:4) in Family 2, seven family members (I:1,I:2,II:1,II:3,II:5,III:1,III:2) in Family 3 participated in the gene sequencing study. Through direct gene sequencing of the coding regions of the candidate genes, we identified transversions of C4T at c.218 (p. S73F) in GJA8 in Family 1 (Figure 1C), A4C at c.125 (p. E42A) in GJA3 in Family 2 (Figure 2C), and C4T at c.268 (p. L90F) in GJA3 in Family 3 (Figure 3C). However, we did not find these mutations in any unaffected family members or in the 100 unrelated controls. We found no further gene mutations in individuals from the studied family, except for a few non-pathogenic single nucleotide polymorphisms.
Bioinformatics Analysis CLC Main Workbench Software revealed that the regions with p.S73F in GJA8 (Figure 1D), p.E42A (Figure 2D) and p.L90F (Figure 3D) in GJA3 are highly conserved among many species. In addition, the structure and function impact of the three mutations were predicted by PolyPhen-2 (http:// genetics.bwh.harvard.edu/pph2/), and the result indicated that p.S73F in GJA8, p.E42A and p.L90F in GJA3 could possibly be damaging, with score of 0.858, 1.000, 1.000, respectively. Hereditary cataracts are genetically heterogeneous. It is known that different mutations in the same gene
can cause similar cataract patterns, while the highly variable morphologies of cataracts within some families suggest that the same mutation in a single gene can lead to different phenotypes.8 The genotype– phenotype correlation of this previously reported mutation provides evidence that other factors, genetic and/or environmental, may influence the development of cataract.11,13 GJA3 and GJA8 function as gap junction that provide pathways for metabolites, ions and signaling molecules, and other molecules smaller than 1 kDa.14 Like other connexins, GJA3 and GJA8 contain four transmembrane domains, referred to as M1, M2, M3, and M4, two extra-cellular loops (E1 and E2), an intracellular loop (CL), and an intra-cellular amino and carboxyl terminus.15 GJA8 is expressed in lens epithelial and fiber cells, and it is essential for lens growth and transparency.7 To date, more than 33 mutations in human GJA8 gene have been reported to induce genetic cataracts (Figure 4A).16 S73F is located in E1 in GJA8. Extra-cellular loops (E1 and E2) play a key role in both mediating hemichannel docking and regulating voltage gating of the channel. Moreover, S73F is highly conserved among many species, so S73F is possibly be damaging. GJA3 is primarily expressed in lens fiber cells, and it is important for lens transparency.17,18 So far, more than 25 different mutations in GJA3 have been reported (Figure 4B).19 E42A is located in M1 in GJA3, and L90F is located in M2 in GJA3. M1 and M2 are conserved domains among connexins. E42A and L90F are highly conserved among many species, PolyPhen-2 indicated that E42A and L90F in GJA3 are possibly be damaging. Current Eye Research
Mutation Analysis in Hereditary Cataracts In conclusion, we identified two novel mutations (c.125 A4C in GJA3 and c.268 C4T in GJA3), one previously reported mutation (c.218 C4T in GJA8) in three Chinese families with hereditary cataracts. These mutations support the role of GJA8 and GJA3 genes in human cataract formation and provide more evidence of genetic heterogeneity of hereditary cataracts.
ACKNOWLEDGEMENTS
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We thank the family members for participation in the project.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
REFERENCES 1. Reddy MA, Francis PJ, Berry V, Bhattacharya SS, Moore AT. Molecular genetic basis of inherited cataract and associated phenotypes. Surv Ophthalmol 2004;49:300–315. 2. Deng H, Yuan L. Molecular genetics of congenital nuclear cataract. Eur J Med Genet 2014;57:113–122. 3. Holmes JM, Leske DA, Burke JP, Hodge DO. Birth prevalence of visually significant infantile cataract in a defined U.S. population. Ophthalmic Epidemiol 2003;10: 67–74. 4. Ionides A, Francis P, Berry V, Mackay D, Bhattacharya S, Shiels A, et al. Clinical and genetic heterogeneity in autosomal dominant cataract. Br J Ophthalmol 1999;83: 802–808. 5. Vanita, Singh JR, Singh D. Genetic and segregation analysis of congenital cataract in the Indian population. Clin Genet 1999;56:389–393. 6. Francis PJ, Moore AT. Genetics of childhood cataract. Curr Opin Ophthalmol 2004;15:10–15.
