Skip to main content

Comparative sequence analyses of rhodopsin and RPE65 reveal patterns of selective constraint across hereditary retinal disease mutations


Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (d N/d S) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur.

Corresponding author
*Address correspondence to: Dr. Belinda Chang, Department of Cell & Systems Biology, Department of Ecology & Evolutionary Biology, University of Toronto, 25 Harbord St, Toronto, Ontario M5S 3G5, Canada. E-mail:
Hide All
Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasimova, A., Bork, P., Kondrashov, A.S. & Sunyaev, S.R. (2010). A method and server for predicting damaging missense mutations. Nature Methods 7, 248249.
Anasagasti, A., Irigoyen, C., Barandika, O., López de Munain, A. & Ruiz-Ederra, J. (2012). Current mutation discovery approaches in retinitis pigmentosa. Vision Research 75, 117129.
Anisimova, M. & Gascuel, O. (2006). Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Systematic Biology 55, 539552.
Bereta, G., Kiser, P.D., Golczak, M., Sun, W., Heon, E., Saperstein, D.A. & Palczewski, K. (2008). Impact of retinal disease-associated RPE65 mutations on retinoid isomerization. Biochemistry 47, 98569865.
Bowne, S.J., Sullivan, L.S., Koboldt, D.C., Ding, L., Fulton, R., Abbott, R.M., Sodergren, E.J., Birch, D.G., Wheaton, D.H., Heckenlively, J.R., Liu, Q., Pierce, E.A., Weinstock, G.M. & Daiger, S.P. (2011). Identification of disease-causing mutations in autosomal dominant retinitis pigmentosa (adRP) using next-generation DNA sequencing. Investigative Ophthalmology & Visual Science 52, 494503.
Breikers, G., Portier-VandeLuytgaarden, M.J.M., Bovee-Geurts, P.H.M. & DeGrip, W.J. (2002). Retinitis pigmentosa-associated rhodopsin mutations in three membrane-located cysteine residues present three different biochemical phenotypes. Biochemical and Biophysical Research Communications 297, 847853.
Briscoe, A.D., Gaur, C. & Kumar, S. (2004). The spectrum of human rhodopsin disease mutations through the lens of interspecific variation. Gene 332, 107118.
Burk-Herrick, A., Scally, M., Amrine-Madsen, H., Stanhope, M.J. & Springer, M.S. (2006). Natural selection and mammalian BRCA1 sequences: Elucidating functionally important sites relevant to breast cancer susceptibility in humans. Mammalian Genome 17, 257270.
Castellana, S., Rónai, J. & Mazza, T. (2015). MitImpact: An exhaustive collection of pre-computed pathogenicity predictions of human mitochondrial non-synonymous variants. Human Mutation 36, E2413E2422.
Choe, H-W., Kim, Y.J., Park, J.H., Morizumi, T., Pai, E.F., Krauß, N., Hofmann, K.P., Scheerer, P. & Ernst, O.P. (2011). Crystal structure of metarhodopsin II. Nature 471, 651655.
Choi, Y., Sims, G.E., Murphy, S., Miller, J.R. & Chan, A.P. (2012). Predicting the functional effect of amino acid substitutions and indels. PLoS One 7, e46688.
Cideciyan, A.V. (2010). Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy. Progress in Retinal and Eye Research 29, 398427.
Cooper, G.M., Brudno, M.; NISC Comparative Sequencing Program, Green, E.D., Batzoglou, S. & Sidow, A. (2003). Quantitative estimates of sequence divergence for comparative analyses of mammalian genomes. Genome Research 13, 813820.
Crottini, A., Madsen, O. & Poux, C. (2012). Vertebrate time-tree elucidates the biogeographic pattern of a major biotic change around the K–T boundary in Madagascar. Proceedings of the National Academy of Sciences of the United States of America 109, 53585363.
Delport, W., Poon, A.F.Y., Frost, S.D.W. & Kosakovsky Pond, S.L. (2010). Datamonkey 2010: A suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26, 24552457.
Dimitrieva, S. & Anisimova, M. (2014). Unraveling patterns of site-to-site synonymous rates variation and associated gene properties of protein domains and families. PLoS One 9, e95034.
