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Biochemical Genetics

, Volume 57, Issue 4, pp 540–554 | Cite as

A Novel CNGA1 Gene Mutation (c.G622A) of Autosomal Recessive Retinitis Pigmentosa Leads to the CNGA1 Protein Reduction on Membrane

  • Qing Gao
  • Yifan Liu
  • Xinlan Lei
  • Qinqin Deng
  • Yongqing Tong
  • Lique Du
  • Yin ShenEmail author
Original Article
  • 123 Downloads

Abstract

CNGA1 encodes a membrane protein on rod photoreceptor related to phototransduction. The present study was to identify a novel mutation of CNGA1 associated with autosomal recessive retinitis pigmentosa by using next generation sequencing of a Chinese family. Next generation sequencing and Sanger sequencing has identified a compound heterozygous mutation in CNGA1 gene, c0.472 del C (reported) and c0.829G>A (novel mutation, same as c0.622G>A according to NM_000087.3) of the proband. SIFT and Polyphen-2 predicted the CNGA1 G622A site to be possibly deleterious. Evolutionary conservation analysis of amino acid residues showed this aspartic acid is highly conserved between species, and protein structure prediction by I-TASSER server indicated that the D208N mutation induced a large disappear of interactions between S2 and S4. Flag-tagged CNGA1 and mutant G622A cDNA were generated and inserted into pCIG-eGFP vectors. Transfection of human embryonic kidney 293T cells was performed with lipofectamine. Interestingly, western blot and immunofluorescence results indicated that the expression of mutant CNGA1 (D208N) decreased significantly, especially on the membrane of transfected HEK293T cells. The novel variant c0.622G>A (p. D208N) in this study enriched the CNGA1 mutation spectrum. Besides, this mutant was predicted “possibly damaging” due to bioinformatics analysis and validated by laboratorial experiments. Our study suggests that this mutation lead to the CNGA1 protein reduction from the cell membrane.

Keywords

Retinitis pigmentosa Cyclic nucleotide gated channel alpha 1 Next generation sequencing 

Notes

Acknowledgements

We thank Ting Xie, the professor of the Stowers Institute for Medical Research for proof reading. We thank professor Xing Jian for the technique support in molecular modeling. Work was funded by The National Nature Science Foundation of China (Grant No. 81470628), International Science and Technology Cooperation Program of China (2017YFE0103400) and Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science (2017B030314025).

Compliance with Ethical Standards

Conflict of interest:

The authors declare that they have no conflict of interest.

Supplementary material

10528_2019_9907_MOESM1_ESM.docx (1.7 mb)
Supplementary file1 (DOCX 1780 kb)

