Genetics Mpdz Null Allele in an Avian Model of Degeneration and Mutations in Human Leber Congenital Amaurosis and

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 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 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 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 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 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

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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 White Leghorn or rdd chicks were harvested for www.ncbi.nlm.nih.gov/sites/entrez?dbϭOMIM), and PubMed (http:// retinal protein extraction according to standard methods. Ten micro- www.ncbi.nlm.nih.gov/pubmed/) Web sites. grams of total protein extract was resolved under reducing conditions by polyacrylamide gel electrophoresis (XCell SureLock System; Invit- Reverse Transcription–Polymerase rogen) and Tris-acetate SDS running buffer. The gel were Chain Reaction transferred onto a 0.45-␮m polyvinylidene difluoride filter membrane (XCell II Blot Module; Invitrogen) and analyzed using the chicken Total chicken retina RNA was extracted (Trizol; Invitrogen) and re- mpdz antibody, anti-rabbit immunoglobulin conjugated to horseradish verse transcribed with an oligonucleotide (dT)15 primer and reverse peroxidase as a secondary antibody, followed by chemiluminescence transcriptase (Superscript II; Invitrogen). For each candidate gene to detection (Fischer Scientific UK Ltd., Loughborough, UK) according to be analyzed, the full-length chicken cDNA was directly downloaded standard methods. from the Ensembl Web site (http://www.ensembl.org/index.html). Where the full-length cDNA was not available, the corresponding Immunofluorescence and Confocal Microscopy human ortholog was identified by synteny at the UCSC Web site, the human cDNA was downloaded, and a BLASTN search was performed Adult rat eyes were harvested and cryopreserved according to standard against the chicken cDNA database at the Ensembl Web site. A series procedures. Twenty-micrometer retinal cross-sections were incubated of overlapping fragments was aligned to construct a putative full-length with a polyclonal rabbit antibody against Mpdz (Invitrogen) or Crb1 (a gift from Jan Wijnholds, Netherlands Institute for Neuroscience, Am- chicken cDNA. Specific primer pairs were designed using Primer3, as 8 a series of approximately 500-bp overlapping fragments to span the sterdam, The Netherlands), followed by an Alexa Fluor 488–conju- entire coding region. An aliquot of total cDNA was used in a standard gated secondary anti-rabbit immunoglobulin (Invitrogen). Nuclei were PCR to amplify the cDNA fragment of interest. counterstained with propidium iodide (Invitrogen). Slides were ana- To test the levels of chicken transcripts in various tissues, specific lyzed using an inverted confocal microscope (Eclipse TE2000–4; primer pairs for mpdz (dAGT CAT CGT CCA GCC AAG TC and dCAA Nikon) with Nikon software (EC-1). TCT GTA ACC GGC CAT CT) and gapdh (dGGA AAG TCA TCC CTG AGC TG and dCAT CAA AGG TGG AGG AAT GG) were used in the PCR Patients on total cDNA for 25 cycles with an annealing temperature of 57°C. Patients with retinitis pigmentosa (RP) or Leber congenital amaurosis The reaction products of 243 bp and 237 bp, respectively, were (LCA) were recruited by ophthalmologists or clinical geneticists in resolved by electrophoresis through a 2% agarose gel. charge of their treatment. Blood samples were taken for DNA extrac- For the investigation of MPDZ expression in human blood lympho- tion with the patients’ informed consent according to the principles of cytes, specific primer pairs (dCAT TGA AAT GCA GGT GGT TG and the Declaration of Helsinki using a process approved by the Leeds dCGG CTG AAC ACA GCA TAA AA) were used in the PCR on total Teaching Hospitals Trust Research Ethics Committee. cDNA for 40 cycles at an annealing temperature of 55°C. Reaction products of 493 bp were predicted after agarose gel electrophoresis. Mutation Screening in Archival DNA Samples Patient DNA samples were PCR amplified with master mix (HotShot; DNA Sequencing Clent Life Science, Stourbridge, UK) in the presence of an intercalating PCR-amplified products were digested (ExoSAP-IT; GE Healthcare, dye (LCGreen-Plus; Clent Life Science) and MPDZ oligonucleotide Chalfont St. Giles, UK) and sequenced (Big Dye Terminator, version primers (Supplementary Table S1, http://www.iovs.org/lookup/suppl/ 3.1; Cycle Sequencing Kit; Applied Biosystems, Foster City, CA) on a doi:10.1167/iovs.11-7872/-/DCSupplemental) according to the supplier’s

