December 2015
Volume 56, Issue 13
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Genetics  |   December 2015
Exome Sequencing Reveals AGBL5 as Novel Candidate Gene and Additional Variants for Retinitis Pigmentosa in Five Turkish Families
Author Affiliations & Notes
  • Simone Kastner
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
  • Ina-Janine Thiemann
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
  • Gabriele Dekomien
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
  • Elisabeth Petrasch-Parwez
    Ruhr-University Bochum, Neuroanatomy and Molecular Brain Research, Bochum, Germany
  • Sabrina Schreiber
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
  • Denis A. Akkad
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
  • Wanda M. Gerding
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
  • Sabine Hoffjan
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
  • Sezgin Güneş
    Ondokuz Mayis University, Faculty of Medicine, Department of Medical Biology, Samsun, Turkey
  • Selçuk Güneş
    Samsun Education and Research Hospital, Department of Ophthalmology, Samsun, Turkey
  • Hasan Bagci
    Ondokuz Mayis University, Faculty of Medicine, Department of Medical Biology, Samsun, Turkey
  • Jörg T. Epplen
    Ruhr-University Bochum, Human Genetics, Bochum, Germany
    University Witten/Herdecke, Faculty of Health, Witten, Germany
  • Correspondence: Gabriele Dekomien, Ruhr-Universität Bochum, Humangenetik, Gebäude MA5/39, Universitätsstr. 150, 44780 Bochum, Germany; Gabriele.Dekomien@rub.de
Investigative Ophthalmology & Visual Science December 2015, Vol.56, 8045-8053. doi:10.1167/iovs.15-17473
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      Simone Kastner, Ina-Janine Thiemann, Gabriele Dekomien, Elisabeth Petrasch-Parwez, Sabrina Schreiber, Denis A. Akkad, Wanda M. Gerding, Sabine Hoffjan, Sezgin Güneş, Selçuk Güneş, Hasan Bagci, Jörg T. Epplen; Exome Sequencing Reveals AGBL5 as Novel Candidate Gene and Additional Variants for Retinitis Pigmentosa in Five Turkish Families. Invest. Ophthalmol. Vis. Sci. 2015;56(13):8045-8053. doi: 10.1167/iovs.15-17473.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Retinitis pigmentosa (RP) is the most common inherited retinal disease with high genetic heterogeneity and variable phenotypes. Characteristic symptoms include night blindness and progressive loss of visual field, leading to blindness. Mutations in >60 genes have been identified to date as causative for RP, and additional candidate genes are assumed.

Methods: To find the disease-causing mutations in the affected members of five Turkish families, we sequenced whole exomes using an Illumina platform.

Results: Among all candidate genes for retinal degeneration we found two previously known sequence variations: a 4 bp deletion in the RPGR gene (c.1662_1665delAGAA; p.Glu555Glyfs*14) and a recently described USH1-causing missense mutation in MYO7A (c.472G>A, p.Gly158Arg). Furthermore, a novel 1 bp deletion in the VCAN gene (c.5118delA; p.Ser1707Valfs*44) was detected as well as a large deletion in EYS, spanning ∼ 400kb and comprising exons 16-26 (p.fs*). In one family, exome analyses of two affected individuals revealed a homozygous missense mutation (c.883G>A; p.Asp295Asn) in the AGBL5 (Agbl5; CCP5) gene, previously not reported to be associated with RP. RNA and protein analyses showed expression in human retina, as well as in mouse retina, brain and testis. Furthermore, cDNA analyses indicate the existence of tissue-specific AGBL5 splice variations in humans. AGBL5/CCP5 immunoreactivity was also visualized in human and mouse retinae.

Conclusion: Due to the characteristic RP phenotype in patients carrying the AGBL5 missense mutation we suggest this gene as a candidate for a new form of autosomal recessively inherited RP and recommend further investigation to confirm this hypothesis.

Retinitis pigmentosa (RP; OMIM #268000) is the most common inherited retinal dystrophy (IRD) with a worldwide prevalence of one in 4000 and approximately two million affected individuals.1 It is characterized by initial degeneration of the photoreceptors followed by progressive degeneration of the other retinal layers and an alteration of the pigment epithelium. At advanced stage funduscopy shows the so-called bone spicules, attenuation of retinal vessels and a waxy pale optic disc.2 Initially, the impairment of rods leads to night blindness and loss of peripheral visual field (tunnel vision). At a later stage, patients also suffer from loss of central vision due to degeneration of the cones and leading to complete blindness.3 RP may show a high degree of clinical heterogeneity with different ages of onset, varying clinical signs and diagnostic characteristics, even within a given family.1,4 It can also occur as a part of complex syndromes, the so-called syndromic RP. Usher syndrome, characterized by RP in combination with congenital or early-onset deafness, is the most frequently described syndromic form and accounts for approximately 10–15% of all RP cases.5 It is divided into three subtypes (USH1, USH2, and USH3) all including retinal degeneration but mainly differing in severity and progression of neurosensory deafness as well as variable impairment of the vestibular system.6 
Both non-syndromic RP and Usher syndrome have been shown to be highly heterogeneous. At present, mutations in >60 genes have been identified as causative for non-syndromic RP and twelve genes are known for Usher syndrome.7 While all known types of Usher syndrome show an autosomal recessive mode of inheritance, isolated RP can be transmitted in an autosomal dominant, autosomal recessive or X-linked mode. Rarely, digenic and mitochondrially (maternally) inherited cases have been reported.5 So far five different genes (PRCD; ABCA4, RP1/RHO, MAK) for autosomal dominant or recessive RP were identified in Turkish families.811 For heterogeneous diseases such as IRDs and especially RP, next-generation sequencing (NGS) techniques have increasingly been used for the detection of new disease causing genes and mutations lately.12 Here we performed exome sequencing in five extended Turkish families with RP. Besides sequence variations of highly probable pathogenic relevance in known candidate genes for RP, we identified a novel homozygous missense mutation in a gene so far not connected to human disease (AGBL5) in one of the families displaying autosomal recessive inheritance. Previous studies investigated the characteristics of the encoded protein in zebrafish13,14 and mice,15,16 where it is called CCP5 (zebrafish, mice) or AGBL5 (mice). Moderate expression of Agbl5 was confirmed in mouse eye and brain as well as its abundant presence in testis.16 We performed additional analyses including expression studies and immunohistochemistry in, both, human and mouse tissues, to further evaluate the role of AGBL5 for RP. 
Material and Methods
Mutation Analysis
Patient Data.
Altogether 36 DNA samples of 17 affected and 19 healthy members from five Turkish families (Fig. 1) were available. Nine affected members, all displaying an inherited form of IRD and two of them suffering from congenital hearing loss (see Table), were investigated by means of exome sequencing. The families were recruited at the Samsun Education and Research Hospital in Samsun (Turkey). Routine ophthalmological examinations were performed on all patients as well as hearing tests in family D. Best-corrected visual acuities were measured with the Snellen chart, and the anterior segment was evaluated with slit lamp biomicroscopy. Fundus examinations were done with both direct and indirect ophthalmoscopy. In suspicious cases, eyes were examined with the Goldmann triple-mirror contact lens for peripheral retinal lesions, and visual field tests were performed (Carl Zeiss, Jena, Germany; 750 visual field perimeter). The clinical information of all investigated individuals is summarized in Table. For clinical and molecular genetic studies informed consent was obtained from all participants. The study has been approved by the Ethics committee of Samsun Education and Research Hospital (Reference number 01, 08/19/2010). This study involves human subjects, and it adheres to the tenets of the Declaration of Helsinki. 
Figure 1
 
