Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa

Suzanne E de Bruijn, Alessia Fiorentino, Daniele Ottaviani, Stephanie Fanucchi, Uirá S Melo, Julio C Corral-Serrano, Timo Mulders, Michalis Georgiou, Carlo Rivolta, Nikolas Pontikos, Gavin Arno, Lisa Roberts, Jacquie Greenberg, Silvia Albert, Christian Gilissen, Marco Aben, George Rebello, Simon Mead, F Lucy Raymond, Jordi Corominas, Claire E L Smith, Hannie Kremer, Susan Downes, Graeme C Black, Andrew R Webster, Chris F Inglehearn, L Ingeborgh van den Born, Robert K Koenekoop, Michel Michaelides, Raj S Ramesar, Carel B Hoyng, Stefan Mundlos, Musa M Mhlanga, Frans P M Cremers, Michael E Cheetham, Susanne Roosing, Alison J Hardcastle, Suzanne E de Bruijn, Alessia Fiorentino, Daniele Ottaviani, Stephanie Fanucchi, Uirá S Melo, Julio C Corral-Serrano, Timo Mulders, Michalis Georgiou, Carlo Rivolta, Nikolas Pontikos, Gavin Arno, Lisa Roberts, Jacquie Greenberg, Silvia Albert, Christian Gilissen, Marco Aben, George Rebello, Simon Mead, F Lucy Raymond, Jordi Corominas, Claire E L Smith, Hannie Kremer, Susan Downes, Graeme C Black, Andrew R Webster, Chris F Inglehearn, L Ingeborgh van den Born, Robert K Koenekoop, Michel Michaelides, Raj S Ramesar, Carel B Hoyng, Stefan Mundlos, Musa M Mhlanga, Frans P M Cremers, Michael E Cheetham, Susanne Roosing, Alison J Hardcastle

Abstract

The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.

Keywords: GDPD; Hi-C; RP17; dominant retinitis pigmentosa; ectopic expression; photoreceptor precursors cells; retinal organoids; stem cells; structural variants; topologically associated domains; whole-genome sequencing.

