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Splice-Site Mutations Identified in PDE6A Responsible for Retinitis Pigmentosa in Consanguineous Pakistani Families

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Splice-Site Mutations Identified in PDE6A Responsible for Retinitis Pigmentosa in Consanguineous Pakistani Families Molecular Vision 2015; 21:871-882 <http://www.molvis.org/molvis/v21/871> © 2015 Molecular Vision Received 1 July 2014 | Accepted 15 August 2015 | Published 18 August 2015 Splice-site mutations identified in PDE6A responsible for retinitis pigmentosa in consanguineous Pakistani families Shahid Y. Khan,1 Shahbaz Ali,2 Muhammad Asif Naeem,2 Shaheen N. Khan,2 Tayyab Husnain,2 Nadeem H. Butt,3 Zaheeruddin A. Qazi,4 Javed Akram,3,5 Sheikh Riazuddin,2,3,5 Radha Ayyagari,6 J. Fielding Hejtmancik,7 S. Amer Riazuddin1 (The first two and last two authors contributed equally to this work.) 1The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore MD; 2National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan; 3Allama Iqbal Medical College, University of Health Sciences, Lahore, Pakistan; 4Layton Rahmatulla Benevolent Trust Hospital, Lahore Pakistan; 5National Centre for Genetic Diseases, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad Pakistan;6 Shiley Eye Institute, University of California San Diego, La Jolla CA; 7Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda MD Purpose: This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin. Methods: Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1. Results: A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthal- mic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromo- some 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028–1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408–2A>G. In silico analysis suggested that these mutations will result in the elimination of wild-type splice acceptor sites that would result in either skipping of the respective exon or the creation of a new cryptic splice acceptor site; both possibilities would result in retinal photoreceptor cells that lack PDE6A wild-type protein. Conclusions: we report two splice acceptor site variations in PDE6A in consanguineous Pakistani families who mani- fested cardinal symptoms of RP. Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well. The term retinitis pigmentosa (RP) refers to bone spicule- structures in the retinal vascular layer [2]. The typical fundus like pigmentation in the mid-peripheral fundus that stimulates appearance includes attenuated arterioles, bone spicule-like inflammation and was first used by the German physician pigmentation, and waxy pallor of the optic disc. Affected Donders in 1857 [1]. RP is a progressive rod-cone dystrophy individuals often have severely abnormal or non-detectable that primarily affects the rod photoreceptors whereas the rod responses in electroretinogram (ERG) recordings even in function of the cone receptors is compromised as the disease the early stage of the disease [2]. progresses [2]. Ocular findings comprise atrophic changes RP is the most common inherited retinal dystrophy, in the photoreceptors and in the retinal pigment epithelium affecting approximately 1 in 5,000 individuals worldwide (RPE) followed by the appearance of melanin-containing [3]. RP is clinically and genetically heterogeneous, which is inherited as a dominant, recessive, as well as an X-linked trait Correspondence to: S. Amer Riazuddin, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, [4]. RP presents intra- and interfamilial variations in expres- Maumenee 840, Baltimore, MD 21287; Phone: (410) 955-3656; FAX: sivity, penetrance, progression, and age of onset [5]. Among (410) 955-3656; email: [email protected] the several causes of variable phenotypes are alternative 871 Molecular Vision 2015; 21:871-882 <http://www.molvis.org/molvis/v21/871> © 2015 Molecular Vision alleles, environmental factors, modifier genes, or a combina- All participating members voluntarily provided approxi- tion of these factors [6,7]. Autosomal recessive RP (arRP) mately 10 ml of blood sample that was stored in 50 ml is the most common form of RP worldwide, and to date, 36 Sterilin® falcon tubes containing 400 µl of 0.5 M EDTA. genes/loci have been associated with autosomal recessive RP Blood samples were stored at −20 °C for long-term storage. (RetNet). Genomic DNA extraction: Genomic DNA was extracted Phosphodiesterase 6 (PDE6) is a critical enzyme of the from white blood cells using a non-organic modified proce- visual phototransduction cascade in the vertebrate retina dure as described previously [16,17]. Approximately, 10 ml [8,9]. The protein is a heterotrimer that consists of an alpha, blood samples were mixed with 35 ml of TE buffer (10 mM a beta, and two gamma subunits. PDE6A (Gene ID: 5145; Tris-HCl, 2 mM EDTA, pH 8.0) and the TE-blood mixture OMIM: 180071) encodes for the α-subunit of cyclic guanosine was centrifuged at 2,000 x g for 20 min. The red blood cells monophosphate (cGMP)-phosphodiesterase that is primarily were discarded and the pellet was re-suspended in 35 ml of expressed in rod photoreceptors [10,11]. PDE6A consists of 22 TE buffer. The TE washing was repeated for 2–3 times and coding exons and encodes for 860 amino acids [12,13]. Dryja the washed pellet was re-suspended in 2 ml of TE buffer. and colleagues were the first to report causal mutations in Next, 6.25 ml of protein digestion cocktail (50 μl [10 mg/ml] PDE6A in patients with arRP [14]. of proteinase K, 6 ml TNE buffer [10 mM Tris HCl, 2 mM We previously reported three mutations in PDE6A EDTA, 400 mM NaCl] and 200 μl of 10% sodium dodecyl including a splice acceptor site mutation in three families of sulfate) was added to the re-suspended pellets and incubated Pakistani origin [15]. Here, we report two additional familial overnight in a shaker (250 rpm) at 37 °C. The digested proteins cases with multiple members manifesting symptoms of RP. were precipitated by adding 1 ml of 5 M NaCl, followed by The clinical phenotype was established with fundus photog- vigorous shaking and chilling on ice for 15 min. The precipi- raphy and electroretinography while genome-wide linkage tated proteins were pelleted by centrifugation at 2,000 x g for analyses localized the disease interval to chromosome 5q. 20 min and removed. The supernatant was mixed with equal Subsequently, Sanger sequencing identified splice acceptor volumes of phenol/chloroform/isoamyl alcohol (25:24:1) and site mutations in these two families that were absent in ethni- the aqueous layer containing the genomic DNA was carefully cally matched control chromosomes. collected. The DNA was precipitated with isopropanol and pelleted by centrifugation at 3,500 x g for 15 min. The DNA METHODS pellets were washed with 70% ethanol and dissolved in TE buffer. The concentration of the extracted genomic DNA was Clinical ascertainment: A total of >300 consanguineous Paki- estimated with a SmartSpec plus Bio-Rad Spectrophotometer stani families with non-syndromic retinal dystrophies were (Bio-Rad, Hercules, CA). recruited to identify new disease loci responsible for inherited visual diseases. The institutional review boards (IRBs) of the Genotype analysis: Applied Biosystems MD-10 linkage National Centre of Excellence in Molecular Biology (Lahore, mapping panels (Foster City, CA) were used to complete the Pakistan), National Eye Institute (Bethesda, MD), and Johns genome-wide scan for family PKRP133. Multiplex PCR was Hopkins University (Baltimore, MD) approved the study. completed as described previously [15]. The PCR products All participating family members provided informed written were mixed with a loading cocktail that contained HD-400 consent that has been endorsed by the respective IRBs and size standards (Applied Biosystems) and were resolved in an is consistent with the tenets of the Declaration of Helsinki. Applied Biosystems 3100 DNA Analyzer. Genotypes were assigned using the GeneMapper software from Applied
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