Identification of a novel homozygous nonsense mutation in EYS in a Chinese family with autosomal recessive retinitis pigmentosa
BMC Medical Genetics
Identification of a novel homozygous nonsense mutation in EYS in a Chinese family with autosomal recessive retinitis pigmentosa
Yukan Huang 0
Jing Zhang 0
Chang Li 0
Mugen Liu 0
Qing K Wang 0
Zhaohui Tang 0
0 Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology , Wuhan, Hubei , PR China
Background: Retinitis pigmentosa is the most important hereditary retinal degenerative disease, which has a high degree of clinical and genetic heterogeneity. More than half of all cases of retinitis pigmentosa are autosomal recessive (arRP), but the gene(s) causing arRP in most families has yet to be identified. The purpose of this study is to identify the genetic basis of severe arRP in a consanguineous Chinese family. Methods: Linkage and haplotype analyses were used to define the chromosomal location of the pathogenic gene in the Chinese arRP family. Direct DNA sequence analysis of the entire coding region and exon-intron boundaries of EYS was used to determine the disease-causing mutation, and to demonstrate that the mutation co-segregates with the disease in the family. Results: A single nucleotide substitution of G to T at nucleotide 5506 of EYS was identified in the Chinese arRP family. This change caused a substitution of a glutamic acid residue at codon 1,836 by a stop codon TAA (p.E1836X), and resulted in a premature truncated EYS protein with 1,835 amino acids. Three affected siblings in the family were homozygous for the p.E1836X mutation, while the other unaffected family members carried one mutant allele and one normal EYS allele. The nonsense mutation p.E1836X was not detected in 200 unrelated normal controls. Conclusions: The EYS gene is a recently identified disease-causing gene for retinitis pigmentosa, and encodes the orthologue of Drosophila spacemaker. To date, there are only eight mutations in EYS that have been identified to cause arRP. Here we report one novel homozygous nonsense mutation of EYS in a consanguineous Chinese arRP family. Our study represents the first independent confirmation that mutations in EYS cause arRP. Additionally, this is the first EYS mutation identified in the Chinese population.
Retinitis pigmentosa (RP; OMIM 268000) is
characterized by the constriction of the visual fields, night
blindness, changes of the fundi including bone corpuscle
lumps of pigment, and the loss of central vision. The
worldwide prevalence of RP is about one in 4,000 .
RP is inherited most frequently (50-60% of cases) as an
autosomal recessive trait, followed by autosomal
dominant (30-40%) and then X-linked (5-15%) [1-6]. It is the
most common hereditary retinal dystrophy causing
irreversible blindness [1-3]. About 30 genes and loci
have been implicated in isolated cases of arRP to date
htm]. Mutations in ABCA4, CDHR1, CERKL, CNGA1,
CNGB1, CRB1, IDH3B, LRAT, MERTK, NR2E3, NRL,
PDE6A, PDE6B, PRCD, PROM1, RBP3, RGR, RHO,
RLBP1, RP1, RPE65, SAG, SPATA7, TULP1, USH2A and
most recently EYS have been identified to be the cause
for arRP [7,8]. However, the gene(s) causing sporadic
arRP in most families has yet to be discovered. The
identification of genotypes and phenotypes and the
physiological roles of novel RP genes in the retina should
provide valuable information for the diagnosis and
classification of retinal degeneration. Consequently, genetic
studies will result in the scientific basis for the
prevention and treatment of RP.
RP25, a genetic locus for arRP, was mapped to a ~ 16
cM region on chromosome 6p12.1-q15 in four Spanish
families by Ruiz et al. in 1998 . Then, the pathogenic
genes for several arRP families with various ancestral
origins, including one Pakistani family and three
Chinese families, were also been mapped to this locus
[10-12]. By using the 10K genechip array, Barragn I et
al. refined the disease interval from the original 16 cM
to only a 2.67 cM region between D6S257 and D6S1557
. Most recently, the disease-causing gene at this
locus has been identified as the EYS gene. EYS is
predicted to be a 3,165 amino acid multi-domain protein,
which contains at least 28 epidermal growth factor
(EGF)-like domains in its N-terminus, and C-terminal
laminin A G-like domains. It is an evolutionarily
conserved protein from Drosophila to humans. Finally,
eight EYS mutations were detected in eight arRP
families from Spanish and Dutch origins at almost the
same time in 2008 [7,8].
In this study, we characterized a consanguineous
Chinese family with a severe form of arRP. After linkage
analysis with known arRP causative genes and loci, we
excluded all other arRP loci except for the RP25 locus.
Further direct DNA sequence analysis revealed a
homozygous mutation in the EYS. Our data confirmed the
conclusion from the two original reports that mutations
in EYS can cause arRP, and expanded the mutation
spectrum of EYS to the Chinese population.
