X-Linked Retinitis Pigmentosa: Mutation Spectrum of the RPGR and RP2 Genes and Correlation with Visual Function

Abstract

purpose. To assess the frequency of RPGR and RP2 mutations in a set of 85 patients with X-linked retinitis pigmentosa (XLRP) and to compare the visual function of patients with mutations in RPGR versus RP2.

methods. Eighty-five unrelated patients with XLRP were ascertained, mainly from North America. The single-strand conformation polymorphism (SSCP) and a direct sequencing technique were used to screen their DNA for mutations in the coding region and splice sites of RPGR and RP2. The Snellen visual acuities, visual field areas, and 0.5-Hz and 30-Hz electroretinograms (ERGs) were measured in male patients. The visual function parameters were compared using multiple regression analysis.

results. A wide spectrum of mutations was found in both genes, including missense, nonsense, splice-site, and frameshift mutations. Twenty putative pathogenic mutations in RPGR, 15 of which were novel, were found in 22 patients (26%), whereas 6 mutations in RP2, 4 of which were novel, were found in 6 patients (7%). A high fraction of the mutations in both genes affected amino acid residues within or adjacent to presumed functional domains. Comparison of visual function between comparably aged patients with mutations in RPGR versus RP2 showed that, on average, patients with RPGR mutations have lower ERG amplitudes and smaller visual field areas.

conclusions. Mutations in RPGR and RP2 genes together account for approximately 33% of cases of XLRP in North America. Patients with RPGR mutations have less overall retinal function on average than those with RP2 mutations, on the basis of measurements of visual field areas and full-field ERG amplitudes.

Retinitis pigmentosa (RP) affects 50,000 to 100,000 people in the United States and approximately 1.5 million people worldwide. ¹ The condition can be inherited as a dominant, recessive, X-linked, digenic, or mitochondrial trait. The X-linked form accounts for 6% to 17% of all RP cases, ^{2 3 4 5} and at least five X-chromosome loci have been mapped. Previous linkage analyses have suggested that RP3 (Mendelian Inheritance in Man [MIM] 312610), located in Xp21.1, causes the highest fraction of cases of X-linked RP (56%–90%), ^{6 7 8 9} and that RP2 (MIM 312600) located in Xp11.23, accounts for most of the remainder (26% of XLRP-affected families). ^{7 8} Three other XLRP loci have been inferred on the basis of linkage studies of only one family each: RP15 assigned to Xp22.13-p22.11, ¹⁰ RP23 assigned to Xp22, ¹¹ and RP24 assigned to Xq26-27. ¹²  

Positional cloning efforts resulted in the identification of the RPGR (RP guanosine triphosphatase [GTPase] regulator) gene in the RP3 region ^{13 14} and the RP2 gene in the RP2 region. ¹⁵ Although the spectrum of mutations in RPGR clearly indicates that this gene is a cause of RP, ^{9 16 17} the reported frequency of mutations observed in families with XLRP (only 15%–20%) is much lower than the 56% to 90% previously predicted by linkage analyses. ^{9 16} The fraction of cases with mutations in the RP2 gene (10%–18%) is slightly smaller than the 26% predicted by previous linkage studies. ^{15 18 19} The RPGR and RP2 proteins are expressed ubiquitously. RPGR shows similarity to the guanine-nucleotide exchange factor (GEF) regulator of chromosome condensation (RCC1), ¹⁴ whereas RP2 shows similarity to human cofactor C. ¹⁵  

Prior clinical studies of patients with mutations in RPGR have not revealed a consistent pattern. ^{17 20} Pedigrees that showed linkage to the RP2 region, ^{21 22 23} or with identified RP2 mutations, ^{24 25} exhibited a wide range of severity and variation in the amount of cone loss relative to rod loss. No average differences have been reported between patients with RPGR versus RP2 mutations. 

In the present study we provide the results of screening a large set of patients with XLRP from across the United States and Canada who were clinically evaluated by us. Our results increase the knowledge of the spectrum of mutations in RPGR and RP2 that cause XLRP and provide evidence indicating different patterns of disease in patients carrying RPGR versus RP2 mutations. 

Methods

Ascertainment of Patients

This study, which involved human subjects, conformed to the tenets of the Declaration of Helsinki. All 85 index cases were diagnosed through ophthalmologic examination that included electroretinography (ERG). Most patients resided in the United States and Canada. All index patients had an unaffected father and came from families with no evidence of male-to-male transmission. Most of the families had multiple affected males. In most of the cases, the mother of the index patient was examined and showed signs of the XLRP carrier state. ²⁶ Up to 96 unrelated individuals (58 females and 38 males; 154 X chromosomes) without symptoms of or a family history of RP were used as normal control subjects. Informed consent was obtained from all participants before they donated 10 to 20 ml of venous blood for this research. Leukocyte nuclei were prepared from the blood samples and stored at −70^οC before DNA was purified from them. 