Du, J., Dungan, S.Z., Sabouhanian, A. & Chang, B.S.W. (2014). Selection on synonymous codons in mammalian rhodopsins: A possible role in optimizing translational processes. BMC Evolutionary Biology 14, 96.
Ferrari, S., Di Iorio, E., Barbaro, V., Ponzin, D., Sorrentino, F.S. & Parmeggiani, F. (2011). Retinitis pigmentosa: Genes and disease mechanisms. Current Genomics 12, 238249.
Flanagan, S.E., Patch, A-M. & Ellard, S. (2010). Using SIFT and PolyPhen to predict loss-of-function and gain-of-function mutations. Genetic Testing and Molecular Biomarkers 14, 533537.
Fong, J.J., Brown, J.M., Fujita, M.K. & Boussau, B. (2012). A phylogenomic approach to vertebrate phylogeny supports a turtle-archosaur affinity and a possible paraphyletic lissamphibia. PLoS One 7, e48990.
Gaucher, E.A., De Kee, D.W. & Benner, S.A. (2006). Application of DETECTER, an evolutionary genomic tool to analyze genetic variation, to the cystic fibrosis gene family. BMC Genomics 7, 44.
Greenblatt, M.S., Beaudet, J.G., Gump, J.R., Godin, K.S., Trombley, L., Koh, J. & Bond, J.P. (2003). Detailed computational study of p53 and p16: Using evolutionary sequence analysis and disease-associated mutations to predict the functional consequences of allelic variants. Oncogene 22, 11501163.
Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59, 307321.
Hamosh, A. (2005). Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic Acids Research 33, D514D517.
Hartong, D.T., Berson, E.L. & Dryja, T.P. (2006). Retinitis pigmentosa. Lancet 368, 17951809.
Hicks, S., Wheeler, D.A., Plon, S.E. & Kimmel, M. (2011). Prediction of missense mutation functionality depends on both the algorithm and sequence alignment employed. Human Mutation 32, 661668.
Hollingsworth, T.J. & Gross, A.K. (2013). The severe autosomal dominant retinitis pigmentosa rhodopsin mutant Ter349Glu mislocalizes and induces rapid rod cell death. Journal of Biological Chemistry 288, 2904729055.
Iannaccone, A., Man, D., Waseem, N., Jennings, B.J., Ganapathiraju, M., Gallaher, K., Reese, E., Bhattacharya, S.S. & Klein-Seetharaman, J. (2006). Retinitis pigmentosa associated with rhodopsin mutations: Correlation between phenotypic variability and molecular effects. Vision Research 46, 45564567.
Janz, J.M., Fay, J.F. & Farrens, D.L. (2003). Stability of dark state rhodopsin is mediated by a conserved ion pair in intradiscal loop E-2. Journal of Biological Chemistry 278, 1698216991.
Kirwan, J.D., Bekaert, M., Commins, J.M., Davies, K.T.J., Rossiter, S.J. & Teeling, E.C. (2013). A phylomedicine approach to understanding the evolution of auditory sensory perception and disease in mammals. Evolutionary Applications 6, 412422.
Kiser, P.D. & Palczewski, K. (2010). Membrane-binding and enzymatic properties of RPE65. Progress in Retinal and Eye Research 29, 428442.
Kiser, P.D., Golczak, M., Lodowski, D.T., Chance, M.R. & Palczewski, K. (2009). Crystal structure of native RPE65, the retinoid isomerase of the visual cycle. Proceedings of the National Academy of Sciences of the United States of America 106, 1732517330.
Kosakovsky Pond, S.L. (2005). Not so different after all: A comparison of methods for detecting amino acid sites under selection. Molecular Biology and Evolution 22, 12081222.
Kosakovsky Pond, S.L., Frost, S.D.W. & Muse, S.V. (2005). HyPhy: Hypothesis testing using phylogenies. Bioinformatics 21, 676679.
Kumar, S., Dudley, J.T., Filipski, A. & Liu, L. (2011). Phylomedicine: An evolutionary telescope to explore and diagnose the universe of disease mutations. Trends in Genetics 27, 377386.
Li, S., Hu, J., Jin, R.J., Aiyar, A., Jacobson, S.G., Bok, D. & Jin, M. (2015). Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis. Journal of Biochemistry 158, 115125.
