- Email: [email protected]
Association between sensitized to food allergens and childhood allergic respiratory diseases in Taiwan
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Journal of the Formosan Medical Association xxx (xxxx) xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.jfma-online.com
Original Article
Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract Kai Jie Wang, Jin Da Wang, Dou Dou Chen, Ming Yang Wang, Bo Yun, Si Quan Zhu* Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology &Visual Sciences Key Lab, Beijing, 100730, China Received 15 March 2018; received in revised form 2 September 2018; accepted 25 February 2019
KEYWORDS Gap junction; Mutation; Nuclear cataract
Background/purpose: A three-generation Chinese family with autosomal dominant congenital nuclear cataract was recruited. This study aimed to identify the disease-causing gene for nuclear cataract with functional dissections of the identified mutant. Methods: Detailed clinical data and family history were recorded. Candidate gene sequencing was performed to identify the disease-causing mutation. Recombinant connexin50 (Cx50) wild type and mutant constructs were synthesized. Triton X-100 solubility and subcellular localization of the recombinant Cx50 proteins were analyzed in HeLa cells. Apoptosis was assayed as the percentage of fragmented nuclei in transfected cells. Results: All affected individuals in the family displayed clear phenotypes of dense nuclear cataracts. A c.227 G > A variation was found in the coding region of Cx50, which arginine residue at position 76 was substituted by histidine (p.R76H). This mutation was co-segregated with the disease in the family, and was not observed in 110 unrelated Chinese controls. No statistically significant differences were found in the Triton X-100 solubility and apoptosis rate between wild type and mutant Cx50 in HeLa cells. However, Cx50 mutant was unable to form gap junctional plaques between adjacent cells as the wild type proteins did. Conclusions: This study identified a novel cataract phenotype caused by the p.R76H mutation in Cx50, providing evidence of further phenotypic heterogeneity associated with this mutation. Functional analysis showed that the mutation affected the formation of gap junction channels and led to opacity in the lens. Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
* Corresponding author. Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, 1 Dong Jiao Min Xiang, Beijng, 100730, China. Fax: þ8610 85110023. E-mail address: [email protected] (S.Q. Zhu). https://doi.org/10.1016/j.jfma.2019.02.015 0929-6646/Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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Introduction Congenital cataract refers to any opacity of lens presented at birth or during early childhood, which is responsible for approximately 10% childhood blindness worldwide.1 Various etiological factors have been identified, including infection, metabolic disorders and gene defects. About one third of isolated congenital cataracts are genetically determined. Autosomal dominant congenital cataract (ADCC) is the most common mode of inheritance, although autosomal recessive and X-linked inheritance are also reported for congenital cataract.2 To date, at least 35 genes have been strongly associated with inherited cataract without other systemic anomalies.3 Of the disease-causing mutations reported, about half are located in crystallins, 15% in connexins, 10% in transcription factors, 5% each in intermediate filaments or aquaporin 0, and 10% in a variety of other genes.4 Therefore, the crystalline and connexin genes appear to be the most common genes associated with congenital cataract. It is appropriate to consider these genes as the top list of candidate genes for screening studies in congenital cataracts.5 Gap junctions are intercellular channels and important for lens homeostasis, function and transparency.6 Gap junction proteins oligomerize to form hexameric hemichannels (connexons) in the membranes. The hemichannels of two adjacent cells dock with each other to form a gap junction channel allowing for intercellular communications in the lens. The functions of gap junctions are not only important for the maintenance of lens transparency but also crucial to the lens development and differentiation.6 Three different gap junction proteins have been identified in the human lens, including connexin43 (Cx43), connexin46 (Cx46) and connexin50 (Cx50).7 Both Cx46 and Cx50 predominantly co-localize in lens fibers, and multiple mutations in these genes have been reported to be associated with congenital cataracts.8 In the present study, we screened the 8 crystalline and 2 connexin genes using the same strategy as described previously.9 A missense mutation in Cx50 was identified to be responsible for dense nuclear cataract, and its functional consequences were investigated to gain further insights into the pathogenesis of inherited cataracts.
Materials and methods Patients and DNA specimens This study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of our institution. A three-generation family from Shandong province with autosomal dominant nuclear cataract was identified after informed consents. All participants underwent complete ophthalmic examinations including visual acuity, slit lamp examination, fundus examination and ultrasonography. The phenotypes were documented by slit lamp photography. A total of 110 unrelated ethnically matched controls with no family history of congenital
K.J. Wang et al. cataracts were also recruited. Peripheral venous blood of all participants was collected for genomic DNA extraction using QIAamp DNA kit (Qiagen, Valencia, CA).
Mutation analysis Mutation screening was performed in 10 candidate genes: 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 (GenBank NM_017541), Cx46 (Genbank NM_021954) and Cx50 (Genbank NM_005267). All coding exons and splice sites of the candidate genes were amplified by polymerase chain reactions (PCR) using previously reported primer sequences.9 The PCR products obtained from the proband and one unaffected member were sequenced on an ABI3730 Automated Sequencer (PE Biosystems, Foster City, CA). Direct sequencing was also used to screen the mutation identified in Cx50 on the sample of all available family members and 110 controls to confirm the mutation.
