HUMAN MUTATION 28(1),81^91,2007
Comprehensive Survey of Mutations in RP2 and
RPGR in Patients Affected With Distinct Retinal
Dystrophies: Genotype–Phenotype Correlations
and Impact on Genetic Counseling
Vale ´rie Pelletier,1Marguerite Jambou,1Nathalie Delphin,1Elena Zinovieva,1Morgane Stum,1
Nadine Gigarel,1He ´le `ne Dollfus,2Christian Hamel,3Annick Toutain,4Jean-Louis Dufier,5Olivier Roche,5
Arnold Munnich,1Jean-Paul Bonnefont,1Josseline Kaplan,1?and Jean-Michel Rozet1
1Unite ´ de Recherches Ge ´ne ´tique et Epige ´ne ´tique des Maladies Me ´taboliques, neurosensorielles et du De ´veloppement; Institut Nationale de la Sante ´
et de la Recherche Me ´dicale (INSERM) U781, Ho ˆpital Necker–Enfants Malades, Paris, France;2Service de Ge ´ne ´tique Me ´dicale,
Ho ˆpital de Haute-Pierre, Strasbourg, France;3Unite ´ INSERM 583, Ho ˆpital Saint-Eloi, Montpellier, France;4Service de Ge ´ne ´tique Me ´dicale,
Centre Hospitalo-Universitaire, Tours, France;5Servive d’Ophtalmologie, Ho ˆpital Necker–Enfants Malades, Paris, France
Communicated by Daniel F. Schorderet
X-linked forms of retinitis pigmentosa (RP) (XLRP) account for 10 to 20% of families with RP and are mainly
accounted for by mutations in the RP2 or RP GTPase regulator (RPGR) genes. We report the screening
of these genes in a cohort of 127 French family comprising: 1) 93 familial cases of RP suggesting X-linked
inheritance, including 48 out of 93 families with expression in females but no male to male transmission;
2) seven male sibships of RP; 3) 25 sporadic male cases of RP; and 4) two cone dystrophies (COD). A total
of 5 out of the 93 RP families excluded linkage to the RP2 and RP3 loci and were removed form the cohort. A
total of 14 RP2 mutations, 12 of which are novel, were identified in 14 out of 88 familial cases of RP and 1 out
of 25 sporadic male case (4%). In 13 out of 14 of the familial cases, no expression of the disease was noted in
females, while in 1 out of 14 families one woman developed RP in the third decade. A total of 42 RPGR
mutations, 26 of which were novel, were identified in 80 families, including: 69 out of 88 familial cases
(78.4%); 2 out of 7 male sibship (28.6%); 8 out of 25 sporadic male cases (32.0%); and 1 out of 2 COD. No
expression of the disease was noted in females in 41 out of 69 familial cases (59.4%), while at least one severely
affected woman was recognized in 28 out of 69 families (40.6%). The frequency of RP2 and RPGR mutations in
familial cases of RP suggestive of X-linked transmission are in accordance to that reported elsewhere (RP2:
15.9% vs. 6–20%; RPGR: 78.4% vs. 55–90%). Interestingly, about 30% of male sporadic cases and 30% of male
sibships of RP carried RP2 or RPGR mutations, confirming the pertinence of the genetic screening of XLRP
genes in male patients affected with RP commencing in the first decade and leading to profound visual
impairment before the age of 30 years. Hum Mutat 28(1), 81–91, 2007.
rrrr2006 Wiley-Liss, Inc.
KEY WORDS: retinitis pigmentosa; RP; X-linked; XLRP; cone dystrophy; RP2; RPGR
Retinitis pigmentosa (RP) is a group of progressive hereditary
disorders of the retina in which various modes of inheritance
have been described. The X-linked forms of RP (XLRP; OMIM:
www.ncbi.nlm.nih.gov/omim; MIM] 268000) account for about
10 to 20% of families with RP [Bird, 1975; Fishman, 1978; Kaplan
et al., 1990] and are among the most severe due to their early
onset leading to significant visual loss before the fourth decade.
Five XLRP loci have been localized by linkage: RP2 (Xp11.4;
MIM] 312600) [Bhattacharya et al., 1984]; RP3 (Xp21.1; MIM]
312310) [Musarella et al., 1987]; RP6 (Xp21.3–p21.2; MIM]
312612) [Ott et al., 1990]; RP23 (Xp22; MIM] 300424)
[Hardcastle et al., 2000]; and RP24 (Xq26–q27; MIM] 300155)
[Gieser et al., 1998]. On the basis of linkage analyses it has been
suggested that RP2 accounts for 10 to 20% and RP3 for 70 to 90%
of XLRP [Musarella et al., 1990; Ott et al., 1990; Teague et al.,
1994; Fujita et al., 1997]. The disease genes at these two loci have
been cloned but their function remain unknown (RP2 [Schwahn
et al., 1998]; RP GTPase regulator (RPGR) [Meindl et al., 1996;
Roepman et al., 1996]).
