Genetics Screening of SPATA7 in Patients with Leber Congenital Amaurosis and Severe Childhood-Onset Retinal Dystrophy Reveals Disease-Causing Mutations Donna S. Mackay,1 Louise A. Ocaka,1 Arundhati Dev Borman,1,2 Panagiotis I. Sergouniotis,1,2 Robert H. Henderson,2 Phillip Moradi,2 Anthony G. Robson,1,2 Dorothy A. Thompson,3,4 Andrew R. Webster,1,2 and Anthony T. Moore1,2 PURPOSE. To investigate the prevalence of sequence variants in loss, but may have some preservation of the photoreceptor the gene SPATA7 in patients with Leber congenital amaurosis structure in the central retina. (Invest Ophthalmol Vis Sci. (LCA) and autosomal recessive, severe, early-onset retinal dys- 2011;52:3032–3038) DOI:10.1167/iovs.10-7025 trophy (EORD) and to delineate the ocular phenotype associ- ated with SPATA7 mutations. eber congenital amaurosis (LCA), first described by The- METHODS. Patients underwent standard ophthalmic evaluation odor Leber in 1869,1 is a generalized retinal dystrophy after providing informed consent. One hundred forty-one DNA L which presents at birth, or soon after, with severe visual samples from patients with LCA and EORD had been analyzed impairment and nystagmus. LCA accounts for 3% to 5% of for mutations by using a microarray, with negative results. One childhood blindness in the developed world and has an inci- additional patient underwent SPATA7 screening due to a re- dence of 2 to 3 per 100,000 live births.2 The clinical features gion of autozygosity surrounding this gene. A further patient include severe visual loss, sluggish pupillary responses, and was screened who had a compatible ocular phenotype. The roving eye movements. The retinal appearance may be normal entire SPATA7 coding sequence was assayed, including the at diagnosis, or there may be a variety of abnormalities, includ- intron–exon junctions, by using a combination of direct DNA ing macular atrophy, peripheral white dots at the level of the sequencing and high-resolution melting screening. retinal pigment epithelium (RPE), RPE atrophy, and/or retinal RESULTS. Screening of SPATA7 identified several known and pigmentation. The full-field electroretinogram (ERG) is usually novel single-nucleotide polymorphisms (SNPs). Affected indi- unrecordable.3,4 viduals from five unrelated families were identified to have Most forms of LCA are inherited as an autosomal recessive coding changes. Clinical features demonstrated a severe infan- (AR) trait, although rare dominant forms have been reported. tile onset retinal dystrophy, similar to Leber congenital amau- To date, 14 causative genes (GUCY2D,5 AIPL1,6 RPE65,7 rosis. The retina had widespread retinal pigment epithelial RPGRIP1,8 CRX,9 TULP1,10 CRB1,11 RDH12,12 CEP290,13 atrophy, with minimal pigment migration into the neurosen- LCA5,14 SPATA7,15 LRAT,16 MERTK17 and IQCB118) with one sory retina. Fundus autofluorescence imaging showed a para- further locus, LCA9,19 have been reported to be associated foveal annulus of increased autofluorescence. High-definition with autosomal recessive LCA. Mutations in some of these optical coherence tomography showed preservation of the genes are also associated with severe rod–cone dystrophies inner segment/outer segment junction at the fovea. presenting later in childhood. 20 CONCLUSIONS. Mutations in SPATA7 are a rare cause of child- The LCA3 locus was identified in 1998 by linkage analysis hood retinal dystrophy accounting for 1.7% of disease in this in a large consanguineous family from Saudi Arabia. A second cohort. Affected patients present in infancy with severe visual family, also from Saudi Arabia, was found to map to the same region of chromosome 14. RDH12, a positional candidate, was excluded as the disease gene, suggesting that this locus repre- sented a novel gene for LCA.21 In 2009, SPATA7 was identified From the 1Department of Genetics, Institute of Ophthalmology, as the causative gene at this locus after further fine mapping of London, United Kingdom; 2Moorfields Eye Hospital, London, United one of the linked families.15 Four novel mutations were found 3 Kingdom; the Clinical and Academic Department of Ophthalmology, in five families with LCA or juvenile onset retinitis pigmentosa Great Ormond Street Hospital for Children, London, United Kingdom; 4 (RP). Recently, a further four families with six mutations (four and the Institute of Child Health, University College London, United 22 Kingdom. of which were novel) have been identified. SPATA7 (spermatogenesis associated protein 7) was first Supported by Fight for Sight Grant 1624 (UK), the National Insti- 23 tute for Health Research UK (Moorfields Eye Hospital Biomedical cloned in 2003 from the rat testis and called RSD-3/HSD-3.1. Research Centre, London, UK), Alexander S. Onassis Public Benefit The corresponding human cDNA was also found to be ex- Foundation (Greece), the Foundation Fighting Blindness (USA), and pressed in the human testis. The human gene (MIM 609868; the Ulverscroft foundation. Mendelian Inheritance in Man; National Center for Biotechnol- Submitted for publication December 9, 2010; revised January 