Bioinformatics analysis The CLC Free Workbench 4.5.1 software (CLC bio, Aarhus, Denmark) was used to align the protein sequences of different species. The possible impact of an amino acid substitution on the structure and function of the protein was predicted by Polyphen-2 (http://genetics.bwh.harvard.edu/ pph2/) and Mutation Taster (http://mutationtaster.org/).
Expression constructs and mutagenesis Human lenses total cDNA was obtained using the standard methods as described previously.10 The coding sequence of human Cx50 isolated from the human lens cDNA library by PCR using the following primers: sense 50 -GAACATCTTGGAGGAGGTGAATG-30 , antisense 50 -TCATCCCCCCACACGAACT-30 . After digestion with XhoI and EcoRI (NEB, Ipswich, MA, USA), the PCR product was cloned into the expression vector p3XFLAG-myc-CMV00-25 (Sigma, St Louis, MO). Correctness of construct sequence was confirmed by direct sequencing. The mutant Cx50 R76H plasmid was constructed by a site-directed mutagenesis kit (Stratagene, La Jolla, CA) using primers: sense 50 GCCTTTCCCATCTCCCACATTCACCTCTGGGTGCTGCAGATCATC-30 , antisense 50 -GATGATCTGCAGCACCCAGAGGTGAATGTGGGAGATGGGAAAGGC-30 . Wild type (WT) and mutant constructs were verified by direct sequencing.
Cell culture and transfection The HeLa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics in a 37 C incubator with 5% CO2. Cells were seeded with 5 105 cells in a 60 mm (diameter) culture dish for overnight before transfection. Expression construct containing WT or mutant Cx50 was mixed with FuGene HD
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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A p.R76H mutation in congenital nuclear cataract reagent (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Cells were collected for protein analysis 24 h after transfection.
Triton X-100 solubility analysis Cells were washed twice with ice-cold PBS and lyzed at 2.5 106 cells/ml lysis buffer containing 0.5% Triton X-100 (Tx) (Sigma). After centrifugation, the supernatant containing Tx-soluble protein was denatured in buffer with 2% Na dodecylsulfate (SDS) and 50 mM DL-dithiothreitol (DTT; Sigma). The pellet containing Tx-insoluble protein was washed twice with ice-cold PBS, sonicated and denatured in urea-SDS buffer. The samples were analyzed by western blot using antibodies against FLAG (recognizing Cx50), GAPDH or b-actin (Sigma). Each experiment was repeated at least three times. Immunofluorescence: Cells were fixed with 2% freshly prepared neutral buffered paraformaldehyde, permeabilized with 0.05% Tx and detected with mouse monoclonal anti-FLAG (Sigma) followed by rhodamine Red-X goat anti-mouse IgG (Invitrogen). The nuclei were contraststained with 40 ,6-diamidino-2-phenylindole (DAPI).
3 equipped with Spot RT color system (Diagnostic Instruments, Sterling Heights, MI). Terminal apoptosis rate was represented as the percentage of cells with fragmented nuclei. For each experiment (n Z 3), ten random images (40 objective) were analyzed.11
Results Clinical findings We identified a three-generation Chinese family with autosomal dominant nuclear cataract (Fig. 1A). Total of 10 family members (5 affected and 5 unaffected) participated in the study. All affected members had been diagnosed with non-progressive bilateral congenital cataracts within the first year after birth. The lens opacity caused obvious visual acuity decreased ranging from 0.04 to 0.3. The proband (III: 1) was 4 years old and presented dense white opacities distributed throughout the embryonic and fetal nuclei in both eyes (Fig. 1B). According to the medical records, the other affected individuals had undergone cataract extraction before the age of 10 years. There were no other ocular or systemic abnormalities in this family.
Terminal apoptosis assay Mutation analysis Fixed cells were stained for FLAG and red X-conjugated secondary antibody and nuclei counterstained with DAPI. Samples were examined by fluorescence microscopy
A heterozygous c. 227G > A variation in Cx50 was identified by direct sequencing of the coding regions of the candidate
Figure 1 Pedigrees and phenotypes. (A) Pedigrees of a cataract family. The black arrow indicated the proband. The asterisk indicated family members who attended this study. (B) Slit lamp photograph of the proband showed dense white opacities distributed throughout the embryonic and fetal nuclei.
Figure 2 Mutation analysis in Cx50. (A) DNA sequence chromatograms of an unaffected member and an affected member in the family showed a single alteration at position 227 (G > A) as a G/A double peak (indicated by an arrow). (B) A multiple-sequence alignment in Cx50 from different species indicated that the Arg at position 76 was highly conserved in different species.
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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4 genes (Fig. 2A), which resulted in a substitution of arginine to histidine at codon 76 (p. R76H). The substitution was not found in any of the unaffected family members or in the 110 unrelated controls.