Published online 12 September 2006 in Wiley InterScience (www.
ique et EpigeŁneŁtique des Maladies MeŁtaboliques, neurosensorielles et
du DeŁveloppement. INSERMU781. HoŒpital Necker^Enfants Malades,
149 rue de SeØvres,75743 Paris Cedex15, France.
Grant sponsor: Association Retina France.
ValeŁrie Pelletier and MargueriteJambou contributed equally to this
rrrr2006 WILEY-LISS, INC.
Mutations in the RP2 gene are observed in 7 to 20% of recessive
XLRP (RXLRP) [Hardcastle et al., 1999; Mears et al., 1999;
Breuer et al., 2002; Sharon et al., 2003]. An RP2 mutation has
also been found to segregate with an atypical phenotype of macular
and peripapillary retinal atrophy [Dandekar et al., 2004]. RPGR
ORF15 exon mutations account for 30 to 60% of RXLRP and
mutations in other RPGR exons account for 11 to 26% of RXLRP
[Hardcastle et al., 1999; Mears et al., 1999; Vervoort et al., 2000;
Breuer et al., 2002; Sharon et al., 2003]. In addition to RXLRP ,
RPGR mutations have been described in families segregating RP
in both males and females [Rozet et al., 2002], X-linked recessive
atrophic macular degeneration or cone degeneration [Ayyagari
et al., 2002], X-linked cone-rod dystrophy [Mears et al., 2000;
Demirci et al., 2002; Ebenezer et al., 2005], RP and Coats’-like
exudative vasculopathy [Demirci et al., 2006], or even RP
associated with extraocular ciliary disorders, including recurrent
respiratory infections [Iannaccone et al., 2003], deafness,
sinorespiratory infections [Zito et al., 2003], and primary ciliary
dyskinesia [Moore et al., 2006].
The aim of this work was to give a comprehensive survey of RP2
and RPGR mutations in French families with: 1) nonambiguous
RXLRP; 2) XLRP with expression in women; 3) male sibships
without any previous history of RP in the maternal branch; and
4) sporadic male cases with a suggestive natural history of XLRP
with or without minor symptoms in mothers and/or sisters.
A total of 127 families segregating RP were ascertained in three
different medical centers in France: the Ophthalmologic and
Genetic consultations of Necker–Enfants Malades Hospital in
Paris, the Hospices Civils in Strasbourg, and the Centre Hospitalo-
Universitaire in Montpellier. In all families, male patients were
affected with early-onset severe RP of rapid progression character-
ized by: 1) severe bilateral peripheral visual field depression; 2)
severely reduced rod electroretinogram (ERG) amplitudes; and 3)
final visual acuity lower than 2/10 in the third decade. The 127
families were classified as a function of the familial segregation of
the disease. In 93 out of 127 families, the segregation of the disease
as an X-linked trait was supported by maternal transmission over
at least two generations and absence of male to male transmission.
These 93 families were split into two groups according to the
phenotype of carriers. In the first group, made up of 45 out of
93 families, obligate carriers either had no clinical symptoms or
displayed minor ophthalmoscopic changes (perimacular tapetal
sheen reflex and/or sectorial pigmentary deposits) and/or slight
electroretinographic alterations. Severe refraction errors, often
asymmetric, were frequently noted. In the second group of 48 out
of 93 remaining families at least one carrier, and often more,
displayed notable retinal degeneration leading to significant visual
loss, prompting them to consult an ophthalmologist.
In 7 out of 127 families, at least two males of the same sibship
were affected with RP but no other relative was affected with
the disease. In 4 out of 7 of these families, the mother or closely
related females (sisters or daughters) were unavailable or unwilling
to have an eye examination to evaluate whether they showed
the carrier state. In 1 out of 7 families, the clinical examination of
the two sisters and the mother of the affected males revealed no
sign of XLRP carrier status. Conversely, in 2 out of 7 families, the
mothers and/or the sisters of affected males were found to display
minor symptoms of retinal dysfunction and/or refraction errors.
In 25 out of 127 families, isolated male cases were suspected of
having XLRPon the basis of clinical risk factors (e.g., severe RP with
onset in the first decade, rapid progression, and visual acuity r2/
10). The mother and/or closely related females of the affected male
accepted to undergo an ophthalmoscopic examination in 12 out of
25 families. In 9 out of 12 families, clinical symptoms suggestive
of the XLRP carrier state were evidenced in at least one female.
Finally, a large multiplex family segregating X-linked cone
dystrophy (XLCOD) and a sporadic case with cone dystrophy
(COD) were ascertained in Necker Hospital. In the familial case,
in the first decade of life the affected males developed signs of
central retinal dysfunction with preservation of the peripheral
retina: graduate decrease of visual acuity, photophobia, and
severely altered color perception. Fundus examination revealed
macular atrophy with inconstant ‘‘bulls eye’’ aspect, confirmed by
fluorescein angiograms. Retinal vessels were normal and no
pigmentary changes were noted in the peripheral retina. Visual
field measurement showed an absolute central scotoma. Electro-
retinography recordings showed severely reduced cone ERG
amplitudes with normal rod ERG amplitudes. Clinical data were
available for three obligate carriers in this family. Two of them were
slightly myopic and one displayed a severe unilateral myopia. All
complained of photophobia. Absence of foveal reflex and presence
of a metallic-sheen reflex in the perimacular region were noted at
the fundus for all three women. ERG recordings were not available
for these women.