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FIGURE 1. Diagram of the refined interval for the rdd locus after genotyping analysis. Genotyping was performed using standard techniques. The image shown was downloaded from the UCSC Genome Browser and depicts the refined interval on chromosome Z. The known chicken protein-coding genes (RefSeq Genes) and the predicted genes based on alignment with the (Human Proteins mapped by chained tBLASTn) are shown.

instructions. PCR products were subjected to high-resolution melting- RESULTS curve analysis (Lightscanner; Clent Life Science), and the results were inspected using the manufacturer’s software. Melting curves that deviated Identifying the rdd Mutation from the normal pattern were identified, and a PCR aliquot of the sample that gave the variant band was sequenced. The rdd phenotype has been shown to be inherited as a Z-linked recessive trait.3 The avian sex differ Comparative Genomics from those in mammals in that the male is homogametic, denoted ZZ, and the female is heterogametic, denoted ZW. We Protein sequence conservation across mammalian and nonmammalian have previously bred 77 progeny from a cross of a carrier male vertebrates was investigated using the UCSC Genome Browser. This (rdd/ϩ) with an unaffected female (rdd/W) and used haplo- study used the Vertebrate Multiz Alignment and Conservation package type analysis to locate the rdd locus to within a 12.5-Mb region across 15 species. on chromosome Z between the markers MCW0055 and

FIGURE 2. Molecular analysis of mpdz in the rdd chicken. (A) Se- quence analysis of the mpdz cDNA in White Leghorn (WL) and rdd birds. Note the C3T substitution (c.1372C3T) changes the arginine residue to a stop codon mutation. (B) Agarose gel photographs of TaqI-di- gested PCR products from chicken genomic DNA. (i) The C3T muta- tion is unique to rdd affected and carrier birds. rge (retinopathy globe enlarged)11 and beg (blindness en- larged globe)12 represent two other inherited lines of blind chickens. (ii) The C3T mutation segregates with the disease phenotype, as expected, for a sex-linked recessive condition. In both gels, lane 1 represents the 100-bp standard ladder.