Figure 1 Pedigree structure and haplotype analysis at the RP25 locus. Blackened bars indicate the disease haplotype. Filled squares or filled
circles represent male or female individuals affected with RP, respectively. Arrow points to the proband (II2). All patients in the arRP family carry
the homozygous haplotype between single nucleotide polymorphisms rs4710292 and rs4710437. The genomic positions of two markers are
from Human (Homo sapiens) Genome Browser Gateway, the GRCh37 build version, and six SNPs are from NCBI B37.1 assembly.
The study subjects from a consanguineous Chinese arRP
family were recruited from Hubei Province, P. R. China.
Informed consent was obtained from the participants in
accordance with the study protocols approved by the
Ethics Committee of Huazhong University of Science
and Technology. Diagnosis of RP was carried out by
clinical and ophthalmological examinations. Peripheral
blood was collected from the family members and 200
normal Chinese Han controls. Total human genomic
DNA was isolated with the DNA Isolation Kit for
Mammalian Blood (Roche Diagnostic Co., Indianapolis, IN).
Linkage and haplotype analyses were applied to test
the linkage of the family to 22 arRP loci, including
ABCA4, CERKL, CNGA1, CNGB1, CRB1, EYS, I8DH3B,
LRAT, MERTK, NR2E3, NRL, PDE6A, PDE6B, PRCD,
PROM1, RGR, RHO, RLBP1, RPE65, SAG, TULP1, and
USH2A. The microsatellite markers flanking these loci
were selected from Linkage Mapping Set MD-10
(Applied Biosystems, Inc., Foster City, CA), and
genotyped using an ABI 3100 Genetic Analyzer. Genotypes
were analyzed by the GeneMapper 2.5 Software program
(Applied Biosystems, Inc., Foster City, CA). PCR and
genotyping were performed as previously described .
When the pathogenic gene was mapped to chromosome
6q12 that harbors the EYS gene, additional SNPs
(rs4710437, rs1057530, rs4710457, rs66462731,
rs10944813, rs4710292) were employed for the linkage
analysis to get unambiguous linked haplotype. The EYS
was then subjected to mutational analysis. All coding
exons and splice sites of EYS were amplified by PCR
and sequenced. The intronic primers and corresponding
PCR conditions were as described in the paper of Abd
El-Aziz et al. .
The RP in the consanguineous Chinese family is
inherited in an autosomal recessive mode (figure 1). Parents
I1 and I2 are third cousins. The proband II2 and his
two sisters had deteriorating vision, while the parents
and young brother II4 did not show any RP features.
The family history was negative for deteriorating vision
or night blindness.
The proband exhibited night blindness at the age of 15
years, and progressively lost his visual acuity to 20/400 at
approximately the age of 35-40 years. Funduscopic
examinations revealed obvious attenuation of the retinal
arteries, waxen color of discs, and bone-spicule
pigmentation in the peripheral-mid retina (figure 2A).
Electrophysiological examination showed an extinguished or
very low amplitude ERG in both eyes (figure 2B).
The other two affected individuals also had the similar
symptoms as the proband. Night blindness appeared late
Figure 2 Fundus photographs and ERG of the proband in the
Chinese arRP family. The features of waxy-pale disc, arteriolar
attenuation, and bone-spicule pigment deposit in the
midperipheral retina are shown in A. The full-field ERG in the proband
in B, either a-wave or b-wave has obvious reduced amplitude and
prolonged response time in both eyes.
in the second decade of life, low visual acuity between
20/400 and hand movement, and a flat ERG confirmed
the diagnosis and severity of the RP disease. No
additional symptoms or ocular defects were observed in the
affected individuals of the family except for patient II5,
who was affected with cataract in both right and left
After linkage analysis for known arRP causative genes
and loci, there was only the RP25 locus that showed a
definite linkage with the disease in the family.
Homozygosity mapping with haplotype analysis revealed that all
three affected siblings (II1, II2, II5) inherited the affected
alleles separately from their carrier parents, whereas
their unaffected brother II4 inherited two normal
chromosomes, and the probands daughter III1 only one
affected allele (Figure 1). These results suggest that EYS
may be the pathogenic gene in this family.
Direct DNA sequence analysis for the entire coding
region of EYS (Genbank accession No: FM209056)
found a single nucleotide transversion of G to T at
nucleotide 5506 of EYS, and it resulted in a substitution
of a glutamic acid residue at codon 1836 of EYS by a
stop codon TAA (p.E1836X) (Figure 3). The nonsense
p.E1836X mutation generates a truncated EYS protein
with only 1835 amino acid residues. Three affected
siblings are homozygous for the p.E1836X mutation,
whereas their mother, and III1, the daughter of proband,
carry only one affected allele. The p.E1836X mutation
was not detected in 200 unrelated normal controls.