Screening for Mutations

The single-strand conformation polymorphism (SSCP) technique was used to screen all five RP2 exons and 20 RPGR exons (exons 1–19 and exon 15a), as well as the immediately flanking intron sequences, for point mutations and other small-scale sequence changes. Each exon was individually amplified from leukocyte DNA samples by polymerase chain reaction (PCR) using previously published primer pairs. ^{13 15 27} PCRs were performed in the wells of 96-well microtiter plates. In each well was 20 ng of leukocyte DNA in 20 μl of a buffer containing 20 mM tris-HCl (pH 8.4 or 8.6), 0.25 to 1.5 mM MgCl₂; 50 mM KCl; 0.02 mM each of dATP, dTTP, and dGTP; 0.002 mM of dCTP; 0.6 μCi [α-³²P]dCTP (3000 Ci/mmol); 0.1 mg/ml bovine serum albumin; 0% or 10% dimethyl sulfoxide; and 0.25 units of Taq polymerase (Perkin Elmer, Norwalk, CT). The pH, Mg²⁺ concentration, annealing temperature, and presence or absence of 10% dimethyl sulfoxide were tailored to each primer pair to yield optimal amplification. After an initial denaturation (94^οC for 5 minutes), 25 cycles of PCR amplification were performed. Each cycle consisted of denaturation (94^οC for 30 seconds), primer annealing (50^οC–62^οC for 30 seconds), and extension (71^οC for 30 seconds). The final extension was at 71^οC for 5 minutes. The amplified DNA fragments were heat denatured, and aliquots of the single-stranded fragments were separated through polyacrylamide gels. Four different gels were used for RP2 fragments: 6% polyacrylamide in tris-borate-EDTA (TBE) buffer, 6% polyacrylamide with 10% glycerol in TBE buffer, 6% polyacrylamide in tris-2-(morpholino)ethanesulfonic acid [MES]-EDTA (TME) buffer (30 mM tris, 35 mM MES, 1 mM Na₂EDTA [pH 6.8]), ²⁸ and 0.5× mutation detection enhancement (MDE) gel (FMC Bioproducts, Rockland, ME) in 0.6× TBE buffer. For the RPGR gene, only the first two types of gels were used. Gels were run at 6 to 18 W for 5 to 18 hours at room temperature before drying and autoradiography. Variant bands were analyzed by sequencing the corresponding PCR-amplified DNA segments (Thermosequenase cycle kit; Amersham, Arlington Heights, IL) or with a dye terminator sequencing kit (Perkin Elmer) on an automated sequencer (model 373; Perkin Elmer–Applied Biosystems, Foster City, CA). Participating relatives of index cases with selected sequence anomalies were evaluated for the same sequence changes by SSCP or sequence analysis of DNA. 

Clinical Evaluation

We recorded the following measures of visual function from each affected male patient examined: Snellen visual acuity (38 patients), a kinetic visual field on a Goldmann perimeter (spot size V-4e; Haag-StreitAG, Liebefeld, Switzerland; 32 patients), and full-field electroretinograms (34 patients for 0.5-Hz ERG and 35 patients for 30-Hz ERG) obtained with computer averaging and narrow band-pass filtering, as described previously. ^{29 30} ERG amplitudes were measured from the trough to the peak of each response, and the area of visual field was expressed in units of degrees squared. ERG amplitudes elicited in response to white light flashes at 0.5 Hz were used as a measure of rod-plus-cone function, and responses to white flashes flickering at 30 Hz were used as a measure of cone function. 

Statistical Analysis

Data on ERG amplitudes and visual field areas were transformed to the log_e scale to approximate a normal distribution. Snellen visual acuities were expressed as decimals. For each patient, values from both eyes were averaged. Multiple regression analyses were performed on all available data with log_e ERG amplitude, log_e visual field area, or Snellen visual acuity as the dependent variable and gene (RPGR versus RP2), age, and refractive error as the independent variables. In this way the relationship of each dependent variable to gene was adjusted for differences in patient age and refractive error. Multiple regression analyses were repeated after eliminating statistical outliers (two patients for the 0.5-Hz ERG, two patients for the 30-Hz ERG, three patients for the visual field area, and one patient for visual acuity). The outlying values were more than four SDs from the multivariate mean derived from estimates of the mean, SD, and correlation matrix that did not include the data points in question. Analyses were performed by computer (JMP, ver. 3.2; SAS Institute, Cary, NC; Macintosh Powerbook G3; Apple Computer, Cupertino, CA). 

