Novel USH2A compound heterozygous mutations cause RP/USH2 in a Chinese family.

Abstract

To identify the disease-causing gene in a four-generation Chinese family affected with retinitis pigmentosa (RP).
Linkage analysis was performed with a panel of microsatellite markers flanking the candidate genetic loci of RP. These loci included 38 known RP genes. The complete coding region and exon-intron boundaries of Usher syndrome 2A (USH2A) were sequenced with the proband DNA to screen the disease-causing gene mutation. Restriction fragment length polymorphism (RFLP) analysis and direct DNA sequence analysis were done to demonstrate co-segregation of the USH2A mutations with the family disease. One hundred normal controls were used without the mutations.
The disease-causing gene in this Chinese family was linked to the USH2A locus on chromosome 1q41. Direct DNA sequence analysis of USH2A identified two novel mutations in the patients: one missense mutation p.G1734R in exon 26 and a splice site mutation, IVS32+1G>A, which was found in the donor site of intron 32 of USH2A. Neither the p.G1734R nor the IVS32+1G>A mutation was found in the unaffected family members or the 100 normal controls. One patient with a homozygous mutation displayed only RP symptoms until now, while three patients with compound heterozygous mutations in the family of study showed both RP and hearing impairment.
This study identified two novel mutations: p.G1734R and IVS32+1G>A of USH2A in a four-generation Chinese RP family. In this study, the heterozygous mutation and the homozygous mutation in USH2A may cause Usher syndrome Type II or RP, respectively. These two mutations expand the mutant spectrum of USH2A.

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Novel USH2A compound heterozygous mutations cause RP/USH2
in a Chinese family
Xiaowen Liu,1,2 Zhaohui Tang,2 Chang Li,2 Kangjuan Yang,3 Guanqi Gan,2 Zibo Zhang,3 Jingyu Liu,2
Fagang Jiang,1 Qing Wang,2 Mugen Liu2
(The first two authors contributed equally to this work)
1The Union Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China; 2Key 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, China; 3Departments of Cell Biology and Medical Genetics, Yanbian University,
Yanji, China
Purpose: To identify the disease-causing gene in a four-generation Chinese family affected with retinitis pigmentosa
(RP).
Methods: Linkage analysis was performed with a panel of microsatellite markers flanking the candidate genetic loci of
RP. These loci included 38 known RP genes. The complete coding region and exon-intron boundaries of Usher syndrome
2A (USH2A) were sequenced with the proband DNA to screen the disease-causing gene mutation. Restriction fragment
length polymorphism (RFLP) analysis and direct DNA sequence analysis were done to demonstrate co-segregation of the
USH2A mutations with the family disease. One hundred normal controls were used without the mutations.
Results: The disease-causing gene in this Chinese family was linked to the USH2A locus on chromosome 1q41. Direct
DNA sequence analysis of USH2A identified two novel mutations in the patients: one missense mutation p.G1734R in
exon 26 and a splice site mutation, IVS32+1G>A, which was found in the donor site of intron 32 of USH2A. Neither the
p.G1734R nor the IVS32+1G>A mutation was found in the unaffected family members or the 100 normal controls. One
patient with a homozygous mutation displayed only RP symptoms until now, while three patients with compound
heterozygous mutations in the family of study showed both RP and hearing impairment.
Conclusions: This study identified two novel mutations: p.G1734R and IVS32+1G>A of USH2A in a four-generation
Chinese RP family. In this study, the heterozygous mutation and the homozygous mutation in USH2A may cause Usher
syndrome Type II or RP, respectively. These two mutations expand the mutant spectrum of USH2A.
Retinitis
characterized by constriction of the visual fields, night
blindness, changes of fundi including 'bone corpuscle' lumps
of pigment, and loss of central vision. RP is inherited most
frequently (84%) as an autosomal recessive trait, followed by
autosomal dominant (10%) and has X-linked recessive (6%)
patterns in the white USA population. The worldwide
prevalence of RP is about 1 in 4,000. RP is the most common
hereditary retinal dystrophy causing irreversible blindness.
Several loci or genes responsible for RP have been reported.
Retinitis pigmentosa (RP) is a group of hereditary retinal
diseases, which are characterized by the degeneration of rod-
cone photoreceptors with resultant night blindness and visual
field loss. About 20%–30% RP patients have extra-ocular
diseases, which are included in more than 30 different
syndromes [1]. The most common form of syndromic RP is
pigmentosa (RP; OMIM 268000) is
Correspondence to: Dr. Mugen Liu, Huazhong University of Science
and Technology Human Genome Research Center, Wuhan, Hubei,
430074, P.R. China; Phone:
86-27-87794549; email: lium@mail.hust.edu.cn
86-27-87794549; FAX:
Usher syndrome, which usually includes both recessive
retinitis pigmentosa and hearing loss [2].