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7. Li J, Wang Q, Fu Q, Zhu Y, Zhai Y, Yu Y, et al. A novel connexin 50 gene (gap junction protein, alpha 8) mutation associated with congenital nuclear and zonular pulverulent cataract. Mol Vis 2013;19:767–774. 8. Santana A, Waiswo M. The genetic and molecular basis of congenital cataract. Arq Bras Oftalmol 2011;74: 136–142. 9. Yang Z, Su D, Li Q, Ma Z, Yang F, Zhu S, et al. A R54L mutation of CRYAA associated with autosomal dominant nuclear cataracts in a Chinese family. Curr Eye Res 2013;38: 1221–1228. 10. Yang Z, Su D, Li Q, Yang F, Ma Z, Zhu S, et al. A novel T– 4G splice site mutation of CRYBA1/A3 associated with autosomal dominant nuclear cataracts in a Chinese family. Mol Vis 2012;18:1283–1288. 11. Hansen L, Mikkelsen A, Nurnberg P, Nurnberg G, Anjum I, Eiberg H, et al. Comprehensive mutational screening in a cohort of Danish families with hereditary congenital cataract. Invest Ophthalmol Vis Sci 2009;50:3291–3303. 12. Yang Z, Li Q, Ma Z, Guo Y, Zhu S, Ma X. A G–4T splice site mutation of CRYBA1/A3 associated with autosomal dominant suture cataracts in a Chinese family. Mol Vis 2011;17: 2065–2071. 13. Richter L, Flodman P, Barria von-Bischhoffshausen F, Burch D, Brown S, Nguyen L, et al. Clinical variability of autosomal dominant cataract, microcornea and corneal opacity and novel mutation in the alpha A crystallin gene (CRYAA). Am J Med Genet A 2008;146A:833–842. 14. Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev 2003;83: 1359–1400. 15. Yeager M, Harris AL. Gap junction channel structure in the early 21st century: facts and fantasies. Curr Opin Cell Biol 2007;19:521–528. 16. Ge XL, Zhang Y, Wu Y, Lv J, Zhang W, Jin ZB, et al. Identification of a novel GJA8 (Cx50) point mutation causes human dominant congenital cataracts. Sci Rep 2014;4:4121. 17. Jiang JX. Gap junctions or hemichannel-dependent and independent roles of connexins in cataractogenesis and lens development. Curr Mol Med 2010;10:851–863. 18. Gong X, Cheng C, Xia CH. Connexins in lens development and cataractogenesis. J Membr Biol 2007;218:9–12. 19. Beyer EC, Ebihara L, Berthoud VM. Connexin mutants and cataracts. Front Pharmacol 2013;4:43.
RESEARCH REPORT
Mutation Analysis in Chinese Families with Autosomal Dominant Hereditary Cataracts Zhenfei Yang1, Qian Li2,3, Xu Ma2,3,4 and Si Quan Zhu1
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1
Beijing Ophthalmology & Visual Sciences Key Lab, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China, 2Graduate School, Peking Union Medical College, Beijing, China, 3 National Research Institute for Family Planning, Beijing, China, and 4World Health Organization Collaborating Center for Research in Human Reproduction, Beijing, China
ABSTRACT Purpose: To identify the molecular basis and clinical phenotype in three Chinese families with hereditary cataracts. Methods: Detailed family history and clinical data were recorded. The phenotypes were documented using slitlamp photography. Candidate genes sequencing was performed to screen out the disease causing mutation. Bioinformatics analysis was performed to predict the function of mutant genes. Results: The phenotypes of the families were identified as nuclear cataract in Family 1, pulverulent cataract in Family 2, and nuclear cataract in Family 3. Direct sequencing revealed transversions of C4T at c.218 (p. S73F) in GJA8 in Family 1, A4C at c.125 (p. E42A) in GJA3 in Family 2, and C4T at c.268 (p. L90F) in GJA3 in Family 3. These mutations co-segregated with all affected individuals in the family and were not found in unaffected family members nor in the 100 unrelated controls. Bioinformatics analysis indicated that S73F in GJA8, E42A and L90F in GJA3 are highly conserved. S73F in GJA8, E42A and L90F in GJA3 could possibly be damaging predicted by PolyPhen-2, with score of 0.858, 1.000, 1.000, respectively. Conclusions: This study identified three mutations in three Chinese families with hereditary cataracts. Of the three mutations, two were novel (c.125 A4C in GJA3 and c.268 C4T in GJA3), one was previously reported (c.218 C4T in GJA8). Keywords: E42A, GJA8, GJA3, hereditary cataract, L90F, S73F
INTRODUCTION
to morphology, hereditary cataracts can be classified into several subtypes: sutural, pulverulent, whole lens, nuclear, lamellar, cortical, polar, cerulean, coralliform, and other minor subtypes.1,6 These subtypes of cataracts can result from mutations at different genetic loci and can have different inheritance patterns. Depending on the population studied, approximately half of the reported inherited cataract families have crystalin mutations,7 including crystalin alpha-A (CRYAA), crystalin alpha-B (CRYAB), crystalin beta A1 (CRYBA1/A3), crystalin beta B1 (CRYBB1), crystalin beta B2 (CRYBB2), crystalin gamma C (CRYGC), crystalin gamma D (CRYGD), crystalin gamma S