Results

Mutations and Polymorphisms in RPGR

Twenty-two of the 85 patients with XLRP were found to carry 20 different mutations that were classified as highly likely to be pathogenic (Table 1 A). The screen also revealed 17 polymorphisms and rare nonpathogenic variants located in coding regions and intron sequences (Table 1B) . 

Frameshift, Nonsense, and Splice-Site Mutations.

Nine frameshift mutations, of which six are novel, were found in 10 patients (Table 1A) . For example, a deletion of one base (delT@Phe279; exon 8) was found in one patient and is illustrated in Figure 1A . This frameshift mutation would encode a truncated protein that is without a part of the region showing similarity to RCC1 as well as the protein’s C terminus. A 2-bp deletion, delTC@Val459, was found in two apparently unrelated patients, 004-165 and 039-082. This frameshift mutation produces a stop two codons downstream (Fig. 1A ; right panel) and, if expressed, would encode a truncated protein with an intact RCC1 domain. Five other small deletions of 1 to 4 bp were found in our set of patients, two of which (delA@Glu290 and delACAA@Thr528) have been previously reported in patients with XLRP. ¹⁶ Two of the nine frameshift mutations are small insertions in exons 10 and 15. The latter, insA@Asn612, is the most 3′ frameshift mutation reported so far in RPGR (affecting the last 212 residues of the coding region). One patient was found to carry a novel nonsense mutation at amino acid 202. This would lead to a truncated protein that is missing a portion of the RCC1 domain. 

Four mutations, of which three are novel, were found to affect intron/exon splice-sites. Three of these occurred in the canonical donor or acceptor sites of introns 1, 4, and 13. The fourth splice-site mutation affected the sixth base of the acceptor site of intron 3 (IVS3-6T→A). A neural-network computer program for recognizing splice sites (available at http://www.fruitfly.org/seq-tools/splice.html) ³¹ predicted that the mutant sequence has a substantially reduced likelihood of having a functional splice acceptor site (probability reduced from 0.54 to 0.03). This splice-site mutation was found to cosegregate with the disease in four additional family members (Fig. 2A ; family 0598). 

Missense Mutations.

Six missense changes interpreted as likely to be pathogenic were found in the RPGR gene in seven patients with XLRP. None of these was found in 140-154 X chromosomes from control subjects. One of the mutations (Gly60Val) has been reported in two patients with XLRP ¹⁶ and affects a residue that is highly conserved in RCC1 proteins from several species. ¹³ Two novel missense mutations were found in codon 43 (Gly43Arg and Gly43Glu). Gly43Glu cosegregated with the disease in four of the index patient’s relatives who were available (Fig. 2A ; family 410). No family members were available to study the inheritance of the second mutation affecting codon 43 (Gly43Arg). Three other novel missense mutations (Arg127Gly, Cys302Tyr, and Gly436Asp) were found in one patient each, and all cosegregated with the disease in the available family members (Fig. 2A ; families 3933, 5784, and 1591, respectively). The Arg127Gly mutation will change a positively charged residue to a neutral residue and affects a nonconserved residue within the RCC1 region. Residue 302 is located within the RCC1 region as well, but both cysteine and tyrosine are polar amino acids. The missense mutation Gly436Asp changes a neutral residue outside the RCC1 region to a negatively charged residue. 

Polymorphisms.

Seventeen polymorphisms and nonpathogenic rare variants in the RPGR gene were found in this study (Table 1B) , 10 of which have been reported as nonpathogenic polymorphisms. ^{16 32 33} A novel missense change (Asn345Asp) was found in one patient with XLRP and not in 150 X chromosomes from normal control subjects; however, the patient’s affected brother did not carry this sequence change, and we therefore consider it to be a nonpathogenic variant. Seven sequence variants, located in intron 10 (IVS10-13delC), exon 11 (Arg425Lys and Ile431Val), intron 12 (IVS12-101-4delAAAT and IVS12-97T→C), intron 13 (IVS13+11A→G), and exon 14 (Gly566Glu) appear to be in linkage disequilibrium, because all were found in each of three patients. Two other polymorphisms, located in distant exons (introns 10 and 18) appear to be in linkage disequilibrium as well, because 17 of the 20 patients (85%) carrying IVS10+16A→G also carried IVS18+11T→C. 

Mutations and Polymorphisms in RP2

Seven sequence changes were found in 9 of 85 patients with XLRP (Table 2) . Five of the seven changes were single-base substitutions, and two were small deletions. The efficiency of SSCP screening methods was evaluated during the screen of the RP2 gene. The two deletions could be detected by all types of SSCP gels (data not shown). The MDE gel detected all five single-base substitutions. An A-to-T transversion, IVS1+3A→T, was detected only by the MDE gel, whereas a transition, C844T, could be detected by all gel types except the nonglycerol TBE gel. In summary, the MDE gel could detect all seven sequence changes, whereas the glycerol and TME gels could detect six of the seven, and the nonglycerol TBE gel could detect five of the seven. 