Pedigrees of the five Turkish families (AE). RP phenotypes are indicated by filled symbols while the terms in [brackets] specify the genotypes. Arrows point at the index patients. The different inheritance modes and variants are given on the upper left hand side of the individual pedigree. N.A., no available DNA sample; [M][W], heterozygous carrier; [m], hemizygous individual; [W][W], person with two wild-type alleles; [M][M], homozygous family member.
Figure 1
 
Pedigrees of the five Turkish families (AE). RP phenotypes are indicated by filled symbols while the terms in [brackets] specify the genotypes. Arrows point at the index patients. The different inheritance modes and variants are given on the upper left hand side of the individual pedigree. N.A., no available DNA sample; [M][W], heterozygous carrier; [m], hemizygous individual; [W][W], person with two wild-type alleles; [M][M], homozygous family member.
Table
 
Clinical Data and Putative Variants of All Affected Family Members
Table
 
Clinical Data and Putative Variants of All Affected Family Members
Mutation Screening.
DNA was extracted from peripheral blood leukocytes following the salting out method.17 The DNA of the nine selected probands was prepared for whole exome sequencing using Roche NimbleGen SeqCap EZ Human Exome (Version 2.0; Roche NimbleGen, Inc. Madison, WI, USA) at ATLAS Biolabs GmbH (Berlin, Germany) following the manufacturer's protocol (Roche NimbleGen, Inc.). These samples were then sequenced using the Illumina HiSequation 2000 platform (Illumina, Inc., Chesterford, UK; paired-end: 2x100bp). The platform works with the sequencing-by-synthesis method and massively parallel bridge amplification on flow cells.18 Our data was aligned to human genome 19 (hg19) using the Illumina ELANDv2 Software (Illumina, Inc.). The variant comparison tool (NextGENe; SoftGenetics, LLC, State College, PA, USA) was chosen to compare the exomes of two affected relatives from each family. By creating a bedfile (UCSC) it was possible to screen all genes which were known to cause retinal disorders. Altogether, 289 genes were scanned (see Supplementary Table S1). Since the pedigree information was not absolutely complete for all five families at the beginning of the investigation and thus the mode of inheritance was not always clearly visible from the start, all candidate genes for retinal degeneration were included for each family. The focus was on likely deleterious mutations, mainly missense and nonsense mutations in coding regions and splice sites. For discovering larger deletions the CNV tool (Copy number variation-tool; NextGENe) was used. To reveal the causal mutation in pedigree E, we extended the search to the entire exomes of the two affected family members. Individual mutations were evaluated in more detail by visualization of the bam-files (binary version of sequence alignment map) with the integrative genomics viewer (IGV).19 Known single nucleotide polymorphisms (SNPs) were filtered out based on the information offered in online variation data bases: NCBI dbSNP, OMIM and HGMD.2022 Online disease prediction tools and genome browser were also used providing useful details about SNP's23 and frequencies in the population: MutationTaster (http://doro.charite.de/MutationTaster/index.html), Polyphen (http://genetics.bwh.harvard.edu/pph2/), UCSC Genome Browser (http://genome.cse.ucsc.edu/), Ensembl (http://www.ensembl.org/index.html), and GenAtlas (http://genatlas.medecine.univ-paris5.fr/).2327 
Verification and Validation of Variants.
To verify the results of our analyses and to eliminate false positive findings, polymerase chain reaction (PCR), Sanger sequencing, restriction fragment length polymorphism (RFLP), denaturing high performance liquid chromatography (DHPLC) as well as fragment length analyses were performed (for primers and enzymes see Supplementary Table S2). Sanger sequencing results were analyzed with the SeqMan Sequencing Software (DNAstar, Inc., Madison, WI, USA). The Beckman Coulter system with the CEQ (Beckman Coulter, Inc., Krefeld, Germany) was used for fragment length analyses. DHPLC analysis was performed with the Transgenomic system (Transgenomic, Inc., Omaha, NE, USA). All available family members were analyzed for the putative disease-causing variant in each family and co-segregation with the disease was evaluated. Additionally, for validation of previously undescribed mutations in pedigrees B, C and E, RFLP analysis was performed in ∼ 350 Turkish control samples. To further characterize the large deletion in EYS (pedigree D), intronic primers were designed using the Primer3web software (version 4.0.0, http://primer3.ut.ee/; see Supplementary Table S3). 
AGBL5/Agbl5/AGBL5 Expression in Mouse and Human Tissues
Mouse and Human Tissue.
C57BL/6 mice were bred in-house (Ruhr-University Bochum, Germany) and grown up in an artificial light/dark cycle of 12 hours while having food and water ad libitum. Human retina and muscle were obtained from a female (71y) who had died from lung cancer. All mice experimental procedures complied with the German guidelines for animal care and were approved by the regional authority (LANUV, Northrhine Westphalia, Germany) and the guidelines of the ARVO statement for the use of animals in ophthalmic and vision research were adhered to. Studies on human tissues and cells were carried out in accordance with the Ethics Committee of the Medical Faculty of the Ruhr-University Bochum (Bochum, Germany; Reference number 3386-09). 
Quantitative PCR (qRT-PCR) and Western Blot.
Retina, remaining eye tissue (without retina), brain, testes and skeletal muscle were dissected from six CO2-anaesthetized and decapitated mice (P10 and P28, 3 animals per developmental stage). Tissue sample were equally divided for RNA and protein analyses, immediately frozen on dry ice and stored at −80°C for further processing. 
