Molecular Vision 2005; 11:535-541 <>
Received 5 January 2005 | Accepted 7 July 2005 | Published 19 July 2005

Novel deletion spanning RCC1-like domain of RPGR in Japanese X-linked retinitis pigmentosa family

Zi-Bing Jin,1,2 Xiao-Qiang Liu,1,2 Asuka Uchida,1 Raf Vervoort,3 Kazuhiro Morishita,4 Mutsuko Hayakawa,5 Akira Murakami,5 Naomichi Matsumoto,6 Norio Niikawa,6 Nobuhisa Nao-i1

Departments of 1Ophthalmology and 4Biochemistry, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan; 2School of Ophthalmology and Optometry, WenZhou Medical College, Zhejiang, China; 3Department of Human Genetics, Katholieke Universiteit Leuven, Leuven, Belgium; 5Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan; 6Department of Human Genetics, Nagasaki University, Nagasaki, Japan

Correspondence to: Nobuhisa Nao-i, Department of Ophthalmology, Miyazaki Medical College, Kihara 5200, Kiyotake, Miyazaki, 889-1692 Japan; Phone: (+81) 985-85-2806; FAX: (+81) 985-84-2065; email:


Purpose: To describe a macrodeletion spanning entire RCC1-like doman in the RPGR gene in one Japanese family with X-linked retinitis pigmentosa (XLRP).

Methods: Clinical ophthalmologic examinations were performed and genomic DNA was extracted from blood samples. Genomic DNA was analyzed by Southern blot and PCR amplification with specific primers.

Results: Patients had severe symptoms with early onset and rapid deterioration. PCR amplification and Southern blot analysis revealed the absence of the 5' half of the RPGR gene. The deletion was confirmed and characterized by designing flanking PCR primers: the deletion start point was located 80 bp upstream of the translation start site in exon 1, the end point was 42 bp downstream of exon 11.

Conclusions: This 30 kb deletion contains the exons coding for the RCC1-like domain of RPGR. It is the first report of a macrodeletion that spans the entire RCC1-like domain of RPGR in X-linked retinitis pigmentosa patients, and suggests that loss of function of this domain disrupts the function of RPGR in human retina.


Retinitis pigmentosa (RP) is a disease with progressive degeneration of retinal photoreceptors, characterized by night blindness, progressive loss of peripheral vision, and pigmentary alterations in the retina with the appearance of "bone spicules". Retinitis pigmentosa is X-linked (XLRP) in about 15-30% of all RP cases, and seems more severe. Retinitis Pigmentosa GTPase Regulator (RPGR) on Xp21.1 is one of the most frequently mutated genes in XLRP, and accounts for about 30-70% of XLRP cases [1-3]. Positional cloning of the RPGR gene originally revealed a 2784-nucleotide (nt) ubiquitously expressed transcript that includes 19 exons coding for 815 amino acids [4,5]. Exon ORF15 was identified later, encodes 567 amino acids, and was shown to be a mutation hot spot [1]. Disease-causing mutations reported so far are localized in the 5' exons and ORF15, while none have been reported in exons 16-19. The N-terminal sequence of RPGR spanning exons 1-10 shows homology to the regulator of chromatin condensation (RCC1), a nuclear protein that catalyzes guanine nucleotide exchange for the small GTPase Ran and regulates nuclear import and export [6]. Mutations in RPGR identified in RP patients predominantly affect the RCC1-like domain (RLD) and exon ORF15, which suggests that these regions contribute to the physiological role of RPGR in the retina. Although a growing number of novel mutations in RPGR are being reported around the world, its function in vivo is still unclear. Through yeast two-hybrid screens, two proteins, the δ subunit of rod cyclic GMP phosphodiesterase (PDE δ) and RPGR-interaction protein 1 (RPGRIP1), were identified that interact with the RCC1-like domain (RLD) of RPGR, further demonstrating its functional importance [7,8]. Interestingly, mutations in RPGRIP1 are associated with the retina disease, Leber congenital amaurosis [9]. Here we report a novel macrodeletion within the RPGR gene, extending from exon 1 to exon 11. The breakpoints were identified by PCR amplification with flanking primers. This is the first deletion spanning the entire RLD identified in RP patients.


Patient protection

Ethical approval for this study and agreement by all patients were obtained from the Miyazaki Medical College. Each subject signed an agreement of participation in this study that was approved by Miyazaki Medical College. The protocol of the study adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee.

Family ascertainment

This family with X-linked Retinitis Pigmentosa (XLRP) was previously reported by one of the authors [10]. Clinical examinations included visual field testing, electroretinograms (ERG), and ocular examination. Detailed family history and pedigree were obtained through personal interviews with patients. Blood samples were collected from two affected male patients (IV-14 and IV-18) and controls (n=118).

