Whole genome sequencing and in vitro splice assays reveal genetic causes for inherited retinal diseases.
Journal
NPJ genomic medicine
ISSN: 2056-7944
Titre abrégé: NPJ Genom Med
Pays: England
ID NLM: 101685193
Informations de publication
Date de publication:
18 Nov 2021
18 Nov 2021
Historique:
received:
20
05
2021
accepted:
21
10
2021
entrez:
19
11
2021
pubmed:
20
11
2021
medline:
20
11
2021
Statut:
epublish
Résumé
Inherited retinal diseases (IRDs) are a major cause of visual impairment. These clinically heterogeneous disorders are caused by pathogenic variants in more than 270 genes. As 30-40% of cases remain genetically unexplained following conventional genetic testing, we aimed to obtain a genetic diagnosis in an IRD cohort in which the genetic cause was not found using whole-exome sequencing or targeted capture sequencing. We performed whole-genome sequencing (WGS) to identify causative variants in 100 unresolved cases. After initial prioritization, we performed an in-depth interrogation of all noncoding and structural variants in genes when one candidate variant was detected. In addition, functional analysis of putative splice-altering variants was performed using in vitro splice assays. We identified the genetic cause of the disease in 24 patients. Causative coding variants were observed in genes such as ATXN7, CEP78, EYS, FAM161A, and HGSNAT. Gene disrupting structural variants were also detected in ATXN7, PRPF31, and RPGRIP1. In 14 monoallelic cases, we prioritized candidate noncanonical splice sites or deep-intronic variants that were predicted to disrupt the splicing process based on in silico analyses. Of these, seven cases were resolved as they carried pathogenic splice defects. WGS is a powerful tool to identify causative variants residing outside coding regions or heterozygous structural variants. This approach was most efficient in cases with a distinct clinical diagnosis. In addition, in vitro splice assays provide important evidence of the pathogenicity of rare variants.
Identifiants
pubmed: 34795310
doi: 10.1038/s41525-021-00261-1
pii: 10.1038/s41525-021-00261-1
pmc: PMC8602293
doi:
Types de publication
Journal Article
Langues
eng
Pagination
97Subventions
Organisme : Foundation Fighting Blindness (Foundation Fighting Blindness, Inc.)
ID : PPA-0517-0717-RAD
Organisme : Foundation Fighting Blindness (Foundation Fighting Blindness, Inc.)
ID : PPA-0517-0717-RAD
Organisme : Foundation Fighting Blindness (Foundation Fighting Blindness, Inc.)
ID : PPA-0517-0717-RAD
Organisme : EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)
ID : EJP RD COFUND-EJP N° 825575
Organisme : EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)
ID : EJP RD COFUND-EJP N° 825575
Organisme : Health Research Board (HRB)
ID : HRB; POR/2010/97
Organisme : Health Research Board (HRB)
ID : HRB; POR/2010/97
Organisme : Health Research Board (HRB)
ID : HRB; POR/2010/97
Organisme : Irish Research Council (An Chomhairle um Thaighde in Éirinn)
ID : IRC; GOIPG/2017/1631
Organisme : Irish Research Council (An Chomhairle um Thaighde in Éirinn)
ID : IRC; GOIPG/2017/163
Organisme : Irish Research Council (An Chomhairle um Thaighde in Éirinn)
ID : IRC; GOIPG/2017/1631
Organisme : Science Foundation Ireland (SFI)
ID : SFI; 16/1A/4452
Organisme : Science Foundation Ireland (SFI)
ID : SFI; 16/1A/4452
Informations de copyright
© 2021. The Author(s).