A novel EYS nonsense mutation p.E1836X in a
consanguineous Chinese arRP family was identified, which is
the first EYS mutation discovered in the Chinese
population. The p.E1836X mutation is predicted to generate
an abnormal EYS protein with only 1,835 of 3,165
amino acid residues. EYS is a multi-domain protein
containing 28 epidermal growth factor (EGF)-like domains,
and 5 LamG domains at the C-terminus. All 5 LamG
domains and 7 EGF-like domains in the C-terminus of
EYS were truncated in the arRP family, which may
Figure 3 Identification of the homozygous mutation in the
consanguineous Chinese arRP family. A and B show the exon 26
of EYS sequences from a normal individual and an affected
individual with p.E1836X mutation, respectively. The sequence for
individual III1, who is heterozygous for the mutation, is shown in C.
significantly disrupt the normal physiological function of
EYS in the retina.
The EYS gene has recently been identified as the
disease-causing gene for retinitis pigmentosa, and encodes an
orthologue of Drosophila spacemaker. So far, only eight
mutations have been reported in arRP families by two
groups in 2008. El-Aziz et al identified six mutations,
including four homozygous mutations p.D904QfsX17,
p.S754AfsX6, p.T657TfsX5, p.W2640X and two
compound heterozygous mutations p.E1953X and
p.R589RfsX5 . Collin et al. reported a p.Y3156X in two
unrelated Dutch families and a p.P2238PfsX16 in an
isolated RP patient from Dutch origin . Interestingly, all of
these reported mutations, including our finding, resulted
in a truncated EYS protein. This suggests that the
Cterminus of EYS is essential for its function in the retina,
or these EYS mutations (nonsense mutations, deletions)
may cause RP by degrading the EYS mRNA through a
nonsense-mediated decay mechanism.
RP 25 could be a major locus for arRP because
1020% of Spanish typical arRP families were found to have
mutations in EYS [9,13]. Consequently, it is interesting
to study the relationship between the clinical features of
RP and different EYS mutations. Until recently,
mutations of EYS have been detected in eight families [7,8].
Three Chinese families were linked to the RP25 locus,
but no mutations were identified in the EYS gene .
Similarly, in one Pakistani arRP family, RP was
suggested to be caused by an EYS mutation, too . All
the patients in these families displayed typical
characteristic RP symptoms, including night blindness as the
initial symptom, retinal bone-spicule pigmentations,
attenuated retinal vessels constriction of visual fields,
and absent or flat ERG. However, the age of onset
displayed interfamilial differences, ranging from the early
second decade to the late forth decade. It is notable that
two patients (described by Collin et al.) from two
different families with mutation p.Y3156X had combined RP
and cataract . One patient II5 in this study was also
affected with cataract in both right and left eyes. Further
work on deciphering if EYS plays an important role in
the cataractogenesis in addition to retinal dystrophy
may be useful to understand the function of EYS.
EYS contains 44 exons covering 2.0 Mb, and it is
considered to be the largest gene identified and expressed
in the human eye as well as the fifth largest overall in
the human genome. EYS is highly expressed in the
retina, but its biological function remains unknown. In
Drosophila, the EYS homological protein spam, and its
interactor protein prom, plays a critical role in retina
morphogenesis. Drosophila mutant lines for spam and
prom can result in a failure of inter-rhabdomeral-space
separation . Mutations in human PROM1 were
associated with arRP, macular degeneration, and cone-rod
dystrophy [16-19]. EYS mutations causing arRP and the
data from Drosophila strongly suggest that EYS may
have an important function in the photoreceptor
morphogenesis. Further investigations of the biochemical
function of EYS in retinas will lead to a significant
understanding of the pathological mechanism(s) of
A novel homozygous nonsense mutation in EYS, p.
E1836X, was responsible for using arRP in the
consanguineous Chinese family. This is the first EYS mutation
found in the Chinese population. This research confirms
that mutations of EYS can cause arRP. It has also
expanded the spectrum of EYS mutations in arRP.
Note Added in Proof
Very recently, three groups of investigators described the identification of
EYS novel mutations in patients with retinal dystrophy. Abd El-Aziz et al. 
identified novel EYS mutations in arRP patients from British and Chinese
origins. Bandah-Rozenfeld et al.  reported EYS mutations in Israeli and
Palestinian families with autosomal recessive retinitis pigmentosa (arRP).
Audo I et al.  found EYS mutations in French patients with rod-cone
dystrophies. These studies supported that EYS is critical for retinal function
and confirmed that EYS is a major causative gene for retinal dystrophies in
populations of various ethnicities.
YH recruited the members of arRP pedigree, performed clinical studies for
whole family members, and relationship studies between genotype and
phenotype. JZ carried out the extraction of genomic DNA and genotyping
assay. CL was responsible for the extraction of genomic DNA and
sequencing for mutation detection. GY was involved in linkage analysis and
the discussion of the manuscript. ML participated in the original conception
of this study, and contributed to the revision of the draft. QKW conceived
and designed the experiments and helped the critical revision of the
manuscript. ZT designed the experiments, analyzed the data, interpreted the
results, and drafted the manuscript. All authors read and approved the final
We are grateful to the arRP family for their participation in this study. We
appreciate Dr. Stephen R Archacki for his critical reading and revising of the
manuscript. This study was supported by grants from Chinese National
Natural Science Foundation grant No. 30771199, 30700455, and 30871386.
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