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ALD0250.3 Using the same progeny DNAs generated for the A Defect in the Outer Limiting Membrane Causes initial linkage screen, we further refined the locus with previ- the rdd Phenotype ously uncharacterized microsatellite markers (data not shown) To investigate the precise consequence of the mpdz premature to an approximately 3-Mb region between 28,186,873 and stop codon mutation, we performed immunohistologic analy- 31,157,461 bp on the chicken May 2006 assembly (galGal3) of sis on rdd and White Leghorn retinas with a polyclonal rabbit the UCSC Genome Browser (Fig. 1). This region contained 14 anti-chicken mpdz (Fig. 3A). We analyzed histologic sections predicted genes. Bioinformatics analysis revealed that the from 17-day-old embryos because there was minimal retinal mouse homolog of one of these, Mpdz (multiple PDZ domain disorganization in rdd leading up to this developmental stage. protein), also known as Mupp1 (multi-PDZ domain protein-1), We confirmed that the lack of full-length mpdz protein in rdd 8 had been shown to interact with Crb1 (crumbs homolog 1). caused the observed phenotype. However, in wild-type birds, Mutations in CRB1 (MIM ϩ604,210) cause recessively inher- mpdz localized to the outer limiting membrane (OLM) be- ited RP and LCA in human patients.9,10 Mpdz was therefore tween the photoreceptor inner segment and the outer nuclear considered a strong candidate gene. layer. To confirm the specificity of our antibody, we performed Given that retinal RNA was readily available from the birds, Western blot analysis of retinal protein extracts from 1-day-old we used direct cDNA sequencing to search for the pathogenic birds using the anti-chicken mpdz (Supplementary Fig. S3, mutation in rdd, as opposed to analyzing genomic DNA, to http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.11-7872/-/ cover the possibility of identifying noncoding changes that DCSupplemental). We observed that only the wild-type retinas affect expression or splicing. The coding sequence of the contained an immunoreactive species of approximately 210 White Leghorn chicken mpdz gene was identified in retinal kDa of the expected size for mpdz. To confirm whether local- RNA by a combination of reverse-transcription PCR and DNA ization of mpdz at the OLM was specific, we purchased a sequencing (Supplementary Fig. S1, http://www.iovs.org/ commercially available polyclonal rabbit anti-mouse Mpdz for lookup/suppl/doi:10.1167/iovs.11-7872/-/DCSupplemental). analysis by immunofluorescence and confocal microscopy on Sequence analysis of mpdz cDNA in rdd birds identified a 12-week-old rat retinal cross-sections (Fig. 3B). We observed intense immunostaining at the OLM that overlapped with the C3T substitution that creates a premature stop codon muta- localization of the Mpdz interactant, Crb1. This expression tion (c.1372C3T, p.R458X) (Fig. 2A). This mutation does not coincided with the site of the earliest observed pathology in appear to affect the level of mpdz transcripts in rdd retina the rdd chicken4 and implied that the primary defect was a significantly, suggesting a lack of nonsense-mediated decay of poorly formed OLM. the mutated transcript (Supplementary Fig. S2, http://www.iovs. org/lookup/suppl/doi:10.1167/iovs.11-7872/-/DCSupplemental). Screening for MPDZ Mutations in Human Disease Using a restriction digest assay on genomic DNA, the 3 Because rdd pathology resembles human RP5,6 and mutations c.1372C T mutation was absent from the DNA of other breeds 9 10 of chickens and was shown to be unique to the rdd-affected and in the known MPDZ interactant CRB1 cause RP and LCA, we investigated the possibility that mutations in the 46-exon carrier birds, segregating with the disease phenotype in a sex- human MPDZ (MIM *603785) gene cause RP or LCA. A panel of linked recessive manner (Fig. 2B). archival DNA from 276 patients with retinal dystrophies (149

FIGURE 3. Localization of mpdz in the chicken retina. (A) 4% paraformaldehyde-fixed paraffin-embedded sections (4 ␮m) of (i) White Leghorn and (ii) rdd retinas from 17-day-old embryos immunostained with polyclonal rabbit anti-chicken mpdz using standard methods. Note mpdz immunoreactivity was observed as intense brown staining at the OLM. (B) Cryosections (20 ␮m) of adult rat retinas stained with polyclonal rabbit antibody against (i) Mpdz, (ii) Crb1, or (iii) rabbit IgG negative control are shown. Bound primary antibody was visualized with Alexa Fluor 488–conjugated secondary antibody (green), and the nuclei were counterstained with propidium iodide (red). The rat retinas detached from the nucleated retinal pigment epithelial layer during tissue preparation. Intense green fluorescence corresponding to Mpdz (i) and Crb1 (ii) immunoreactivity was observed at the OLM, whereas the IgG control (iii) was immunon- egative. Scale bars, 50 ␮m. RPE (retinal pigment epithelium), OLM, ONL (outer nuclear layer), OPL (outer plexiform layer), INL (inner nuclear layer), IPL (inner plexiform layer), and GCL (ganglion cell layer).