On the basis of the severity or progression of the hearing
loss, as well as the presence or absence of vestibular
dysfunction, Usher syndrome is divided into three clinical
subcategories: USH1, USH2, and USH3. Among them,
Usher syndrome Type II (USH2) accounts for over 50% of
Usher syndrome cases. It is regarded as the most common
type, which is characterized by moderate to severe congenital
hearing loss, intact vestibular function and postpuberal onset
of retinitis pigmentosa [3-5]. Because of mild hearing
impairment, USH2 is often misdiagnosed as nonsyndromic
RP. To date, three loci have been identified for USH2:
USH2A,USH2C (GPR98), USH2D (WHRN), and the
mutations in the USH2A gene are responsible for 74%–90%
of USH2 cases [6-10].
The USH2A gene is located on human chromosome 1q41,
and two USH2A transcripts were identified: the shorter one
consists of 21 exons and the longer one consists of 72 exons
[7,11]. In 2004,van Wijk et al. [11] identified additional 51
exons of USH2A. The longer isoform of USH2A encodes
usherin, a transmembrane protein of 5,202 amino acids and
Molecular Vision 2010; 16:454-461 <http://www.molvis.org/molvis/v16/a51>
Received 10 November 2009 | Accepted 9 March 2010 | Published 17 March 2010
© 2010 Molecular Vision
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contains 68 additional fibronectin repeats [11]. In the inner
ears, usherin is transiently expressed in cochlea as an ankle-
link complex that connects cochlear hair cells [12,13]. In the
retina, usherin is predicted to be a fibrous link that connects
the photoreceptor inner segment plasma membrane to the
ciliary surface [14,15].
To date, over 80 different mutations in exons 2–21 and
40 mutations in exons 22–72 of the USH2A gene have been
reported to be associated with Usher syndrome type II, most
of which are missense mutations or truncating mutations [7,
9,11,16-24]. One of them, c.2299delG, may be the most
common mutation among patients because it has been found
in 16%–77% of USH2A families [7,18,19,21,23-26]. USH2A
mutations have been identified in the patients with a typical
USH2 phenotype or nonsyndromic RP [26-29].
In this study, we investigated a four-generation Chinese
family with retinitis pigmentosa. After linkage analysis, we
mapped the disease-causing gene in the USH2A region. Using
direct DNA sequence analysis of exons 2–72 and exon-intron
boundaries of USH2A, we found two novel compound
heterozygous mutations: one missense mutation and one
splicing site mutation. These two mutations co-segregated
with the affected members in the family and were not present
in the 100 normal controls. Meanwhile, all members of this
family who were initially misdiagnosed as having
nonsyndromic RP received an audiometric vestibular test.
Results showed that some patients in the family display
hearing impairment.
METHODS
Study subjects and isolation of human genomic DNA: The
participants of this study were diagnosed and enrolled at
Union Hospital. Informed written consent was obtained from
the study subjects. Whole peripheral blood was collected from
all participants, and genomic DNA was isolated using the
DNA isolation kit for Mammalian Blood (Tiangen Biotech
Co., Ltd., Beijing, China). All patients in the family underwent
careful ophthalmologic examination, including visual acuity,
slit-lamp, fundus ophthalmoscopy, visual field test, and
electroretinogram (ERG). Initial RP diagnosis was based on
the description of night blindness, typical RP fundus
appearance, non-detectable electroretinogram and loss of
peripheral visual fields. When the disease gene was mapped
to chromosome 1q41 where USH2A harbors, patients were
given audiometric and vestibular tests. Audiometric tests
Figure 1. Pedigree structure of the family with USH2/RP. Affected males and females are shown with filled squares and circles, respectively.
Normal individuals are shown with empty symbols. The deceased individual is shown with slash. The proband (III:5) is indicated by an arrow.
Results from genotyping and haplotype analysis of two markers on chromosome 1 are displayed below each symbol. The disease haplotype
is shown with a black or a gray box and the normal haplotypes are shown with open boxes. The USH2A gene is located between D1S213 and
D1S425. The homozygous individual (IV:1), whose haplotype is different from other patients, is indicated by an asterisk. Four patients (III:
1, III:3, III:5) are affected with USH2, and patient (IV:1) displays nonsyndromic RP symptom.
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included otoscopy and standard pure-tone audiometry.
