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1 From the Ocular Molecular Genetics Institute and the 3 Berman–Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston; 2 Genetics Division, Children’s Hospital, Boston, Massachusetts.
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Abstract |
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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.
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Introduction |
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Positional cloning efforts resulted in the identification of the RPGR (RP guanosine triphosphatase [GTPase] regulator) gene in the RP3 region13 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.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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 [
-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
(94oC for 5 minutes), 25 cycles of PCR
amplification were performed. Each cycle consisted of denaturation
(94oC for 30 seconds), primer annealing
(50oC–62oC for 30
seconds), and extension (71oC for 30 seconds).
The final extension was at 71oC 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 0.5x mutation detection enhancement (MDE)
gel (FMC Bioproducts, Rockland, ME) in 0.6x 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 loge 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
loge ERG amplitude, loge
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).
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Results |
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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).
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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 IVS12-97TC), intron 13
(IVS13+11AG), 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+16AG also carried IVS18+11TC.
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+3AT, 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.
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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 carriers26 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.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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
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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+3AT 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 RCC113
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 cones37
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 ß-tubulin and may function as a chaperone for
ß-tubulin.38
The - and ß-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.
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Acknowledgements |
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Footnotes |
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Submitted for publication November 12, 1999; revised January 13 and February 23, 2000; accepted March 3, 2000.
Commercial relationships policy: N.
Corresponding author: Thaddeus P. Dryja, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114. dryja{at}helix.mgh.harvard.edu
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