Li, S., Izumi, T., Hu, J., Jin, H.H., Siddiqui, A-A.A., Jacobson, S.G., Bok, D. & Jin, M. (2014). Rescue of enzymatic function for disease-associated RPE65 proteins containing various missense mutations in non-active sites. Journal of Biological Chemistry 289, 1894318956.
Li, W.H., Wu, C.I. & Luo, C.C. (1985). A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Molecular Biology and Evolution 2, 150174.
Liu, M.Y., Liu, J., Mehrotra, D., Liu, Y., Guo, Y., Baldera-Aguayo, P.A., Mooney, V.L., Nour, A.M. & Yan, E.C.Y. (2013). Thermal stability of rhodopsin and progression of retinitis pigmentosa: Comparison of S186W and D190N rhodopsin mutants. Journal of Biological Chemistry 288, 1769817712.
Löytynoja, A. & Goldman, N. (2005). An algorithm for progressive multiple alignment of sequences with insertions. Proceedings of the National Academy of Sciences of the United States of America 102, 1055710562.
Löytynoja, A. & Goldman, N. (2008). Phylogeny-aware gap placement prevents errors in sequence alignment and evolutionary analysis. Science 320, 16321635.
Magrane, M. & Uniprot Consortium (2011). UniProt Knowledgebase: A hub of integrated protein data. Database 2011, bar009.
McKeone, R., Wikstrom, M., Kiel, C. & Rakoczy, P.E. (2014). Assessing the correlation between mutant rhodopsin stability and the severity of retinitis pigmentosa. Molecular Vision 20, 183199.
Mendes, H.F., van der Spuy, J., Chapple, J.P. & Cheetham, M.E. (2005). Mechanisms of cell death in rhodopsin retinitis pigmentosa: Implications for therapy. Trends in Molecular Medicine 11, 177185.
Meredith, R.W., Janečka, J.E., Gatesy, J., Ryder, O.A., Fisher, C.A., Teeling, E.C., Goodbla, A., Eizirik, E., Simão, T.L., Stadler, T., Rabosky, D.L., Honeycutt, R.L., Flynn, J.J., Ingram, C.M., Steiner, C., Williams, T.L., Robinson, T.J., Burk-Herrick, A., Westerman, M., Ayoub, N.A., Springer, M.S. & Murphy, W.J. (2011). Impacts of the cretaceous terrestrial revolution and KPg extinction on mammal diversification. Science 334, 521524.
Miller, M.P. & Kumar, S. (2001). Understanding human disease mutations through the use of interspecific genetic variation. Human Molecular Genetics 10, 23192328.
Moiseyev, G., Chen, Y., Takahashi, Y., Wu, B.X. & Ma, J-X. (2005). RPE65 is the isomerohydrolase in the retinoid visual cycle. Proceedings of the National Academy of Sciences of the United States of America 102, 1241312418.
Mooney, S.D. & Klein, T.E. (2002). The functional importance of disease-associated mutation. BMC Bioinformatics 3, 24.
Morgan, C.C., Mc Cartney, A.M., Donoghue, M.T.A., Loughran, N.B., Spillane, C., Teeling, E.C. & O’Connell, M.J. (2013). Molecular adaptation of telomere associated genes in mammals. BMC Evolutionary Biology 13, 251.
Morrow, J.M. & Chang, B.S.W. (2015). Comparative mutagenesis studies of retinal release in light-activated zebrafish rhodopsin using fluorescence spectroscopy. Biochemistry 54, 45074518.
Murrell, B., Moola, S., Mabona, A., Weighill, T., Sheward, D., Kosakovsky Pond, S.L. & Scheffler, K. (2013). FUBAR: A fast, unconstrained bayesian approximation for inferring selection. Molecular Biology and Evolution 30, 11961205.
Neidhardt, J., Barthelmes, D., Farahmand, F., Fleischhauer, J.C. & Berger, W. (2006). Different amino acid substitutions at the same position in rhodopsin lead to distinct phenotypes. Investigative Ophthalmology & Visual Science 47, 16301635.
Neveling, K., Collin, R.W., Gilissen, C., van Huet, R.A., Visser, L., Kwint, M.P., Gijsen, S.J., Zonneveld, M.N., Wieskamp, N., de Ligt, J., Siemiatkowska, A.M., Hoefsloot, L.H., Buckley, M.F., Kellner, U., Branham, K.E., den Hollander, A.I., Hoischen, A., Hoyng, C., Klevering, B.J., van den Born, L.I., Veltman, J.A., Cremers, F.P. & Scheffer, H. (2012). Next-generation genetic testing for retinitis pigmentosa. Human Mutation 33, 963972.