K.J. Wang et al. plasma membrane and formed gap junction plaques on appositional membranes. In contrast, the mutant was able to localize in the cytoplasm and plasma membrane, but failed to formed gap junction plaques on appositional membranes (Fig. 4A).
Bioinformatics analysis Quantification of apoptosis rate The Arg at position 76 of human Cx50 was located within a phylogenetically conserved region by multiple-sequence alignment (Fig. 2B). The p. R76H was predicted to be “probably damaging” by Polyphen-2 analysis with a score of 1.000. This was also predicted to be a disease-causing mutation by Mutation Taster with a score of 29.
Expression of Cx50 in HeLa cells Recombinant FLAG -tagged WT or mutant Cx50 was expressed in HeLa cells. Tx soluble and insoluble fractions were western blotted for FLAG to detect WT and mutant Cx50. Results showed that WT Cx50 remained almost Txsoluble and a little mutant Cx50 protein became Txinsoluble (Fig. 3A). Band densitometry followed by normalization with housekeeping proteins (GAPDH for Txsoluble fractions and b-actin for Tx-insoluble fractions) showed that about 84% of Cx50 R76H was Tx soluble compared to 96% of WT Cx50 (Fig. 3B). Similar observations were obtained in triplicate experiments. Hence, the solubility of Cx50 R76H was slightly reduced, but there was no significant difference between WT and mutant Cx50.
Localization of Cx50 in HeLa cells The immunofluorescence study showed that the WT Cx50 proteins predominantly localized in the cytoplasm and
The apoptosis rate in WT and mutant Cx50 was approximately 2.7% and 3.2%, respectively, and there was no significant difference between them (Fig. 4B).
Discussion In this study, we identified a mutation (p.R76H) in Cx50 associated with dense nuclear cataract in a Chinese family, which was co-segregated in affected members and not detected in 110 unrelated ethnically matched controls. Although the Cx50 R76H mutation had been previously reported, it was associated with zonular/lamellar punctate cataracts in another Chinese family.12 Our study first identified of the p.R76H mutation in a large pedigree with dense nuclear cataract, and different with the phenotype reported. In this family, all affected individuals showed non-progressive bilateral nuclear cataract, with dense white opacities accumulated in the fetal and embryonic nucleus. The dense nuclear opacities led to obvious visual acuity decreased and early cataract surgery. In contrast, the phenotype in the previously reported family showed a perinuclear cataract with fine punctate opacities involving the central zone (2 mm) of the lens, which was described as lamellar punctate cataract. However, there are also some similarities in the inheritance pattern and age at diagnosis between the two families. The p.R76H mutation in Cx50
Figure 3 Detergent solubility of WT and mutant Cx50. (A) The amount of Cx50 R76H in Tx-soluble fractions was similar to that of WT protein. A little of mutant was detected in Tx-insoluble fractions. (B) There was no significant difference between WT and R76H mutant amounts after normalization by the housekeeping protein expression.
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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A p.R76H mutation in congenital nuclear cataract
Figure 4 The subcellular localization and apoptosis assay of Cx50 in HeLa cells. (A) Both the WT and mutant proteins were predominantly localized in the cytoplasm and plasma membrane, but Cx50 R76H failed to formed gap junction plaques on appositional membranes. The red fluorescence indicated the expression of Cx50, which was overlaid with DAPI-stained nucleus in blue color. Scale bar: 20 mm. (B) There was no significant difference in apoptosis rate between WT and mutant Cx50.
displays a remarkable inter-familial clinical variability, supporting the idea that environmental and/or other genetic factors may influence the expression and function of Cx50 in lens development. There have been previous suggestions of modifier genes involved in the development of congenital cataract, although such genes have not yet been identified.13,14 Cx50 comprises two exons with exon-2 coding for the entire 433 amino acid residues of gap junction protein a8 (GJA8) which is essential for maintaining lens transparency. Like other connexins, Cx50 contains four transmembrane domains (M1-M4), two extracellular loops (E1 and E2), and three intracellular regions (the NH2-terminus, a cytoplasmic loop and COOH-terminus).15 However, much less is known about the functions of different domains of the connexin proteins. It has been shown that integrity of the transmembrane domains is essential for correct transport of connexins into plasma membrane.16 The extracellular loops are critical for the docking of intercellular connexin hemichannels,17 and the E1 domain lines the pore and can facilitate/limit the rate of ion permeation by a combination of pore size and their electrostatic properties.18 The NH2terminus may play a role in voltage-gating of gap junctional channels.17 The COOH-terminus may not be essential for normal function of connexin, but this is the only part of connexin that contain phosphorylation sites for different