RNA was isolated using TriFast reagent (Peqlab, Erlangen, Germany) according to the manufacturer's instructions up to the point of phase separation. The aqueous phase was transferred to an RNeasy column (Qiagen, Hilden, Germany) and processed according to the manufacturer's protocol, RNA quality was measured with the Nanodrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Quantitative real-time PCR (qRT-PCR) was performed using the StepOne Plus system (Applied Biosystems, Darmstadt, Germany) and the PowerSYBR green RNA-to-CT TM kit (Applied Biosystems). Primers were designed with the Primer3 software28 and were designed to span exon boundaries in order to avoid amplification of contaminating genomic DNA (see Supplementary Table S4). Human GAPDH,29 mouse Gapdh30 as well as human/mouse 18s rRNA29 primer sequences for internal standard have been reported previously. Using 30ng of RNA per reaction, PCR cycling conditions for reverse transcription one-step PCR were as recommended by the manufacturer (StepOne SoftwareTM v2.1; Applied Biosystems). The relative RNA expression level was determined from triplicates of three independent assays by the ΔΔCt method and expressed as 2ΔΔCt for fold change. Statistical analysis was performed using Student's t-test (Fold changes >2 were considered as statistical significant). Western blot analysis was conducted as described previously.30 Briefly, whole protein lysates from human and mouse tissues were extracted in ice-cold lysis buffer (50 mMTris–HCl [pH 8.0], 150 mM NaCl, 1% [v/v] NP-40, 1 g/L SDS, 1 g/L Na-Desoxycholate) with protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA) on ice for 10 minutes centrifuged at 600g for 20 minutes and then supernatants were harvested and stored at −20°C. Protein quantification was performed according to a standard method (BCA Protein Assay Reagent, Thermo Scientific). For each gel lane, 40 μg protein was denatured in 5 x Laemmli buffer at 95°C for 5 minutes. Disulfide bonds were reduced by adding 1M Dithiothreitol (DTT, Sigma-Aldrich Corp.). Proteins were loaded on 10% SDS–PAGE, transferred onto nitrocellulose (Hybond C, GE Healthcare, Freiburg, Germany) and incubated with the polyclonal goat anti-CCP5 antibody (human tissue: T-20; Santa Cruz Biotechnology, Inc., Heidelberg, Germany; 1:300 dilution, mouse tissue: G-19, Santa Cruz Biotechnology, Inc.; 1:250 dilution). Using mouse anti-goat secondary antibody (1:10,000 dilution; Thermo Scientific) as a conjugate, detection was carried out using ECL plus (Thermo Scientific) and the Fusion SL imaging system (Peqlab). Blots were stripped using Western blot stripping solution (Thermo Scientific) and re-probed with rabbit anti-GAPDH (1:1000 [human] and 1:500 [mouse] dilution, ab9485; Abcam, Cambridge, UK) as a loading control. 
Immunohistochemistry.
Human eyes were immersion-fixed in 4% Paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4), sagittally halved at the level of the optic nerve and embedded in Paraffin.31 P10 and P28 mice were anaesthesized with pentobarbital (720 mg/kg Nembutal i.p.) and transcardially perfused with 4% paraformaldehyde as described.32 After fixation, eyes were rinsed in phosphate-buffered saline (PBS), immersed overnight in 30% saccharose in PBS, covered with Tissue-Tek OCT (Sakura Finetek, Heppenheim, Germany), shock-frozen in 8% methylcylcohexan in 2-methylbutan (v/v; −80°C) and stored at −80°C until used. Peroxidase immunohistochemistry was performed on 12μm Paraffin (human retinae) and cryosections (mice retinae) with the polyclonal anti-AGBL5 antibody (1:100; NBP1-83616, Novus Biologicals, Littleton, CO, USA) and the polyclonal goat anti-CCP5-antibody (1:1000; T-20, Santa Cruz Biotechnology, Inc.). The incubation was performed as previously described.31 Both antibodies showed the same immunoreactivity. In order to control the specificity of the CCP5 antibody, the corresponding peptide was used to eliminate immunoreactivity by prior absorption of the antibody. The antibody was mixed with a 10-fold excess of the peptide in 1:1000 CCP5 antibody dilution for two hours at RT°C. The incubation with the peptide blocking solution was performed with the same concentration as used for the primary antibody. For the AGBL5 antibody a blocking peptide was not available. Adjacent sections were stained with Hematoxylin-Eosin for morphological reference. 
Characterization of AGBL5/Agbl5 Splice-variants.
RNA isolation from human retina, muscle, lymphocytes and mouse retina was performed as mentioned above. mRNA was purified using Oligotex mRNA mini kit (Qiagen) followed by cDNA synthesis by reverse transcription of mRNA using a AGBL5 R-primer located in the 3′ region in order to allow amplification of the whole coding transcript (see Supplementary Table S5), following the manufacturer‘s instructions (Sensiscript RT Kit, Qiagen). Due to the length of the AGBL5/Agbl5 coding region, five sequencing primers (see Supplementary Table S5) were designed, spanning the cDNA to reveal previously undescribed splice variants by sequencing amplification products. cDNA sequencing was performed using an automated Sanger didoxy method33 (Big dye terminator kit 3.1; Applied Biosystems). Sequences were evaluated with SeqManII (DNAstar, Inc.). 
Results
Exome Sequencing Reveals the Putative Disease Causing Variants in Five Turkish Families
Exome sequencing with subsequent screening of all candidate genes for retinal degeneration revealed a 4 bp deletion in exon 14 of the RP GTPase regulator (RPGR) gene on the X chromosome (c.1662_1665delAGAA; p.Glu555Glyfs*14) in both investigated patients in pedigree A (Fig. 1; Table). This deletion, which has previously been described as pathogenic in a Jordanian patient with a mild disease course,34 leads to a stop codon after 14 amino acids. It was subsequently verified here by fragment length analysis in all affected family members. Two female carriers were clinically affected, but the deletion was also verified in a healthy woman without RP symptoms in this family. All other healthy family members as well as 160 German and 60 Turkish controls harbored wild type RPGR genotypes (Fig. 1). 
In pedigree B, a heterozygous 1 bp deletion was identified in exon 8 of the VCAN gene on chromosome 5 that results in a frame shift and a stop codon after 44 amino acids (c.5118delA; p.Ser1707Valfs*44; Fig. 1; Table). This deletion, which has not been reported before, was subsequently confirmed in all affected family members, but it was not detected in 160 German and 60 Turkish controls. 
Searching for a putative disease causing variant in pedigree C by screening all candidate genes with the integrative genomics viewer (IGV) showed areas in the EYS (Eyes Shut homolog) gene on chromosome 6 without alignment or with low coverage. This observation was reviewed in more detail with the CNV tool, revealing a homozygous deletion between exons 16 and 26 which was subsequently confirmed in all affected members of pedigree C while all examined healthy descendants carried the deletion in heterozygous state (Fig. 1; Table). PCR analysis with intronic primers (intronic primer: see Supplementary Table S3) confirmed the deletion between intron 15 and intron 26 and revealed the breakpoint (c.2381+5961_5644-24572del; p.fs*). The deletion spans 377,282 bp and leads to a stop codon (Fig. 2). 
Figure 2
 