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RP and 127 LCA) was screened using high-resolution melting (http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.11- curve analysis. Primer sequences used are depicted in Supple- 7872/-/DCSupplemental). Even though the numbers were mentary Table S1 (http://www.iovs.org/lookup/suppl/doi: limited, there appeared to be no obvious correlation be- 10.1167/iovs.11-7872/-/DCSupplemental). This approach iden- tween the mutation type and the age of disease onset or tified 10 heterozygous MPDZ sequence changes consisting of severity. five variants in patients with RP (c.132–133delCT, The lack of a second mutation in human patients carrying p.F45QfsX27; c.322C3 G, p.P108A; c.658A3 C, p.I220L; MPDZ variants argues against recessive inheritance. However, the c.3685C3 T, p.R1229W; c.5255C3 G, p.S1752X) and five size of the MPDZ gene (46 exons spread over 145 kb) means that variants in patients with LCA (c.1788–1790AGA3 GG, deletions encompassing whole exons, which would give rise to p.D597AfsX5; c.2344G3 A, p.G782R; c.4220A3 G, alleles that fail to amplify, cannot be excluded and may account p.Y1407C; c.4793C3 T, p.P1598L; c.5072A3 G, p.D1691G) for the second allele in some cases. Mutations in the promoter (Fig. 4). These variants, comprising three null alleles and region or in would also have been missed. To investigate seven missense changes, were not present in the SNP data- these possibilities, an RNA-based strategy focusing on expression base (http://www.ncbi.nlm.nih.gov/snp) and were absent levels and cDNA sequencing was considered. We initially per- from 190 ethnically matched control DNAs (380 chromo- formed reverse-transcription PCR on blood lymphocyte total somes). Assuming recessive inheritance, we sequenced the RNA, extracted from healthy persons, to assess MPDZ expression. entire coding region, and splice recognition signals, of However, the absence of an amplification product (data not MPDZ in the 10 patients but did not identify a second shown) suggested that MPDZ was not expressed in blood lym- mutation. To investigate the significance of the seven mis- phocytes and that an alternative source of RNA, from cells that sense substitutions, we compared the evolutionary conser- express MPDZ, would be needed from patients before this expla- vation of the corresponding wild-type amino acids (Supple- nation could be explored. mentary Fig. S4, http://www.iovs.org/lookup/suppl/doi: Alternatively, heterozygous MPDZ variants may cause dom- 10.1167/iovs.11-7872/-/DCSupplemental). Residues p.G782, inant disease in humans, so we analyzed any families from p.R1229, p.Y1407, and p.D1691, were found to be totally whom DNA was available for segregation of the variant with conserved, suggesting their functional importance. How- the disease phenotype (Supplementary Fig. S5, http://www.iovs. ever, p.P108, p.I220, and p.P1598 were not fully conserved, org/lookup/suppl/doi:10.1167/iovs.11-7872/-/DCSupplemental). suggesting that their variants may represent rare polymor- Using this approach, we showed that p.F45QfsX27 was inher- phisms and should be interpreted with caution. The clinical ited by an affected sibling and by an unaffected child who was history of the patients carrying a heterozygous MPDZ null younger than the age of onset of RP in this family (see Supple- allele or a missense substitution in an evolutionarily con- mentary Table S2, http://www.iovs.org/lookup/suppl/doi: served residue are depicted in Supplementary Table S2 10.1167/iovs.11-7872/-/DCSupplemental) and that p.I220L was

FIGURE 4. MPDZ mutation screen in human RP and LCA. (A) Schematic representation of the human MPDZ protein showing the distribution of the novel human sequence changes that were identified in RP and LCA. (B) Sequence chromatograms showing the 10 heterozygous MPDZ variants (i–x) in the DNA of human RP and LCA patients.