Conflict of interest statement

The authors declare no competing interests.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1
Figure 1
Mapping of the RP17 Locus in Two Unrelated Families (A) Pedigree of Dutch NL1 family. (B) Pedigree of UK1 family. (C) Pedigrees of additional UK families with the founder haplotype on Chr17q. WGS or WES was performed in individuals highlighted in blue or red, respectively. (D) SNP haplotyping results for NL1. The refined RP17 locus (rs8078110–rs9910672) is shared by all affected individuals (n = 35) and not present in unaffected individuals (n = 28, only individuals with recombination close to or refining the critical region are depicted) with a maximum LOD score of 15.0. The horizontal numbers represent the number of individuals with this haplotype. (E) UK founder haplotype refining the RP17 locus in UK families. Representative haplotypes from several unrelated families are shown with affected (aff) individuals compared to an unaffected (unaff) individual. Black lines and arrows indicate recombination events. Shared haplotype in individuals is shaded red. (F) Overlap of refined RP17 loci in UK, NL, and previously described SA families.
Figure 2
Figure 2
Overview of Structural Variants within the RP17 Locus in adRP-Affected Families Breakpoints are indicated with dashed lines. Blue segments represent duplicated or triplicated regions, whereas inversions are highlighted in purple. (A) Wild-type (WT) chromosomal organization. (B) Structural variants identified in NL1 (NL-SV1) and UK founder haplotype families (UK-SV2). (C) Structural variants identified in adRP-affected families that were previously linked to the RP17 locus; SA-SV3 and CA-SV4 (unpublished data). (D) Structural variants found in a cohort of unsolved adRP-affected families; NL-SV5, UK-SV6, UK-SV7, and UK-SV8. Letters A–AI depict the genomic intervals for each SV used to analyze and annotate SV breakpoints. (E) Overview of all SV breakpoints identified in the RP17 locus. An overlapping genomic region that is duplicated or triplicated in all SVs was identified (chr17: 57,499,214–57,510,765) and is highlighted by a light blue vertical bar. The size of DHX40 is reduced, and CLTC is partially shown for the purpose of this figure.
Figure 3
Figure 3
YPEL2 is Located within a Structured Active Compartment that Contains Retinal-Specific Enhancers (A) The TAD landscape of the genomic region disrupted by the RP17 SVs. Hi-C map of control retinal organoids revealed a structured domain containing YPEL2. (B) YPEL2 TAD boundaries correspond with CTCF sites identified in human retina. Analysis of RNA-seq and assay for transposase-accessible chromatin using seqencing (ATAC-seq) data across the YPEL2 region shows YPEL2 retinal expression and an accessible chromatin configuration. Analysis of H3K27Ac ChIP-seq data in the same region revealed several active enhancers located within the YPEL2 TAD, which are enriched for retinal transcription factor binding sites, including NRL, CRX, and OTX2. These enhancers were located 5′ of the CTCF boundary site within LINC01476. (C) Schematic representation of the YPEL2 TAD structure.
Figure 4
Figure 4
RP17 SVs Create Novel Domains (neo-TADs) and Hyper-activation of Retinal Enhancers (A) Schematic modeling of the genome architecture spanning the RP17 region using Hi-C maps. The wild-type Hi-C map derived from neuronal tissue shows a TAD with CTCF boundaries containing YPEL2 and retinal enhancers, flanked by unstructured domains. TAD models of NL-SV1 and UK-SV2 (dotted vertical lines represent SV breakpoints) predict the formation of neo-TADs and ectopic interactions of the retinal enhancer with GDPD1. (B) Hi-C performed on retinal organoids (ROs) derived from control (top) and RP17 UK-SV2 individuals (bottom) (10 kb resolution; raw count map). The chromatin organization in control ROs shows the YPEL2 TAD (indicated by dashed lines). Two novel domains (neo-TAD 1 and 2) are visible in the UK-SV2 ROs, and neo-TAD 2 allows ectopic retinal enhancer contacts to GDPD1 and SMG8. The dashed circle indicates the strong chromatin contact between retinal enhancers and the GDPD1 promoter. (C) qPCR revealed significantly upregulated retinal enhancer RNA expression in UK-SV2 ROs compared to controls (n = 3, mean ± standard error of the mean, **p ≤ 0.01).
Figure 5
Figure 5
Convergent Mechanism of Ectopic Retinal Enhancer-GDPD1 Interaction Caused by RP17 SVs (A) In wild-type genomic context, YPEL2 expression in retina is driven by retinal enhancers in a TAD with CTCF boundaries. Neighboring genes are insulated from retinal enhancer activation. (B) The NL-SV1 duplication creates a neo-TAD with a full-length copy of YPEL2, GDPD1, and the retinal enhancers. This enables retinal-specific enhancers to ectopically interact with GDPD1, which drives its misexpression. (C) qPCR analysis of photoreceptor precursor cells (PPCs) revealed a significant upregulation of GDPD1 in NL-SV1 PPCs compared to controls. (D) The UK-SV2 duplication and inversion creates a neo-TAD with a full-length copy of GDPD1 and SMG8 and the retinal enhancers bounded by CTFC sites. (E) qPCR analysis ROs revealed a significant upregulation of GDPD1 expression in UK-SV2 ROs compared to controls (n = 3 independent ROs, mean ± standard error of the mean, **p ≤ 0.01, ****p ≤ 0.0001).