A novel deletion in EYS revealed in Turkish family members with RP symptoms. The black arrow on the right indicates the missing number of sequence copies as displayed by the CNV tool. Length and position of the deletion is indicated. The grey area below the deletion indicates the homologous region within introns 15 and 26 which comprise the breakpoints. The genomic position of the breakpoints is shown to the left (intron 15) and to the right (intron 26) of this area.
Figure 2
 
A novel deletion in EYS revealed in Turkish family members with RP symptoms. The black arrow on the right indicates the missing number of sequence copies as displayed by the CNV tool. Length and position of the deletion is indicated. The grey area below the deletion indicates the homologous region within introns 15 and 26 which comprise the breakpoints. The genomic position of the breakpoints is shown to the left (intron 15) and to the right (intron 26) of this area.
In pedigree D, a homozygous missense mutation (c.472G>A; p.Gly158Arg) was identified in both analyzed patients in exon 6 of MYO7A located on chromosome 11 (Table). This missense mutation was recently described in compound heterozygous state together with a truncating mutation in a patient with USH1 and was classified as UV3 (unclassified variant) based on in silico analyses.35 All affected examined individuals in our family were found to carry the missense mutation in homozygous state while the descendants of one patient harbored a heterozygous genotype (Fig. 1). This variant was not found in 350 Turkish controls. 
In pedigree E, screening of all known candidate genes for retinal degeneration did not lead to the detection of a mutation. Therefore we extended the investigation and evaluated the entire exome of the two affected relatives and thereby identified a novel homozygous missense mutation (c.883G>A; p. Asp295Asn) in exon 5 of the AGBL5 gene on chromosome 2 (Fig. 1; Table). This missense mutation is located in a highly conserved region and in silico analyses predicted a pathogenic effect of this variant with high probability (PolyPhen: “probably damaging,” MutationTaster: “disease causing,” score 0.999). It was subsequently confirmed in homozygous state in all affected individuals in the family, and the consanguineous parents carried the mutation in heterozygous state (Fig. 1). The variant was not found in 367 Turkish controls. 
AGBL5/Agbl5 Expression in Human and Mouse Retina
Comparing the relative quantitation scores (RQ) of RNA expression levels in human tissues AGBL5 mRNA was detected in retinal tissue, whereas expression in skeletal muscle was significantly lower (RQ: 0.2; P < 0.05, Fig. 3A). In the mouse, retinal Agbl5 expression was detected at P10 and P28 in the retina, differences in expression levels were observed in P28 mouse testis (+8.4 fold change compared to retina P10; P < 0.01) and P10 testis (+1.76 fold compared to retina P10; P < 0.01). Similar expression levels were found in P10 and P28 mouse brain when compared to the retina at P10 (P10 brain: RQ: 0.7; P28 brain: RQ: 0.5) as well as in eye tissue without retina at P10 (RQ: 0.7). In contrast, Agbl5 expression was barely detectable in eye tissue without retina at P28 (RQ: 0.06; P < 0.05) and muscle (P10: RQ: 0.03; P28: RQ: 0.09; P < 0.05). In general, highest Agbl5 mRNA expression levels were detected at the P10 stage in retina, brain and testis (Fig. 3A). 
Figure 3
 