Ng, P.C. & Henikoff, S. (2003). SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Research 31, 38123814.
Nishiguchi, K.M., Tearle, R.G., Liu, Y.P., Oh, E.C., Miyake, N., Benaglio, P., Harper, S., Koskiniemi-Kuendig, H., Venturini, G., Sharon, D., Koenekoop, R.K., Nakamura, M., Kondo, M., Ueno, S., Yasuma, T.R., Beckmann, J.S., Ikegawa, S., Matsumoto, N., Terasaki, H., Berson, E.L., Katsanis, N. & Rivolta, C. (2013). Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene. Proceedings of the National Academy of Sciences of the United States of America 110, 1613916144.
Okada, T., Sugihara, M., Bondar, A-N., Elstner, M., Entel, P. & Buss, V. (2004). The retinal conformation and its environment in rhodopsin in light of a new 2.2Å crystal structure. Journal of Molecular Biology 342, 571583.
Opefi, C.A., South, K., Reynolds, C.A., Smith, S.O. & Reeves, P.J. (2013). Retinitis pigmentosa mutants provide insight into the role of the N-terminal cap in rhodopsin folding, structure, and function. Journal of Biological Chemistry 288, 3391233926.
Palczewski, K., Kumasaka, T., Hori, T., Behnke, C.A., Motoshima, H., Fox, B.A., Le Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E., Yamamoto, M. & Miyano, M. (2000). Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739745.
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C. & Ferrin, T.E. (2004). UCSF Chimera—A visualization system for exploratory research and analysis. Journal of Computational Chemistry 25, 16051612.
Philpa, A.R., Jin, M., Li, S., Schindler, E.I., Iannaccone, A., Lam, B.L., Weleber, R.G., Fishman, G.A., Jacobson, S.G., Mullins, R.F., Travis, G.H. & Stone, E.M. (2009). Predicting the pathogenicity of RPE65 mutations. Human Mutation 30, 11831188.
Piechnick, R., Ritter, E., Hildebrand, P.W., Ernst, O.P., Scheerer, P., Hofmann, K.P. & Heck, M. (2012). Effect of channel mutations on the uptake and release of the retinal ligand in opsin. Proceedings of the National Academy of Sciences of the United States of America 109, 52475252.
Pierrottet, C.O., Zuntini, M., Digiuni, M., Bazzanella, I., Ferri, P., Paderni, R., Rossetti, L.M., Cecchin, S., Orzalesi, N. & Bertelli, M. (2014). Syndromic and non-syndromic forms of retinitis pigmentosa: A comprehensive Italian clinical and molecular study reveals new mutations. Genetics and Molecular Research 13, 88158833.
Plotkin, J.B. & Kudla, G. (2011). Synonymous but not the same: The causes and consequences of codon bias. Nature Reviews Genetics 12, 3242.
Pope, A., Eilers, M., Reeves, P.J. & Smith, S.O. (2014). Amino acid conservation and interactions in rhodopsin: Probing receptor activation by NMR spectroscopy. Biochimica et Biophysica Acta 1837, 683693.
Porter, M.L., Blasic, J.R., Bok, M.J., Cameron, E.G., Pringle, T., Cronin, T.W. & Robinson, P.R. (2011). Shedding new light on opsin evolution. Proceedings of the Royal Society B: Biological Sciences 279, 314.
Redmond, T.M., Poliakov, E., Yu, S., Tsai, J-Y., Lu, Z. & Gentleman, S. (2005). Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proceedings of the National Academy of Sciences of the United States of America 102, 1365813663.
Rishishwar, L., Varghese, N., Tyagi, E., Harvey, S.C., Jordan, I.K. & McCarty, N.A. (2012). Relating the disease mutation spectrum to the evolution of the Cystic Fibrosis transmembrane conductance regulator (CFTR). PLoS One 7, e42336.
Rivolta, C., Sharon, D., DeAngelis, M.M. & Dryja, T.P. (2002). Retinitis pigmentosa and allied diseases: Numerous diseases, genes, and inheritance patterns. Human Molecular Genetics 11, 12191227.