References

    1. Haer-Wigman L., van Zelst-Stams W.A., Pfundt R., van den Born L.I., Klaver C.C., Verheij J.B., Hoyng C.B., Breuning M.H., Boon C.J., Kievit A.J. Diagnostic exome sequencing in 266 Dutch patients with visual impairment. Eur. J. Hum. Genet. 2017;25:591–599.
    1. Verbakel S.K., van Huet R.A.C., Boon C.J.F., den Hollander A.I., Collin R.W.J., Klaver C.C.W., Hoyng C.B., Roepman R., Klevering B.J. Non-syndromic retinitis pigmentosa. Prog. Retin. Eye Res. 2018;66:157–186.
    1. Hamel C. Retinitis pigmentosa. Orphanet J. Rare Dis. 2006;1:40.
    1. Sullivan L.S., Bowne S.J., Reeves M.J., Blain D., Goetz K., Ndifor V., Vitez S., Wang X., Tumminia S.J., Daiger S.P. Prevalence of mutations in eyeGENE probands with a diagnosis of autosomal dominant retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2013;54:6255–6261.
    1. Daiger S.P., Bowne S.J., Sullivan L.S. Genes and Mutations Causing Autosomal Dominant Retinitis Pigmentosa. Cold Spring Harb. Perspect. Med. 2014;5:a017129.
    1. Bardien S., Ebenezer N., Greenberg J., Inglehearn C.F., Bartmann L., Goliath R., Beighton P., Ramesar R., Bhattacharya S.S. An eighth locus for autosomal dominant retinitis pigmentosa is linked to chromosome 17q. Hum. Mol. Genet. 1995;4:1459–1462.
    1. den Hollander A.I., van der Velde-Visser S.D., Pinckers A.J.L.G., Hoyng C.B., Brunner H.G., Cremers F.P.M. Refined mapping of the gene for autosomal dominant retinitis pigmentosa (RP17) on chromosome 17q22. Hum. Genet. 1999;104:73–76.
    1. Rebello G., Ramesar R., Vorster A., Roberts L., Ehrenreich L., Oppon E., Gama D., Bardien S., Greenberg J., Bonapace G. Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa. Proc. Natl. Acad. Sci. USA. 2004;101:6617–6622.
    1. Köhn L., Burstedt M.S.I., Jonsson F., Kadzhaev K., Haamer E., Sandgren O., Golovleva I. Carrier of R14W in carbonic anhydrase IV presents Bothnia dystrophy phenotype caused by two allelic mutations in RLBP1. Invest. Ophthalmol. Vis. Sci. 2008;49:3172–3177.
    1. Golovleva I., Köhn L., Burstedt M., Daiger S., Sandgren O. Mutation spectra in autosomal dominant and recessive retinitis pigmentosa in northern Sweden. Adv. Exp. Med. Biol. 2010;664:255–262.
    1. Rao S.S.P., Huntley M.H., Durand N.C., Stamenova E.K., Bochkov I.D., Robinson J.T., Sanborn A.L., Machol I., Omer A.D., Lander E.S., Aiden E.L. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159:1665–1680.
    1. Spielmann M., Lupiáñez D.G., Mundlos S. Structural variation in the 3D genome. Nat. Rev. Genet. 2018;19:453–467.
    1. Franke M., Ibrahim D.M., Andrey G., Schwarzer W., Heinrich V., Schöpflin R., Kraft K., Kempfer R., Jerković I., Chan W.-L. Formation of new chromatin domains determines pathogenicity of genomic duplications. Nature. 2016;538:265–269.
    1. Lupiáñez D.G., Kraft K., Heinrich V., Krawitz P., Brancati F., Klopocki E., Horn D., Kayserili H., Opitz J.M., Laxova R. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell. 2015;161:1012–1025.
    1. Ibrahim D.M., Mundlos S. Three-dimensional chromatin in disease: What holds us together and what drives us apart? Curr. Opin. Cell Biol. 2020;64:1–9.
    1. Albert S., Garanto A., Sangermano R., Khan M., Bax N.M., Hoyng C.B., Zernant J., Lee W., Allikmets R., Collin R.W.J., Cremers F.P.M. 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.
    1. Sangermano R., Bax N.M., Bauwens M., van den Born L.I., De Baere E., Garanto A. Photoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T>C Mutation in Stargardt Disease. Ophthalmology. 2016;123:1375–1385.
    1. Schwarz N., Lane A., Jovanovic K., Parfitt D.A., Aguila M., Thompson C.L., da Cruz L., Coffey P.J., Chapple J.P., Hardcastle A.J., Cheetham M.E. Arl3 and RP2 regulate the trafficking of ciliary tip kinesins. Hum. Mol. Genet. 2017;26:2480–2492.
    1. Díaz N., Kruse K., Erdmann T., Staiger A.M., Ott G., Lenz G., Vaquerizas J.M. Chromatin conformation analysis of primary patient tissue using a low input Hi-C method. Nat. Commun. 2018;9:4938.
    1. Durand N.C., Shamim M.S., Machol I., Rao S.S.P., Huntley M.H., Lander E.S., Aiden E.L. Juicer Provides a One-Click System for Analyzing Loop-Resolution Hi-C Experiments. Cell Syst. 2016;3:95–98.
    1. Melo U.S., Schöpflin R., Acuna-Hidalgo R., Mensah M.A., Fischer-Zirnsak B., Holtgrewe M., Klever M.K., Türkmen S., Heinrich V., Pluym I.D. Hi-C identifies complex genomic rearrangements and TAD-shuffling in developmental diseases. Am. J. Hum. Genet. 2020;106:872–884.