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transmitted from an affected mother. We also showed that MPDZ (MIM *603785) contains one L27 domain at the p.P1598L did not fully segregate with LCA in a pedigree with amino terminus and 13 PDZ domains. The L27 domain is a multiple affected persons, confirming that this variant is not protein interaction module usually found in scaffold pro- pathogenic. teins,20 consisting of three alpha helices that form a hetero- A third possibility to consider was that heterozygous MPDZ meric complex with other L27 domain-containing proteins.21 variants could act as modifiers of disease severity caused by PDZ domains, named after the first three PDZ domain-contain- mutations in other retinal dystrophy genes, or act in concert ing proteins identified (Postsynaptic density protein-95/ with mutations in other genes to cause digenic or trigenic Drospohila Discs large tumor suppressor/tight junction pro- disease.13,14 The MPDZ variant, p.P1598L, that did not segre- tein Zona occludins-1), are composed of approximately 90 gate with LCA (Supplementary Fig. S5, http://www.iovs.org/ amino acids that form six beta strands and two alpha helices.22 lookup/suppl/doi:10.1167/iovs.11-7872/-/DCSupplemental) PDZ domain-containing proteins are usually targeted to sub- could act as a modifier, leading to a more severe phenotype. membranous structures, where they bind to the carboxyl ter- However, detailed clinical data were not available for this mini or internal peptide sequences of receptors and chan- family, which was recruited in rural Pakistan; hence, no useful nels.23,24 Here they form a platform for mediating the assembly comparison could be made between family members with and of signaling and cell polarity complexes. MPDZ interacts di- without the MPDZ allele. Given the known interaction be- rectly with many different proteins (see GeneCards version 3, tween MPDZ and CRB1, this would appear the most likely http://www.genecards.org/), including claudins and associ- locus/modifier pairing. However, because RPE65 (MIM ated tight junction proteins.25–30 Different PDZ domains ϩ180,069) is similar to CRB1 in that mutations can cause within MPDZ are involved in these reactions, suggesting that either RP or LCA, this gene was also considered a potential there is scope for multiple simultaneous interactions, the na- partner.9,10,15–17 The possibility of digenic or trigenic inheri- ture of which depends entirely on the tissue in which the tance with CRB1 or RPE65 mutations was investigated by protein is expressed. sequencing the entire coding region and splice recognition MPDZ is found in the central nervous system,26,31–33 ret- signals of these genes in the 10 patients with MPDZ sequence ina,8 and many other tissues, where it has been shown to changes. Primer sequences used are depicted in (Supplemen- localize to tight,25,28,34,35 gap,33 neuromuscular,26 synap- tary Table S3, http://www.iovs.org/lookup/suppl/doi:10.1167/ tic,36–38 and adherens junctions.8 There MPDZ is involved in a iovs.11-7872/-/DCSupplemental). We identified homozygous diverse range of pathways that maintain cell integrity and CRB1 gene mutations in two LCA patients and RPE65 muta- function. These include regulating the conductive permeability tions in another two. GUCY2D (MIM *600179) mutations had of tight junctions,27,35 preventing spontaneous acrosomal ex- been found in the fifth LCA case (SGJ, unpublished data, 2010). ocytosis,39 regulating AMPA receptor clustering in hippocam- 36 The results are summarized in Table 1. Notably, the RP patients pal neuron synapses, maintaining GABAB and Kir4.2 receptor with MPDZ variants did not have CRB1 or RPE65 mutations. stabilities,40,41and regulating intracellular signaling in response This could imply that patients with certain CRB1, RPE65,or to osmotic stress in kidney cells,34 melatonin receptor signal- GUCY2D mutations have more severe disease (LCA) when a ing,42 and olfactory receptor signaling.43 heterozygous MPDZ variant is also present, whereas the MPDZ In the eye, MPDZ localizes to the OLM between the photo- variants seen in RP patients could be dominant alleles or could receptor inner segment and outer nuclear layer.8 The OLM is a modify the severity of mutations in retinal genes other than specialized site of adhesion that links the photoreceptors with CRB1 and RPE65 leading to RP. Mu¨ller glial cells, maintaining the correct photoreceptor shape and orientation, and is thought to contain both adherent and tight junctions.44 However, although the RPE cells are also DISCUSSION enriched with tight junctions, we did not observe any apparent Here we report that a premature stop codon mutation in the localization of MPDZ to this layer (SF and WJC, personal com- mpdz gene causes sex-linked, recessively inherited retinal de- munication, 2010). Expression of MPDZ at the OLM coincides generation and dysplasia (rdd) in chickens, and we describe with the site of the earliest observed pathology in the rdd heterozygous sequence changes in the corresponding human chicken and implies that the primary defect is a poorly formed ortholog in five patients with RP and five patients with LCA. OLM. The Mpdz interactant Crb1 also localizes to the OLM, as The lack of a second mutation in human patients carrying do the interactants Pals1 and Mpp4.8 Crb1 knockout mice MPDZ variants argues against recessive inheritance, though the develop lesions in the OLM that become progressively worse deletion of whole exons, intronic or promoter mutations, and during exposure to light.8,45 No other animal models with loss gene rearrangements would have been missed. Alternatively, of Mpdz function have been reported before this study. our findings in human patients with LCA suggest that heterozy- To conclude, our findings highlight the importance of gous MPDZ variants may act as modifiers of mutations in CRB1, MPDZ function in normal eye development. Because chicken RPE65,orGUCY2D. In RP, such changes could either cause eyes are relatively large, rdd birds could be used to test ther- dominant disease or act in concert with alleles at other loci. In apies for human blindness based on small molecules that pro- cases of dominant inheritance in humans, the contrast with mote stop-codon read-through.46 Furthermore, it has been sug- sex-linked recessive inheritance in rdd chickens (where the gested that disruption of the OLM may improve the chance of male is the homogametic sex, denoted ZZ, and the female is successful integration of transplanted photoreceptor precursor heterogametic, denoted ZW) could be due to inactivation of cells.47,48 Given that disruption of the OLM is one of the the mutated Z chromosome in half of all cells in male chick- earliest pathologic observations in the rdd chicken, rdd would ens.19 Thus, in a heterozygous human patient, regardless of be a strong model in which to test this theory. sex, each retinal cell expresses both normal and affected MPDZ alleles from chromosome 9p23; in contrast, in the Acknowledgments heterozygous male chicken retina, half of all cells express only a single normal allele of mpdz from the Z chromosome, having The authors thank David Morrice and Graeme Robertson for technical inactivated the other Z chromosome carrying the mutated assistance; and Frans P. M. Cremers and Anneke I. den Hollander allele. These cells may then derive a clonal advantage over cells (Department of Human Genetics, Radboud University Nijmegen Med- that have inactivated the normal mpdz allele and form a ical Centre, The Netherlands), Robert Koenekoop (McGill University, healthy OLM. Montreal, Canada), and Carmen Ayuso and Marta Perez Corton (Genet-