5 kinases and the functional role of phosphorylation for some of these connexin has been proved.19 The c. 227G > A substitution observed in the present study results in the replacement of arginine to histidine at codon 76 (p.R76H), localized in the E1 loop of the Cx50. The R76 residue of Cx50 is highly conserved in different species, and the substitution is predicted to be probably damaging by using the bioinformatic algorithms. These data indicate that the arginine is likely to be functionally important and that the mutation may have a detrimental effect on the normal docking of intercellular connexons. To elucidate the pathogenic mechanism of the p.R76H mutation, further cell expression studies are conducted in HeLa cells to characterize the functional consequences of this mutation. In this study, we have shown that the solubility and apoptosis rate were similar between wide type and mutant Cx50, indicating that the mutation did not affect the stability of the protein. The immunofluorescence study showed that both WT and mutant Cx50 predominantly localized in the cytoplasm and plasma membrane, but the mutant failed to formed gap junctional plaques on appositional membranes. However, in the previous study, the p.R76H mutation showed no disruptive effect on oligomerization and trafficking, with the result that no cellular or functional consequence was found when expressed in Hek293 cells.12 The difference in results may be due to the fact that there are many different derangements in the cellular behaviors of Cx50 among different transfected cells. To date, total of 8 mutations (p.D47H, p.D47N, p.D47Y, p.E48K, p.P59A, p.V64G, p.S73F and p.R76H) in the E1 loop of Cx50 have been reported to be associated with congenital cataracts in human,20e25,12 which highlights the functional importance of this domain for lens growth. Besides p.R76H, there are 3 missense mutations (p.D47N, p.E48K and p.P59A) in the E1 loop of Cx50 characterized functionally by cell expression studies. Interestingly, the three mutations share a similar mechanism that affected the formation of intercellular coupling.12,21,23 The effects of p.R76H in our study are similar to those of the three Cx50 mutations, which further validated the importance of the E1 loop in the docking of intercellular connexin hemichannels to form gap junctions. Moreover, these data indicate that the amino acids at the interface between the extracellular loop and transmembrane domain (D47 and R76) play an important role in the formation of functional Cx50 gap junction channels and are most likely mutational hot spots. However, whether the R76H mutation affects the electrophysiological properties of the hemichannels or gap junction channels, or exerts any effects on lens cell differentiation remain to be further investigated. In summary, we reported a novel cataract phenotype caused by the p.R76H mutation of Cx50, providing evidence of further phenotypic heterogeneity associated with this variant. Functional studies showed that the mutation affected the formation of gap junction channels, disrupted the intercellular communication and thereby led to nuclear cataract formation.
Conflicts of interest The authors declare no conflict of interest.
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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Acknowledgements We thank the family and all participants for taking part in this study. This study was supported by National Natural Science Foundation of China (81200673; 51573101), Beijing Nova Program (Z151100000315096), Beijing Natural Science Foundation (7172056) and the priming scientific research foundation for the senior researcher in Beijing Tongren Hospital, Capital Medical University (2016-YJJ-GGL-010).
References 1. Shiels A, Hejtmancik JF. Mutations and mechanisms in congenital and age-related cataracts. Exp Eye Res 2017;156: 95e102. 2. Pichi F, Lembo A, Serafino M, Nucci P. Genetics of congenital cataract. Dev Ophthalmol 2016;57:1e14. 3. Messina-Baas O, Cuevas-Covarrubias SA. Inherited congenital cataract: a guide to suspect the genetic etiology in the cataract genesis. Mol Syndromology 2017;8(2):58e78. 4. Shiels A, Hejtmancik JF. Genetics of human cataract. Clin Genet 2013;84(2):120e7. 5. Wang KJ, Li SS, Yun B, Ma WX, Jiang TG, Zhu SQ. A novel mutation in MIP associated with nuclear cataract in a Chinese family. Mol Vis 2011;17:70e7. 6. Mathias RT, White TW, Gong X. Lens gap junctions in growth, differentiation, and homeostasis. Physiol Rev 2010;90(1): 179e206. 7. Berthoud VM, Ngezahayo A. Focus on lens connexins. BMC Cell Biol 2017;18(Suppl. 1):6. 8. Jiang JX. Gap junctions or hemichannel-dependent and independent roles of connexins in cataractogenesis and lens development. Curr Mol Med 2010;10(9):851e63. 9. Wang KJ, Wang BB, Zhang F, Zhao Y, Ma X, Zhu SQ. Novel betacrystallin gene mutations in Chinese families with nuclear cataracts. Arch Ophthalmol 2011;129:337e43. 10. Gu F, Luo W, Li X, Wang Z, Lu S, Zhang M, et al. A novel mutation in AlphaA-crystallin (CRYAA) caused autosomal dominant congenital cataract in a large Chinese family. Hum Mutat 2008; 29:769. 11. Yam GH, Gaplovska-Kysela K, Zuber C, Roth J. Aggregated myocilin induces russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary openangle glaucoma. Am J Pathol 2007;170(1):100e9. 12. Yu Y, Wu M, Chen X, Zhu Y, Gong X, Yao K. Identification and functional analysis of two novel connexin 50 mutations
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associated with autosome dominant congenital cataracts. Sci Rep 2016;6:26551. Maeda YY, Funata N, Takahama S, Sugata Y, Yonekawa H. Two interactive genes responsible for a new inherited cataract (RCT) in the mouse. Mamm Genome 2001;12:278e83. Burdon KP, Wirth MG, Mackey DA, Russell-Eggitt IM, Craig JE, Elder JE, et al. A novel mutation in the Connexin 46 gene causes autosomal dominant congenital cataract with incomplete penetrance. J Med Genet 2004;41:e106. Yeager M, Nicholson BJ. Structure and biochemistry of gap junction. Adv Mol Cell Biol 2000;30:31e98. Kronengold J, Trexler EB, Bukauskas FF, Bargiello TA, Verselis VK. Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels. J Gen Physiol 2003;122(4):389e405. Verselis VK, Ginter CS, Bargiello TA. Opposite voltage gating polarities of two closely related connexins. Nature 1994; 368(6469):348e51. Tong X, Aoyama H, Sudhakar S, Chen H, Shilton BH, Bai D. The first extracellular domain plays an important role in unitary channel conductance of Cx50 gap junction channels. PLoS One 2015;10(12):e0143876. Krutovskikh V, Yamasaki H. Connexin gene mutations in human genetic diseases. Mutat Res 2000;462(2e3):197e207. 