Références
Berger, W., Kloeckener-Gruissem, B. & Neidhardt, J. The molecular basis of human retinal and vitreoretinal diseases. Prog. Retin. Eye Res. 29, 335–375 (2010).
pubmed: 20362068
doi: 10.1016/j.preteyeres.2010.03.004
Ellingford, J. M. et al. Whole genome sequencing increases molecular diagnostic yield compared with current diagnostic testing for inherited retinal disease. Ophthalmology 123, 1143–1150 (2016).
pubmed: 26872967
doi: 10.1016/j.ophtha.2016.01.009
Ellingford, J. M. et al. Pinpointing clinical diagnosis through whole exome sequencing to direct patient care: a case of Senior-Loken syndrome. Lancet 385, 1916 (2015).
pubmed: 25987160
doi: 10.1016/S0140-6736(15)60496-2
Albert, S. et al. Identification and rescue of splice defects caused by two neighboring deep-intronic ABCA4 mutations underlying Stargardt disease. Am. J. Hum. Genet. 102, 517–527 (2018).
pubmed: 29526278
pmcid: 5985352
doi: 10.1016/j.ajhg.2018.02.008
Buermans, H. P. & den Dunnen, J. T. Next generation sequencing technology: advances and applications. Biochim. Biophys. Acta 1842, 1932–1941 (2014).
pubmed: 24995601
doi: 10.1016/j.bbadis.2014.06.015
Whelan, L. et al. Findings from a genotyping study of over 1000 people with inherited retinal disorders in Ireland. Genes 11, https://doi.org/10.3390/genes11010105 (2020).
Consugar, M. B. 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. 17, 253–261 (2015).
pubmed: 25412400
doi: 10.1038/gim.2014.172
Patel, A. et al. The Oculome Panel Test: next-generation sequencing to diagnose a diverse range of genetic developmental eye disorders. Ophthalmology 126, 888–907 (2019).
pubmed: 30653986
doi: 10.1016/j.ophtha.2018.12.050
Dockery, A. et al. Target 5000: target capture sequencing for inherited retinal degenerations. Genes 8, https://doi.org/10.3390/genes8110304 (2017).
Lin, X. et al. Applications of targeted gene capture and next-generation sequencing technologies in studies of human deafness and other genetic disabilities. Hear. Res. 288, 67–76 (2012).
pubmed: 22269275
doi: 10.1016/j.heares.2012.01.004
Tucker, T., Marra, M. & Friedman, J. M. Massively parallel sequencing: the next big thing in genetic medicine. Am. J. Hum. Genet. 85, 142–154 (2009).
pubmed: 19679224
pmcid: 2725244
doi: 10.1016/j.ajhg.2009.06.022
Choi, M. et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc. Natl Acad. Sci. USA 106, 19096–19101 (2009).
pubmed: 19861545
pmcid: 2768590
doi: 10.1073/pnas.0910672106
Lewis, C. A. et al. Tubby-like protein 1 homozygous splice-site mutation causes early-onset severe retinal degeneration. Invest. Ophthalmol. Vis. Sci. 40, 2106–2114 (1999).
pubmed: 10440267
Stojic, J., Stöhr, H. & Weber, B. H. Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression. BMC Mol. Biol. 8, 42 (2007).
pubmed: 17521428
pmcid: 1890297
doi: 10.1186/1471-2199-8-42
Khan, M. et al. Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics. Genet. Med. 22, 1235–1246 (2020).
pubmed: 32307445
doi: 10.1038/s41436-020-0787-4
Haer-Wigman, L. et al. Diagnostic exome sequencing in 266 Dutch patients with visual impairment. Eur. J. Hum. Genet. 25, 591–599 (2017).
pubmed: 28224992
pmcid: 5437915
doi: 10.1038/ejhg.2017.9
Anna, A. & Monika, G. Splicing mutations in human genetic disorders: examples, detection, and confirmation. J. Appl. Genet. 59, 253–268 (2018).
pubmed: 29680930
pmcid: 6060985
doi: 10.1007/s13353-018-0444-7
Fadaie, Z. et al. Identification of splice defects due to noncanonical splice site or deep-intronic variants in ABCA4. Hum. Mutat. 40, 2365–2376 (2019).
pubmed: 31397521
pmcid: 6899986
doi: 10.1002/humu.23890
Carss, K. J. et al. Comprehensive rare variant analysis via whole-genome sequencing to determine the molecular pathology of inherited retinal disease. Am. J. Hum. Genet. 100, 75–90 (2017).