In the simplex family, the patient complained from photophobia
since the age of 3 years. At 11 years of age, he developed a severe
myopia (–8D). Visual acuity was 5/10 with correction while
fundus, color vision, and visual field recordings were slightly
altered. Full-field photopic ERG was nonrecordable, while scotopic
ERG was normal. The multifocal ERG showed a significant
impairment of the central function. The mother, the only sister,
and the maternal grandmother of the patient had complained from
photophobia for a long time. His mother and sister were astigmatic
and their visual acuity was equal to 9/10 and 7/10, respectively.
Scotopic ERGs were normal while photopic recordings showed a
slight reduction of amplitude.
Linkage Analysis at the RP2 and RP3 Loci
Fluorescent markers containing short tracks of dinucleotide
repeats were chosen form the Ge ´ne ´thon Linkage Map [Dib et al.,
1996] to flank the RP2 and RP3 loci: DXS110, DXS1068,
DXS1058, DXS1055, DXS1003, and DXS1039. In addition, primers
flanking dinucleotide repeats located 125 kb upstream and 17 kb
downstream of the RPGR gene, respectively, were designed from the
Working Draft of the Human Genome available at the University of
California Santa Cruz (UCSC) Genome Browser (http://genome.
ucsc.edu; data not shown, available on request).
Amplified fragments were electrophoresed on an automatic
sequencer (ABI 3100; Applied Biosystems, Foster City, CA) and
analyzed using the GeneScan Analysis 3.7 (Applied Biosystems)
and Genotyper (Applied Biosystems) software.
For each marker, the heterozygote frequency and the size range
of alleles were either available from the Ge ´ne ´thon Linkage Map
or determined by the present study (data not shown, available
Mutational Screening of the RP2 and RPGR Genes
The mutational screening of the RP2 and RPGR gene was
performed using primers designed to flank the splice-site junctions
of the five exons of the RP2 gene (1F-1R to 5F-5R) and the
19 exons of the RPGR gene (1F-1R to 19F-19R), respectively.
82 HUMAN MUTATION 28(1),81^91,2007
Human Mutation DOI 10.1002/humu
The ORF15 exon of the RPGR gene was screened using primers
designed to generate six overlapping fragments (15.1F/15.1R,
15.2F/15.2R, 15.3F/15.3R, 15.4F/15.4R, 15.5F/15.7R, and 15.7F/
Genomic DNA (100ng) was submitted to PCR amplification in
a buffer containing: 200mM of each dNTP , 1mM of each primer,
0.5 Unit of Taq DNA Polymerase (Invitrogen, Cergy Pontoise,
France) and either 1.5mM MgCl2, 20mM Tris-HCl (pH8.4),
50mM KCl for RP2, RPGR, and the 15.1F/15.1R–15.4F/15.4R
fragments of the RPGR exon ORF15 or 1?GC-RICH PCR
reaction buffer and 1? GC-RICH resolution solution of the
GC-Rich PCR System (Roche Diagnostics GmbH, Penzberg,
Germany) for the 15.5F/15.7R and 15.7F/15.9R fragments.
Amplification products were loaded onto a 1.5% low melting
agarose gel, purified by phenol-chloroform extraction and recovered
by ethanol precipitation. Purified fragments were directly sequenced
using reverse primers (3.2 pmol) and the Big Dye Terminator Cycle
Sequencing Kit version 3.1 (Applied Biosystems, Foster City, CA)
on a 3100 automated sequencer for all PCR fragments. Additionally,
the second exon of the RP2 gene and the 15.5F/15.7R and 15.7F/
15.9R PCR fragments of the RPGR ORF15 exon were sequenced
using additional primers (RP2: 2.1F, 2.2F, and 2.2R; RPGR ORF15:
15.5R, 15.6R, 15.7R, and 15.8R).
All PCR conditions and primer sequences are not shown but
can be obtained on request.
Mutations were confirmed by analyzing family members when
available. Sequence changes were considered as mutations
when they were predicted to result in a truncated protein, had
not been reported as a polymorphism and were not found in a
cohort of 100 control males.
XChromosome Inactivation Analysis
DNA was extracted from peripheral blood cells according to
standard procedure. The pattern of X chromosome inactivation
was determined by PCR analysis of genomic DNA using
fluorescent primers designed to flank of a polymorphic CAG
repeat in the first exon of the androgen receptor (AR). Since the
methylation of sites close to this repeat correlates with
X-chromosome inactivation, only the inactive X chromosome
could be amplified after digestion with the methylation-sensitive
restriction enzyme HpaII. In females heterozygous for the
polymorphic AR repeat, this assay allowed the distinction between
the maternal and paternal alleles and identifies their methylation
status. For each DNA sample, two parallel reactions were set:
in the first, 200ng of genomic DNA was digested for 2hr at
371C with 10 U of HpaII in the buffer recommended by the
manufacturer (New England Biolabs, Ipswich, MA); in the second,
200ng of genomic DNA was incubated only with the enzyme
digestion buffer containing no enzyme. DNA was recovered by
ethanol precipitation prior to PCR amplification (standard
conditions, available on request). PCR products from undigested
and digested DNA were separated on a 3100 automated sequencer
and analyzed by GeneScan.
