Abstract
Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients’ features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.
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All data are fully available without restriction. Nucleotide sequence data reported have been submitted to GenBank to obtain the corresponding accession numbers.
References
Ali M, McKibbin M, Booth A, Parry DA, Jain P, Riazuddin SA, Hejtmancik JF, Khan SN, Firasat S, Shires M, Gilmour DF, Towns K, Murphy AL, Azmanov D, Tournev I, Cherninkova S, Jafri H, Raashid Y, Toomes C, Craig J, Mackey DA, Kalaydjieva L, Riazuddin S, Inglehearn CF (2009) Null mutations in LTBP2 cause primary congenital glaucoma. Am J Hum Genet 84:664–671
Alsaif HS, Khan AO, Patel N, Alkuraya H, Hashem M, Abdulwahab F, Ibrahim N, Aldahmesh MA, Alkuraya FS (2019) Congenital glaucoma and CYP1B1: an old story revisited. Hum Genet 138:1043–1049. https://doi.org/10.1007/s00439-018-1878-z
Aroca-Aguilar J-D, Martinez-Redondo F, Martin-Gil A, Pintor J, Coca-Prados M, Escribano J (2013) Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin. PLoS ONE. https://doi.org/10.1371/journal.pone.0054385
Bhattacharya D, Marfo CA, Li D, Lane M, Khokha MK (2015) CRISPR/Cas9: an inexpensive, efficient loss of function tool to screen human disease genes in Xenopus. Dev Biol 408:196–204. https://doi.org/10.1016/j.ydbio.2015.11.003
Campos-Mollo E, Lopez-Garrido M-P, Blanco-Marchite C, Garcia-Feijoo J, Peralta J, Belmonte-Martinez J, Ayuso C, Escribano J (2009) CYP1B1 mutations in Spanish patients with primary congenital glaucoma: phenotypic and functional variability. Mol Vis 15:417–431
Ceroni F et al (2019) New GJA8 variants and phenotypes highlight its critical role in a broad spectrum of eye anomalies. Hum Genet 138:1027–1042. https://doi.org/10.1007/s00439-018-1875-2
Chavarria-Soley G, Michels-Rautenstrauss K, Caliebe A, Kautza M, Mardin C, Rautenstrauss B (2006) Novel CYP1B1 and known PAX6 mutations in anterior segment dysgenesis (ASD). J Glaucoma 15:499–504
Cheong SS et al (2016) Mutations in CPAMD8 cause a unique form of autosomal-recessive anterior segment dysgenesis. Am J Hum Genet 99:1338–1352. https://doi.org/10.1016/j.ajhg.2016.09.022
Coca-Prados M, Escribano J (2007) New perspectives in aqueous humor secretion and in glaucoma: the ciliary body as a multifunctional neuroendocrine gland. Prog Retin Eye Res 26:239–262
Coca-Prados M, Escribano J, Ortego J (1999) Differential gene expression in the human ciliary epithelium. Prog Retin Eye Res 18:403–429
Colomb E, Kaplan J, Garchon HJ (2003) Novel cytochrome P450 1B1 (CYP1B1) mutations in patients with primary congenital glaucoma in France. Hum Mutat 22:496
Creuzet S, Vincent C, Couly G (2005) Neural crest derivatives in ocular and periocular structures. Int J Dev Biol 49:161–171. https://doi.org/10.1387/ijdb.041937sc
El-Brolosy MA et al (2019) Genetic compensation triggered by mutant mRNA degradation. Nature 568:193–197. https://doi.org/10.1038/s41586-019-1064-z
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516. https://doi.org/10.1080/01926230701320337
Escribano J, Ortego J, Coca-Prados M (1995) Isolation and characterization of cell-specific cDNA clones from a subtractive library of the ocular ciliary body of a single normal human donor: transcription and synthesis of plasma proteins. J Biochem (Tokyo) 118:921–931
Ferre-Fernández JJ et al (2017) Whole-exome sequencing of congenital glaucoma patients reveals hypermorphic variants in GPATCH3, a new gene involved in ocular and craniofacial development. Sci Rep 7:46175. https://doi.org/10.1038/srep46175
Garcia-Anton MT et al (2017) Goniodysgenesis variability and activity of CYP1B1 genotypes in primary congenital glaucoma. PLoS ONE. https://doi.org/10.1371/journal.pone.0176386
Goel R et al (2013) Characterizing the normal proteome of human ciliary body. Clin Proteomics 10:9. https://doi.org/10.1186/1559-0275-10-9
Gonias SL (1992) Alpha 2-macroglobulin: a protein at the interface of fibrinolysis and cellular growth regulation. Exp Hematol 20:302–311
Gould DB, John SW (2002) Anterior segment dysgenesis and the developmental glaucomas are complex traits. Hum Mol Genet 11:1185–1193
Gould DB, Smith RS, John SW (2004) Anterior segment development relevant to glaucoma. Int J Dev Biol 48:1015–1029
Hanson IM et al (1994) Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly. Nat Genet 6:168–173. https://doi.org/10.1038/ng0294-168
Hendee KE et al (2018) PITX2 deficiency and associated human disease: insights from the zebrafish model. Hum Mol Genet 27:1675–1695. https://doi.org/10.1093/hmg/ddy074
Hollmann AK et al (2017) Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development. PLoS ONE 12:e0180665. https://doi.org/10.1371/journal.pone.0180665
Janssen SF, Gorgels TG, Ten Brink JB, Jansonius NM, Bergen AA (2014) Gene expression-based comparison of the human secretory neuroepithelia of the brain choroid plexus and the ocular ciliary body: potential implications for glaucoma. Fluids Barriers CNS 11:2. https://doi.org/10.1186/2045-8118-11-2
Jordan T et al (1992) The human PAX6 gene is mutated in two patients with aniridia. Nat Genet 1:328–332. https://doi.org/10.1038/ng0892-328
Kaur K et al (2005) Myocilin gene implicated in primary congenital glaucoma. Clin Genet 67:335–340
Kupfer C, Kaiser-Kupfer MI (1978) New hypothesis of developmental anomalies of the anterior chamber associated with glaucoma. Trans Ophthalmol Soc UK 98:213–215
Kupfer C, Kaiser-Kupfer MI (1979) Observations on the development of the anterior chamber angle with reference to the pathogenesis of congenital glaucomas. Am J Ophthalmol 88:424–426
Li ZF, Wu XH, Engvall E (2004) Identification and characterization of CPAMD8, a novel member of the complement 3/alpha2-macroglobulin family with a C-terminal Kazal domain. Genomics 83:1083–1093. https://doi.org/10.1016/j.ygeno.2003.12.005
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
López-Garrido M-P, Medina-Trillo C, Morales-Fernandez L, Garcia-Feijoo J, Martínez-De-La-Casa J-M, García-Antón M, Escribano J (2013) Null CYP1B1 genotypes in primary congenital and nondominant juvenile glaucoma. Ophthalmology 120:716–723. https://doi.org/10.1016/j.ophtha.2012.09.016
Medina-Trillo C et al (2016) Rare FOXC1 variants in congenital glaucoma: identification of translation regulatory sequences. Eur J Hum Genet 24:672–680. https://doi.org/10.1038/ejhg.2015.169
Medina-Trillo C et al (2015) Hypo- and hypermorphic FOXC1 mutations in dominant glaucoma: transactivation and phenotypic variability. PLoS ONE 10:e0119272. https://doi.org/10.1371/journal.pone.0119272
Murthy KR et al (2015) Proteomics of human aqueous humor. OMICS 19:283–293. https://doi.org/10.1089/omi.2015.0029
Nishimura DY et al (1998) The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nat Genet 19:140–147
Ortego J, Escribano J, Coca-Prados M (1997) Gene expression of proteases and protease inhibitors in the human ciliary epithelium and ODM-2 cells. Exp Eye Res 65:289–299
Pagani F et al (2003) New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12. Hum Mol Genet 12:1111–1120. https://doi.org/10.1093/hmg/ddg131
Papadopoulos M et al (2013) Establishing the diagnosis and determining glaucoma progression. In: Weinreb R, Grajewski A, Papadopoulos M, Grigg J, Freedman S (eds) Childhood glaucoma. Kluger Publications, Amsterdam, pp 15–41
Polansky JR, Wood IS, Maglio MT, Alvarado JA (1984) Trabecular meshwork cell culture in glaucoma research: evaluation of biological activity and structural properties of human trabecular cells in vitro. Ophthalmology 91:580–595. https://doi.org/10.1016/s0161-6420(84)34241-5
Rehman AA, Ahsan H, Khan FH (2013) α-2-Macroglobulin: a physiological guardian. J Cell Physiol 228:1665–1675. https://doi.org/10.1002/jcp.24266
Reis LM, Semina EV (2011) Genetics of anterior segment dysgenesis disorders. Curr Opin Ophthalmol 22:314–324. https://doi.org/10.1097/ICU.0b013e328349412b
Ricklin D, Reis ES, Mastellos DC, Gros P, Lambris JD (2016) Complement component C3: the "Swiss Army Knife" of innate immunity and host defense. Immunol Rev 274:33–58. https://doi.org/10.1111/imr.12500
Sarfarazi M, Stoilov I (2000) Molecular genetics of primary congenital glaucoma. Eye 14(Pt 3B):422–428
Semina EV et al (1996) Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet 14:392–399
Siggs OM et al (2020) Biallelic CPAMD8 variants are a frequent cause of childhood and Juvenile open-angle glaucoma. Ophthalmology. https://doi.org/10.1016/j.ophtha.2019.12.024
Souma T et al (2016) Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity. J Clin Invest 126:2575–2587. https://doi.org/10.1172/JCI85830
Sowden JC (2007) Molecular and developmental mechanisms of anterior segment dysgenesis. Eye (Lond) 21:1310–1318
Stoilov I, Akarsu AN, Sarfarazi M (1997) Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum Mol Genet 6:641–647
Strausberg RL et al (2002) Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci USA 99:16899–16903. https://doi.org/10.1073/pnas.242603899