pubmed: 28041643
doi: 10.1016/j.ajhg.2016.12.003
Belkadi, A. et al. Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants. Proc. Natl Acad. Sci. USA 112, 5473–5478 (2015).
pubmed: 25827230
pmcid: 4418901
doi: 10.1073/pnas.1418631112
Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016).
pubmed: 27535533
pmcid: 5018207
doi: 10.1038/nature19057
Karczewski, K. J. et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581, 434–443 (2020).
pubmed: 32461654
pmcid: 7334197
doi: 10.1038/s41586-020-2308-7
Richards, S. et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405–424 (2015).
pubmed: 25741868
pmcid: 4544753
doi: 10.1038/gim.2015.30
Wallis, Y. et al. Practice Guidelines for the Evaluation of Pathogenicity and the Reporting of Sequence Variants in Clinical Molecular Genetics 1–16 (Association for Clinical Genetic Science & Dutch Society of Clinical Genetic Laboratory Specialists, 2013).
Gardiner, S. L. et al. Large normal-range TBP and ATXN7 CAG repeat lengths are associated with increased lifetime risk of depression. Transl. Psychiatry 7, e1143 (2017).
pubmed: 28585930
pmcid: 5534943
doi: 10.1038/tp.2017.116
Stingl, K. et al. CDHR1 mutations in retinal dystrophies. Sci. Rep. 7, 6992 (2017).
pubmed: 28765526
pmcid: 5539332
doi: 10.1038/s41598-017-07117-8
Khan, M. et al. Detailed phenotyping and therapeutic strategies for intronic ABCA4 variants in Stargardt disease. Mol. Ther. Nucleic Acids 21, 412–427 (2020).
pubmed: 32653833
pmcid: 7352060
doi: 10.1016/j.omtn.2020.06.007
Braun, T. A. et al. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136–5145 (2013).
pubmed: 23918662
pmcid: 3842174
doi: 10.1093/hmg/ddt367
Cremers, F. P. et al. Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt’s disease gene ABCR. Hum. Mol. Genet. 7, 355–362 (1998).
pubmed: 9466990
doi: 10.1093/hmg/7.3.355
Sangermano, R. et al. Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides. Genet. Med. 21, 1751–1760 (2019).
pubmed: 30643219
pmcid: 6752325
doi: 10.1038/s41436-018-0414-9
Weisschuh, N. et al. Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation. Hum. Mutat. 41, 255–264 (2020).
pubmed: 31544997
doi: 10.1002/humu.23920
Weisschuh, N., Buena-Atienza, E. & Wissinger, B. Splicing mutations in inherited retinal diseases. Prog. Retin. Eye Res. 80, 100874 (2021).
pubmed: 32553897
doi: 10.1016/j.preteyeres.2020.100874
Di Scipio, M. et al. Phenotype driven analysis of whole genome sequencing identifies deep intronic variants that cause retinal dystrophies by aberrant exonization. Invest. Ophthalmol. Vis. Sci. 61, 36 (2020).
pubmed: 32881472
pmcid: 7443117
doi: 10.1167/iovs.61.10.36
Di, Y. et al. Whole-exome sequencing analysis identifies mutations in the EYS gene in retinitis pigmentosa in the Indian population. Sci. Rep. 6, 19432 (2016).
pubmed: 26787102
pmcid: 4726297
doi: 10.1038/srep19432
Schiff, E. R. et al. A genetic and clinical study of individuals with nonsyndromic retinopathy consequent upon sequence variants in HGSNAT, the gene associated with Sanfilippo C mucopolysaccharidosis. Am. J. Med. Genet. C 184, 631–643 (2020).
doi: 10.1002/ajmg.c.31822
Gerber, S. et al. Complete exon-intron structure of the retina-specific ATP binding transporter gene (ABCR) allows the identification of novel mutations underlying Stargardt disease. Genomics 48, 139–142 (1998).