The X inactivation pattern was recorded as the relative amount
of PCR product of the smallest allele. The X inactivation pattern
was classified as skewed, when 90% or more of the cells
preferentially expressed one X chromosome.
Indirect Studies at the RP2 and RP3 Loci
Linkage analyses were performed using polymorphic markers
flanking the RP2 and RP3 loci in familial cases when blood
samples of the affected patient relatives were available; i.e., in 46
out of 93 familial cases of RP suggesting X-linked transmission,
4 out of 7 male sibships, and 1 out of 2 families affected with
Exclusion of both the RP2 and RP3 loci was evidenced in 4 out
of 46 families. Conversely, haplotypes were consistent with linkage
to the RP3 locus in 13 out of 46 families (28.3%) with dominant
RP while a hit of linkage to the RP2 locus was found in 6 out of
46 families (13.0%). In 23 out of 46 families (50%) we could not
decide between RP2 and RP3 as no recombination event occurred
between the two loci neither in males nor in obligate carriers.
Concerning the four male sibships, one was compatible with
linkage to RP2, one with RP3, and two with both RP2 and RP3.
Finally, the familial XLCOD excluded the RP2 locus but was
compatible with linkage to the RP3 locus.
Spectrum of RPGR Mutations
The RP2 and RPGR genes were screened for mutations on the
basis of the linkage analyses orientation when available.
With regard to the RP2 gene, 14 different disease-alleles were
identified in 15 out of 127 families (11.8%; Table 1; Fig. 1). These
14 mutations are made of four missense mutations, two nonsense
mutations, one splice-site mutation, four microdeletions, two
microinsertions, and one deletion of the whole gene (Table 1; Fig.
Of the 14 mutant alleles, 10 lie in exon 2 of the gene and no
mutation was found in exons 4 and 5 (Fig. 1). Of the 14 different
disease-alleles, 12 have never been reported elsewhere, while 2 out
of 14 were referenced in the Human Gene Mutation Database
A total of 42 different RPGR disease-associated mutations were
identified in 65 out of 127 families (51.2%; Tables 2 and 3; Fig. 2).
Of the 42 mutations, 14 lie in exons 1–14 of the gene (33.3%) and
were identified in 14 out of 65 families (21.5%; Table 2). These 14
mutations were made of six missense mutations, one nonsense
mutation, two splice-site mutations; four microdeletions, and one
deletion of two codons (Table 2; Fig.
missense mutations, c.154G4A, has been shown to result in the
deletion of the exon 3 [Demirci et al., 2004]. Of the 14 different
mutations, 12 have never been reported elsewhere and 2 out of 14
were referenced in the Human Gene Mutation Database.
A total of 28 out of the 42 RPGR mutations lie in the ORF15
exon (66.7%; Table 3; Fig. 2). These 28 mutations were identified
in 51 families (51/65 families with RPGR mutations; 78.4%). Two
of them accounted for by the disease in 21 out of 51 families
(41.2%): the ORF151652_653delAG and ORF151483_484del-
GA mutations identified in 12 and 9 unrelated families,
respectively (Table 2; Fig. 2). Haplotype analyses were carried
out using at least two highly polymorphic markers closely flanking
the RPGR gene in individuals harboring one or the other of the
two mutations. No linkage disequilibrium was found for any of
them (not shown; available on request). All 28 ORF15 mutations
were null alleles (Table 2; Fig.
referenced in the Human Gene Mutation Database and 17 out of
28 were novel mutations.
2). One out of the six
2), 11 out of 28 which were
Families With Suggestive XLRP
Five families among the 93 familial cases of RP originally
implying X-linked transmission were excluded from the study. In 4
out of 5, indirect analyses excluded linkage to both the RP2 and
RP3 loci. Besides, in 2 out of 5 families (one excluding RP2 and
RP3), ophthalmologic investigations in a boy born to an affected
HUMAN MUTATION 28(1),81^91,2007 83
Human Mutation DOI 10.1002/humu
male revealed clinical symptoms of RP , ruling out X-linked
RPGR and RP2 mutations were identified in 69 out of 88
familial cases of RP implying X-linked transmission (78.4%), RP2
mutations accounted for 14 out of 69 families (20.3%), while
RPGR mutations were involved in 55 out of 69 families (79.7%;
Table 4). Among these latter 55 families, 43 carried a mutation in
the ORF15 exon (78.2%), while 12 carried a mutation in other
RPGR exons (21.8%; Table 4).
In 28 out of 55 families segregating RPGR mutations, several
carrier females complained of severe visual impairment. The X
inactivation was studied in 55 women belonging to these 28
families independent of their phenotype. A total of 54 of these
women showed random inactivation of the X chromosome while 1
out of 55 displayed the skewed X inactivation (Fig. 3). This latter
woman (Patient 37; Fig. 3) belonged to a large multiplex family in
which all five carrier females complained of severe visual loss. The
4 out of 5 other affected females were available for analysis and
displayed a random X-inactivation (Patients 35, 36, 38, and 39;
Fig.3). Besides, in one family segregating a RP2 mutation
(c.540_541delGT), nine women were either obligate carrier or
were shown to harbor the mutation but only one was affected with
RP . This woman was not informative at the AR locus, preventing
from studying the X-inactivation in her lymphocytes.
The multiplex family with XLCOD included in this study
because of strong linkage with RP3 was found to harbor a mutation
at the 30end of the ORF15 exon of the RPGR gene, ORF151
1641_1642delAA, expected to result in a protein shortened of
nine amino acids (Table 3; Fig. 2). This mutation has never been
Concerning the simplex family affected with COD, no mutation
was identified in any of the two XLRP genes.
Among the seven families with male sibships, two were found to
carry a mutation in the RPGR gene (Table 4). One of them carried
a RPGR splice-site mutation (Table 2; Fig. 2). The second one
harbored a mutation truncating the ORF15 exon (Table 3; Fig. 2).
In the two families, several female relatives displayed the minimal
clinical signs for XLRP carrier status and were found to harbor the
Eight out of the 25 male sporadic cases selected with regard to
the early onset and the severe progression of the RP (32%;
Table 4) carried mutations in either RP2 (n51; Table 1; Fig. 1) or
RPGR (n57; Tables 2 and 3; Fig. 2). Ophthalmologic data were
TABLE 1. Mutations Identi¢edin theRP2 Gene
Pedigree type (number
aNucleotide positions are based on GenBank sequence (www.ncbi.nlm.nih.gov/entrez, accession number NM_006915.1), with theA of the ¢rst ATG
in the ORFdesignated as base1.
XLRP, familial case of RP with nonambiguous X-linked transmission; XLRP1, familial cases of RP segregating in males and females over at least
two generations with no male to male transmission; SC, male sporadic case; MS, male sibship; UR, unreported elsewhere; CM], mutation referred
in the HumanGene Mutation Database (www.hgmd.cf.ac.uk).
FIGURE 1. Location of RP2 mutations identi¢ed in this study.
Familial case of RP with nonambiguous X-linked transmission
(J), familialcase of RPsegregatingin males and females over at
least two generations with no male to male transmission (?; in
this case only 1 out of 9 carrier women complained from severe
visualimpairment), malesporadiccase (&).Thetubulin-speci¢c
cofactor-C-like and the NDK domains of the protein are
horizontally andobliquely hatched, respectively.
84HUMAN MUTATION 28(1),81^91,2007
Human Mutation DOI 10.1002/humu
available for female relatives of 5 out of 8 male sporadic cases (1/5
carrying a missense mutation in exon 8; 4/5 harboring ORF15
truncating mutations). In all five families, the mother and/or a
sister had minimal clinical signs compatible with being an XLRP
XLRP are among the most frequent and severe forms of
inherited retinal dystrophies. Linkage analyses indicate that there
are at least five XLRP genes (The Retinal Information Network;
www.sph.uth.tmc.edu/Retnet). RP2 and RPGR are by far the
major XLRP genes.
A number of studies have reported mutations in XLRP genes
[Schwahn et al., 1998; Hardcastle et al., 1999; Mears et al., 1999;
Thiselton et al., 2000; Miano et al., 1999, 2001; Breuer et al.,
2002; Sharon et al., 2000, 2003 Roepman et al., 1996; Meindl
et al., 1996; Vervoort et al., 2000; Zito et al., 1999; Buraczynska
et al., 1997; Vervoort and Wright 2002; Demirci et al., 2002].
These mutations have recently been referenced in the Human
Gene Database. In this study, 14 RP2 mutations were identified,
12 of which are novel, and 42 RPGR mutations divided into: 14
exon 1–14 mutations, nine of which are novel, and 28 ORF15
mutations, 17 of which are novel.
RP2 encodes a 350–amino acid ubiquitously-expressed protein
targeted to the cytoplasmic face of the plasma membrane
[Schwahn et al., 1998]. Targeting is mediated by posttranslational
acyl modifications of myristoylation and palmitoylation at the
N-terminus that were shown to be critical for its function [Chapple
et al., 2000]. Besides, the N-terminal domain of RP2 shares amino
acid sequence similarity to the tubulin-specific chaperone protein
cofactor C and, like cofactor C itself, RP2 stimulates the GTPase
activity of tubulin. RP2 has also been shown to interact with
adenosine diphosphate (ADP) ribosylation factor-like 3 (Arl3),
a retrovirus-associated DNA (Ras)-related small GTP-binding
protein [Bartolini et al., 2002]. Arl3 is localized to the
photoreceptor connecting cilium of both rods and cones [Grayson
et al., 2002], where it could regulate the GTPase activity of RP2
[Bartolini et al., 2002]. Finally, the C-terminus of RP2 consists of a
domain with similarity to nucleoside diphosphate kinases (NDKs)
that, in addition to their kinase activity, have direct DNA
processing functions. It has recently been shown that RP2 exhibits
exonuclease activity and translocates to the nucleus in response to
various DNA damaging agents most notably simulated solar
ultraviolet (UV) light and ultraviolet radiation [Yoon et al., 2006].
Interestingly, 12 out of the 14 RP2 mutations identified (85.7%)
are expected to result in null alleles, highly truncated, and/or
mislocalized products: three were expected to carry away two-
thirds of the protein and are likely resulting in nonsense-mediated
decay of mRNA; four mutations were expected to carry away one-
third of the protein, including part of the beta-tubulin folding
cofactor C domain and the NDK; the p.Gly2Val mutation
disrupted the consensus sequence for N-terminal myristoylation
of the protein (M1GCFFS4M1VCFFS) and is expected to result
in incorrect target of RP2 to the plasma membrane as it has been
shown for the dS6 mutation [Chapple et al., 2002; Evans et al.,
2006] and the p.Arg118His mutation has recently been shown to
drastically reduce the affinity of RP2 to Arl3 [Kuhnel et al., 2006].
One of the 2 out of 14 remaining mutations affected a cystine
conserved in cofactor C [Schwahn et al., 1998]. The last one
resembles the p.Arg118His as it changed the arginine at position
211 into a histidine.
RPGR was originally reported to include 19 exons resulting in
the constitutive or default transcript expressed in multiple tissues
[Meindl et al., 1996; Roepman et al., 1996, Yan et al., 1998;
Kirschner et al., 1999]. The N terminal region of RPGR is similar
to RCC1, a guanine nucleotide exchange factor for Ran-GTPase,
and is predicted to be a guanine exchange factor for small GTP-
binding proteins [Meindl et al., 1996; Roepman et al., 1996].
Besides, the RCC1-like domain has been shown to interact with
TABLE 2. Mutations Identi¢edinExons1^19 of theRPGRGene
p.[Gly52Arg;Gly52_Lys82del]XLRP (1) Demirciet al. 
p.Ser41X XLRP1(1) UR
aNucleotide positions are based onGenBank sequence (accession numberNM_000328.2),with theA of the ¢rstATG in the ORFdesignated as base1.
XLRP, familial case of RP with nonambiguous X-linked transmission; XLRP1, familial case of RP segregating in males and females over at least
two generations with no male to male transmission; SC, male sporadic case; MS, male sibship; UR, unreported elsewhere; CD], CM], CS], mutation
referenced in the Human Gene Mutation Database (the asterisks indicate that the mutation has been labeled di¡erently in the database because the
position of the deletion in thesequencecould be interpreted in di¡erent ways).
HUMAN MUTATION 28(1),81^91,2007 85
Human Mutation DOI 10.1002/humu
TABLE 3. Mutations Identi¢edin theORF15 Exonof theRPGRGene?
aNucleotide positions are based onGenBank sequenceNM_001034853.1for approved nomenclature.
bNucleotide positions are based onVervoort et al.  for the traditional ORF15 nomenclature.
XLRP, familial case of RP with nonambiguous X-linked transmission; XLRP1, familial case of RP segregating in males and females over at least two generations with no male to male transmission; SC, male
sporadic case; MS, male sibship; UR, unreported elsewhere; CD], CM], mutation referred in the Human Gene Mutation Database (the asterisk indicates that the mutation name in the database does not follow
the approved nomenclature).
86HUMAN MUTATION 28(1),81^91,2007
Human Mutation DOI 10.1002/humu
several retinal-specific proteins including the delta subunit of the
phosphodiesterase [Linari et al., 1999; Hong et al., 2001] and the
Leber congenital amaurosis-associated RPGRIP1 protein [Hong
et al., 2001; Dryja et al., 2001; Gerber et al., 2001]. The retinal
enriched ORF15 transcript, shares the same exons 1 through 13,
but utilizes exon 14 through part of intron 15 as a large terminal
exon (ORF15 exon [Vervoort et al., 2000]). The ORF15 exon has
a highly repetitive purine-rich internal region that appears to act
as a splicing enhancer in mice [Hong and Li, 2002]. Additional
alternative spicing is found in both the constitutive and the
ORF15 transcript [Yan et al., 1998; Kirschner et al., 1999; Hong
and Li, 2002]. At the protein level, the ORF15 variant is found in
only photoreceptors, whereas the constitutive variant is expressed
at higher levels outside of photoreceptors, suggesting that the
ORF15 variant may be the functionally significant isoform in
Most RPGR exon 1–14 mutations (12/14; 86%) are expected to
either concern conserved amino acid or truncate the conserved
RCC1-like domain and are predicted to alter the GTPase activity
of the protein and/or its interaction with its functional partners in
the retina. The 2 out of 14 remaining mutations lie outside the
RCC1-like domain but are expected to truncate the protein.