Screening of RPGR

DNA was extracted using the DNA Extractor WB Kit (Wako, Osaka Japan). The coding sequence and intron/exon boundaries of RPGR, including exon 1-exon 19, exon 15a, and exon ORF15, were amplified by PCR as described previously [4,11,12]. To verify the products before sequencing, PCR products were electrophoresed on agarose gel, and then purified by the Amicon-PCR Centrifugal Filter Device. Purified DNA samples were cycle-sequenced with the Big Dye Terminator cycle sequencing kit. Reactions were electrophoresed on an ABI 310 automated Genetic Analyzer (Applied Biosystems, Foster City, CA). As some PCR products could not be sequenced neatly through previous methods, cloning into plasmid vector was carried out (Invitrogen, pCR4-TOPO, Carlsbad, CA) and plasmids were purified using the Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI), digested with EcoRI and electrophoresed on agarose gel for verification, and sequenced as described above.

Southern blot analysis

Genomic DNA of IV-14 was extracted from leukocytes, and digested with BamHI, EcoRI, and HindIII, and separated by electrophoresis on a 0.75% agarose gel. The DNA was transferred on Hybond N+ nylon membrane (Amersham Biosciences, Tokyo, Japan). PCR products corresponding to the following exons and their intron boundaries were used as probes: exon 4 (208 bp), exon 8 (352 bp), exon 10 (292 bp), and exon 11 (327 bp). All probes were labeled with α-32P-dCTP by Random Primer DNA Labeling Kit (Takara, Otsu, Japan) and hybridized to the membrane following the manufacturers' protocols. Washes were performed with 2X SSC once and 0.1X SSC twice. Finally, radiolabeled bands were detected on X-ray film.

Determination of deletion breakpoints

To define the deletion breakpoints within RPGR, primers flanking the deletion were designed to amplify the gene, including a 3 kb region upstream of exon 1 where the promoter may be located (Table 1). PCR products of patients and controls were electrophoresed on 1% agarose gel. Sequence analyses were performed as described previously. The protocol of sequence analysis was same as the description in the "Screening of RPGR".


Clinical assessment

Two patients affected with retinitis pigmentosa exhibited X-linked inheritance (XLRP) through pedigree evaluation (Figure 1). The onset of disease of patient IV-14 was with night blindness at 10 years of age and with visual acuity of 0.6/0.8 and 15° concentric and temporal island fields. At the age of 17, the patient had progressed to 10° concentric fields and 0.6/0.6 visual acuity. The visual disorder in patient IV-18 was diagnosed at an age of 3 years. His visual acuity at 17 years of age was 0.8/0.7, 0.4/0.4 at 24 years associated with 15° concentric fields, and 0.2/0.4 at 36 years old with visual field less than 10°. Recent visual fields are shown in Figure 2. Clinical examinations showed extinguished ERG (Figure 3) and fundus changes with severe degeneration and pigmentary alterations (Figure 4).

Myopia was frequently observed in the pedigree, and best corrected visual acuity was insufficient in carrier females. The most recent available data including two affected males and four female carriers in this family are shown in Table 2.

RPGR screening

We were unable to amplify exons 1 to 11 with PCR from either patient sample (IV-14 or IV-18), while exons 12 to 19, 15a, and ORF15 were successfully amplified. All amplified PCR products were electrophoresed and were the same size as controls (data not shown). After cloning, digestion with EcoRI, verification of vector and target DNA fragments by electrophoresis, the fragments were sequenced, and sequences were compared to the published entry in GenBank. No sequence alterations were found except a polymorphism (g.ORF15+1675A>G) which had been detected in 10 individuals in our group of 118 normal controls.

Deletion breakpoints

Failure to amplify exons 1 to 11 suggested a partial deletion of RPGR in both patients. Southern blotting of patient IV-14 DNA compared with normal controls confirmed the absence of all exons tested (exons 4, 8, 10, and 11; Figure 5A). We then designed a set of specific primers (Table 1) to localize the deletion with a PCR-based approach. PCR products US-1, US-2, and US-3, corresponding to the upstream region of the gene were amplified and sequenced and were the same as controls. Products A and C could not be amplified while product D was successfully amplified. After amplifying fragments spanning the deletion with a specifically designed primer set (Del-f and Del-r), the deletion start-point was determined at 80 bp upstream of the translation start codon in exon 1, and the endpoint was located at 42 bp downstream of exon 11 (Figure 5B). The deletion found in this family spanned about 30 kb, and contains the entire RCC1-like domain of the RPGR gene. When sequences near the deletion breakpoints were searched by NCBI blastn software on the web, we did not find any direct or inverted repeats, transposable elements, or reiterated sequences near the deletion breakpoints. However, sequences at the breakpoint before exon 1 (TGC TTTA) were very similar to those after exon 11 (TGC [CT] TTTA). Therefore, it is possible that the region of the breakpoint (TGCC TTTA) was homologously recombined, although we do not know the actual mechanism.