Ronquist, F. & Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 15721574.
Sauna, Z.E. & Kimchi-Sarfaty, C. (2011). Understanding the contribution of synonymous mutations to human disease. Nature Reviews Genetics 12, 683691.
Sauna, Z.E., Kimchi-Sarfaty, C., Ambudkar, S.V. & Gottesman, M.M. (2007). Silent polymorphisms speak: How they affect pharmacogenomics and the treatment of cancer. Cancer Research 67, 96099612.
Scheffler, K., Martin, D.P. & Seoighe, C. (2006). Robust inference of positive selection from recombining coding sequences. Bioinformatics 22, 24932499.
Schott, R.K., Refvik, S.P., Hauser, F.E., López-Fernández, H. & Chang, B.S.W. (2014). Divergent positive selection in rhodopsin from lake and riverine cichlid fishes. Molecular Biology and Evolution 31, 11491165.
Sherry, S.T., Ward, M.H., Kholodov, M., Baker, J., Phan, L., Smigielski, E.M. & Sirotkin, K. (2001). dbSNP: The NCBI database of genetic variation. Nucleic Acids Research 29, 308311.
Stenson, P.D., Mort, M., Ball, E.V., Shaw, K., Phillips, A.D. & Cooper, D.N. (2013). The human gene mutation database: Building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Human Genetics 133, 19.
Stover, D.A. & Verrelli, B.C. (2010). Comparative vertebrate evolutionary analyses of type I collagen: Potential of COL1a1 gene structure and intron variation for common bone-related diseases. Molecular Biology and Evolution 28, 533542.
Sugawara, T., Imai, H., Nikaido, M., Imamoto, Y. & Okada, N. (2010). Vertebrate rhodopsin adaptation to dim light via rapid meta-II intermediate formation. Molecular Biology and Evolution 27, 506519.
Takahashi, Y., Moiseyev, G. & Ma, J.X. (2014). Identification of key residues determining isomerohydrolase activity of human RPE65. Journal of Biological Chemistry 289, 2674326751.
Ueyama, H., Miraki-Oda, S., Yamade, S., Tanabe, S., Yamashita, T., Shichida, Y. & Ogita, H. (2012). Unique haplotype in exon 3 of cone opsin mRNA affects splicing of its precursor, leading to congenital color vision defect. Biochemical and Biophysical Research Communications 424, 152157.
Vincent, A.L., Carroll, J., Fishman, G.A., Sauer, A., Sharp, D., Summerfelt, P., Williams, V., Dubis, A.M., Kohl, S. & Wong, F. (2013). Rhodopsin F45L allele does not cause autosomal dominant retinitis pigmentosa in a large caucasian family. Translational Vision Science and Technology 2, 4.
Webb, A.E., Gerek, Z.N., Morgan, C.C., Walsh, T.A., Loscher, C.E., Edwards, S.V. & O’Connell, M.J. (2015). Adaptive evolution as a predictor of species-specific innate immune response. Molecular Biology and Evolution 32, 17171729.
Weitz, C.J. & Nathans, J. (1992). Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II. Neuron 8, 465472.
Yang, G., Xie, S., Feng, N., Yuan, Z., Zhang, M. & Zhao, J. (2014). Spectrum of rhodopsin gene mutations in Chinese patients with retinitis pigmentosa. Molecular Vision 20, 11321136.
Yang, Z. (2005). Bayes empirical bayes inference of amino acid sites under positive selection. Molecular Biology and Evolution 22, 11071118.
Yang, Z. (2007). PAML 4: Phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution 24, 15861591.
Zhao, H., Ru, B., Teeling, E.C., Faulkes, C.G., Zhang, S. & Rossiter, S.J. (2009). Rhodopsin molecular evolution in mammals inhabiting low light environments. PLoS One 4, e8326.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Visual Neuroscience
  • ISSN: 0952-5238
  • EISSN: 1469-8714
  • URL: /core/journals/visual-neuroscience
Please enter your name
Please enter a valid email address
Who would you like to send this to? *


Type Description Title
Supplementary materials

Hauser supplementary material
Figures S1-S8 and Tables S1-S9

 PDF (2.3 MB)
2.3 MB


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed