Official journal of the American College of Medical Genetics and Genomics ORIGINAL RESEARCH ARTICLE Open GUCA1A mutation causes maculopathy in a five-generation family with a wide spectrum of severity Xue Chen, MD, PhD1,2,3, Xunlun Sheng, MD, PhD4, Wenjuan Zhuang, MD, PhD4, Xiantao Sun, MD5, Guohua Liu, MD6, Xun Shi, MD7, Guofu Huang, MD7, Yan Mei, MD7, Yingjie Li, MD7, Xinyuan Pan, MSc2, Yani Liu, MD4, Zili Li, MD4, Qingshun Zhao, PhD8, Biao Yan, PhD9 and Chen Zhao, MD, PhD1,2,3,5,10 Purpose: The aim of this study was to investigate the genetic basis Those phenotypes could not be fully rescued by exogenous wild- and pathogenic mechanism of variable maculopathies, ranging from type GUCA1A, suggesting a likely gain-of-function mechanism for mild photoreceptor degeneration to central areolar choroidal dystro- p.R120L. GUCA1A p.D100E, another mutation previously implicated phy, in a five-generation family. in cone dystrophy, also impaired the retinal pigment epithelium and photoreceptors in zebrafish, but probably via a dominant negative Methods: Clinical characterizations, whole-exome sequencing, and effect. genome-wide linkage analysis were carried out on the family. Zebraf- ish models were used to investigate the pathogenesis of GUCA1A Conclusion: We conclude that GUCA1A mutations could cause sig- mutations. nificant variability in maculopathies, including central areolar cho- roidal dystrophy, which represents a severe pattern of maculopathy. Results: A novel mutation, GUCA1A p.R120L, was identified in the The diverse pathogenic modes of GUCA1A mutations may explain family and predicted to alter the tertiary structure of guanylyl cyclase- the phenotypic diversities. activating protein 1, a photoreceptor-expressed protein encoded by the GUCA1A gene. The mutation was shown in zebrafish to cause Genet Med advance online publication 26 January 2017 significant disruptions in photoreceptors and retinal pigment epithe- Key Words: GUCA1A; maculopathy; medical genetics; lium, together with atrophies of retinal vessels and choriocapillaris. ­ophthalmology; pathogenic mechanism INTRODUCTION regulating the retinal guanylate cyclase 1 (retGC1) –medicated Inherited retinal degenerations (IRDs) include a group of cyclic guanosine monophosphate production in a calcium- diverse retinal degenerative diseases presenting both genetic sensing manner.5,6 RetGC1 is encoded by the guanylate cyclase and clinical heterogeneities. To date, according to RetNet 2D gene (GUCY2D; MIM 600179). The important roles of (https://sph.uth.edu/retnet/), 293 loci (including 256 identified GCAP1 and retGC1 in regulating hemostasis of calcium and genes) have been associated with IRDs. Among the 256 identi- cyclic guanosine monophosphate in photoreceptors have been fied genes, the guanylate cyclase activator 1A gene (GUCA1A; well addressed. MIM 600364) has been implicated in dominant cone dystro- In this study we found a novel GUCA1A mutation to be dis- phy, cone-rod dystrophy, and macular dystrophy.1–3 GUCA1A, ease causative in a five-generation family affected with variable located in 6p21.1, encodes the guanylyl cyclase-activating pro- maculopathies ranging from mild photoreceptor degeneration tein 1 (GCAP1), a photoreceptor-specific protein with more to central areolar choroidal dystrophy (CACD). CACD is a spe- expression in the inner segment/outer segment (OS) layer of cial form of IRD that mainly affects the maculae and is char- cones than rods in mammals.4 GCAP1 is a critical component acterized by a well-defined atrophic region of retinal pigment in the phototransduction cascade, which acts as a calcium sen- epithelium (RPE) and choriocapillaris at the latest stage.7–9 sor in the recovery of photoreceptors from photon capture by Before our study, mutations in two other genes, peripherin-2 The first three authors contributed equally to this work. 1Department of Ophthalmology and Vision Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China; 2Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, State Key Laboratory of Reproductive Medicine, Nanjing, China; 3Key Laboratory of Myopia of State Health Ministry (Fudan University) and Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China; 4Department of Ophthalmology, Ningxia Eye Hospital, People Hospital of Ningxia Hui Autonomous Region (First Affiliated Hospital of Northwest University for Nationalities), Yinchuan, China; 5Department of Ophthalmology, Children’s Hospital of Zhengzhou, Zhengzhou, China; 6Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan, China; 7Department of Ophthalmology, The Third Affiliated Hospital of Nanchang University, Nanchang, China; 8MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing, China; 9Research Center, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China; 10State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat Sen University, Guangzhou, China. Correspondence: Chen Zhao ([email protected]) or Biao Yan ([email protected]) Submitted 6 July 2016; accepted 18 November 2016; advance online publication 26 January 2017. doi:10.1038/gim.2016.217 GENETICS in MEDICINE | Volume 19 | Number 8 | August 2017 945 ORIGINAL RESEARCH ARTICLE CHEN et al | GUCA1A mutation causes variable maculopathy (PRPH2; MIM 179605) and GUCY2D, were implicated in Exome sequencing and bioinformatics analysis CACD etiology.8,10–12 We also found that diverse pathogenic Two patients, DC-IV:3 and DC-IV:13, were selected for whole- mechanisms of GUCA1A mutations might correlate with the exome sequencing using the SureSelect Human All Exon phenotypic diversity of IRDs. 50Mb Kit (Agilent Technologies, Santa Clara, CA) and the HiSeq 2000 platform (Illumina, San Diego, CA).15 Illumina MATERIALS AND METHODS base-calling software v1.7 was then applied to turn raw image Participants and clinical assessments files into 90-base-paired-end reads. Bioinformatics were sub- Our study conformed to the Declaration of Helsinki and sequently analyzed, and mutations validated.16,17 Briefly, all was prospectively reviewed and approved by the ethics com- detected variants were initially filtered against six single-nucle- mittee of Ningxia Eye Hospital. Written informed consent otide polymorphism databases, including dbSNP144 (http:// was obtained from all participants before their enrollment. hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp144. Eighteen patients and 18 unaffected family members from txt.gz), the HapMap project (ftp://ftp.ncbi.nlm.nih.gov/hap- family DC were included in the study (Figure 1). Medical map), the 1000 Genomes Project (ftp://ftp.1000genomes.ebi. records from each participant were reviewed. Routine oph- ac.uk/vol1/ftp), the YanHuang database (http://yh.genomics. thalmic examination was conducted for all participants, and org.cn/), the Exome Variant Server (http://evs.gs.washington. all patients received comprehensive ophthalmic examinations, edu/EVS/), and the Exome Aggregation Consortium database including best-corrected visual acuity, slit-lamp examination, (http://exac.broadinstitute.org/). Noncoding variants were dis- visual field test, funduscopic evaluations, and electroretino- carded, and only variants located within an annotated exon grams. Fundus autofluorescence, fundus fluorescein angiog- or within 10 base pairs on either side were analyzed further. raphy, optical coherence tomography, and electroretinography Intrafamilial cosegregation analysis and prevalence testing in were performed when possible. Another 423 unrelated Chinese the 423 unrelated controls were then conducted with the prim- controls free of maculopathy and other major ocular diseases ers detailed in Supplementary Table S1 online. were also recruited. Samples of peripheral venous blood (5 ml) were collected from each participant for genomic DNA extrac- In silico analyses tion using a QIAmp DNA Mini Blood Kit (Qiagen, Hilden, Evolutionary conservation of the mutated residues was analyzed Germany). with Vector NTI Advance 2011 (Invitrogen, Carlsbad, CA) by aligning the protein sequence of human GCAP1 (NP_000400.2) Linkage analysis with sequences of the following orthologous proteins: Pan Thirteen patients from family DC (II:11, II:13, III:7, III:14, troglodytes (ENSPTRP00000031039), Canis lupus familia- III:16, III:18, III:20, III:31, IV:3, IV:6, IV:9, IV:13, and V:3) and ris (ENSCAFP00000002385), Bos taurus (NP_776971.1), Sus four unaffected members from the same family (III:23, III:35, scrofa (ENSSSCP00000001774), Mus musculus (NP_032215.2), IV:12, and IV:18) were carefully examined and genotyped. Gallus gallus (NP_989651), and Danio rerio (NP_571945.1). Genome-wide linkage analysis was conducted in collaboration Crystal structural models of the wild-type (WT) and mutant with Genesky Biotech (Shanghai, China) using a total of 366 GCAP1 were constructed using the SWISS-MODEL online microsatellite markers spaced at about 10 cM (Weber set 6.0) server. Predicted structures were displayed by PyMol software and distributed throughout all autosomes; these markers were (version 1.5). amplified by polymerase chain reaction (PCR) using primers labeled by FAM.13,14 PCR products were appropriately pooled Plasmids construction and messenger RNA synthesis according to allele size and labeling, mixed with GeneScan-500 The open reading frame sequence of WT human GUCA1A TAMRA standard (Applied Biosystems, Foster