Small Deletions and Splice-Site Mutations.

A novel, in-frame, 3-bp deletion (delATT@Ile137) in exon 2 of the RP2 gene was found in one patient (004-233; Fig. 1B , left). It leads to a deletion of an isoleucine that is conserved in homologous protein from Caenorhabditis elegans, whereas a valine is at this position in human cofactor C and Arabidopsis thaliana (Fig. 3) . Cosegregation analysis involving six additional family members (seven informative meioses) showed that the mutation cosegregates with the disease (Fig. 2B ; family F842). A deletion of 5 bp in exon 2 (delAAGAG@Lys230) was found in one patient (004-110); it would lead to a premature stop two codons downstream (codon 232). This mutation has been reported in another patient with XLRP. ¹⁸ In our study, two additional family members were available (one female carrier and one affected male) and both carried the defect (Fig. 2B ; family 2799). 

One splice-site mutation was found (patient 004-215) in the RP2 gene. It was in the third base of the donor site of intron 1 (IVS1+3A→T). A neural-network computer program for predicting splice sites predicted that this mutation substantially reduced the likelihood of a functional splice donor site at this location (probability reduced from 0.89 to 0.06). The mutation was present in the index patient, his affected brother, and their carrier mother (Fig. 2B ; family D202); the mutation was not found in 146 X chromosomes from control individuals. 

Missense Mutations.

Four missense changes were found in the RP2 gene in patients with XLRP. One of these (Arg118His) had been reported in two families with XLRP. ^{15 18} In our study, this mutation was found in one patient (004-176) and not in 145 X chromosomes from control individuals. The mutation cosegregated with the disease in 15 other family members (Fig. 2B ; family 0844). The homologous amino acid is conserved in the human cofactor C protein, but not in the homologous proteins in C. elegans or A. thaliana (Fig. 3) . 

A novel missense mutation in exon 2 (Cys86Tyr) was found in one patient (004-149; Fig. 1B ), but not in 133 X chromosomes from control individuals. No additional informative relatives were available for segregation analysis from this patient’s family. Cys86 is located within the region homologous to human cofactor C and is conserved in an A. thaliana–homologous sequence but not in human cofactor C (Fig. 3) . 

A novel missense change in exon 2 (Pro95Leu; Table 2B ) was found in one patient (004-229) but not in 133 X chromosomes from control individuals. Evaluation of the index patient’s relatives showed that all affected members and obligate carriers studied carry this mutation (Fig. 2B ; family D993). However, one female (IV-2) with normal ERG amplitudes at age 21 also carried the mutation. In this family member the absence of the subnormal or delayed ERG amplitudes characteristically found in XLRP carriers ²⁶ could be explained as an example of unbalanced Lyonization leading to retinas in which most cells have their mutant X chromosomes inactivated. Alternatively, the missense change may not be pathogenic, and the true mutation causing XLRP in this family is in another gene. Pro95 is conserved within the cofactor C-homologous region, but not in the homologous proteins in C. elegans or A. thaliana (Fig. 3) . 

Three index patients were found to share the same missense change in exon 3 (Arg282Trp), which is not located within the cofactor C–homologous region. It was also detected in 3 of 145 control chromosomes (two females were heterozygous and one male was hemizygous). Thus, this sequence change is likely to be a polymorphism with an approximate frequency of 3.5% (Table 2C) . 

Ophthalmologic Features of Patients with RPGR and RP2 Mutations

Retinal function was measured in 27 affected males carrying RPGR mutations (age 12–47; mean, 30 years), and 11 affected males carrying RP2 mutations (age 10–62, mean, 23 years). Findings for each patient are shown in Table 3 . All had delayed implicit times in response to 30-Hz flicker (i.e., greater than 32 msec). Statistical calculations were performed separately including and excluding the patients with the RP2 sequence variant Pro95Leu of uncertain pathogenicity. The levels of statistical significance were not substantially different in either case; P values shown in Table 4 are calculated to include patients with this sequence change. 

In both RPGR and RP2 groups, ERG amplitudes and visual field areas were severely reduced. Patients with RPGR mutations had, on average, less retinal function than those with RP2 mutations, when measured by full-field ERG amplitudes and visual field areas (Table 4 , Fig. 4 ). For Snellen visual acuity, patients with RPGR mutations had, on average, better visual acuity than patients with mutations in RP2, but the group difference was of borderline statistical significance (P = 0.084 including Pro95Leu, P = 0.057 excluding Pro95Leu). 