Characterization of AGBL5/Agbl5/AGBL5 in human and mouse. (A) Quantitative real-time PCR revealed higher AGBL5 RNA expression in human retina than in muscle. In the mouse, Agbl5 RNA expression was detectable in the retina, remaining eye tissue, brain and testis at P10 and likewise at P28 with the difference of a reduced expression in the eye tissue without retina. Expression levels of Agbl5 were very low in skeletal muscle tissue at both ages. For RQ calculation, retinal expression levels in human retina and mouse (P10) were set to 1 and GAPDH and 18SrRNA were used as house-keeping controls. Mean relative quantitation score (RQ) from three independent experiments was calculated by the ΔCT-method; standard deviation is indicated by error bars. The RQ of retinae were compared to other tissues and statistically evaluated using Student's t-test (*P < 0.05). n = 1 human retina and tissue from n = 3 mice was analyzed. (B) AGBL5 protein expression in human and mouse tissue at P10 and P28 was analyzed by Western blot analyses. AGBL5 protein was detected in human and mouse retina. AGBL5 protein expression was detected in mouse retina, brain and testis at P10 and P28. A signal was lacking in mouse eye tissues without retina and skeletal muscle GAPDH signals represent loading controls. The molecular weight of the AGBL5/CCP5 protein is ∼ 97 kDa as expected (human and mouse, UniProtKB database [http://www.uniprot.org/]). 40 μg protein/lane was applied on a 10% SDS gel. (Ew/oR): mouse eye without retina, (C) AGBL5/CCP5 immunohistochemistry in human and mouse retinae. AGBL5 immunoreactivity is distributed all over in the human retina though varying in expression. Staining is most prominent in the cone inner segments (black arrowheads, IS), ganglion cells (GCL, white arrowheads) and in nerve fiber layer (NFB). Sections of P10 and P28 mouse retinae show prominent AGBL5/CCP5 immunoreactivity in the outer plexiform (OPL), inner plexiform (IPL), GCL and NFL, the latter more expressed in the P28 mouse retina. Faint immunoreaction is observed in the developing IS of the photoreceptors (PR) in P10 retina, more distinct in the IS of the P28 mouse with faint staining also observed in the outer segments. More staining is detected in the inner nuclear layer (INL). Control sections lack reactivity, when treated with CCP5 and blocking peptide or when the primary antibody has been omitted. Hemotoxylin-Eosin (HE-) shows morphology of the respective retina sections. (ONL) outer nuclear layer, (OS) outer segments. Scale bars in the control sections represent 20μm, also for the respective adjacent sections.
Figure 3
 
