Article | Published:

Contribution of noncoding pathogenic variants to RPGRIP1-mediated inherited retinal degeneration



With the advent of gene therapies for inherited retinal degenerations (IRDs), genetic diagnostics will have an increasing role in clinical decision-making. Yet the genetic cause of disease cannot be identified using exon-based sequencing for a significant portion of patients. We hypothesized that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and evaluated patients with single coding pathogenic variants in RPGRIP1 to test this hypothesis.


IRD families underwent targeted panel sequencing. Unsolved cases were explored by exome and genome sequencing looking for additional pathogenic variants. Candidate pathogenic variants were then validated by Sanger sequencing, quantitative polymerase chain reaction, and in vitro splicing assays in two cell lines analyzed through amplicon sequencing.


Among 1722 families, 3 had biallelic loss-of-function pathogenic variants in RPGRIP1 while 7 had a single disruptive coding pathogenic variants. Exome and genome sequencing revealed potential noncoding pathogenic variants in these 7 families. In 6, the noncoding pathogenic variants were shown to lead to loss of function in vitro.


Noncoding pathogenic variants were identified in 6 of 7 families with single coding pathogenic variants in RPGRIP1. The results suggest that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and RPGRIP1-mediated IRDs are more common than previously thought.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Farrar GJ, Carrigan M, Dockery A, et al. Toward an elucidation of the molecular genetics of inherited retinal degenerations. Hum Mol Genet. 2017;26:R2–R11. R1

  2. 2.

    Ellingford JM, Barton S, Bhaskar S, et al. Molecular findings from 537 individuals with inherited retinal disease. J Med Genet. 2016;53:761–767.

  3. 3.

    Riera M, Navarro R, Ruiz-Nogales S, et al. Whole exome sequencing using ion proton system enables reliable genetic diagnosis of inherited retinal dystrophies. Sci Rep. 2017;7:42078.

  4. 4.

    Bujakowska KM, Fernandez-Godino R, Place E, et al. Copy-number variation is an important contributor to the genetic causality of inherited retinal degenerations. Genet Med. 2017;19:643–651.

  5. 5.

    Liquori A, Vache C, Baux D, et al. Whole USH2A gene sequencing identifies several new deep intronic mutations. Hum Mutat . 2016;37:184–193.

  6. 6.

    Russell S, Bennett J, Wellman JA, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390:849–860.

  7. 7.

    Pawlyk BS, Bulgakov OV, Liu X, et al. Replacement gene therapy with a human RPGRIP1 sequence slows photoreceptor degeneration in a murine model of Leber congenital amaurosis. Hum Gene Ther. 2010;21:993–1004.

  8. 8.

    Lhériteau E, Petit L, Weber M, et al. Successful gene therapy in the RPGRIP1-deficient dog: a large model of cone–rod dystrophy. Mol Ther. 2014;22:265–277.

  9. 9.

    Institute of Medical Genetics. Human Gene Mutation Database (HGMD) [database online]. Cardiff, UK: Cardiff University; 2017. Accessed 23 July 2018.

  10. 10.

    Shu X, Fry A, Tulloch B, et al. RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin. Hum Mol Genet. 2005;14:1183–1197.

  11. 11.

    Zhao Y, Hong D-H, Pawlyk B, et al. The retinitis pigmentosa GTPase regulator (RPGR)-interacting protein: subserving RPGR function and participating in disk morphogenesis. Proc Natl Acad Sci U S A. 2003;100:3965–3970.

  12. 12.

    Mavlyutov TA, Zhao H, Ferreira PA. Species-specific subcellular localization of RPGR and RPGRIP isoforms: implications for the phenotypic variability of congenital retinopathies among species. Hum Mol Genet. 2002;11:1899–1907.

  13. 13.

    Castagnet P, Mavlyutov T, Cai Y, et al. RPGRIP1s with distinct neuronal localization and biochemical properties associate selectively with RanBP2 in amacrine neurons. Hum Mol Genet. 2003;12:1847–1863.