With regard to the ORF15 exon that encodes the plaid domain
of the protein, 100% of the mutations were either nonsense
mutations or small-scale rearrangements leading to a long stretch
of mutant codons downstream from the site of the mutation prior
to a termination codon. It has been proposed that the longer the
encoded wild-type ORF15 amino acid sequence, the milder the
disease, and that the longer the encoded abnormal amino acid
sequence, the more severe the disease [Sharon et al., 2003]. This
observation seems to be corroborated by dogs harboring mutations
in ORF15 [Zhang et al., 2002].
Mutations in RP2 and RPGR were identified in 69 out of 88
French families of RP consistent with X-linked transmission
(78.4%). A total of 28 out of the 69 families with mutations
belonged to the subset of 43 out of 88 families of our series in
which the disease segregated in males and females over at least
two generations without male to male transmission (27/28 RPGR;
The description of inconstant severe visual impairment in
women carrying RPGR mutations is not novel [Rozet et al., 2002].
However, only ORF15 exon mutations were reported to be
associated with this phenotype [Rozet et al., 2002]. Here, in an
enlarged series, we show that the proportion of variants in this
exon and exons 1–14 are similar in families with (77.7% in
ORF15; 22.2% in other exons) and without affected women
(78.5% in ORF15; 21.4% other exons). Besides, we confirm that
RPGR mutations in these two family subsets are localized in same
regions of the gene, are similar in nature, and are expected to have
similar effects at the protein level. With regard to the ORF15
exon, no relation between the size of the wild-type ORF15 amino
acid sequence or the size of the long stretch of mutant codons
downstream from the site of the mutation prior to a termination
codon, and the phenotype of women could be determined. No
skewed X-inactivation was noted in families with affected women
in our series (n527). Only one affected woman belonging to a
multiplex family showed a bias in inactivation, but other affected
females of this family showed random inactivation, questioning the
FIGURE 2. Location of RPGR mutations identi¢ed in this study.
Familial case of RP with nonambiguous X-linked transmission
(J), familialcase of RPsegregating in males andfemales over at
least two generations with no male to male transmission (?),
malesibship (&), malesporadic (&), familialXLCOD (m). Each
symbol represents one family.The RCC1-like domain of the pro-
tein is hatched.The nonstandard traditional ORF15 mutations
nomenclature have been used in this ¢gure. See Table 3 for
HUMAN MUTATION 28(1),81^91,2007 87
Human Mutation DOI 10.1002/humu
significance of the X-inactivation bias identified in this family.
Thus, it is likely that modifying genetic factors (RPGR
polymorphisms in cis or in trans of the mutation, variants in other
genes located on chromosome X, or autosomes) might explain the
range of severity of the clinical signs in the women of these
Conversely to RPGR, RP2 was not known to be involved in
XLRP affecting women. Here, we report the identification of a
2-bp deletion in exon 2 of the gene (c.540_541delGT) in a large
multiplex family with five carriers, one of whom only developed an
authentic and severe RP since the age of 30 years. A skewed X-
inactivation or a karyotypic abnormality could have explained the
disease in this woman. The karyotype of this woman is an
X-autosome translocation. The X-inactivation could not be
investigated as she was not informative at the AR locus. Whatever
the mechanism resulting in the severe clinical manifestation in this
woman, this observation appears anecdotal in light of the very
large number of asymptomatic women reported to harbor RP2
mutations. On the contrary, the probability of the occurrence of
RP in women carrying RPGR mutations is not infrequent and
should be considered in genetic counseling.
With regard to the 19 out of 88 families with no mutation, a
distinction has to be made between families unambiguously
segregating RP as an X-linked trait and families in which this
mode of inheritance can be questioned, in particular in families
with severely affected women.
In one of the 19 families, the X-linked inheritance was based on
the affected status of the deceased maternal grandfather of an
isolated male case. In this family, a possible misdiagnosis cannot
be excluded for the grandfather. In 3 out of 19 other families, the
X-linked inheritance was unambiguous. Linkage analyses were
possible in 2 out of 3 of these families and showed that the
localization of the disease gene was compatible at the RP2 and
RP3 loci. In these two families, and in the third one as well, the
disease mutation could lie in an unscreened region of either the
RP2 or RPGR genes (untranslated regions, intronic sequences,
etc.). It is also possible that the disease mutation lies in a gene at
another XLRP locus.
FIGURE 3. X-inactivationstudy inRPGRcarrier females.TheX-inactivationwasinvestigatedon lymphocyteDNA in 55 RPGRcarrier
women informative at the androgen receptor (AR) locus.These women belong to 27 families segregating RP in males and females
over at least two generations with no male to male transmission. Foreachwoman, numbered from1to 55, results are means7SD of
three distinct experiments. Each bar represents oneAR allele. Avalue of 50% indicates‘‘random’’X-inactivation,with equal number
of cells having each X chromosome active; the X-inactivation pattern was classi¢ed as skewed only when 90% or more of the cells
TABLE 4. DistributionofRP2 andRPGR Mutations inDi¡erentGroups ofFamilies?
