Genetics Mpdz Null Allele in an Avian Model of Retinal Degeneration and Mutations in Human Leber Congenital Amaurosis and Retinitis Pigmentosa Manir Ali,1 Paul M. Hocking,2 Martin McKibbin,3 Sorcha Finnegan,4 Mike Shires,5 James A. Poulter,1 Katrina Prescott,6 Adam Booth,1,7 Yasmin Raashid,8 Hussain Jafri,9 Jonathan B. Ruddle,10 David A. Mackey,11 Samuel G. Jacobson,12 Carmel Toomes,1 Douglas H. Lester,13 David W. Burt,2 William J. Curry,4 and Chris F. Inglehearn1 PURPOSE. To identify the defective gene in the sex-linked, re- retinal outer limiting membrane, where it may have a role in cessively inherited retinal dysplasia and degeneration (rdd) the interactions between photoreceptors and Mu¨ller glia cells. chicken and to search for the human equivalent disease. The screen to identify the human equivalent disease found 10 METHODS. Microsatellites from chicken chromosome Z were heterozygous variants in the orthologous gene in patients with genotyped in 77 progeny of a carrier male (rdd/ϩ) and an RP (three missense and two null alleles) and LCA (four mis- affected female (rdd/W), and candidate genes were sequenced. sense and one null allele). Retinal cross-sections from rdd and wild-type birds were ana- CONCLUSIONS. These findings reveal that MPDZ is essential for lyzed by immunohistology. The human orthologous gene was normal development of the retina and may have a role in screened in a panel of archival DNAs from 276 patients with maintaining photoreceptor integrity. The identification of hu- retinitis pigmentosa (RP) or Leber congenital amaurosis (LCA) man mutations suggests that MPDZ plays a role in human using melting curve analysis and DNA sequencing. retinal disease, but the precise nature of this role remains to be RESULTS. The rdd locus was refined to an approximately 3-Mb determined. (Invest Ophthalmol Vis Sci. 2011;52:7432–7440) region on chromosome Z. Sequence analysis identified a C3T DOI:10.1167/iovs.11-7872 change in the mpdz gene that created a premature stop codon (c.1372C3T, p.R458X), which segregated with the disease he retinal dysplasia and degeneration (rdd) phenotype phenotype. As expected, the full-length mpdz protein was Tarose spontaneously in commercial chicken stocks over 30 absent in rdd retinas, but in wild-type birds, it localized to the years ago,1,2 and the associated eye defect was shown to be inherited in a sex-linked recessive manner.3 Rdd chicks are sighted at hatch, but vision gradually deteriorates until by 10 From the Sections of 1Ophthalmology and 5Pathology, Leeds In- weeks all the birds are blind. The earliest pathologic signs are stitute of Molecular Medicine, and the 3Eye Department, St. James’s holes in the retinal pigment epithelial (RPE) layer and undula- University Hospital, Leeds, United Kingdom; 2Division of Genetics and tions in the outer nuclear, outer plexiform, and inner nuclear Genomics, The Roslin Institute and Royal School of Veterinary Studies, layers in prehatched embryos.1,2,4 As the disease develops, University of Edinburgh, Midlothian, United Kingdom; 4Centre for there is widespread pigmentary disturbance, progressive thin- Vision Sciences, Institute of Clinical Science, Queen’s University of 6 ning of all the retinal layers, and degeneration of the photore- Belfast, Belfast, Northern Ireland, United Kingdom; Department of ceptor and inner nuclear layers, causing the retina to become Clinical Genetics, Chapel Allerton Hospital, Leeds, United Kingdom; 2 7Peninsula Medical School, Plymouth, United Kingdom; 8Department buckled. It has been suggested that the rdd chicken has features that resemble a severe early-onset pigmentary retinop- of Obstetrics and Gynaecology, King Edward Medical University, La- 5,6 hore, Pakistan; 9Gene Tech Lab 146/1, Lahore, Pakistan; 10Department athy in humans. of Ophthalmology, University of Melbourne, Melbourne, Australia; Retinal dystrophies are a common cause of human morbidity 11Lions Eye Institute, University of Western Australia, Perth, Australia; and many have a genetic basis. Thus far, 214 unique Mendelian 12Department of Ophthalmology, Scheie Eye Institute, University of human retinal diseases have been mapped to specific chromo- Pennsylvania, Philadelphia, Pennsylvania; and 13Molecular Vision somal loci, and the defective genes for 172 of these have been Group, School of Contemporary Sciences, University of Abertay, identified (RetNet; http://www.sph.uth.tmc.edu/retnet/). How- Dundee, United Kingdom. ever, identification of the genes underlying recessively inherited Supported by Medical Research Council Grant G0501050 and Yorkshire Eye Research Grant 022 and by Biotechnology and Biological diseases has tended to lag behind their dominant and X-linked Sciences Research Council Core Grant (The Roslin Institute). MA