pubmed: 9503029
doi: 10.1006/geno.1997.5164
González-Del Pozo, M. et al. Unmasking retinitis pigmentosa complex cases by a whole genome sequencing algorithm based on open-access tools: hidden recessive inheritance and potential oligogenic variants. J. Transl. Med. 18, 73 (2020).
pubmed: 32050993
pmcid: 7014749
doi: 10.1186/s12967-020-02258-3
Bhatia, S., Goyal, S., Singh, I. R., Singh, D. & Vanita, V. A novel mutation in the PRPF31 in a North Indian adRP family with incomplete penetrance. Doc. Ophthalmol. 137, 103–119 (2018).
pubmed: 30099644
doi: 10.1007/s10633-018-9654-x
Rose, A. M. & Bhattacharya, S. S. Variant haploinsufficiency and phenotypic non-penetrance in PRPF31-associated retinitis pigmentosa. Clin. Genet. 90, 118–126 (2016).
pubmed: 26853529
doi: 10.1111/cge.12758
Vithana, E. N. et al. Expression of PRPF31 mRNA in patients with autosomal dominant retinitis pigmentosa: a molecular clue for incomplete penetrance? Invest. Ophthalmol. Vis. Sci. 44, 4204–4209 (2003).
pubmed: 14507862
doi: 10.1167/iovs.03-0253
Brambillasca, F. et al. Promoter analysis of TFPT (FB1), a molecular partner of TCF3 (E2A) in childhood acute lymphoblastic leukemia. Biochem. Biophys. Res. Commun. 288, 1250–1257 (2001).
pubmed: 11700047
doi: 10.1006/bbrc.2001.5906
Rak, M. & Rustin, P. Supernumerary subunits NDUFA3, NDUFA5 and NDUFA12 are required for the formation of the extramembrane arm of human mitochondrial complex I. FEBS Lett. 588, 1832–1838 (2014).
pubmed: 24717771
doi: 10.1016/j.febslet.2014.03.046
Rose, A. M., Mukhopadhyay, R., Webster, A. R., Bhattacharya, S. S. & Waseem, N. H. A 112 kb deletion in chromosome 19q13.42 leads to retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 52, 6597–6603 (2011).
pubmed: 21715351
doi: 10.1167/iovs.11-7861
Radziwon, A. et al. Single-base substitutions in the CHM promoter as a cause of choroideremia. Hum. Mutat. 38, 704–715 (2017).
pubmed: 28271586
doi: 10.1002/humu.23212
Bauwens, M. et al. ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants. Genet. Med. 21, 1761–1771 (2019).
pubmed: 30670881
pmcid: 6752479
doi: 10.1038/s41436-018-0420-y
Runhart, E. H. et al. Association of sex with frequent and mild ABCA4 alleles in Stargardt disease. JAMA Ophthalmol. 138, 1035–1042 (2020).
pubmed: 32815999
pmcid: 7441467
doi: 10.1001/jamaophthalmol.2020.2990
Maggi, J. et al. De novo assembly-based analysis of RPGR exon ORF15 in an Indigenous African Cohort overcomes limitations of a standard next-generation sequencing (NGS) data analysis pipeline. Genes 11, https://doi.org/10.3390/genes11070800 (2020).
Diekstra, A. et al. Translating sanger-based routine DNA diagnostics into generic massive parallel ion semiconductor sequencing. Clin. Chem. 61, 154–162 (2015).
pubmed: 25274553
doi: 10.1373/clinchem.2014.225250
Weisschuh, N. et al. Molecular and clinical analysis of 27 German patients with Leber congenital amaurosis. PLoS ONE 13, e0205380 (2018).
pubmed: 30576320
pmcid: 6303042
doi: 10.1371/journal.pone.0205380
Sharon, D. et al. A nationwide genetic analysis of inherited retinal diseases in Israel as assessed by the Israeli inherited retinal disease consortium (IIRDC). Hum. Mutat. 41, 140–149 (2020).
pubmed: 31456290
doi: 10.1002/humu.23903
Okonechnikov, K., Conesa, A. & García-Alcalde, F. Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics 32, 292–294 (2016).
pubmed: 26428292
doi: 10.1093/bioinformatics/btv566
McLaren, W. et al. The Ensembl variant effect predictor. Genome Biol. 17, 122 (2016).
pubmed: 27268795
pmcid: 4893825
doi: 10.1186/s13059-016-0974-4
The Genome of the Netherlands Consortium. Whole-genome sequence variation, population structure and demographic history of the Dutch population. Nat. Genet. 46, 818–825 (2014).