Discussion

Our results indicate that mutations in the RPGR and RP2 genes account for approximately 33% of the cases of XLRP in North America, with RPGR accounting for more cases than RP2. The proportion of patients with RPGR mutations (26% of 85) is higher than that found in five previous reports: 11% of 74 patients, ¹³ 18% of 28 patients, ¹⁴ 21% of 80 patients, ¹⁶ 17% of 29 patients, ³³ and 16% of 49 patients. ³² In contrast, the RP2 mutation frequency in our set of patients was 7% (6 of 85), or approximately 10%, if the 22 patients with RPGR mutations are excluded. This is equal to that found in two previous reports that excluded RPGR cases: 10% of 51 patients, ¹⁹ and 10% of 59 patients. ¹⁸ It is not clear whether a third study (18% of 38 patients) ¹⁵ excluded the RPGR cases. These differences may reflect differences between the frequencies of the various genetic types of XLRP in North America and Europe, ^{13 14 15 32} a different subset of patients (with or without prior linkage information), and/or different sensitivity of mutation detection methods used. Regarding RPGR, other groups and our group have found that the frequency of mutations is much lower than that predicted by linkage analyses. ^{9 14 16 33} The discrepancy could be reconciled if RPGR contains a considerable number of mutations in regions that were not evaluated (i.e., the promoter region, the 5′ and 3′ untranslated regions, and deep in the introns), undiscovered exons, or if there are one or more additional XLRP genes closely linked to RPGR. 

We report here 15 novel RPGR mutations (of which 10 are frameshifts, splice-site, or nonsense mutations and are likely null) and confirm five that have already been reported. The mutation spectrum of RPGR (Fig. 5A ) shows that the majority of the mutations found in our study and by others are within the RCC1 domain (12 of the 13 missense mutations located within the RCC1 domain region; P = 0.02). This may indicate that the RCC1 domain in the RPGR protein is important for photoreceptor cell function and viability. Other evidence consistent with this assumption is the reduced interaction between the delta subunit of rod phosphodiesterase (PDE-delta), and three RPGR variants with mutations in the RCC1 domain found in some patients with XLRP. ³⁴  

Four of the novel RPGR mutations are 3′ to the RCC1 domain, including a 1-bp insertion (insA@Asn612) in exon 15. A new RPGR splice variant described recently ²⁷ contains a new exon, 15a, and is missing downstream exons (16-19); exon 15a was found to be deleted in one of 23 patients with XLRP. ²⁷ We found no variants in exon 15a in our set of 85 patients. Of note, no mutations have been reported downstream to this exon (in exons 16-19), that may indicate that the residues encoded by the last four exons are not essential for the protein’s function in the retina. 

We also report four novel RP2 mutations and confirm two that have already been reported. Most of the mutations found so far in RP2 (Fig. 5B) interrupt the region showing similarity to human cofactor C. The missense polymorphism presented in the current study, Arg282Trp, is located outside this region and is currently the only polymorphism known in the RP2 gene. Although mainly nonsense and frameshift mutations were found in other studies, ^{15 18 19} we provide evidence that splice-site mutations also occur in RP2. The IVS1+3A→T mutation reported in this study is the first splice-site mutation documented in the RP2 gene. 

To our knowledge this is the first report in which the visual function of patients with identified mutations in RPGR is compared with that of RP2. The available data suggest that there is a difference in the severity of retinal degeneration in patients with RPGR versus RP2 mutations. Specifically, the patients with RPGR mutations have on average smaller visual fields and more severely reduced full-field ERGs than the patients with RP2 mutations. In a recent study, ³⁵ no clear phenotypic differences were found between patients with RP2 and RP3 haplotypes. However, the responsible mutations were not identified, and, at least for the RP3 subset of patients, other genes beside RPGR may be the cause of the disease. None of our patients with RP2 mutations had normal cone ERG implicit times or a predominant cone dysfunction as previously reported in two families showing linkage to the RP2 region. ^{21 23}  

Both RP2 and RPGR are expressed ubiquitously, ^{14 15} but mutations in either cause nonsyndromic XLRP, indicating that the protein products have an important and specific role in the retina. The similarity of the RPGR protein to RCC1 ^{13 14} may provide a clue to its function. RCC1 is the GEF for the GTPase Ran, which is important in nucleocytoplasmic transport. ³⁶ Recent articles have shown that the RPGR protein is concentrated in the connecting cilia of rods and cones ³⁷ and forms a complex with the delta subunit of PDE. ³⁴ Its location in the cilia and its homology to RCC1 suggest a role in intracellular transport. The primary structure of RP2 is similar to human cofactor C, a protein that has a role in folding β-tubulin and may function as a chaperone forβ -tubulin. ³⁸ The α- and β-tubulins form the tubulin heterodimer from which microtubules are assembled. Microtubules are involved in a diversity of biologic functions including cell division, intracellular transport, and the maintenance of cellular architecture. The RP2 protein may be involved in retina-specific microtubule functions. Of interest, the nucleation of microtubules around chromatin is induced by a high local concentration of Ran-GTP generated by RCC1. ³⁹ Thus, RPGR and RP2 may be involved in the same retina-specific microtubule pathway. 