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;19:767e74. Rubinos C, Villone K, Mhaske PV, White TW, Srinivas M. Functional effects of Cx50 mutations associated with congenital cataracts. Am J Physiol Cell Physiol 2014;306(3):C212e20. Lin Y, Liu NN, Lei CT, Fan YC, Liu XQ, Yang Y, et al. [A novel GJA8 mutation in a Chinese family with autosomal dominant congenital cataract]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2008;25(1):59e62. Banks EA, Toloue MM, Shi Q, Zhou ZJ, Liu J, Nicholson BJ, et al. Connexin mutation that causes dominant congenital cataracts inhibits gap junctions, but not hemichannels, in a dominant negative manner. J Cell Sci 2009;122(Pt 3):378e88. Zheng JQ, Ma ZW, Sun HM. A heterozygous transversion of connexin 50 in a family with congenital nuclear cataract in the northeast of China. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2005; 22(1):76e8. Yang Z, Li Q, Ma X, Zhu SQ. Mutation analysis in Chinese families with autosomal dominant hereditary cataracts. Curr Eye Res 2015;40(12):1225e31.
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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Journal of the Formosan Medical Association xxx (xxxx) xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.jfma-online.com
Original Article
Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract Kai Jie Wang, Jin Da Wang, Dou Dou Chen, Ming Yang Wang, Bo Yun, Si Quan Zhu* Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology &Visual Sciences Key Lab, Beijing, 100730, China Received 15 March 2018; received in revised form 2 September 2018; accepted 25 February 2019
KEYWORDS Gap junction; Mutation; Nuclear cataract
Background/purpose: A three-generation Chinese family with autosomal dominant congenital nuclear cataract was recruited. This study aimed to identify the disease-causing gene for nuclear cataract with functional dissections of the identified mutant. Methods: Detailed clinical data and family history were recorded. Candidate gene sequencing was performed to identify the disease-causing mutation. Recombinant connexin50 (Cx50) wild type and mutant constructs were synthesized. Triton X-100 solubility and subcellular localization of the recombinant Cx50 proteins were analyzed in HeLa cells. Apoptosis was assayed as the percentage of fragmented nuclei in transfected cells. Results: All affected individuals in the family displayed clear phenotypes of dense nuclear cataracts. A c.227 G > A variation was found in the coding region of Cx50, which arginine residue at position 76 was substituted by histidine (p.R76H). This mutation was co-segregated with the disease in the family, and was not observed in 110 unrelated Chinese controls. No statistically significant differences were found in the Triton X-100 solubility and apoptosis rate between wild type and mutant Cx50 in HeLa cells. However, Cx50 mutant was unable to form gap junctional plaques between adjacent cells as the wild type proteins did. Conclusions: This study identified a novel cataract phenotype caused by the p.R76H mutation in Cx50, providing evidence of further phenotypic heterogeneity associated with this mutation. Functional analysis showed that the mutation affected the formation of gap junction channels and led to opacity in the lens. Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
* Corresponding author. Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, 1 Dong Jiao Min Xiang, Beijng, 100730, China. Fax: þ8610 85110023. E-mail address: [email protected] (S.Q. Zhu). https://doi.org/10.1016/j.jfma.2019.02.015 0929-6646/Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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Introduction Congenital cataract refers to any opacity of lens presented at birth or during early childhood, which is responsible for approximately 10% childhood blindness worldwide.1 Various etiological factors have been identified, including infection, metabolic disorders and gene defects. About one third of isolated congenital cataracts are genetically determined. Autosomal dominant congenital cataract (ADCC) is the most common mode of inheritance, although autosomal recessive and X-linked inheritance are also reported for congenital cataract.2 To date, at least 35 genes have been strongly associated with inherited cataract without other systemic anomalies.3 Of the disease-causing mutations reported, about half are located in crystallins, 15% in connexins, 10% in transcription factors, 5% each in intermediate filaments or aquaporin 0, and 10% in a variety of other genes.4 Therefore, the crystalline and connexin genes appear to be the most common genes associated with congenital cataract. It is appropriate to consider these genes as the top list of candidate genes for screening studies in congenital cataracts.5 Gap junctions are intercellular channels and important for lens homeostasis, function and transparency.6 Gap junction proteins oligomerize to form hexameric hemichannels (connexons) in the membranes. The hemichannels of two adjacent cells dock with each other to form a gap junction channel allowing for intercellular communications in the lens. The functions of gap junctions are not only important for the maintenance of lens transparency but also crucial to the lens development and differentiation.6 Three different gap junction proteins have been identified in the human lens, including connexin43 (Cx43), connexin46 (Cx46) and connexin50 (Cx50).7 Both Cx46 and Cx50 predominantly co-localize in lens fibers, and multiple mutations in these genes have been reported to be associated with congenital cataracts.8 In the present study, we screened the 8 crystalline and 2 connexin genes using the same strategy as described previously.9 A missense mutation in Cx50 was identified to be responsible for dense nuclear cataract, and its functional consequences were investigated to gain further insights into the pathogenesis of inherited cataracts.