Characterization of AGBL5/Agbl5/AGBL5 in human and mouse. (A) Quantitative real-time PCR revealed higher AGBL5 RNA expression in human retina than in muscle. In the mouse, Agbl5 RNA expression was detectable in the retina, remaining eye tissue, brain and testis at P10 and likewise at P28 with the difference of a reduced expression in the eye tissue without retina. Expression levels of Agbl5 were very low in skeletal muscle tissue at both ages. For RQ calculation, retinal expression levels in human retina and mouse (P10) were set to 1 and GAPDH and 18SrRNA were used as house-keeping controls. Mean relative quantitation score (RQ) from three independent experiments was calculated by the ΔCT-method; standard deviation is indicated by error bars. The RQ of retinae were compared to other tissues and statistically evaluated using Student's t-test (*P < 0.05). n = 1 human retina and tissue from n = 3 mice was analyzed. (B) AGBL5 protein expression in human and mouse tissue at P10 and P28 was analyzed by Western blot analyses. AGBL5 protein was detected in human and mouse retina. AGBL5 protein expression was detected in mouse retina, brain and testis at P10 and P28. A signal was lacking in mouse eye tissues without retina and skeletal muscle GAPDH signals represent loading controls. The molecular weight of the AGBL5/CCP5 protein is ∼ 97 kDa as expected (human and mouse, UniProtKB database [http://www.uniprot.org/]). 40 μg protein/lane was applied on a 10% SDS gel. (Ew/oR): mouse eye without retina, (C) AGBL5/CCP5 immunohistochemistry in human and mouse retinae. AGBL5 immunoreactivity is distributed all over in the human retina though varying in expression. Staining is most prominent in the cone inner segments (black arrowheads, IS), ganglion cells (GCL, white arrowheads) and in nerve fiber layer (NFB). Sections of P10 and P28 mouse retinae show prominent AGBL5/CCP5 immunoreactivity in the outer plexiform (OPL), inner plexiform (IPL), GCL and NFL, the latter more expressed in the P28 mouse retina. Faint immunoreaction is observed in the developing IS of the photoreceptors (PR) in P10 retina, more distinct in the IS of the P28 mouse with faint staining also observed in the outer segments. More staining is detected in the inner nuclear layer (INL). Control sections lack reactivity, when treated with CCP5 and blocking peptide or when the primary antibody has been omitted. Hemotoxylin-Eosin (HE-) shows morphology of the respective retina sections. (ONL) outer nuclear layer, (OS) outer segments. Scale bars in the control sections represent 20μm, also for the respective adjacent sections.
These findings were supported by qualitative Western blot analyses, revealing AGBL5 protein expression in human retina as well as mouse retina, brain and testis visualized at ∼ 97 kDa (Fig. 3B) but barely detectable in eye tissue without retina and muscle tissue (Fig. 3B). 
cDNA Analysis Emphasizes the Presence of Different Splice Variants
Amplifying and evaluating cDNA from human tissues (retina and muscle) by comparing them to the longest known AGBL5 transcript (AGBL5 004; ENS00000360131; Ensembl) revealed a possible AGBL5 splice variant in the retina missing exon 2. Furthermore, Agbl5 cDNA was found in lymphocytes and muscle comprising an additional exon, which is part of a splice variant, that was not proven experimentally before (AGBL5 001; ENST00000487078, exon 14). In mouse retina, a splice variant missing exon 3 was detected by comparing sequences to the longest Agbl5 transcript known in mouse (Agbl5-004; ENSMUST00000114700; Ensembl). 
AGBL5/CCP5 Immunohistochemistry in Human and Mouse Retinae
Human retina showed AGBL5 immunoreactivity in all layers, most prominent in the cone inner segments, ganglion cells and nerve fiber layer (Fig. 3C). AGBL5 reactivity was also detected in the nerve fiber, ganglion cell, inner and outer plexiform layers of P10 and P28 mice. Moderate staining was observed in the inner nuclear layer and faint staining was detected in the outer nuclear and inner segment layers of the photoreceptors, the latter slightly more expressed in the P28 retina according to the advanced development. As in humans the outer segment layer showed faint AGBL5 reactivity in P28 mice retina. Omission of the primary antibody and pre-absorption of the CCP5 antibody with the respective peptide confirmed the specificity of the antibody used as documented in the control sections (Fig. 3C). The normal structure of the human retina and the stage of retinal development in P10 and P28 mice used for immunohistochemistry were documented by adjacent Hematoxylin-Eosin-stained sections (Fig. 3C). 
Discussion
This study is based on the analysis of nine patients' exomes using NGS. For highly heterogeneous diseases such as IRDs and especially RP, NGS has become a faster and much more cost-effective method than the standard Sanger sequencing approach.12 We performed whole exome sequencing (WES) which reduces time and costs compared to whole genome sequencing (WGS) but requires an enrichment procedure.36 This treatment was reported to result in a skewed coverage37 but did not impair our examinations here. By using an RP panel, exclusively genes linked to IRDs had to be evaluated. The DNA sequence screening was expanded in one family in which no candidate could be defined among the examined genes (Pedigree E, see below). Besides the advantages of NGS in general, the ethical aspects should be kept in mind38 as well as the possibility to detect false positive or negative single nucleotide variants (SNVs)37 which demands the verification of all findings. We started with targeted analysis of all genes linked to retinal disorders, which led to the identification of two previously described as well as two novel sequence variants of highly probable pathogenic relevance in RP-related genes in four of the five families. In detail, the 4 bp deletion in exon 14 of the RPGR gene identified in pedigree A has previously been reported in a family from Jordan.34 In our Turkish family, both males and carrier females were equally affected, but the deletion was also found in a healthy woman suggesting highly variable clinical expression in carrier females as described for other X-chromosomal disorders.39 The RPGR protein is localized in photoreceptor-connecting cilia ensuring the survival of the photoreceptors, and it also plays an important role in protein transport.40 
In pedigree B, a novel 1 bp deletion leading to a frameshift was identified in the VCAN gene on chromosome 5. VCAN encodes the chondroitin sulfate proteoglycan versican, which is a component of the vitreous body and important for maintaining the vitreous matrix.41 Up to now, only one missense mutation in exon 8 of VCAN has been identified as the causative mutation in a family with autosomal dominant RP.42 Furthermore, mutations in splice sites were reported as disease causing in families with Wagner syndrome (WS, OMIM #143200), a rare IRD with several symptoms related to RP.43 Since the affected individuals in our family do not show typical features of WS, our results support to the hypothesis that mutations in the VCAN gene may result in different ophthalmic phenotypes. 