  14. 14.

    Gerber S, Perrault I, Hanein S, et al. Complete exon-intron structure of the RPGR-interacting protein (RPGRIP1) gene allows the identification of mutations underlying Leber congenital amaurosis. Eur J Hum Genet. 2001;9:561.

  15. 15.

    Patnaik SR, Raghupathy RK, Zhang X, et al. The role of RPGR and its interacting proteins in ciliopathies. J Ophthalmol . 2015;2015:414781.

  16. 16.

    Boylan JP, Wright AF. Identification of a novel protein interacting with RPGR. Hum Mol Genet. 2000;9:2085–2093.

  17. 17.

    Consugar MB, Navarro-Gomez D, Place EM, et al. Panel-based genetic diagnostic testing for inherited eye diseases is highly accurate and reproducible and more sensitive for variant detection than exome sequencing. Genet Med. 2015;17:253.

  18. 18.

    Lek M, Karczewski KJ, Minikel EV, et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016;536:285–291.

  19. 19.

    Falk MJ, Zhang Q, Nakamaru-Ogiso E, et al. NMNAT1 mutations cause Leber congenital amaurosis. Nat Genet. 2012;44:1040–1045.

  20. 20.

    Handsaker RE, Van Doren V, Berman JR, et al. Large multiallelic copy number variations in humans. Nat Genet . 2015;47:296–303.

  21. 21.

    Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013;29:15–21.

  22. 22.

    Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–26.

  23. 23.

    Buenrostro JD, Giresi PG, Zaba LC, et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013;10:1213–1218.

  24. 24.

    Andzelm MM, Cherry TJ, Harmin DA, et al. MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers. Neuron. 2015;86:247–263.

  25. 25.

    Li T. Leber congenital amaurosis caused by mutations in RPGRIP1. Cold Spring Harb Perspect Med. 2015;5:a017384.

  26. 26.

    Baker KE, Parker R. Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr Opin Cell Biol . 2004;16:293–299.

  27. 27.

    Usher CL, McCarroll SA. Complex and multi-allelic copy number variation in human disease. Brief Funct Genomics. 2015;14:329–338.

  28. 28.

    Farkas MH, Grant GR, White JA, et al. Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes. BMC Genomics. 2013;14:486.

  29. 29.

    Calarco JA, Superina S, O’Hanlon D, et al. Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein. Cell. 2009;138:898–910.

  30. 30.

    McFall RC, Sery TW, Makadon M. Characterization of a new continuous cell line derived from a human retinoblastoma. Cancer Res. 1977;37:1003–1010.

  31. 31.

    Dryja TP, Adams SM, Grimsby JL, et al. Null RPGRIP1 alleles in patients with Leber congenital amaurosis. Am J Hum Genet. 2001;68:1295–1298.

  32. 32.

    Sangermano R, Bax NM, Bauwens M, et al. Photoreceptor progenitor mRNA analysis reveals exon skipping resulting from the ABCA4 c.5461-10T-->C mutation in Stargardt disease. Ophthalmology. 2016;123:1375–1385.

  33. 33.

    Cummings BB, Marshall JL, Tukiainen T, et al. Improving genetic diagnosis in Mendelian disease with transcriptome sequencing. Sci Transl Med. 2017;9:eaal5209.

  34. 34.

    Albert S, Garanto A, Sangermano R, et al. Identification and rescue of splice defects caused by two neighboring deep-intronic ABCA4 mutations underlying Stargardt disease. Am J Hum Genet . 2018;102:517–527.

  35. 35.

    Kimberling WJ. Estimation of the frequency of occult mutations for an autosomal recessive disease in the presence of genetic heterogeneity: application to genetic hearing loss disorders. Hum Mutat . 2005;26:462–470.

  36. 36.

    Zallocchi M, Binley K, Lad Y, et al. EIAV-based retinal gene therapy in the shaker1 mouse model for Usher syndrome type 1B: development of UshStat. PLoS One. 2014;9:e94272.

  37. 37.