was equivalents because of a lack of large pedigrees for mapping the recipient of a New Investigator Award. studies. The prospect of identifying the disease-causing genes in Submitted for publication May 13, 2011; revised July 21, 2011; chickens, with large “family” sizes of 50 or more progeny, has accepted July 23, 2011. been simplified with the publication of the chicken genome and Disclosure: M. Ali, None; P.M. Hocking, None; M. McKibbin, the revelation that its physical size is only one-third that of the None; S. Finnegan, None; M. Shires, None; J.A. Poulter, None; K. human.7 This is further supported by the higher density of recom- Prescott, None; A. Booth, None; Y. Raashid, None; H. Jafri, None; bination in the chicken genome and the remarkable conservation J.B. Ruddle, None; D.A. Mackey, None; S.G. Jacobson, None; C. of synteny between the genomes. Furthermore, the development Toomes, None; D.H. Lester, None; D.W. Burt, None; W.J. Curry, None; C.F. Inglehearn, None of specific treatments for these conditions based on the genetic Corresponding author: Manir Ali, Section of Ophthalmology and information generated has been limited in part by the lack of Neuroscience, Leeds Institute of Molecular Medicine, Wellcome Trust suitable animal models to test therapies. Brenner Building, St. James’s University Hospital, Leeds LS9 7TF, UK; Here we report the molecular defect that causes the rdd [email protected]. phenotype in chickens and attempt to identify the analogous Investigative Ophthalmology & Visual Science, September 2011, Vol. 52, No. 10 7432 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from jov.arvojournals.org on 09/27/2021 IOVS, September 2011, Vol. 52, No. 10 Mpdz Null Allele Causes Rdd 7433 human condition so that the value of this animal model for genetic analyzer (ABI3130xl; Applied Biosystems) according to the targeted therapy can be assessed. manufacturer’s instructions. Mutation Restriction Analysis MATERIALS AND METHODS For detecting the c.1372C3T mutation in chicken genomic DNA, Animals oligonucleotide primer pair dGAA GAA AAA TAT TTC AAG TCC AAT The rdd flock and sighted White Leghorn birds were maintained at the TT and dTCT GGA CAT TTT CTA TCT TTC TGC T was used in the PCR Roslin Institute, as described elsewhere.3 All animal husbandry and exper- with an annealing temperature of 53°C, followed by incubation with 2 imental procedures were conducted under the appropriate Home Office the restriction endonuclease TaqI (T CGA) (Invitrogen) at 65°C. project and personal licenses in a manner consistent with the ARVO Reaction products were resolved through a 2% agarose gel to give Statement for the Use of Animals in Ophthalmic and Vision Research and digestion bands of 169 and 123 bp for the wild-type sequence and a in compliance with the UK Animals (Scientific Procedures) Act 1986. single undigested band of 292 bp for the mutant sequence. Genotyping Immunohistology Genomic DNA was extracted from chicken blood using the DNAzol Eyes were dissected from 17-day-old embryos, fixed in buffered 4% method (Invitrogen, Paisley, UK). High-resolution genotyping was per- paraformaldehyde (Sigma-Aldrich Ltd, Gillingham, UK), and embedded formed with previously uncharacterized microsatellite markers down- in paraffin wax. Four-micrometer chicken retina cross-sections were loaded as simple repeat sequences from the chicken genome database incubated with polyclonal rabbit anti-chicken mpdz, followed by rab- at the University of California at Santa Cruz (UCSC; http:// bit immunoglobulin detection system (EnVision; Dako, Cambridge, www.genome.ucsc.edu/). Primer sequences were designed using UK). The rabbit anti-chicken mpdz serum had been raised using the Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/), and DNA oligonu- synthetic peptide KDSSETEQGSPSQPC-CONH2 corresponding to cleotides were labeled with fluorochrome FAM, HEX, or TET (Invitro- amino acid residues 497 to 511 in chicken mpdz as immunogen and gen). PCR products, generated with fluorescence-labeled polymorphic affinity-purified before use (Eurogentech S.A., Liege, Belgium). The microsatellite markers on chicken DNA, were size fractionated on a rationale for designing a peptide immunogen C-terminal to the prema- 3130xl Genetic Analyzer (Applied Biosystems, Warrington, UK) and ture stop codon mutation (residue 458) was to use rdd tissue as a viewed using GeneMapper version 4.0 (Applied Biosystems). negative control. The sections were analyzed using a fluorescence microscope (Eclipse E1000; Nikon, Tokyo, Japan). Bioinformatic Analysis of the Candidate Genes Western Blot Analysis The gene and protein information for each candidate was found using the GeneCards version 3 (http://www.genecards.org/), OMIM (http:// Eyes from 1-day-old