Total numberof families
Number (%) of familieswith mutations
TotalGroup RP2 RPGRexon1^19RPGRORF15
14 (15.9%) [1/14 RPLX1]
12 (13.6%) [6/12 RPLX1]
?Note that among the 93 families suggestingX-linkedinheritance, ¢vewereexcludedfromthestudy:3 out of 5 wereunlinked to theRP2/RP3 loci and
in 2 out of 5 families an a¡ected boy was born to an a¡ected father.
XLRP, familial case of RP with nonambiguous X-linked transmission; XLRP1, familial case of RP segregating in males and females over at least two
generations with no male to male transmission.
88HUMAN MUTATION 28(1),81^91,2007
Human Mutation DOI 10.1002/humu
Thirteen out of the 19 families with no mutation belonged to
the subset of 43 families segregating the disease in males and
females with no male to male transmission. The location of the
disease gene in 1 out of 13 of the families was compatible at the
RP3 locus (RP2 excluded) but no indirect study was possible in
the remaining 12 out of 13 families (samples were unavailable).
With regard to these last 12 families, the disease segregated over
two generations (with transmission from the affected mothers to
their sons) and over three generations in 2 out of 12 and 10 out of
12 families, respectively. In these 13 families, though the onset of
the disease in women was delayed compared to men and though
no male to male transmission was noted, the autosomal dominant
mode of inheritance cannot be ruled out.
Altogether, the frequency of RPGR and RP2 mutations in
French XLRP families is in accordance with that reported in other
studies (RP2: 15.9% vs. 6–20% [Schwahn et al., 1998; Hardcastle
et al., 1999; Mears et al., 1999; Thiselton et al., 2000; Miano et al.,
2001; Breuer et al., 2002; Sharon et al., 2003]; RPGR: 78.4% vs.
55–90% [Roepman et al., 1996; Meindl et al., 1996; Zito et al.,
1999; Vervoort et al., 2000; Buraczynska et al., 1997; Vervoort and
Wright 2002; Demirci et al., 2002; Breuer et al., 2002; Sharon
et al., 2003]).
With regard to sporadic male cases, we report here a high
prevalence of mutations in either RPGR or RP2 (8/25 patients;
32%). This frequency is significantly higher than that reported by
Sharon et al.  (13.3%) but close to that reported by Breuer
et al.  (29%). These results confirm the pertinence of
screening for XLRP genes in sporadic male cases selected on the
basis of early onset and severity of the affection (onset in the first
decade, profound visual impairment before the age of 30 years).
Previous studies identified mutations of XLRP genes in 32 to
54% of male sibships affected with RP [Breuer et al., 2002; Sharon
et al., 2003]. Here we report the involvement of these genes in
28.6% of similar families in our series (2/7 families), giving further
support to the screening of XLRP genes in such families.
With regard to the two families segregating COD, the disease
was inherited as a nonambiguous X-linked trait in the first one and
was compatible with linkage to the RP3 locus. A RPGR mutation
was identified in the 30end of the ORF15 exon of the gene
(p.Asn547ArgfsX11). This finding corroborates the notion that
mutations downstream of codon 445 of ORF15 lead to preferential
loss of cone function, although affecting rod function much less
[Sharon et al., 2003; Demirci et al., 2002; Yang et al., 2002].
In the second family, the mother and maternal grandmother of a
11-year-old male affected with COD did not complain of visual
discomfort with the exception of a minor photophobia. In addition
they showed a normal fundus but a slight reduction in the
amplitude of the photopic ERG, contrasting with normal scotopic
ERG. These data are suggestive of possible X-linked inheritance
but no mutation was found in either RPGR or RP2. It is possible
that the disease-causing mutation maps to CORDX2 on Xq27 but
the family is too small to perform linkage studies.
The data presented here have had an important impact on
genetic counseling and render genotyping more accessible. First,
with respect to: 1) the frequency of mutations in the RPGR
ORF15 exon, RPGR exons 1–19 and RP2, respectively; 2) the
involvement of RPGR mutations in a significant subset of XLRP
families with women complaining of severe visual dysfunction;
and 3) the existence of recurrent mutations, we drew a decision-
making flowchart for the molecular diagnosis (Fig.
flowchart lightens the task of genotyping new patients especially
as in 50% of families the RP2 and RP3 loci cosegregate
without recombination events hindering the recognition of the
FIGURE 4. Decisional-making £owchart for the molecular study of families a¡ectedwithX-linked (XL) RP, male sibships (MS), male
sporadic cases (SC), and X-linked recessive COD. On the basis of the phenotype of the patients, the pedigree type and the linkage
analyses at theRP2 andRP3 loci,wepropose a sequenceof regions of theRPGR and/or theRP2 genes (a^h) toscreeninpriority.
HUMAN MUTATION 28(1),81^91,2007 89
Human Mutation DOI 10.1002/humu
We thank Alan Wright for providing us the sequences of primers
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