 Supported by National Eye Institute Grants EY08683 and EY00169; the Foundation Fighting Blindness; National Institute for Child Health and Human Development Grant HD18658; and a gift from the Lawrence J. and Anne Cable Rubenstein Foundation.

 Submitted for publication November 12, 1999; revised January 13 and February 23, 2000; accepted March 3, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Sequence Anomalies Found in the RPGR Gene in Male Patients with XLRP

A. Mutations
DNA Change*	Sequence Change, †	Protein Change	Exon	Patient(s)
Deletion
415delT	TTG to T-G	Leu119; Ter@131	5	004-148
544-5delTT	TTT to T--	Phe162; Ter@165	6	004-139
896delT	TTT to TT-	Phe279; Ter@297	8	004-123
928delA	GAG to G-G	Glu290; Ter@297	8	004-105
1159delC	CCT to C-T	Pro367; Ter@380	10	004-220
1435-6delTC	GTC to G--	Val459; Ter@461	11	004-165 and 039-082
1641-4delACAA	ACA ATT to----TT	Thr528; Ter@531	14	099-056
Insertion
1151-2insT	GCT to TGCT	Ala365; Ter@376	10	099-042
1894-5insA	AAT to AAAT	Asn612; Ter@628	15	004-206
Nonsense
C664A	TCA to TAA	Ser202Ter	6	099-029
Splice site
IVS1+1G → A	CCGgtga to CCGatga		Int1	099-007
IVS3-6T → A	tattttt to tatattt		Int3	004-202
IVS4+1G → C	CAGgtat to CAGctat		Int4	004-169
IVS13-1G → A	acagAAA to acaaAAA		Int13	004-100
Missense
G186A	GGA to AGA	Gly43Arg	2	004-152
G187A	GGA to GAA	Gly43Glu	2	004-223
G238T	GGC to GTC	Gly60Val	3	004-158 and 004-133
A438G	AGA to GGA	Arg127Gly	5	004-157
G964A	TGT to TAT	Cys302Tyr	8	099-008
G1366A	GGC to GAC	Gly436Asp	11	004-264

B. Polymorphisms
DNA Change*	Sequence Change, †	Protein Change	Exon	Allele Frequencies in X-Chromosomes of Index Patients Versus Controls, ‡
Missense
A1092G	AAT to GAT	Asn345Asp	9	0.01 (1:84) vs. 0.007 (1:152)
G1333A	AGG to AAG	Arg425Lys	11	0.04 (3:82) vs. 0.05 (8:144)
A1350G	ATA to GTA	Ile431Val	11	0.04 (3:82) vs. 0.05 (7:145)
G1756A	GGG to GAG	Gly566Glu	14	0.04 (3:82) vs. 0.03 (1:34)
Silent
G791A	AAG to AAA	Lys244Lys	7	0.01 (1:84) vs. 0.007 (1:150)
G1223A	GCG to GCA	Ala388Ala	10	0.14 (12:73) vs. ND
Intronic
IVS1-15A → G	accatct to accgtct		Int1	0.04 (3:82) vs. 0.22 (8:29)
IVS6-41T → C	ccatatg to ccacatg		Int6	0 (0:85) vs. 0.007 (1:150)
IVS10+16A → G	cctattc to cctgttc		Int10	0.24 (20:65) vs. ND
IVS10-13delC	ctgcctt to ctg-ctt		Int10	0.07 (6:79) vs. ND
IVS12-97T → C	atatata to atacata		Int12	0.04 (3:82) vs. ND
IVS12-92-3delTA	atataatt to ata--att		Int12	0.01 (1:84) vs. ND
IVS12-101-4delAAAT	taaaaatata to taa----ata		Int12	0.04 (3:82) vs. ND
IVS12-101T → AA	atatata to ataaaata		Int12	0.01 (1:84) vs. ND
IVS13+11A → G	atgaaga to atggaga		Int13	0.04 (3:82) vs. ND
IVS17+46C → T	acacaca to acataca		Int17	0.11 (9:76) vs. 0.03 (4:150)
IVS18+11T → C	aaatgaa to aaacgaa		Int18	0.27 (23:62) vs. 0.24 (37:117)

 ND, not determined; Int, intron.

*  Nomenclature based on Meindl et al. ¹³

†  Uppercase designates coding regions, and lowercase designates intronic sequences.

‡  Allele counts are in parentheses.