A large homozygous deletion in the EYS gene was found to be segregating with disease in pedigree C. Nearly 5 to 18% of all non-syndromic autosomal recessive RP cases44 as well as one recently described patient with autosomal recessive cone-rod dystrophy45 were linked to different variations in EYS. This gene comprises 43 exons and encodes a protein which harbors at least 31 EGF-like and several LamG domains interrupted by further EGF-like domains. The gene was described as a human orthologue of the Drosophila eyes shut (eys) gene which is important for photoreceptor morphogenesis in insects with an open rhabdomer system.46 In a previous study a large deletion, spanning exons 16-19, was reported to be causative for autosomal recessive RP in a Spanish family.47 The deletion identified in this study is the largest detected so far, spanning approximately 377 kb. The loss of exons 16-26 causes a frame shift leading to a reduction of domains and thus EYS dysfunction. 
In pedigree D, a homozygous missense mutation in MYO7A was detected which was recently described in the compound heterozygous state together with a truncating mutation in a patient with Ush1 and was classified as UV3.35 MYO7A encodes an actin-based molecular motor with ATPase activity which belongs to the class of unconventional myosins.47 Mutations in MYO7A are the most frequent cause of Ush1,48 but several variants were also linked to Leber congenital amaurosis (LCA)49 and different non-syndromic forms of deafness.50 In line with these previous reports, the affected members of this Turkish family D displayed both RP and deafness, suggesting Usher syndrome. The Gly158Arg mutation is located in a highly conserved region and affects the head (motor) domain which enables the transport of cargo by MYO7A moving along the actin filaments.47 We therefore believe that this missense mutation constitutes the disease-causing variant in pedigree D. 
Since targeted analysis of all genes linked to retinal disorders did not reveal a pathogenic mutation in pedigree E, we extended our search to include the entire exomes of the two affected family members. With this approach, a homozygous missense mutation (Asp295Asn) was detected in the deglutamylase gene AGBL5 which was confirmed in all affected family members while the unaffected parents are heterozygous carriers. 
AGBL5/Agbl5 (ATP/GTP-binding protein–like protein) is a member of the cytosolic carboxypeptidases (CCP) protein family, a subgroup of the M14 carboxypeptidases (CPs),51 often called CCP5. Members of the CCP family are involved in posttranslational modifications (PTMs) of α- and β-tubulin, the main component of microtubules.52 AGTBP1 (Nna-1, CCP1), AGBL1 (CCP4) and AGBL4 (CCP6) were described to remove long poly-glutamate chains from mouse brain tubulin, while AGBL5 specifically cleaves branching point glutamates.15 A recent study further reported that AGBL5 also removes longer α-linked glutamate chains from different substrates, albeit more slowly.53 The missense mutation identified here results in an exchange (D > N), thus altering the amino acid sequence NPDG to a NPNG within the zinc finger domain of AGBL5. The NPDG motif was previously found to be highly conserved within the CP family.16 Therefore the catalytic domain may be disturbed. In patients, the impairment of this domain could lead to reduced or impeded AGBL5 deglutamylase activity which in turn may result in an altered tubulin modification pattern, thus influencing tubulin function or binding character. The transport cilium in photoreceptors, which is important for the renewal of the outer segments54 as well as the axons of the different cell types in the retina and brain are composed of tubulin and therefore constitute potential substrates for AGBL5. Interestingly, it was recently revealed that biallelic variants in a glutamylase encoding gene (TTLL5) cause IRDs.55 
Given the potential involvement of AGTBP1/AGBLs in ciliary function, we regarded AGBL5 as a promising candidate gene for RP and aimed at further characterizing expression sites and levels and distribution of this gene. One study reported a smaller eye size in CCP5 zebrafish morphants,14 and knock down of Ccp5 was found to increase cilia tubulin glutamylation which led to ciliopathy phenotypes and interferred multicilia motility.13 In our study we were able to demonstrate the expression of RNA and protein in human retina. Moreover, AGBL5 expression was also found in mouse retina where it was located in the inner nerve fiber and inner ganglion cell layer (GCL), as well as in the inner (IPL) and outer (OPL) plexiform layers. 
A sex specific modification pattern as well as disease manifestation appears possible for AGBL5/Agbl5, as emphasized by the observation that the male patient (family E) is the only individual which shows additional symptoms. That this patient also suffered from mild mental retardation is in line with another study which reported that hyperglutamylation of tubulin caused by deficient AGTBP1 activity is causative for the neurodegeneration in pcd mice.15 
Age-dependent expression levels were found in mouse tissues, revealing increased Agbl5 levels in P10 mouse brain and retina when compared to P28. In zebrafish, Ccp5 was found to be crucial for the embryonic development and ciliogenesis.13,14 We therefore suggest that AGBL5 expression and thus deglutaminase function could be essential for developmental processes in the mouse and possibly in humans. The high RNA expression levels of Agbl5 in testis of P10 and P28 mice are in line with previously reported AGTBP1 expression where stronger antibody signals were detected in mouse testis from P18 onwards when compared to earlier stages.56 This emphasizes an involvement of AGBL5 in spermatogenesis similarly to pcd-mice lacking AGTBP1 expression, showing infertility and abnormal sperm shape and motility.14,16,56 
Several AGBL5/Agbl5 splice variants are comprised in the Ensembl database but most of them are not yet experimentally confirmed. We revealed cDNA sequences in human retina missing exon 2 as well as sequences in human lymphocytes and muscle with an additional exon that is not present in the retina. The additional exon leads to an earlier stop codon which presumably changes protein structure and function. 
In conclusion, by performing analysis on mRNA, protein and cDNA basis, we propose that the above mentioned deglutamylase activity is crucial for maintaining the function of certain tubulin containing structures such as retina and brain, both impaired within the affected individuals of pedigree E. Due to immunohistochemical staining of mouse retina it appears possible that the typical RP symptoms do not have their origin in photoreceptor degeneration primarily, but in the disturbance of different transport pathways within the retinal layers which may lead to the impairment of cones and rods in the peripheral retina. We, therefore, suppose that this disease is a previously unknown form of RP extending the spectrum of candidate genes for IRDs. Whether the discovered impairment of retina and brain refers to a new syndrome should be confirmed by analyzing further tissues of the affected individuals coupled with detailed clinical examinations or the generation of a mouse model lacking Agbl5 expression. 
Acknowledgments
The excellent technical assistance of Hans-Werner Habbes and Marlen Löbbecke-Schumacher are gratefully acknowledged. 
Supported by private funds of the Department of Human Genetics, Ruhr University Bochum. 
Disclosure: S. Kastner, None; I.-J. Thiemann, None; G. Dekomien, None; E. Petrasch-Parwez, None; S. Schreiber, None; D.A. Akkad, None; W.M. Gerding, None; S. Hoffjan, None; S. Güneş, None; S. Güneş, None; H. Bagci, None; J.T. Epplen, None 
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Figure 1
 