Vestibular function was evaluated by caloric test, rotatory
chair and electronystagmography. The final clinical diagnosis
of USH2 was verified based on typical RP symptoms
companied with sensorineural hearing impairment and normal
vestibular function.
Genotyping: A panel of candidate genetic loci for retinitis
pigmentosa, including 38 known RP genes, was selected for
preliminary linkage and
microsatellite markers that flank the 38 known RP genes were
selected from the ABI Prism LMS v2.5-MD 10 marker set
(Applied Biosystems, Foster City, CA). These markers were
genotyped by using an ABI 3100 genetic analyzer (Applied
Biosystems). Genotypes were analyzed through GeneMapper
2.5 software (Applied Biosystems).
Mutation screening: Mutation screening was performed by
direct DNA sequence analysis. The complete coding region
(exons 2–72) and exon-intron boundaries of USH2A were
amplified by polymerase chain reaction (PCR). The methods
of primers design, PCR amplification, and DNA sequence
analysis were performed as previously described [22,23].
RFLP analysis: Exon 26 of wild type USH2A allele contains
an NlaIII restriction enzyme site, which the p.G1734R
mutation disrupts. We used RFLP analysis to confirm this
mutation and test whether the mutation co-segregates with the
disease in the family. The 228 bp fragments of exon 26 in
USH2A gene were amplified from all available family
members and the 100 normal controls. The PCR products
were digested with 2 units of NlaIII restriction enzyme (New
England Biolabs, Inc., Beijing, China) at 37 °C for 5 h. The
digested products were separated by a 2.5% agarose gel and
visualized by ultraviolet light.
To test if the other novel mutation IVS32+1G>A is the
disease-causing mutation, direct DNA sequence analysis was
performed for all the family member and 100 normal controls.
haplotype analysis. The
RESULTS
Clinical examinations: Five individuals of the primary study
family were having RP or USH2 (Figure 1). The proband (III:
5) was a 47-year-old female who experienced night blindness
at the age of 23 years as her initial symptoms of RP, which
was followed by progressive loss of visual acuity. Her best
corrected visual acuity was decreased to finger count level.
Fundus examination showed attenuation of the retinal vessels,
waxy pallor of the optic nerve head, and bone speckle-like
pigmentation clumps in her peripheral retina (Figure 2). The
ERG wave amplitudes were unrecordable under scotopic and
photopic conditions in both eyes. Similar ophthalmologic
examination results were detected in the other two affected
siblings (III:1, III:3) of the proband, but the symptom of RP
in IV:1 and IV:2 were mild. Audiometric tests of the proband
and her siblings indicated mild sensorineural hearing
impairment and normal vestibular function while patient IV:
1 was normal (Figure 3). His younger brother (IV:2) did not
undergo the audiometric test. Detail clinical examination
results are shown in Table 1.
Linkage analysis: Genetic linkage analysis excluded all
previously identified RP genes except for USH2A, which was
located between D1S213 and D1S425. Further haplotype
analysis suggested that USH2A may be the disease-causing
gene of this family (Figure 1).
Mutation analysis: Direct DNA sequence analysis was
performed to identify the USH2A gene mutation that caused
RP in the family. The complete coding region (exons 2–72)
and the exon-intron boundaries of the USH2A gene were
amplified with the proband DNA and then sequenced. Two
novel mutations were found (Figure 4A-D): One was a
p.G1734R mutation in exon 26, which resulted in a
substitution of glycine for arginine at codon 1734
(p.G1734R). RFLP analysis at this site showed that this
Figure 2. Fundus appearance of the proband. The typical RP symptoms including attenuation of the retinal vessels, waxy pallor of the optic
nerve head, and bone speckle-like pigmentation clumps in the peripheral retina were showed.
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mutation co-segregated with the disease in the family (Figure
4E) and was not present in the 100 normal control individuals.
The other mutation was a change from G→A at a 5′ splicing
site in intron 32(IVS32+1G>A). Direct DNA sequence
analysis of each member of the family showed that the splice
site mutation was present in all patients except patient IV:1
and was absent in the 100 normal control individuals. All
affected members had the compound heterozygote
(p.G1734R and IVS32+1G>A) mutation, except for patient
IV:1, who is homozygote for the p.G1734R mutation (Figure
4F).
In addition to the two novel pathogenic mutations
detected in this study, seven nonpathogenic sequence variants
were identified. One of them, c.1935A>T, was novel. The
synonymous variant did not cause an amino acid substitution.
DISCUSSION
In the present study, two novel mutations, p.G1734R and
IVS32+1G>A, were identified in a Chinese RP/USH2 family.