Figure 1.

 

Genomic sequences of RPGR (A) and RP2 (B) mutations. The sequences are presented in the sense direction from bottom (5′) to top (3′).

Figure 2.

 

Segregation analysis of mutations in RPGR (A) or RP2 (B) in families with XLRP. For each pedigree, the pedigree number and the mutation are indicated. The arrows point to the index patients. The genotype of all individuals whose DNAs were available is shown beneath their symbols:+ , wild-type sequences; m, mutant sequences. Circles with dots, female carriers; asterisks, individuals who were clinically evaluated. Patient III-7 of family 1591 had funduscopic and ERG findings suggestive of but not diagnostic of the carrier state.

Table 2.

 

Sequence Anomalies Found in the RP2 Gene in Male Patients with XLRP

A. Mutations
DNA Change*	Sequence Change, †	Protein Change	Exon	Patient
Deletions
409delATT	ATT to ---	Ile137del	2	004-233
688delAAGAG	AAG AGC to-----C	Lys230; Ter@232	2	004-110
Splice site
IVS1+3A → T	Cgtaatg to Cgttatg		1	004-215
Missense
G257A	TGT to TAT	Cys86Tyr	2	004-149
G353A	CGT to CAT	Arg118His	2	004-176
B. Rare Variant with Uncertain Pathogenicity
C284T	CCC to CTC	Pro95Leu	2	004-229

C. Polymorphism
DNA Change*	Sequence Change, †	Protein Change	Exon	Allele Frequencies in X-Chromosomes of Index Patients Versus Controls, ‡
Missense
C844T	CGG to TGG	Arg282Trp	3	0.04 (3:82) vs. 0.03 (5:148)

*  Nucleotide positions are based on sequence in GenBank accession number NM_006915, with the A base of the first ATG in the open reading frame as base 1.

†  Uppercase designates coding regions, and lowercase designates intronic sequences.

‡  Allele counts are in parentheses.

Figure 3.

 

An amino acid sequence alignment of homologues of the RP2 gene. RP2, human RP2 (GenBank accession number NM_006915); TBCC, human tubulin-specific chaperone C (NM_003192); C. elegans, an anonymous C. elegans sequence (AAB97559); and A. thaliana, an A. thaliana putative protein (CAB38905). The amino acid number of the RP2 protein is indicated on the left. The amino acids affected by RP2 mutations (Cys86Tyr, Pro95Leu, Arg118His, I137del), as well as the aligned amino acids, are highlighted in bold.

Table 3.

 

Raw Values of the Clinical Parameters of Patients with RPGR Versus RP2 Mutations

DNA	Family*	Mutation	Age	Sph	0.5 Hz	30 Hz	Field	VA
RPGR
099-007	5955	IVS1+1G → A	41	−2.06	10.40	5.94	4247	0.36
004-152	5843	Gly43Arg	27	−8.12	1.00	0.23	1969	0.44
004-223	0410 (II-4)	Gly43Glu	47	−4.44	1.00	0.11	NA	0.02
004-133	6147	Gly60Val	31	1.50	3.05	0.57	2984	0.27
004-158	6813	Gly60Val	18	−1.75	5.00	1.24	3632	0.40
004-202	0598 (II-1)	IVS3-6T → A	30	−1.51	2.18	0.86	1911	0.68
004-169	6737	IVS4+1G → C	22	−0.57	3.05	0.33	6857	0.58
004-148	1194	Leu119(1-bp del)	36	−0.88	1.00	0.12	1497	0.39
004-157	3933 (II-2)	Arg127Gly	24	0.63	1.00	0.09	2201	0.40
004-171	3933 (II-3)	Arg127Gly	30	−5.25	1.73	0.27	557	0.22
004-139	6208	Phe162(2-bp del)	31	−7.39	1.00	0.20	1217	0.33
099-029	5571	Ser202Ter	30	−7.88	1.00	0.11	133, †	0.29
004-123	6004	Phe279(1-bp del)	27	−1.56	1.14	0.35	2689	0.44
004-105	1053	Glu290(1-bp del)	38	−4.25	NA	0.08	573	0.36
099-008	5784 (II-7)	Cys302Tyr	33	−1.94	1.00	0.46	3342	0.41
099-042	6082	Ala365(1-bp ins)	28	−3.50	1.00	0.09	1659	0.48
004-220	0582	Pro367(1-bp del)	47	−2.25	1.00	0.19	304	0.23
NA	0582	Pro367(1-bp del)	13	−10.7	NA	NA	NA	0.40
004-112	1591 (III-11)	Gly436Asp	26	−0.39	3.64	4.46	4199	0.98
004-264	1591 (III-13)	Gly436Asp	30	−5.31	1.00	0.79	3040	0.36
004-265	1591 (IV-1)	Gly436Asp	15	0.00	5.60	1.44	11857	0.57
004-266	1591 (IV-3)	Gly436Asp	47	−9.88	5.32, †	2.14, †	4310, †	0.10
004-165	7056	Val459(2-bp del)	26	0.38	1.80	0.71	2220	0.60
039-082	2981	Val459(2-bp del)	45	0.38	4.10	0.80	3086	0.73
004-100	3044	IVS13-1G → A	18	−5.69	2.15	0.28	5760	0.48
099-056	5714	Thr528(4-bp del)	33	−4.64	1.00	0.20	2273	0.33
004-206	7273	Asn612(1-bp ins)	12	−3.69	5.86	0.60	3937	0.57
RP2
004-215	D202 (II-2)	IVS1+3A → T	17	−2.25	33.80	6.02	NA	0.20
004-149	7024	Cys86Tyr	21	−10.1	16.00	6.06	11292	0.44
121-418	D993 (II-2)	Pro95Leu	62	−5.25	1.00, †	0.28, †	581, †	0.01, †
004-230	D933 (IV-3)	Pro95Leu	20	−7.88	3.75	3.28	NA	0.41
019-002	0844 (II-11)	Arg118His	30	−7.50	NA	NA	NA	0.03
004-241	0844 (III-8)	Arg118His	23	−8.88	NA	NA	NA	0.13
004-176	0844 (III-10)	Arg118His	19	−10.2	1.00	0.96	9115	0.27
004-276	0844 (IV-1)	Arg118His	14	−10.30	4.49	1.78	6200	0.10
004-275	0844 (IV-2)	Arg118His	12	−10.20	1.00	0.53	7041	0.10
004-233	F842 (III-4)	Ile137(3-bp del)	10	−5.38	12.90	4.35	9381	0.22
004-110	2799 (IV-3)	Lys230(5-bp del)	20	−2.69	3.13	0.42	5089	0.67