References

  1. Adzhubei IA et al (2010) A method and server for predicting damaging missense mutations. Nat Methods 7:248–249.  https://doi.org/10.1038/nmeth0410-248 CrossRefGoogle Scholar
  2. Biel M, Michalakis S (2009) Cyclic nucleotide-gated channels. Handb Exp Pharmacol.  https://doi.org/10.1007/978-3-540-68964-5_7 Google Scholar
  3. Choi Y, Chan AP (2015) PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 31:2745–2747.  https://doi.org/10.1093/bioinformatics/btv195 CrossRefGoogle Scholar
  4. Dias MF, Joo K, Kemp JA, Fialho SL, da Silva Cunha A, Jr., Woo SJ, Kwon YJ (2018) Molecular genetics and emerging therapies for retinitis pigmentosa: basic research and clinical perspectives. Prog Retin Eye Res 63:107–131. https://doi.org/10.1016/j.preteyeres.2017.10.004 CrossRefGoogle Scholar
  5. Dryja TP, Finn JT, Peng YW, McGee TL, Berson EL, Yau KW (1995) Mutations in the gene encoding the alpha subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa. Proc Natl Acad Sci USA 92:10177–10181CrossRefGoogle Scholar
  6. James ZM, Zagotta WN (2018) Structural insights into the mechanisms of CNBD channel function. J Gen Physiol 150:225–244.  https://doi.org/10.1085/jgp.201711898 CrossRefGoogle Scholar
  7. Jin X, Qu LH, Hou BK, Xu HW, Meng XH, Pang CP, Yin ZQ (2016) Novel compound heterozygous mutation in the CNGA1 gene underlie autosomal recessive retinitis pigmentosa in a Chinese family. Biosci Rep 36:e00289.  https://doi.org/10.1042/BSR20150131 CrossRefGoogle Scholar
  8. Kaupp UB, Seifert R (2002) Cyclic nucleotide-gated ion channels. Physiol Rev 82:769–824.  https://doi.org/10.1152/physrev.00008.2002 CrossRefGoogle Scholar
  9. Kramer RH, Molokanova E (2001) Modulation of cyclic-nucleotide-gated channels and regulation of vertebrate phototransduction. J Exp Biol 204:2921–2931Google Scholar
  10. Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4:1073–1081.  https://doi.org/10.1038/nprot.2009.86 CrossRefGoogle Scholar
  11. Larkin MA et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948.  https://doi.org/10.1093/bioinformatics/btm404 CrossRefGoogle Scholar
  12. Leconte L, Barnstable CJ (2000) Impairment of rod cGMP-gated channel alpha-subunit expression leads to photoreceptor and bipolar cell degeneration. Investig Ophthalmol Vis Sci 41:917–926Google Scholar
  13. Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26:589–595.  https://doi.org/10.1093/bioinformatics/btp698 CrossRefGoogle Scholar
  14. Li M et al (2017) Structure of a eukaryotic cyclic-nucleotide-gated channel. Nature 542:60–65.  https://doi.org/10.1038/nature20819 CrossRefGoogle Scholar
  15. Maity S, Mazzolini M, Arcangeletti M, Valbuena A, Fabris P, Lazzarino M, Torre V (2015) Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy. Nat Commun 6:7093.  https://doi.org/10.1038/ncomms8093 CrossRefGoogle Scholar
  16. Marchesi A, Mazzolini M, Torre V (2012) A ring of threonines in the inner vestibule of the pore of CNGA1 channels constitutes a binding site for permeating ions. J Physiol 590:5075–5090.  https://doi.org/10.1113/jphysiol.2012.238352 CrossRefGoogle Scholar
  17. Marchesi A, Arcangeletti M, Mazzolini M, Torre V (2015) Proton transfer unlocks inactivation in cyclic nucleotide-gated A1 channels. J Physiol 593:857–870.  https://doi.org/10.1113/jphysiol.2014.284216 CrossRefGoogle Scholar
  18. Matulef K, Zagotta WN (2003) Cyclic nucleotide-gated ion channels. Ann Rev Cell Dev Biol 19:23–44.  https://doi.org/10.1146/annurev.cellbio.19.110701.154854 CrossRefGoogle Scholar
  19. Mazzolini M, Anselmi C, Torre V (2009) The analysis of desensitizing CNGA1 channels reveals molecular interactions essential for normal gating. J Gen Physiol 133:375–386.  https://doi.org/10.1085/jgp.200810157 CrossRefGoogle Scholar
  20. Mazzolini M et al (2018) The gating mechanism in cyclic nucleotide-gated ion channels. Sci Rep 8:45.  https://doi.org/10.1038/s41598-017-18499-0 CrossRefGoogle Scholar
  21. Meighan SE, Meighan PC, Rich ED, Brown RL, Varnum MD (2013) Cyclic nucleotide-gated channel subunit glycosylation regulates matrix metalloproteinase-dependent changes in channel gating. Biochemistry 52:8352–8362.  https://doi.org/10.1021/bi400824x CrossRefGoogle Scholar
  22. Nair AV, Mazzolini M, Codega P, Giorgetti A, Torre V (2006) Locking CNGA1 channels in the open and closed state. Biophys J 90:3599–3607.  https://doi.org/10.1529/biophysj.105.073346 CrossRefGoogle Scholar
  23. Nair AV, Nguyen CH, Mazzolini M (2009) Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels. Eur Biophys J 38:993–1002.  https://doi.org/10.1007/s00249-009-0491-4 CrossRefGoogle Scholar
  24. Pepe IM (2001) Recent advances in our understanding of rhodopsin and phototransduction. Prog Retin Eye Res 20:733–759CrossRefGoogle Scholar
  25. Pifferi S, Boccaccio A, Menini A (2006) Cyclic nucleotide-gated ion channels in sensory transduction. FEBS Lett 580:2853–2859.  https://doi.org/10.1016/j.febslet.2006.03.086 CrossRefGoogle Scholar
  26. Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682.  https://doi.org/10.1038/nmeth.2019 CrossRefGoogle Scholar
  27. Weitz D, Ficek N, Kremmer E, Bauer PJ, Kaupp UB (2002) Subunit stoichiometry of the CNG channel of rod photoreceptors. Neuron 36:881–889CrossRefGoogle Scholar
  28. Yang L et al (2015) Dependable and efficient clinical molecular diagnosis of chinese rp patient with targeted exon sequencing. PLoS ONE 10:e0140684.  https://doi.org/10.1371/journal.pone.0140684 CrossRefGoogle Scholar
  29. Zhong H, Molday LL, Molday RS, Yau KW (2002) The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry. Nature 420:193–198.  https://doi.org/10.1038/nature01201 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Qing Gao
    • 1
  • Yifan Liu
    • 1
  • Xinlan Lei
    • 1
  • Qinqin Deng
    • 1
  • Yongqing Tong
    • 2
  • Lique Du
    • 3
  • Yin Shen
    • 1
    Email author
  1. 1.Eye CenterRenmin Hospital of Wuhan UniversityWuhanChina
  2. 2.Department of Clinical LaboratoryRenmin Hospital of Wuhan UniversityWuhanChina
  3. 3.BGI-WuhanWuhanChina

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