Materials and methods Patients and DNA specimens This study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of our institution. A three-generation family from Shandong province with autosomal dominant nuclear cataract was identified after informed consents. All participants underwent complete ophthalmic examinations including visual acuity, slit lamp examination, fundus examination and ultrasonography. The phenotypes were documented by slit lamp photography. A total of 110 unrelated ethnically matched controls with no family history of congenital
K.J. Wang et al. cataracts were also recruited. Peripheral venous blood of all participants was collected for genomic DNA extraction using QIAamp DNA kit (Qiagen, Valencia, CA).
Mutation analysis Mutation screening was performed in 10 candidate genes: 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 (GenBank NM_017541), Cx46 (Genbank NM_021954) and Cx50 (Genbank NM_005267). All coding exons and splice sites of the candidate genes were amplified by polymerase chain reactions (PCR) using previously reported primer sequences.9 The PCR products obtained from the proband and one unaffected member were sequenced on an ABI3730 Automated Sequencer (PE Biosystems, Foster City, CA). Direct sequencing was also used to screen the mutation identified in Cx50 on the sample of all available family members and 110 controls to confirm the mutation.
Bioinformatics analysis The CLC Free Workbench 4.5.1 software (CLC bio, Aarhus, Denmark) was used to align the protein sequences of different species. The possible impact of an amino acid substitution on the structure and function of the protein was predicted by Polyphen-2 (http://genetics.bwh.harvard.edu/ pph2/) and Mutation Taster (http://mutationtaster.org/).
Expression constructs and mutagenesis Human lenses total cDNA was obtained using the standard methods as described previously.10 The coding sequence of human Cx50 isolated from the human lens cDNA library by PCR using the following primers: sense 50 -GAACATCTTGGAGGAGGTGAATG-30 , antisense 50 -TCATCCCCCCACACGAACT-30 . After digestion with XhoI and EcoRI (NEB, Ipswich, MA, USA), the PCR product was cloned into the expression vector p3XFLAG-myc-CMV00-25 (Sigma, St Louis, MO). Correctness of construct sequence was confirmed by direct sequencing. The mutant Cx50 R76H plasmid was constructed by a site-directed mutagenesis kit (Stratagene, La Jolla, CA) using primers: sense 50 GCCTTTCCCATCTCCCACATTCACCTCTGGGTGCTGCAGATCATC-30 , antisense 50 -GATGATCTGCAGCACCCAGAGGTGAATGTGGGAGATGGGAAAGGC-30 . Wild type (WT) and mutant constructs were verified by direct sequencing.
Cell culture and transfection The HeLa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics in a 37 C incubator with 5% CO2. Cells were seeded with 5 105 cells in a 60 mm (diameter) culture dish for overnight before transfection. Expression construct containing WT or mutant Cx50 was mixed with FuGene HD
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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A p.R76H mutation in congenital nuclear cataract reagent (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Cells were collected for protein analysis 24 h after transfection.
Triton X-100 solubility analysis Cells were washed twice with ice-cold PBS and lyzed at 2.5 106 cells/ml lysis buffer containing 0.5% Triton X-100 (Tx) (Sigma). After centrifugation, the supernatant containing Tx-soluble protein was denatured in buffer with 2% Na dodecylsulfate (SDS) and 50 mM DL-dithiothreitol (DTT; Sigma). The pellet containing Tx-insoluble protein was washed twice with ice-cold PBS, sonicated and denatured in urea-SDS buffer. The samples were analyzed by western blot using antibodies against FLAG (recognizing Cx50), GAPDH or b-actin (Sigma). Each experiment was repeated at least three times. Immunofluorescence: Cells were fixed with 2% freshly prepared neutral buffered paraformaldehyde, permeabilized with 0.05% Tx and detected with mouse monoclonal anti-FLAG (Sigma) followed by rhodamine Red-X goat anti-mouse IgG (Invitrogen). The nuclei were contraststained with 40 ,6-diamidino-2-phenylindole (DAPI).
