Introduction
X-linked retinoschisis (XLRS, MIM 312700) is a
hereditary retinal disease characterized by a splitting of the
neurosensory retina, with a prevalence of 1:5,000 to 1:25,000 males
worldwide (1). Typical fundus
changes include radiating cysteic maculopathy in most cases and
peripheral retinoschisis in half of the cases (2). However, the disease has a high
degree of phenotypic variability (3–6),
in which genetic testing is of value in confirming the diagnosis
(4).
XLRS accounts for most congenital retinoschisis
(2,7) and is due to mutations in the
retinoschisin gene (RS1, OMIM 312700) localized on Xp22.13
(8,9). The encoded protein, retinoschisin,
is secreted from photoreceptors and bipolar cells as a functional
homo-octameric complex that is thought to play a role in cellular
adhesion and cell-to-cell interaction (10).
Gene transference to mouse models of X-linked
juvenile retinoschisis, which suggest gene replacement may be a
possible future therapy for patients (11–13). Genetic diagnosis is the basis for
gene transference in the future. Therefore, we have to fully
understand the molecular basis of XLRS. To date, more than 160
different RS1 mutations have been identified in patients
with XLRS (http://www.dmd.nl/rs), including small
intragenic deletions, nonsense and missense mutations, frame shift
insertions and deletions, and splice site mutations. However, there
are still some RS1 mutations that remain unknown.
In this study, we analyzed the coding exons and the
adjacent regions of RS1 in patients from 20 unrelated
Chinese families with XLRS. Ten hemizygous mutations, including 4
novel mutations, were detected in 14 families.
Subjects and methods
Probands with XLRS from 20 unrelated families were
enrolled in this study. Written informed consent was obtained from
the participating individuals or their guardians prior to the
collection of clinical data and genomic samples. This study was
approved by the Internal Review Board of the Zhongshan Ophthalmic
Center.
Mutation detection
Genomic DNA was prepared from venous leukocytes. Six
pairs of primers (Table I) were
used to amplify the six coding exons and the adjacent intronic
sequence of RS1 (NCBI human genome build 37.2, NG_008659.1
for genomic DNA, NM_000330.3 for mRNA, and NP_000321.1 for
protein). Touchdown polymerase chain reaction (PCR) was performed
with decreasing 0.5°C per cycle from 64°C for the first 15 cycles
then down to 57°C (the annealing temperature) for the remaining 21
cycles. GC buffer was used. DNA sequences of the amplicons were
identified with ABI BigDye Terminator cycle sequencing kit version
3.1 (Applied Biosystems, Foster City, CA) on an ABI 3130 Genetic
Analyzer (Applied Biosystems). Sequencing results and consensus
sequences from the NCBI human genome database were compared by
using the SeqMan II program of the Lasergene package (DNA Star,
Inc., Madison, WI) and then aligned to identify variations. Each
variation was confirmed by bidirectional sequencing. Mutation
description followed the recommendation of the Human Genomic
Variation Society (HGVS). Variations detected in patients were
further evaluated in controls by sequencing 176 normal
individuals.
| Table IPrimers used for the amplification
and sequencing of RS1. |
Table I
Primers used for the amplification
and sequencing of RS1.
Exon | Direction | Primer sequence
(5′-3′) | Size of amplified
fragment (bp) | Annealing
temperature (°C) |
---|
1 | F |
GGTTAACTTGATGGGGCTCA | 374 | 57 |
R |
AACTGGAAAGCCATCCACAC |
2 | F |
TCTATTTCACTTTTCCATGTAACGA | 243 | 57 |
R |
ACCATGCCCAGCCAAAATA |
3 | F |
GACGATGCATAAGGACTGAGTG | 296 | 57 |
R |
AGCGTTCAGGGGGTTAATTC |
4 | F |
GCAAAGCAGATGGGTTTGTT | 359 | 57 |
R |
CCACCACGCCAGTTAATTTT |
5 | F |
CAGGGGGCTCTTTGGATG | 389 | 57 |
R |
ACAGAGGGCAGTGACAGGAG |
6 | F |
CACCCGCAAACTGCTTTAAC | 384 | 57 |
R |
TGCGAAATATAGCCCTGTCC |
The Sorting Intolerant From Tolerant (SIFT) program
and the Polymorphism Phenotyping (PolyPhen-2) were used to predict
whether an amino acid substitution was likely to affect the protein
function (14,15).
Results
Mutation analysis
Ten hemizygous mutations in RS1 were detected
in patients from 14 of the 20 families with retinoschisis (Table II and Fig. 1), including c:176G>A
(p:Cys59Tyr) in exon 3, c:214G>A (p:Glu72Lys) and c:304C>T
(p:Arg102Trp) in exon 4, c:436G>A (p:Glu146Lys) in exon 5,
c.531T>G (p:Tyr177X), c:544C>T (p:Arg182Cys), c:599G>A
(p:Arg200His), c:607C>G (p:Pro203Ala), c:644A>T (p:Glu215Val)
and c:668G>A (p:Cys223Tyr) in exon 6. Of the 10, the
c:176G>A, c:531T>G, c:607C>G and c:668G>A were novel.
These novel mutations occurred in highly conserved regions
(Fig. 2) and were predicted to be
pathogenic (Table II). They were
absent in 176 normal individuals.
| Table IIThe mutations of the RS1 gene
in XLRS. |
Table II
The mutations of the RS1 gene
in XLRS.
| | | | Computational
prediction | Frequency | | |
---|
| | | |
|
| | |
---|
Exon | Patient ID | Nucleotide
change | Amino acid
change | Blosum62 | PolyPhen | SIFT | Patients | Controls | Note | Ref |
---|
3 | QT42, QT335 | c:176G>A | p:Cys59Tyr | 9→-2 | 0.996 | 0 | 2/20 | 0/176 | Novel | |
4 | QT221, QT232,
QT653 | c:214G>A | p:Glu72Lys | 5→1 | 0.998 | 0 | 3/20 | | Reported | (19) |
4 | MD15 | c:304C>T | p:Arg102Trp | 5→-3 | 1 | 0 | 1/20 | | Reported | (20) |
5 | RP6 | c:436G>A | p:Glu146Lys | 5→1 | 0.961 | 0.17 | 1/20 | | Reported | (21) |
6 | MD30 | c:531T>G | p:Tyr177X | | | | 1/20 | 0/176 | Novel | |
6 | QT417, QT212 | c:544C>T | p:Arg182Cys | 5→-3 | 1 | 0.01 | 2/20 | | Reported | (22) |
6 | QT848 | c:599G>A | p:Arg200His | 5→0 | 1 | 0 | 1/20 | | Reported | (23) |
6 | QT911 | c:607C>G | p:Pro203Ala | 7→-1 | 1 | 0.13 | 1/20 | 0/176 | Novel | |
6 | QT219 | c:644A>T | p:Glu215Val | 5→-3 | 1 | 0 | 1/20 | | Reported | (31) |
6 | QT758 | c:668G>A | p:Cys223Tyr | 9→-2 | 0.996 | 0.01 | 1/20 | 0/176 | Novel | |
All 10 probands with hemizygous RS1 mutations
(the clinical data of 4 probands were not available) had clinical
symptoms and signs of retinoschisis (Table III). The four probands with
novel mutations showed macular and peripheral retinoschisis.
| Table IIIClinical information on individuals
with RS1 variations. |
Table III
Clinical information on individuals
with RS1 variations.