All the affected individuals were compound heterozygote for
p.G1734R and IVS32+1G>A except for patient IV:1, who
was homozygous for the p.G1734R mutation because his
parents were consanguineous. These results suggest that the
compound heterozygous/homozygous mutations in USH2A
may be the disease-causing mutations in this family.
USH2A has been described as the most common locus for
syndromic RP [10]. Its mutations have been identified in
Colombian, Spanish, Israeli, Canadian, Dutch, and British
families with Usher syndrome [21,23,25,30-32]. However,
most of the mutations have been restricted to exons 1–21. To
date, only one study reported mutation analysis of the
complete coding sequence of USH2A in the Chinese
population, and five mutations (p.R34fs, p.S2828fs,
Exon43DEL, p.W3150X, p.T3936P) were identified, four of
which were specific to the longer isoform of USH2A [22]. Our
study is the second report on this subject in the Chinese
population. Both of the novel mutations are located in exons
22–72. Both of these studies suggest that the function of the
Figure 3. Audiometry results for III:5
and IV:1. A and B show the result for
IV:1 who is normal, C and D show mild
hearing loss for III:5.
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C-terminal of usherin is important in the retina, and mutations
in this region may be responsible for most USH2 cases.
It should be noted that the degree of hearing loss was
slightly progressive. Many members in the family-study were
unaware that they had partial hearing loss. Furthermore, they
were initially misdiagnosed as nonsyndromic retinitis
pigmentosa.
We conducted linkage analysis to locate the disease gene
and then sequenced the USH2A gene. Our results demonstrate
that the combination of two mutations affecting the long
usherin isoform have a relatively mild effect on audition.
Interestingly, three patients
heterozygotes for the two mutations showed Usher syndrome
phenotypes, while patient IV:1 was homozygous for the
p.G1734R mutation. Upon clinical examination and pure tone
audiometry, IV:1 was found to have hearing acuity within the
who were compound
normal range, which may suggest that homozygosity for
p.G1734R does not cause hearing loss (Figure 3). Our result
is consistent with the findings of Seyedahmadi et al. [24],
Rivolta et al. [29], and Kaiserman et al. [33]. Their studies
indicated that mutations in USH2A may cause retinitis
pigmentosa without hearing loss [24,29,33]. However,
because IV:1 is 25 years of age, it is possible that he will suffer
from hearing impairment in his late years.
The Gly1734 residue is located in the laminin G-like
domain (LNS) of USH2A and is highly conserved during
evolution. When compared with wild type usherin, the
missense mutation of p.G1734R results in the conversion of
a non-polar hydrophobic amino acid (glycine) to a positively
charged amino acid (arginine). This change might affect the
structure and/or function of USH2A and influence the laminin
G-like domain of the protein. The LNS domains are usually
TABLE 1. CLINICAL FEATURES OF THE PATIENTS FROM THE CHINESE FAMILY WITH USHER SYNDROME TYPE II.
Patient
number
III:1
III:3
III:5
IV:1
IV:2
Age
(year)
55
53
47
25
19
Best corrected visual
activity (R/L)
FC/FC
FC/HM
FC/FC
0.15/0.1
0.6/0.6
Fundus
appearance
RP
RP
RP
RP
RP
Hearing
impairment
Mild
Mild
Mild
Normal
N/A
ERG
extinct
extinct
extinct
N/A
N/A
Vestibular
function
normal
normal
normal
normal
normal
Abbreviations: R represents right eye; L represents left eye; FC represents finger counting; HM represents hand motion; N/A
represents data not available.
Figure 4. Identification of two novel USH2A mutations. DNA sequence analysis for patient III1 showed the presence of compound
heterozygous p.G1734R (c.5200G>C) and c.IVS32+1A>T mutations. A and B show the sequences of a normal and affected family member
with mutation p.G1734R (c.5200G>C) allele, respectively. C and D show the sequences of a normal and affected family member with mutation
c.IVS32+1A>T allele, respectively. E: Restriction fragment length analysis on the p.G1734R (c.5200G>C) mutation in this study. All the
affected individuals (III:1, III:3, III:5, IV:1, IV:2) and the carriers (III:2, IV:3) have three bands (228 bp, 164 bp, and 64 bp), while the
unaffected individuals only have two bands (164 bp and 64 bp).The patient IV1 who is homozygous for the p.G1734R (c.5200G>C) mutation
displayed only one 228 bp band. F: Alignment of the amino acid sequences of laminin G-like domain in the long usherin isoform from different
species. Gly1734 (G1734) is conserved during evolution. The box indicates this mutated residue in USH2A.
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