Here we report the first deletion of the entire RCC1-like domain of RPGR, spanning exons 1 to 11. The deletion is associated with XLRP in a Japanese family, and patients have severe symptoms and rapid progression of the disease. Since we failed to amplify exons 1-11, while exons 12-19, 15a, and ORF15 were amplified successfully, a partial gene deletion was suspected. To confirm our hypothesis, Southern blotting was performed on one patient (IV-14) using 32P labeled probes corresponding to exons 4, 8 10 and 11, and the result again indicated their absence. We then defined the extent of the deletion in the upstream region with five specific PCR fragments, of which two failed to amplify. Finally, we obtained fragments spanning the deletion, with primer set Del-f and Del-r. The deletion start point was found at 80 bp upstream of the translation start codon of exon 1, and the end point was 42 bp downstream of exon 11. The deletion, c.1-80_1414+42delinsC, thus spans about 30 kb, containing the entire RLD of RPGR (Figure 5B).

RPGR is ubiquitously expressed with tissue specific alternative splicing patterns [4,5,13,14]. The N-terminus of RPGR consists of six complete tandem repeats of 52 to 54 amino acids with homology to the repeats of RCC1, a nuclear protein involved in nuclear import and export through its regulation of the small GTPase Ran in eukaryotic cells [6]. The RCC1-like domain (RLD) of RPGR is encoded by exons 1 to 11 [4]. Interestingly, alternative splicing of RPGR in different tissues and in different species, generates a population of proteins with a shared N-terminal core containing the RLD followed by a C-terminal portion of variable length and sequence [1,11,15]. Most reported mutations in RPGR are frameshift and nonsense mutations in exon ORF15, and nonsense, frameshift, missense mutations and in-frame deletions located in the RLD. So far no mutation has been found in exons 16-19, 15a, 15b1, and 15b2. This distribution of mutations suggested that the N-terminal exons 1-14 and ORF15 are necessary for the function of RPGR in the retina. Only six macrodeletions in RPGR have been associated with XLRP, removing exon 8, exon 8-10, exon 14-15, exon 15-15a, exon 10-19 or exon 11-19, respectively.

The RLD interacts with the delta subunit of the rod cGMP phosphodiesterase (PDEδ) [7], which mediates the solubility of rod and cone PDE in the photoreceptors [16]. The RLD also interacts with RPGR-interacting protein 1 (RPGRIP1) [8,17], now known to be a component of the ciliary axoneme [18]. Since mutations in RPGRIP1 have been found in patients with Leber congenital amaurosis, a retinal disease closely related to RP [9,19], gain of function mutations in the RPGR RLD-domain disturbing the RPGRIP1 function could theoretically contribute to the RP phenotype. The macrodeletion presented here is the first to remove the entire RLD, thus excluding any gain of function contribution of mutant RLD protein to the RP phenotype in this family, which can not be excluded in patients expressing truncated RLD, or RLD with missense mutations.

In order to present harmonious and interchangeable data, we followed the HUGO guidelines for mutation nomenclature [20-23] and the latest recommendations of the Human Genome Variation Society (HGVS). The systematic name is based on the genomic sequence of the RPGR gene (GenBank accession numbers NM_000328, AF286472) and follows the convention of numbering the A of the ATG translational initiation codon as +1 [24]. There is no nucleotide 0, and the nucleotide immediately upstream of +1 is designated as -1. We denote the sequence variations in genomic DNA level or cDNA level. The deletion in the present study (c.1-80_1414+42delinsC) denotes the deletion start from 80 bp upstream of the translation start codon in exon 1 to 42 bp downstream of the last nucleotide of exon 11 (nucleotide 1414) and an insertion/substitution of one nucleotide "C". Recently, a nomenclature system has been suggested for the description of sequence variants (mutations, polymorphisms) in DNA and protein sequences [20,22,23,25-28]. Nomenclature of the reported sequence variations of RPGR gene had not been united, most of them named in different ways (data not shown). We recommend a uniform and unequivocal description of sequence variants in RPGR gene following HUGO guidelines for mutation nomenclature and the recommendations of the HGVS.


We thank the subjects with XLRP and their families for their willing and continued participation in this study. We would like to express our gratitude to Dr. Hidenori Sasaki and Dr. Xing-Yun Chen for their helpful suggestions.


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