3 equipped with Spot RT color system (Diagnostic Instruments, Sterling Heights, MI). Terminal apoptosis rate was represented as the percentage of cells with fragmented nuclei. For each experiment (n Z 3), ten random images (40 objective) were analyzed.11
Results Clinical findings We identified a three-generation Chinese family with autosomal dominant nuclear cataract (Fig. 1A). Total of 10 family members (5 affected and 5 unaffected) participated in the study. All affected members had been diagnosed with non-progressive bilateral congenital cataracts within the first year after birth. The lens opacity caused obvious visual acuity decreased ranging from 0.04 to 0.3. The proband (III: 1) was 4 years old and presented dense white opacities distributed throughout the embryonic and fetal nuclei in both eyes (Fig. 1B). According to the medical records, the other affected individuals had undergone cataract extraction before the age of 10 years. There were no other ocular or systemic abnormalities in this family.
Terminal apoptosis assay Mutation analysis Fixed cells were stained for FLAG and red X-conjugated secondary antibody and nuclei counterstained with DAPI. Samples were examined by fluorescence microscopy
A heterozygous c. 227G > A variation in Cx50 was identified by direct sequencing of the coding regions of the candidate
Figure 1 Pedigrees and phenotypes. (A) Pedigrees of a cataract family. The black arrow indicated the proband. The asterisk indicated family members who attended this study. (B) Slit lamp photograph of the proband showed dense white opacities distributed throughout the embryonic and fetal nuclei.
Figure 2 Mutation analysis in Cx50. (A) DNA sequence chromatograms of an unaffected member and an affected member in the family showed a single alteration at position 227 (G > A) as a G/A double peak (indicated by an arrow). (B) A multiple-sequence alignment in Cx50 from different species indicated that the Arg at position 76 was highly conserved in different species.
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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4 genes (Fig. 2A), which resulted in a substitution of arginine to histidine at codon 76 (p. R76H). The substitution was not found in any of the unaffected family members or in the 110 unrelated controls.
K.J. Wang et al. plasma membrane and formed gap junction plaques on appositional membranes. In contrast, the mutant was able to localize in the cytoplasm and plasma membrane, but failed to formed gap junction plaques on appositional membranes (Fig. 4A).
Bioinformatics analysis Quantification of apoptosis rate The Arg at position 76 of human Cx50 was located within a phylogenetically conserved region by multiple-sequence alignment (Fig. 2B). The p. R76H was predicted to be “probably damaging” by Polyphen-2 analysis with a score of 1.000. This was also predicted to be a disease-causing mutation by Mutation Taster with a score of 29.
Expression of Cx50 in HeLa cells Recombinant FLAG -tagged WT or mutant Cx50 was expressed in HeLa cells. Tx soluble and insoluble fractions were western blotted for FLAG to detect WT and mutant Cx50. Results showed that WT Cx50 remained almost Txsoluble and a little mutant Cx50 protein became Txinsoluble (Fig. 3A). Band densitometry followed by normalization with housekeeping proteins (GAPDH for Txsoluble fractions and b-actin for Tx-insoluble fractions) showed that about 84% of Cx50 R76H was Tx soluble compared to 96% of WT Cx50 (Fig. 3B). Similar observations were obtained in triplicate experiments. Hence, the solubility of Cx50 R76H was slightly reduced, but there was no significant difference between WT and mutant Cx50.
Localization of Cx50 in HeLa cells The immunofluorescence study showed that the WT Cx50 proteins predominantly localized in the cytoplasm and
The apoptosis rate in WT and mutant Cx50 was approximately 2.7% and 3.2%, respectively, and there was no significant difference between them (Fig. 4B).
Discussion In this study, we identified a mutation (p.R76H) in Cx50 associated with dense nuclear cataract in a Chinese family, which was co-segregated in affected members and not detected in 110 unrelated ethnically matched controls. Although the Cx50 R76H mutation had been previously reported, it was associated with zonular/lamellar punctate cataracts in another Chinese family.12 Our study first identified of the p.R76H mutation in a large pedigree with dense nuclear cataract, and different with the phenotype reported. In this family, all affected individuals showed non-progressive bilateral nuclear cataract, with dense white opacities accumulated in the fetal and embryonic nucleus. The dense nuclear opacities led to obvious visual acuity decreased and early cataract surgery. In contrast, the phenotype in the previously reported family showed a perinuclear cataract with fine punctate opacities involving the central zone (2 mm) of the lens, which was described as lamellar punctate cataract. However, there are also some similarities in the inheritance pattern and age at diagnosis between the two families. The p.R76H mutation in Cx50
Figure 3 Detergent solubility of WT and mutant Cx50. (A) The amount of Cx50 R76H in Tx-soluble fractions was similar to that of WT protein. A little of mutant was detected in Tx-insoluble fractions. (B) There was no significant difference between WT and R76H mutant amounts after normalization by the housekeeping protein expression.