Pedigrees of the five Turkish families (AE). RP phenotypes are indicated by filled symbols while the terms in [brackets] specify the genotypes. Arrows point at the index patients. The different inheritance modes and variants are given on the upper left hand side of the individual pedigree. N.A., no available DNA sample; [M][W], heterozygous carrier; [m], hemizygous individual; [W][W], person with two wild-type alleles; [M][M], homozygous family member.
Figure 1
 
Pedigrees of the five Turkish families (AE). RP phenotypes are indicated by filled symbols while the terms in [brackets] specify the genotypes. Arrows point at the index patients. The different inheritance modes and variants are given on the upper left hand side of the individual pedigree. N.A., no available DNA sample; [M][W], heterozygous carrier; [m], hemizygous individual; [W][W], person with two wild-type alleles; [M][M], homozygous family member.
Figure 2
 
A novel deletion in EYS revealed in Turkish family members with RP symptoms. The black arrow on the right indicates the missing number of sequence copies as displayed by the CNV tool. Length and position of the deletion is indicated. The grey area below the deletion indicates the homologous region within introns 15 and 26 which comprise the breakpoints. The genomic position of the breakpoints is shown to the left (intron 15) and to the right (intron 26) of this area.
Figure 2
 
A novel deletion in EYS revealed in Turkish family members with RP symptoms. The black arrow on the right indicates the missing number of sequence copies as displayed by the CNV tool. Length and position of the deletion is indicated. The grey area below the deletion indicates the homologous region within introns 15 and 26 which comprise the breakpoints. The genomic position of the breakpoints is shown to the left (intron 15) and to the right (intron 26) of this area.
Figure 3
 
Characterization of AGBL5/Agbl5/AGBL5 in human and mouse. (A) Quantitative real-time PCR revealed higher AGBL5 RNA expression in human retina than in muscle. In the mouse, Agbl5 RNA expression was detectable in the retina, remaining eye tissue, brain and testis at P10 and likewise at P28 with the difference of a reduced expression in the eye tissue without retina. Expression levels of Agbl5 were very low in skeletal muscle tissue at both ages. For RQ calculation, retinal expression levels in human retina and mouse (P10) were set to 1 and GAPDH and 18SrRNA were used as house-keeping controls. Mean relative quantitation score (RQ) from three independent experiments was calculated by the ΔCT-method; standard deviation is indicated by error bars. The RQ of retinae were compared to other tissues and statistically evaluated using Student's t-test (*P < 0.05). n = 1 human retina and tissue from n = 3 mice was analyzed. (B) AGBL5 protein expression in human and mouse tissue at P10 and P28 was analyzed by Western blot analyses. AGBL5 protein was detected in human and mouse retina. AGBL5 protein expression was detected in mouse retina, brain and testis at P10 and P28. A signal was lacking in mouse eye tissues without retina and skeletal muscle GAPDH signals represent loading controls. The molecular weight of the AGBL5/CCP5 protein is ∼ 97 kDa as expected (human and mouse, UniProtKB database [http://www.uniprot.org/]). 40 μg protein/lane was applied on a 10% SDS gel. (Ew/oR): mouse eye without retina, (C) AGBL5/CCP5 immunohistochemistry in human and mouse retinae. AGBL5 immunoreactivity is distributed all over in the human retina though varying in expression. Staining is most prominent in the cone inner segments (black arrowheads, IS), ganglion cells (GCL, white arrowheads) and in nerve fiber layer (NFB). Sections of P10 and P28 mouse retinae show prominent AGBL5/CCP5 immunoreactivity in the outer plexiform (OPL), inner plexiform (IPL), GCL and NFL, the latter more expressed in the P28 mouse retina. Faint immunoreaction is observed in the developing IS of the photoreceptors (PR) in P10 retina, more distinct in the IS of the P28 mouse with faint staining also observed in the outer segments. More staining is detected in the inner nuclear layer (INL). Control sections lack reactivity, when treated with CCP5 and blocking peptide or when the primary antibody has been omitted. Hemotoxylin-Eosin (HE-) shows morphology of the respective retina sections. (ONL) outer nuclear layer, (OS) outer segments. Scale bars in the control sections represent 20μm, also for the respective adjacent sections.
Figure 3
 
Characterization of AGBL5/Agbl5/AGBL5 in human and mouse. (A) Quantitative real-time PCR revealed higher AGBL5 RNA expression in human retina than in muscle. In the mouse, Agbl5 RNA expression was detectable in the retina, remaining eye tissue, brain and testis at P10 and likewise at P28 with the difference of a reduced expression in the eye tissue without retina. Expression levels of Agbl5 were very low in skeletal muscle tissue at both ages. For RQ calculation, retinal expression levels in human retina and mouse (P10) were set to 1 and GAPDH and 18SrRNA were used as house-keeping controls. Mean relative quantitation score (RQ) from three independent experiments was calculated by the ΔCT-method; standard deviation is indicated by error bars. The RQ of retinae were compared to other tissues and statistically evaluated using Student's t-test (*P < 0.05). n = 1 human retina and tissue from n = 3 mice was analyzed. (B) AGBL5 protein expression in human and mouse tissue at P10 and P28 was analyzed by Western blot analyses. AGBL5 protein was detected in human and mouse retina. AGBL5 protein expression was detected in mouse retina, brain and testis at P10 and P28. A signal was lacking in mouse eye tissues without retina and skeletal muscle GAPDH signals represent loading controls. The molecular weight of the AGBL5/CCP5 protein is ∼ 97 kDa as expected (human and mouse, UniProtKB database [http://www.uniprot.org/]). 40 μg protein/lane was applied on a 10% SDS gel. (Ew/oR): mouse eye without retina, (C) AGBL5/CCP5 immunohistochemistry in human and mouse retinae. AGBL5 immunoreactivity is distributed all over in the human retina though varying in expression. Staining is most prominent in the cone inner segments (black arrowheads, IS), ganglion cells (GCL, white arrowheads) and in nerve fiber layer (NFB). Sections of P10 and P28 mouse retinae show prominent AGBL5/CCP5 immunoreactivity in the outer plexiform (OPL), inner plexiform (IPL), GCL and NFL, the latter more expressed in the P28 mouse retina. Faint immunoreaction is observed in the developing IS of the photoreceptors (PR) in P10 retina, more distinct in the IS of the P28 mouse with faint staining also observed in the outer segments. More staining is detected in the inner nuclear layer (INL). Control sections lack reactivity, when treated with CCP5 and blocking peptide or when the primary antibody has been omitted. Hemotoxylin-Eosin (HE-) shows morphology of the respective retina sections. (ONL) outer nuclear layer, (OS) outer segments. Scale bars in the control sections represent 20μm, also for the respective adjacent sections.
Table
 
Clinical Data and Putative Variants of All Affected Family Members
Table
 
Clinical Data and Putative Variants of All Affected Family Members
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