Hereditary cataract is the most common cause of treatable childhood blindness, which is characterized by opacification of all or part of the eye’s crystalin lens within the first year of life.1,2 The prevalence of hereditary cataracts is 1 to 6 per 10,000 live births.3 The cataract may be isolated, may be hereditary or secondary to an intra-uterine event. Approximately one-quarter to one-third of hereditary cataracts is inherited and has been reported with all three types of Mendelian inheritance, including autosomal dominant, autosomal recessive, and X-linked.4,5 According
Received 7 August 2014; revised 19 November 2014; accepted 7 December 2014; published online 30 December 2014 Correspondence: Professor Si Quan Zhu, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, 1 Dong Jiao Min Xiang, Beijing 100730, China. Tel: +8610-58269605. Fax: +8610-85110023. E-mail: [email protected]
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(CRYGS). About one quarter have mutations in gap junctional proteins,7 including gap junction protein, alpha 3 (GJA3), and gap junction protein, alpha 8 (GJA8), with the remainder divided among the genes for heat shock transcription factor-4 (HSF4), aquaporin-0 (MIP), and beaded filament structural protein-2 (BFSP2).2,8–10 In the present study, we sought to identify the genetic defect in three autosomal dominant hereditary cataract Han Chinese pedigrees. Functional candidate approach was used to test the known cataract-causing genes in Chinese family. We identified three transversions of the three mutations, two were novel (c.125 A4C in GJA3 and c.268 C4T in GJA3), one was previously- reported (c.218 C4T in GJA8).11
METHODS Clinical Examination and Isolation of Genomic DNA Three Chinese pedigrees with autosomal dominant hereditary cataract were collected from Beijing Tongren Hospital. We also recruited 100 unrelated subjects without eye diseases except mild myopia from the Ophthalmology Clinic of Beijing Tongren Hospital as normal controls in 2011.12 The ethics committee of Capital Medical University approved the research. All participants from the family gave their informed consent. The study protocol followed the principles of the Declaration of Helsinki. Histories of cataract extraction or ophthalmologic examination were used to determine those whose status was considered to be affected. All participating members underwent ophthalmic examination, including visual acuity, slit-lamp examination, intra-ocular pressure measurement, ultrasonography, and fundus examination of the dilated pupil. The phenotypes were documented by slit-lamp photography, and 5 ml of venous blood was collected in BD Vacutainers (BD, San Jose, CA) containing EDTA from participating family members and controls. Genomic DNA was extracted by QIAamp DNA Blood Mini Kits (Qiagen Science, Germantown, MD).
Mutation Detection All coding exons and intron–exon junction of the candidate genes known to be associated with hereditary cataract including CRYAA (GenBank NM_000394), CRYAB (GenBank NM_001885), CRYBA1 (GenBank NM_005208), CRYBB1 (GenBank NM_001887), CRYBB2 (GenBank NM_000496), CRYGC (GenBank NM_020989), CRYGD (GenBank NM_006891), CRYGS (GenBankNM_017541), GJA3 (GenBank NM_021954), GJA8 (GenBank NM_005267), MIP (GenBank
NM_012064.3), HSF4 (GenBank NM_001040667.2), and BFSP2 (GenBank NM_003571). Each exon and intron–exon junction of the genes were amplified by polymerase chain reaction (PCR) using previously published primer sequences.10 Each reaction mix (25 ml) contained 20 ng of genomic DNA, 1 PCR buffer, 1.5 mM MgCl2, 0.2 mMdNTPs, 0.5 mM each of forward and reverse primers and 2.5U of Taq DNA polymerase (TianGen, Beijing, China). A PCR program was performed for DNA amplifying: 95 C for 5 min; followed by 35 cycles at 95 C for 30 s, 57 C–63 C for 30 s (annealing temperature depending on different primer); 72 C for 30 s; and a final extension at 72 C for 10 min. The PCR products were sequenced using ABI 3730 Automated Sequencer (PE Biosystems, Foster City, CA). The sequencing results were analyzed using Chromas 2.33 (http://www.technelysium.com.au/ chromas.html) and compared to the reference sequence in the NCBI (http://www.ncbi.nlm.nih.gov/) database.