    1. Lukowski S.W., Lo C.Y., Sharov A.A., Nguyen Q., Fang L., Hung S.S., Zhu L., Zhang T., Grünert U., Nguyen T. A single-cell transcriptome atlas of the adult human retina. EMBO J. 2019;38:e100811.
    1. Peng Y.-R., Shekhar K., Yan W., Herrmann D., Sappington A., Bryman G.S., van Zyl T., Do M.T.H., Regev A., Sanes J.R. Molecular Classification and Comparative Taxonomics of Foveal and Peripheral Cells in Primate Retina. Cell. 2019;176:1222–1237.e22.
    1. Dixon J.R., Selvaraj S., Yue F., Kim A., Li Y., Shen Y., Hu M., Liu J.S., Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012;485:376–380.
    1. Cherry T.J., Yang M.G., Harmin D.A., Tao P., Timms A.E., Bauwens M., Allikmets R., Jones E.M., Chen R., De Baere E. Mapping the cis-regulatory architecture of the human retina reveals noncoding genetic variation in disease. Proceedings of the National Academy of Sciences. 2020;117:9001–9012.
    1. Fishilevich S., Nudel R., Rappaport N., Hadar R., Plaschkes I., Iny Stein T., Rosen N., Kohn A., Twik M., Safran M. GeneHancer: genome-wide integration of enhancers and target genes in GeneCards. Database (Oxford) 2017;2017:bax028.
    1. Conrad D.F., Bird C., Blackburne B., Lindsay S., Mamanova L., Lee C., Turner D.J., Hurles M.E. Mutation spectrum revealed by breakpoint sequencing of human germline CNVs. Nat. Genet. 2010;42:385–391.
    1. Sudmant P.H., Rausch T., Gardner E.J., Handsaker R.E., Abyzov A., Huddleston J., Zhang Y., Ye K., Jun G., Fritz M.H.-Y., 1000 Genomes Project Consortium An integrated map of structural variation in 2,504 human genomes. Nature. 2015;526:75–81.
    1. Karczewski K.J., Francioli L.C., Tiao G., Cummings B.B., Alföldi J., Wang Q. Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. Nature. 2020;581:434–443.
    1. MacDonald J.R., Ziman R., Yuen R.K.C., Feuk L., Scherer S.W. The Database of Genomic Variants: a curated collection of structural variation in the human genome. Nucleic Acids Res. 2014;42:D986–D992.
    1. Lohan S., Spielmann M., Doelken S.C., Flöttmann R., Muhammad F., Baig S.M., Wajid M., Hülsemann W., Habenicht R., Kjaer K.W. Microduplications encompassing the Sonic hedgehog limb enhancer ZRS are associated with Haas-type polysyndactyly and Laurin-Sandrow syndrome. Clin. Genet. 2014;86:318–325.
    1. Ngcungcu T., Oti M., Sitek J.C., Haukanes B.I., Linghu B., Bruccoleri R., Stokowy T., Oakeley E.J., Yang F., Zhu J. Duplicated Enhancer Region Increases Expression of CTSB and Segregates with Keratolytic Winter Erythema in South African and Norwegian Families. Am. J. Hum. Genet. 2017;100:737–750.
    1. Gu S., Yuan B., Campbell I.M., Beck C.R., Carvalho C.M.B., Nagamani S.C.S., Erez A., Patel A., Bacino C.A., Shaw C.A. Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3. Hum. Mol. Genet. 2015;24:4061–4077.
    1. Zhang F., Khajavi M., Connolly A.M., Towne C.F., Batish S.D., Lupski J.R. The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans. Nat. Genet. 2009;41:849–853.
    1. Lupski J.R. Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet. 1998;14:417–422.
    1. Lieber M.R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 2010;79:181–211.
    1. Ohshima N., Kudo T., Yamashita Y., Mariggiò S., Araki M., Honda A., Nagano T., Isaji C., Kato N., Corda D. New members of the mammalian glycerophosphodiester phosphodiesterase family: GDE4 and GDE7 produce lysophosphatidic acid by lysophospholipase D activity. J. Biol. Chem. 2015;290:4260–4271.
    1. Tsuboi K., Okamoto Y., Rahman I.A.S., Uyama T., Inoue T., Tokumura A., Ueda N. Glycerophosphodiesterase GDE4 as a novel lysophospholipase D: a possible involvement in bioactive N-acylethanolamine biosynthesis. Biochim. Biophys. Acta. 2015;1851:537–548.
    1. Friedman J.S., Chang B., Krauth D.S., Lopez I., Waseem N.H., Hurd R.E., Feathers K.L., Branham K.E., Shaw M., Thomas G.E. Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice. Proc. Natl. Acad. Sci. USA. 2010;107:15523–15528.
    1. Kmoch S., Majewski J., Ramamurthy V., Cao S., Fahiminiya S., Ren H., MacDonald I.M., Lopez I., Sun V., Keser V., Care4Rare Canada Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness. Nat. Commun. 2015;6:5614.
    1. Lidgerwood G.E., Morris A.J., Conquest A., Daniszewski M., Rooney L.A., Lim S.Y., Hernández D., Liang H.H., Allen P., Connell P.P. Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2018;1863:750–761.

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