| Mutations | Age (years) | | BCVA | | | | | | |
---|
|
|
| |
| | | | | | |
---|
Patient ID | Nucleotide | Protein | Exam | Onset | Family history | OD | OS | Macular change | Peripheral
change | Retinal hole | Strabismus | OCT | ERG(b/a) |
---|
QT042 | 176G>A | Cys59Tyr | N/A | N/A | No | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
QT335 | 176G>A | Cys59Tyr | 11 | 6 | No | 0.4 | 0.2 | mRS | pRS | No | No | RS | N/A |
QT221 | 214G>A | Glu72Lys | 19 | EC | Yes | 0.1 | 0.2 | mRS | PD | No | No | N/A | N/A |
QT232 | 214G>A | Glu72Lys | 18 | 8 | No | 0.4 | 0.2 | mRS | Degenenation | No | No | N/A | N/A |
QT653 | 214G>A | Glu72Lys | 5 | 3 | No | 0.3 | 0.7 | mRS | pRS | Yes | No | N/A | Reduced |
MD015 | 304C>T | Arg102Trp | N/A | 7 | No | 0.2 | 0.3 | PD, FRB | No | No | No | N/A | N/A |
RP006 | 436G>A | Glu146Lys | 5 | 4 | No | FC | 0.03 | PD, FRB | No | No | No | N/A | Reduced |
MD030 | 531T>G | Tyr177X | 6 | 5 | No | 0.3 | FC | mRS | pRS | No | Yes | N/A | Reduced |
QT212 | 544C>T | Arg182Cys | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
QT417 | 544C>T | Arg182Cys | 12 | EC | No | 0.3 | 0.03 | No | pRS | Yes | No | N/A | N/A |
QT848 | 599G>A | Arg200His | 21 | EC | No | 0.6 | 0.4 | mRS | No | No | No | N/A | Reduced |
QT911 | 607C>G | Pro203Ala | 22 | EC | No | 0.2 | 0.4 | mRS | pRS | No | Yes | N/A | N/A |
QT219 | 644A>T | Glu215Val | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
QT758 | 668G>A | Cys223Tyr | 9 | 6 | No | 0.4 | 0.3 | mRS | pRS | Yes | No | RS | N/A |
Discussion
In this study, ten different hemizygous mutations in
RS1 were identified in 14 families with XLRS. These
mutations are predicted to be pathogenic. All patients with
mutations demonstrated typical signs of XLRS. The ten mutations
affected different domains of retinoschisin, including the RS1
domain (1 mutation), discoidin domain (8 mutations) and C-terminal
segment (1 mutation). These mutations were not randomly distributed
over the gene (Fig. 3) because
80% of mutations were clustered in the discoidin domain (16). The two novel mutations, Tyr177X
and Pro203Ala in the discoidin domain, may cause a shorter
retinoschisin form or protein misfolding (13). The cysteine mutations in the
RS1 domain (Cys59Tyr) and C-terminal segment (Cys223Tyr) may
cause failure of the discoidin domain to assemble into a normal
multisubunit complex (17,18).
Most of RS1 mutation loci were hot mutation
spots, while the Cys59, Glu72, Arg102, Glu146, Arg182, Arg200,
Pro203, Glu215 and Cys223 could be substituted by 1–2 other kinds
of amino acids and be reported more frequently (19–30). However, the mutations in the
present study also differed from those reported previously. The
RS1 mutations accounts for 70% of the Chinese retinoschisis
(14/20) cases in our study. The Cys59Tyr, Tyr177X, Pro203Ala,
Glu215Val and Cys223Tyr mutations only are present in the Chinese
population (31), and the
Cys59Tyr mutation was more common (10% frequency in our
retinoschisis cases). The Glu72Lys mutation is the most common
among Chinese (15%) as well as other populations (19,32), while another very common mutation,
Pro192Ser (33), which was
reported from people of different ethnic backgrounds was not found.
We do not know whether the spectrum and frequency of RS1
gene in the Chinese is different from others. Our study contributes
to the current state of knowledge.
In summary, we identified ten mutations in 14 of 20
families with XLRS. Our results expand the mutation spectrum of
RS1 that might enrich our understanding of the molecular
basis of XLRS in the Chinese population.
Acknowledgements
The authors thank all of the patients and controls
subjects for their participation. This study was supported by the
Open Research Fund Program of State Key Laboratory of
Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University,
and in part by grant 30725044 from the National Science Fund for
Distinguished Young Scholars.
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