Bioinformatics Analysis The amino acid sequences of GJA8 and GJA3 from several different species were obtained from NCBI GenBank and conservation analysis was performed by CLC Main Workbench Software (Aarhus, Denmark). The function impact of the mutation was predicted by PolyPhen-2 (http:// genetics.bwh.harvard.edu/pph2/).
RESULTS AND DISCUSSION Clinical Evaluation Family 1 Seven family members (I:2,II:3,II:5,II:6,III:2,III:3,III:4) of a three-generation Chinese family with a history of cataracts participated in the study, including four affected and three unaffected individuals (Figure 1A). All patients in this family had bilateral cataracts. The proband, who was a 14-year-old girl, experienced vision decrease at 2 years old and had been diagnosed with bilateral cataracts at age 3. Slit-lamp examination revealed dense nuclear cataract in the center (Figure 1B). The girl did not have nystagmus, her best corrected visual acuity (no mydriasis) was 0.2/0.3. Family 2 Nine family members (I:1,I:2,II:1,II:3,II:5,II:6,III:2, III:3,III:4) of a three-generation Chinese family with a history of cataracts participated in the study, including three affected and six unaffected individuals (Figure 2A). Most patients experienced decreased visual acuity at 5 years old, and then Current Eye Research
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Mutation Analysis in Hereditary Cataracts
FIGURE 1 The results of Family 1. (A) Pedigree of Family 1 with autosomal dominant cataract. The arrow indicates the proband. The family members diagnosed with hereditary cataract by slit-lamp are marked with black. Individuals marked with a star indicate family members participated in this study. (B) The photograph of the proband. Slit-lamp biomicroscopy photographs showed dense nuclear cataract of proband in the left eye. (C) The mutation C4T at c.218 (p. S73F) in GJA8 was identified in all the affected participants, but was not found in unaffected family members nor in the 100 unrelated control subjects. (D) A multiple-sequence alignment of the amino acid sequence in GJA8 from different species. The alignment data indicate that the Ser at the 73th amino acid position is highly conserved among many species (indicated by an arrow).
their visual acuity decreased gradually until surgery was required. The proband, who was a 5-year-old girl experienced vision decrease and had been diagnosed with bilateral cataracts. Slit-lamp !
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examination revealed pulverulent nuclear cataract (Figure 2B). The girl did not have nystagmus, her best corrected visual acuity was 0.3/0.3 (no mydriasis).
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FIGURE 2 The results of Family 2. (A) Pedigree of Family 2 with autosomal dominant cataract. The arrow indicates the proband. The family members diagnosed with hereditary cataract by slit-lamp are marked with black. Individuals marked with a star indicate family members participated in this study. (B) The photograph of the proband. Diffuse slit-lamp biomicroscopy photographs showed nuclear pulverulent cataract of proband in the right eye. (C) The mutation A4C at c.125 (p. E42A) in GJA3 was identified in all the affected participants, but was not found in unaffected family members nor in the 100 unrelated control subjects. (D) A multiplesequence alignment of the amino acid sequence in GJA3 from different species. The alignment data indicate that the Glu at the 42nd amino acid position is highly conserved among many species (indicated by an arrow).
Family 3 Seven family members (I:1,I:2,II:1,II:3,II:5,III:1,III:2) of a three-generation Chinese family with a history of cataracts participated in the study, including five affected and two unaffected individuals (Figure 3A). All patients in this family had bilateral
cataracts. The proband, who was a 38-year-old man, did not have nystagmus and experienced vision decrease at 6 years old. Slit-lamp examination revealed nuclear cataract with lamellar opacity (Figure 3B). His best corrected visual acuity was 0.3/0.4 (no mydriasis). Current Eye Research
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Mutation Analysis in Hereditary Cataracts
FIGURE 3 The results of Family 3. (A) Pedigree of Family 3 with autosomal dominant cataract. The arrow indicates the proband. The family members diagnosed with hereditary cataract by slit-lamp are marked with black. Individuals marked with a star indicate family members participated in this study. (B) The photograph of the proband. Diffuse slit-lamp biomicroscopy photographs showed nuclear cataract with lamellar opacity of proband in the right eye. (C) The mutation C4T at c.268 (p. L90F) in GJA3 was identified in all the affected participants, but was not found in unaffected family members nor in the 100 unrelated control subjects. (D) A multiplesequence alignment of the amino acid sequence in GJA3 from different species. The alignment data indicate that the Leu at the 90th amino acid position is highly conserved among many species (indicated by an arrow).