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TABLE 1. Summary of the MPDZ Sequences Variants Identified in RP and LCA Patients

Evolutionarily Clinical History DNA from Other Exon Variant Effect Conserved Residue Ethnicity Available Family Members CRB1 or RPE65 Mutations

Human MPDZ Sequences Variants in RP 2 c.132–133delCT p.F45QfsX27 NA White Caucasian Yes Yes None 3 c.322C3G p.P108A No White Caucasian No No None 5 c.658A3C p.1220L No White Caucasian No Yes None 25 c.3685C3T p.R1229W Yes White Caucasian Yes No None 38 c.5255C3G p.S1752X NA White Caucasian Yes No None Human MPDZ Sequence Variants in LCA 13 c.1788–1790AGA3GG p.D597AfsX5 NA White Caucasian Yes No RPE65: c.1022T3C, p.L341S/ c.94–2A3T, splice defect 16 c.2344G3A p.G782R Yes White Caucasian Yes No CRB1:c.609–615delGAAATAG, p.1205DfsX10/c.1437T3C, p.C480R 29 c.4220A3G p.Y1407C Yes White Caucasian Yes No RPE65: c.131G3A, p.R44Q/ c.271C3T, p.R91W 34 c.4739C3T p.P1598L No Asian No Yes* CRB1: c.107C3G, p.S36X/ c.107C3G, p.S36X* 37 c.5072A3G p.D1691G Yes White Caucasian Yes No None†

The exon in which the variant was identified, the change in the human cDNA sequence (NM_003829), the effect on the 2041 amino acid protein (NP_003820), whether a missense change is

an evolutionary conserved residue (Supplementary Fig. S4, http://www.iovs.org/lookup/suppl/doi:10.1167/lovs.11-7872/-/DCSupplemental), the ethnicity of the patient, whether a clinical history is IOVS, available (Supplementary Table S2, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.11-7872/-/DCSupplemental), whether DNA is available from other family members (Supplementrary Fig. S5, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.11-7872/- /DCSupplemental), and the identity of CRB1 or RPE65 mutations are shown. NA, not applicable. * The CRB1 mutation segregates with the disease phenotype and has been previously reported.18 10 No. 52, Vol. 2011, September †The patient has biallelic GUCY2D mutations, c.2302C3T, p.R768W and c.124–129delCTGCTT, p.L42-L43del (SGJ, personal communication, 2010). IOVS, September 2011, Vol. 52, No. 10 Mpdz Null Allele Causes Rdd 7439

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