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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A p.R76H mutation in congenital nuclear cataract
Figure 4 The subcellular localization and apoptosis assay of Cx50 in HeLa cells. (A) Both the WT and mutant proteins were predominantly localized in the cytoplasm and plasma membrane, but Cx50 R76H failed to formed gap junction plaques on appositional membranes. The red fluorescence indicated the expression of Cx50, which was overlaid with DAPI-stained nucleus in blue color. Scale bar: 20 mm. (B) There was no significant difference in apoptosis rate between WT and mutant Cx50.
displays a remarkable inter-familial clinical variability, supporting the idea that environmental and/or other genetic factors may influence the expression and function of Cx50 in lens development. There have been previous suggestions of modifier genes involved in the development of congenital cataract, although such genes have not yet been identified.13,14 Cx50 comprises two exons with exon-2 coding for the entire 433 amino acid residues of gap junction protein a8 (GJA8) which is essential for maintaining lens transparency. Like other connexins, Cx50 contains four transmembrane domains (M1-M4), two extracellular loops (E1 and E2), and three intracellular regions (the NH2-terminus, a cytoplasmic loop and COOH-terminus).15 However, much less is known about the functions of different domains of the connexin proteins. It has been shown that integrity of the transmembrane domains is essential for correct transport of connexins into plasma membrane.16 The extracellular loops are critical for the docking of intercellular connexin hemichannels,17 and the E1 domain lines the pore and can facilitate/limit the rate of ion permeation by a combination of pore size and their electrostatic properties.18 The NH2terminus may play a role in voltage-gating of gap junctional channels.17 The COOH-terminus may not be essential for normal function of connexin, but this is the only part of connexin that contain phosphorylation sites for different
5 kinases and the functional role of phosphorylation for some of these connexin has been proved.19 The c. 227G > A substitution observed in the present study results in the replacement of arginine to histidine at codon 76 (p.R76H), localized in the E1 loop of the Cx50. The R76 residue of Cx50 is highly conserved in different species, and the substitution is predicted to be probably damaging by using the bioinformatic algorithms. These data indicate that the arginine is likely to be functionally important and that the mutation may have a detrimental effect on the normal docking of intercellular connexons. To elucidate the pathogenic mechanism of the p.R76H mutation, further cell expression studies are conducted in HeLa cells to characterize the functional consequences of this mutation. In this study, we have shown that the solubility and apoptosis rate were similar between wide type and mutant Cx50, indicating that the mutation did not affect the stability of the protein. The immunofluorescence study showed that both WT and mutant Cx50 predominantly localized in the cytoplasm and plasma membrane, but the mutant failed to formed gap junctional plaques on appositional membranes. However, in the previous study, the p.R76H mutation showed no disruptive effect on oligomerization and trafficking, with the result that no cellular or functional consequence was found when expressed in Hek293 cells.12 The difference in results may be due to the fact that there are many different derangements in the cellular behaviors of Cx50 among different transfected cells. To date, total of 8 mutations (p.D47H, p.D47N, p.D47Y, p.E48K, p.P59A, p.V64G, p.S73F and p.R76H) in the E1 loop of Cx50 have been reported to be associated with congenital cataracts in human,20e25,12 which highlights the functional importance of this domain for lens growth. Besides p.R76H, there are 3 missense mutations (p.D47N, p.E48K and p.P59A) in the E1 loop of Cx50 characterized functionally by cell expression studies. Interestingly, the three mutations share a similar mechanism that affected the formation of intercellular coupling.12,21,23 The effects of p.R76H in our study are similar to those of the three Cx50 mutations, which further validated the importance of the E1 loop in the docking of intercellular connexin hemichannels to form gap junctions. Moreover, these data indicate that the amino acids at the interface between the extracellular loop and transmembrane domain (D47 and R76) play an important role in the formation of functional Cx50 gap junction channels and are most likely mutational hot spots. However, whether the R76H mutation affects the electrophysiological properties of the hemichannels or gap junction channels, or exerts any effects on lens cell differentiation remain to be further investigated. In summary, we reported a novel cataract phenotype caused by the p.R76H mutation of Cx50, providing evidence of further phenotypic heterogeneity associated with this variant. Functional studies showed that the mutation affected the formation of gap junction channels, disrupted the intercellular communication and thereby led to nuclear cataract formation.
Conflicts of interest The authors declare no conflict of interest.
Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015
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Acknowledgements We thank the family and all participants for taking part in this study. This study was supported by National Natural Science Foundation of China (81200673; 51573101), Beijing Nova Program (Z151100000315096), Beijing Natural Science Foundation (7172056) and the priming scientific research foundation for the senior researcher in Beijing Tongren Hospital, Capital Medical University (2016-YJJ-GGL-010).
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Please cite this article as: Wang KJ et al., Characterization of a p.R76H mutation in Cx50 identified in a Chinese family with congenital nuclear cataract, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.02.015