Vanichkina DP, Schmitz U, Wong JJ, Rasko JEJ (2018) Challenges in defining the role of intron retention in normal biology and disease. Semin Cell Dev Biol 75:40–49. https://doi.org/10.1016/j.semcdb.2017.07.030
Vincent A et al (2006) Further support of the role of CYP1B1 in patients with Peters anomaly. Mol Vis 12:506–510
Vincent A et al (2001) Phenotypic heterogeneity of CYP1B1: mutations in a patient with Peters' anomaly. J Med Genet 38:324–326
Weisschuh N, Wolf C, Wissinger B, Gramer E (2009) A clinical and molecular genetic study of German patients with primary congenital glaucoma. Am J Ophthalmol 147:744–753
Westerfield M (2000) The Zebrafish book: a guide for the laboratory use of Zebrafish (Danio rerio), 5th edn. University of Oregon Press, Eugene
Zhao Y et al (2013) Cyp1b1 mediates periostin regulation of trabecular meshwork development by suppression of oxidative stress. Mol Cell Biol 33:4225–4240. https://doi.org/10.1128/MCB.00856-13
Acknowledgements
We are indebted to the patients and their families for cooperation in this study. We would like to thank Ms. María-José Cabañero for excellent technical assistance and Dr. María-Luisa García-Gil for outstanding electron microscope assistance.
Funding
This study has been supported by research grants from the “Instituto de Salud Carlos III/European Regional Development Fund (ERDF)” (PI15/01193, PI19/00208 and RD16/0008/0019, OFTARED to JE; RD16/0008/0004, OFTARED to JG-F; RD16/0008/0005, OFTARED to A-IR; PI17_01164 to MC and CIBERER 06/07/0036 to CA; https://www.isciii.es), the Ministry of Economy and Competitiveness/ERDF (MINECO, SAF2013-46943-R to MC; https://www.ciencia.gob.es), the Regional Ministry of Science and Technology of the Board of the Communities of “Castilla-La Mancha” (SBPLY/17/180501/000404 to JE; https://www.educa.jccm.es/idiuniv/es) and the University Chair UAMIIS-FJD of Genomic Medicine, the Ramon Areces Foundation and Regional Government of Madrid (CAM, B2017/BMD3721 to CA). MC was sponsored by the Miguel Servet Program (CPII17_00006) from ISCIII, SA-M was sponsored by the Regional Ministry of Science and Technology of the Board of the Communities of “Castilla-La Mancha” (PREJCCM2016/28) and AT was sponsored by the Regional Government of Madrid/European Regional Development Fund (ERDF) (PEJD-2018-PRE/BMD-9453). M-TG-A was the recipient of a fellowship from the “Ministerio de Educación, Cultura y Deporte” (FPU 13/03308). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J-MB-F and J-DA-A share the first authorship. J-MB-F, J-DA-A, performed the genetic analyses, molecular biology and zebrafish experiments. MC designed the gene panel. MC, CV and AT performed targeted DNA sequencing, ddPCR and data analyses. II contributed with bioinformatic SNV and CNV analysis. M-TG-A, A-IR and J-JS carried out the optical and electron microscopy of human samples. J-MB-F, A-IR and J-JS performed optical and electron microscopy of zebrafish samples. SA-M and RA-A carried out immunohistochemistry and western blot analyses. J-JF-F performed exome sequence analyses. C-DM-H, LM-F, CA, J-MM-d-l-C and JG-F recruited, diagnosed and followed-up patients and supplied human samples. MC-P supplied human tissues and contributed to design expression analyses. J-DA-A and JE supervised the experiments. JE conceived and coordinated the study and wrote the manuscript. All authors contributed to the review and approval of the manuscript.
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The human study and informed consent procedures were approved by the Ethics Committee for Human Research of the University Hospital Fundación Jiménez Díaz and Hospital Clínico San Carlos (approval number 13/388-E). The research followed the tenets of the Declaration of Helsinki. Informed written consents were obtained prior to participants’ inclusion in the study. All animal husbandry and experiments were approved and conducted in accordance with the guidelines set forth by the Institutional Animal Research Committee of the University of Castilla-La Mancha (approval number PR-2015-04-10).
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Bonet-Fernández, JM., Aroca-Aguilar, JD., Corton, M. et al. CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix. Hum Genet 139, 1209–1231 (2020). https://doi.org/10.1007/s00439-020-02164-0
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DOI: https://doi.org/10.1007/s00439-020-02164-0