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Z. Yang et al.
FIGURE 4 Schematic diagrams of GJA8 and GJA3 reported mutations. (A) Schematic diagram of GJA8 protein containing all reported human mutations associated with cataracts. The identified mutation (p. S73F) in this study is marked with rectangle. (B) Schematic diagram of GJA3 protein containing all reported human mutations associated with cataracts. The identified two mutations (p. E42A and p.L90F) in this study are marked with rectangle.
Mutation Analysis Seven family members (I:2,II:3,II:5,II:6,III:2,III:3,III:4) in Family 1, nine family members (I:1,I:2,II:1, II:3,II:5,II:6,III:2,III:3,III:4) in Family 2, seven family members (I:1,I:2,II:1,II:3,II:5,III:1,III:2) in Family 3 participated in the gene sequencing study. Through direct gene sequencing of the coding regions of the candidate genes, we identified transversions of C4T at c.218 (p. S73F) in GJA8 in Family 1 (Figure 1C), A4C at c.125 (p. E42A) in GJA3 in Family 2 (Figure 2C), and C4T at c.268 (p. L90F) in GJA3 in Family 3 (Figure 3C). However, we did not find these mutations in any unaffected family members or in the 100 unrelated controls. We found no further gene mutations in individuals from the studied family, except for a few non-pathogenic single nucleotide polymorphisms.
Bioinformatics Analysis CLC Main Workbench Software revealed that the regions with p.S73F in GJA8 (Figure 1D), p.E42A (Figure 2D) and p.L90F (Figure 3D) in GJA3 are highly conserved among many species. In addition, the structure and function impact of the three mutations were predicted by PolyPhen-2 (http:// genetics.bwh.harvard.edu/pph2/), and the result indicated that p.S73F in GJA8, p.E42A and p.L90F in GJA3 could possibly be damaging, with score of 0.858, 1.000, 1.000, respectively. Hereditary cataracts are genetically heterogeneous. It is known that different mutations in the same gene
can cause similar cataract patterns, while the highly variable morphologies of cataracts within some families suggest that the same mutation in a single gene can lead to different phenotypes.8 The genotype– phenotype correlation of this previously reported mutation provides evidence that other factors, genetic and/or environmental, may influence the development of cataract.11,13 GJA3 and GJA8 function as gap junction that provide pathways for metabolites, ions and signaling molecules, and other molecules smaller than 1 kDa.14 Like other connexins, GJA3 and GJA8 contain four transmembrane domains, referred to as M1, M2, M3, and M4, two extra-cellular loops (E1 and E2), an intracellular loop (CL), and an intra-cellular amino and carboxyl terminus.15 GJA8 is expressed in lens epithelial and fiber cells, and it is essential for lens growth and transparency.7 To date, more than 33 mutations in human GJA8 gene have been reported to induce genetic cataracts (Figure 4A).16 S73F is located in E1 in GJA8. Extra-cellular loops (E1 and E2) play a key role in both mediating hemichannel docking and regulating voltage gating of the channel. Moreover, S73F is highly conserved among many species, so S73F is possibly be damaging. GJA3 is primarily expressed in lens fiber cells, and it is important for lens transparency.17,18 So far, more than 25 different mutations in GJA3 have been reported (Figure 4B).19 E42A is located in M1 in GJA3, and L90F is located in M2 in GJA3. M1 and M2 are conserved domains among connexins. E42A and L90F are highly conserved among many species, PolyPhen-2 indicated that E42A and L90F in GJA3 are possibly be damaging. Current Eye Research
Mutation Analysis in Hereditary Cataracts In conclusion, we identified two novel mutations (c.125 A4C in GJA3 and c.268 C4T in GJA3), one previously reported mutation (c.218 C4T in GJA8) in three Chinese families with hereditary cataracts. These mutations support the role of GJA8 and GJA3 genes in human cataract formation and provide more evidence of genetic heterogeneity of hereditary cataracts.
ACKNOWLEDGEMENTS
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We thank the family members for participation in the project.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
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