 The data presented are the average of both eyes for each patient and show spherical equivalent (Sph), 0.5-Hz ERG amplitude (in microvolts), 30-Hz ERG amplitude (in microvolts), visual field area (in degrees squared), and Snellen visual acuity (VA, decimal). Lower norm for 0.5-Hz amplitude = 350 μV, for 30-Hz amplitude = 50μ V, for visual field area = 11,310 deg², and for Snellen visual acuity = 0.80. NA, not available.

*  Family number designates number in the files of the Berman-Gund Laboratory; symbols in parenthesis for some individuals (e.g., II-4) describe the position of patients in families illustrated in Figure 2 .

†  A statistical outlier taking into account age and spherical equivalent.

Table 4.

 

Visual Function of Patients with XLRP

	RPGR			RP2			P *
		n	Mean ± SE	n	Mean ± SE
Loge 0.5-Hz ERG, †	24	0.55 ± 0.18 (1.73 μV)	8	1.86 ± 0.37 (6.42 μV)	0.008
Loge 30-Hz ERG, †	25	−1.02 ± 0.25 (0.36 μV)	8	0.86 ± 0.51 (2.36 μV)	0.006
Loge visual field area, ‡	23	7.83 ± 0.14 (2515 deg2) [57°]	6	8.70 ± 0.35 (6003 deg2) [87°]	0.046
Snellen visual acuity, §	27	0.42 ± 0.03 (20/48)	10	0.27 ± 0.06 (20/74)	0.084

 The data presented in this table were corrected for age and refractive error.

*   P for two-tailed t-test comparing the two groups.

†  0.5-Hz and 30-Hz ERG expressed as log_e amplitude ± SE; geometric means expressed parenthetically, lower norm for 0.5-Hz amplitude = 350 μV and for 30-Hz amplitude = 50 μV.

‡  Visual field area expressed as log_e to a V-4e white test light ± SE; geometric mean areas expressed parenthetically, lower norm = 11,310 deg². Equivalent circular diameters expressed in brackets, lower norm = 120°.

§  Snellen visual acuity expressed as decimal ± SE; geometric means expressed parenthetically, lower norm for Snellen visual acuity = 20/25.

Figure 4.

 

The distribution of raw clinical data for patients with mutations in RPGR versus those with RP2. The data points have been shifted horizontally to facilitate visualization of similar values.

Figure 5.

 

Locations of the mutations identified so far for RPGR (A) or RP2 (B). Asterisk next to the RP2 change Pro95Leu designates this missense change as one with an uncertain pathogenicity. This report, mutations found in the present study; other reports, mutations reported to date in other studies. ^{13 14 16 25 32 33}

The authors thank Robert Eisenman, David Rucinski, Peggy Rodriguez, and Elizabeth Sweklo for technical assistance.