Erikson, G. A. et al. Whole-genome sequencing of a healthy aging cohort. Cell 165, 1002–1011 (2016).
pubmed: 27114037
pmcid: 4860090
doi: 10.1016/j.cell.2016.03.022
Abecasis, G. R. et al. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012).
pubmed: 23128226
doi: 10.1038/nature11632
Pollard, K. S., Hubisz, M. J., Rosenbloom, K. R. & Siepel, A. Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 20, 110–121 (2010).
pubmed: 19858363
pmcid: 2798823
doi: 10.1101/gr.097857.109
Kircher, M. et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet. 46, 310–315 (2014).
pubmed: 24487276
pmcid: 3992975
doi: 10.1038/ng.2892
Grantham, R. Amino acid difference formula to help explain protein evolution. Science 185, 862–864 (1974).
pubmed: 4843792
doi: 10.1126/science.185.4154.862
Jaganathan, K. et al. Predicting splicing from primary sequence with deep learning. Cell 176, 535–548.e524 (2019).
pubmed: 30661751
doi: 10.1016/j.cell.2018.12.015
Farek, J. et al. xAtlas: scalable small variant calling across heterogeneous next-generation sequencing experiments. Preprint at bioRxiv https://doi.org/10.1101/295071 (2018).
Chen, X. et al. Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics 32, 1220–1222 (2016).
pubmed: 26647377
doi: 10.1093/bioinformatics/btv710
Boeva, V. et al. Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data. Bioinformatics 28, 423–425 (2012).
pubmed: 22155870
doi: 10.1093/bioinformatics/btr670
Verbakel, S. K. et al. The identification of a RNA splice variant in TULP1 in two siblings with early-onset photoreceptor dystrophy. Mol. Genet. Genom. Med. 7, e660 (2019).
doi: 10.1002/mgg3.660
Firth, H. V. et al. DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources. Am. J. Hum. Genet. 84, 524–533 (2009).
pubmed: 19344873
pmcid: 2667985
doi: 10.1016/j.ajhg.2009.03.010
Dolzhenko, E. et al. Detection of long repeat expansions from PCR-free whole-genome sequence data. Genome Res. 27, 1895–1903 (2017).
pubmed: 28887402
pmcid: 5668946
doi: 10.1101/gr.225672.117
Allaire, J. RStudio: integrated development environment for R. RStudio 770, 394 (2012).
Shapiro, M. B. & Senapathy, P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15, 7155–7174 (1987).
pubmed: 3658675
pmcid: 306199
doi: 10.1093/nar/15.17.7155
Reese, M. G., Eeckman, F. H., Kulp, D. & Haussler, D. Improved splice site detection in Genie. J. Comput. Biol. 4, 311–323 (1997).
pubmed: 9278062
doi: 10.1089/cmb.1997.4.311
Pertea, M., Lin, X. & Salzberg, S. L. GeneSplicer: a new computational method for splice site prediction. Nucleic Acids Res. 29, 1185–1190 (2001).
pubmed: 11222768
pmcid: 29713
doi: 10.1093/nar/29.5.1185
Yeo, G. & Burge, C. B. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol. 11, 377–394 (2004).
pubmed: 15285897
doi: 10.1089/1066527041410418
Desmet, F. O. et al. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 37, e67 (2009).
pubmed: 19339519
pmcid: 2685110
doi: 10.1093/nar/gkp215
Sangermano, R. et al. ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease. Genome Res. 28, 100–110 (2018).
pubmed: 29162642
pmcid: 5749174
doi: 10.1101/gr.226621.117