11, ogy Information, Bethesda, MD) consisted of 12 exons that 2011; accepted January 11, 2011 spanned approximately 52.8 kb and mapped to chromosome Disclosure: D.S. Mackay, None; L.A. Ocaka, None; A.D. Borman, 14, region q31.3. The gene encodes a protein of 599 amino None; P.I. Sergouniotis, None; R.H. Henderson, None; P. Moradi, None; acids. It is conserved from sea urchin to human, but is absent A.G. Robson, None; D.A. Thompson, None; A.R. Webster, None; A.T. 15 Moore, None in lower eukaryotes. Analysis of the protein using the PSIPred Corresponding author: Donna S. Mackay, Department of Genetics, program (provided in the public domain by Bioinformatics Institute of Ophthalmology, 11-43 Bath Street, London, UK EC1V 9EL; Group, Department of Computer Science, University College [email protected]. London, at http://bioinf.cs.ucl.ac.uk/introduction) showed Investigative Ophthalmology & Visual Science, May 2011, Vol. 52, No. 6 3032 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from jov.arvojournals.org on 10/02/2021 IOVS, May 2011, Vol. 52, No. 6 SPATA7 Mutations 3033 one transmembrane domain but no known functional domains. coherence tomography (SD-OCT; Spectralis Spectral domain OCT scan- Expression in the mouse retina was found in multiple retinal ner; Heidelberg Engineering, Germany) and retinal autofluorescence layers, including the ganglion cell and inner nuclear layers and (AF) imaging with a confocal scanning laser ophthalmoscope (Zeiss in the inner segments of photoreceptors. Expression levels at Prototype; Carl Zeiss Meditec, GmbH, Oberkochen, Germany). Pattern different time points suggested that Spata7 is important for and full-field electroretinography (PERG and ERG) were performed in normal retinal function rather than development.15 A recent consideration of the recommendations of the International Society for report on the Spata7-knockout mouse suggested that the pro- Clinical Electrophysiology of Vision (ISCEV) or using a modified pedi- tein is involved in protein transport (Abulimiti Sr A, et al. IOVS atric ERG protocol with skin electrodes, as previously described.25–28 2010;51:ARVO E-Abstract 723). Blood samples were collected in EDTA tubes, and DNA was ex- Two isoforms of SPATA7 are known to be expressed.24 tracted with a blood-extraction kit (Puregene; Invitrogen, Paisley, UK), Expression analysis of these two isoforms in 22 adult and fetal according to the manufacturer’s instructions. human tissues showed high levels in the retina, cerebellum, From a total of 292 subjects recruited into this study, DNA samples whole brain, and testis. Isoform 1 was more highly expressed from 141 subjects with no known mutations in LCA genes were chosen than was isoform 2 (missing exon 3) in neuronal tissues, for SPATA7 mutation screening. whereas isoform 2 was highly expressed in the testis.22 To evaluate the role of SPATA7 in childhood-onset retinal Microarray SNP6 Autozygosity Mapping dystrophies in the British population, we screened 141 patients Subject 2 from family 3 was chosen for analysis with a gene microarray with LCA or early childhood onset severe retinal dystrophy for (SNP6.0 array; Affymetrix, Santa Clara, CA), according to the manufac- all the coding exons, using a combination of melting curve turer’s instructions. Genotypes for single-nucleotide polymorphisms analysis and Sanger sequencing. We also investigated the phe- (SNPs) were called by gene analysis software (GeneChip DNA Analysis notype associated with SPATA7 mutations. Software; GDAS ver. 3.0; Affymetrix). Regions of homozygosity were also identified on computer (AutoSNPa; provided in the public domain MATERIALS AND METHODS by the Leeds Institute of Molecular Medicine, University of Leeds, UK, at http://dna.leeds.ac.uk/autospna/).29 Clinical Investigations All patients in this study had a clinical diagnosis of LCA or severe AR Mutation Screening retinal dystrophy with symptom onset before 6 years of age. All Primers used to amplify the coding exons and the intron-exon bound- provided informed consent as part of a research project approved by aries of SPATA7 were designed by hand. Primer sequences are shown the local research ethics committee, and all investigations were con- in Table 1. Exons 2 to 5 were initially screened with the high-resolution ducted in accordance with the principles of the Declaration of Hel- melting curve technique (LightScanner; Idaho Technology, Salt Lake sinki. A clinical evaluation, including monocular best corrected visual City, UT), with abnormal melting curves identified and then se- acuity (BCVA), perimetry, and slit lamp biomicroscopy was performed quenced. All other exons were directly sequenced. All standard poly- on all patients who could cooperate with testing. Patients underwent merase chain reactions (PCRs) were performed in a total volume of 30 retinal imaging (TRC 501A retinal camera; Topcon Corp., Tokyo, Ja- ␮L containing 200 ␮M dNTPs (VH Bio, Gateshead, UK), 20 ␮M of each ϫ pan) and, where possible, high-resolution spectral domain optical primer, 1 reaction buffer including 1.5 mM MgCl2 (VH Bio), with 1 TABLE 1.