    Banin E, Gootwine E, Obolensky A, et al. Gene augmentation therapy restores retinal function and visual behavior in a sheep model of CNGA3 achromatopsia. Mol Ther. 2015;23:1423–1433.

  38. 38.

    Komáromy AM, Alexander JJ, Rowlan JS, et al. Gene therapy rescues cone function in congenital achromatopsia. Hum Mol Genet. 2010;19:2581–2593.

  39. 39.

    Bainbridge JW, Smith AJ, Barker SS, et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med. 2008;358:2231–2239.

  40. 40.

    Maguire AM, Simonelli F, Pierce EA, et al. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med. 2008;358:2240–2248.

Download references


This work was supported by grants from the National Eye Institute (RO1EY012910 [EAP], R01EY026904 [KMB/EAP], and P30EY014104 [MEEI core support]), and the Foundation Fighting Blindness (USA, EAP). Sequencing and analysis was provided by the Center for Mendelian Genomics at the Broad Institute of MIT and Harvard and was funded by the National Human Genome Research Institute, the National Eye Institute, and the National Heart, Lung, and Blood Institute grant UM1 HG008900 to Daniel MacArthur and Heidi Rehm. The authors would like to thank the patients and their family members for their participation in this study and the Ocular Genomics Institute Genomics Core members for their experimental assistance. The authors would like to thank the Exome Aggregation Consortium, the Genome Aggregation Database (gnomAD), and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at and

Author information

Correspondence to Eric A. Pierce M.D., Ph.D. or Kinga M. Bujakowska Ph.D..

Ethics declarations


The authors declare no conflict of interest.

Electronic supplementary material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary File

Supplementary Table S1

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark


  • Inherited retinal degeneration
  • Noncoding pathogenic variants
  • Intronic pathogenic variants
  • genome sequencing

Further reading

  • Where are the missing gene defects in inherited retinal disorders? Intronic and synonymous variants contribute at least to 4% of CACNA1F ‐mediated inherited retinal disorders

    • Christina Zeitz
    • , Christelle Michiels
    • , Marion Neuillé
    • , Christoph Friedburg
    • , Christel Condroyer
    • , Fiona Boyard
    • , Aline Antonio
    • , Nassima Bouzidi
    • , Diana Milicevic
    • , Robin Veaux
    • , Aurore Tourville
    • , Axelle Zoumba
    • , Imene Seneina
    • , Marine Foussard
    • , Camille Andrieu
    • , Markus N. Preising
    • , Steven Blanchard
    • , Jean‐Paul Saraiva
    • , Lilia Mesrob
    • , Edith Le Floch
    • , Claire Jubin
    • , Vincent Meyer
    • , Hélène Blanché
    • , Anne Boland
    • , Jean‐François Deleuze
    • , Dror Sharon
    • , Isabelle Drumare
    • , Sabine Defoort‐Dhellemmes
    • , Elfride Baere
    • , Bart P. Leroy
    • , Xavier Zanlonghi
    • , Ingele Casteels
    • , Thomy J. Ravel
    • , Irina Balikova
    • , Rob K. Koenekoop
    • , Fanny Laffargue
    • , Rebecca McLean
    • , Irene Gottlob
    • , Dominique Bonneau
    • , Daniel F. Schorderet
    • , Francis L. Munier
    • , Martin McKibbin
    • , Katrina Prescott
    • , Valerie Pelletier
    • , Hélène Dollfus
    • , Yaumara Perdomo‐Trujillo
    • , Céline Faure
    • , Charlotte Reiff
    • , Bernd Wissinger
    • , Isabelle Meunier
    • , Susanne Kohl
    • , Eyal Banin
    • , Eberhart Zrenner
    • , Bernhard Jurklies
    • , Birgit Lorenz
    • , José‐Alain Sahel
    •  & Isabelle Audo

    Human Mutation (2019)

Fig. 1
Fig. 2: Exon 1 and 2 duplication in OGI-237 and identification of a novel exon.
Fig. 3
Fig. 4