Molecular Vision 2011; 17:1537-1552
Received 25 January 2011 | Accepted 3 June 2011 | Published 9 June 2011
Wenjun Xu, Hanjun Dai, Tingting Lu, Xiaohui Zhang, Bing Dong, Yang Li
Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing, China
Correspondence to: Yang Li, Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Hougou Lane 17, Chong Nei Street, Beijing, 100730, China; Phone: 8610-58265915; FAX: 8610-65288561 or 65130796; email: email@example.com
Purpose: To describe the clinical and genetic findings in one Chinese family with autosomal recessive retinitis pigmentosa (arRP) and in three unrelated Chinese families with Usher syndrome type II (USH2).
Methods: One family (FR1) with arRP and three unrelated families (F6, F7, and F8) with Usher syndrome (USH), including eight affected members and seven unaffected family individuals were examined clinically. The study included 100 normal Chinese individuals as normal controls. After obtaining informed consent, peripheral blood samples from all participants were collected and genomic DNA was extracted. Genotyping and haplotyping analyses were performed on the known genetic loci for arRP with a panel of polymorphic markers in family FR1. In all four families, the coding region (exons 2–72), including the intron-exon boundary of the USH2A (Usher syndrome type −2A protein) gene, was screened by PCR and direct DNA sequencing. Whenever substitutions were identified in a patient, a restriction fragment length polymorphism (RFLP) analysis, single strand conformation polymorphism (SSCP) analysis, or high resolution melt curve analysis (HRM) was performed on all available family members and on the 100 normal controls.
Results: The affected individuals presented with typical fundus features of retinitis pigmentosa (RP), including narrowing of the vessels, bone-spicule pigmentation, and waxy optic discs. The electroretinogram (ERG) wave amplitudes of the available probands were undetectable. Audiometric tests in the affected individuals in family FR1 were normal, while indicating moderate to severe sensorineural hearing impairment in the affected individuals in families F6, F7, and F8. Vestibular function was normal in all patients from all four families. The disease-causing gene in family FR1 was mapped to the USH2A locus on chromosome 1q41. Seven novel mutations (two missenses, one 7-bp deletion, two small deletions, and two nonsenses) were detected in the four families after sequencing analysis of USH2A.
Conclusions: The results further support that mutations of USH2A are also responsible for non-syndromic RP. The mutation spectrum among Chinese patients might differ from that among European Caucasians.
Retinitis pigmentosa (RP) is a heterogeneous group of retinal dystrophies, characterized by progressive degeneration of the photoreceptors. Clinical features include progressive night blindness, constriction and gradual loss of the peripheral visual field, and eventual loss of visual acuity. With an incidence of 1 in 3,500, RP can be inherited as an autosomal recessive (arRP), an autosomal dominant (adRP), or an X-linked recessive (xlRP) pattern [1,2].
RP can be classified as syndromic and nonsyndromic RP, based on whether or not extra-ocular diseases exist. Nonsyndromic arRP is caused by the mutations of 32 identified genes [1,2]. Syndromic RP includes more than 30 different syndromes [1,2]. The most common syndrome is Usher syndrome (USH), which is also an autosomal recessive disorder characterized by sensorineural hearing loss, variable vestibular dysfunction, and visual impairment due to retinitis pigmentosa [2,3]. Clinically, USH is subdivided into three types: USH type I (USH1), USH type II (USH2), and USH type III (USH3). USH1 is the most severe form of this disease and is characterized by congenital profound hearing loss, prepuberal onset of RP, and vestibular dysfunction. Patients with USH2 experience congenital moderate to severe hearing impairment, and postpuberal onset of RP with intact vestibular function. Patients with USH3 show progressive postlingual hearing loss, later onset of RP, and variable vestibular dysfunction. Of the three clinical types, USH2, which accounts for more than half of all patients with USH, is the most common form of USH [2-4]. To date, reports indicate that three genes (USH2A [Usher syndrome type −2A protein], GPR98 [G-protein coupled receptor 98], and DFNB31 [CASK-interacting protein CIP98 isoform 1]) are responsible for USH2, and most USH2 patients have mutations in USH2A [3-9].
USH2A, located on chromosome 1q41, has two alternatively spliced isoforms: a short USH2A isoform a, consisting of 21 exons, and a long USH2A isoform b, consisting of 51 additional exons at the 3′ end of USH2A [5,9]. The protein usherin, encoded by USH2A isoform b, is a transmembrane protein, which has 5,202 amino acids . The usherin is transiently expressed in the stereocilia of cochlear hair cells, suggesting an important role in their maturation [4,9-11]. In mammalian photoreceptors, the usherin is expressed specifically in the connecting cilia, which links the inner and outer retinal segments; this would appear to indicate that it is crucial for the long-term maintenance of photoreceptors [9-11].
This study investigated a Chinese family with non-syndromic arRP. After haplotyping analysis, the disease-causing gene was mapped to the USH2A region. Mutations screening of the USH2A gene, corresponding to the USH2A isoform b, was then performed in this nonsyndromic RP family and in three USH2 families. Seven novel mutations were identified.
This study adhered to the tenets of the Declaration of Helsinki for research involving human subjects. The Beijing Tongren Hospital Joint Committee on Clinical Investigation approved the study. One Chinese family with nonsyndromic RP and three unrelated Chinese families with USH were referred to Beijing Tongren Hospital. After informed consent was obtained, each participant underwent careful ophthalmologic examinations, including best-corrected visual acuity testing using E decimal charts, slit-lamp biomicroscopy, fundus examination with dilated pupils, visual field testing, and electroretinogram (ERG) examination. Three probands from the three families with USH underwent audiometric testing, including otoscopy and standard pure-tone audiometry, and vestibular tests. The patients with nonsyndromic arRP were given audiometric tests after the disease gene was mapped to chromosome 1q41, where the USH2A gene is located. Clinical diagnosis of USH2 was based on the clinical history, typical RP fundus appearance, sensorineural hearing impairment, and intact vestibular function. Peripheral blood was obtained by venipuncture, and genomic DNA was extracted according to standard phenol protocols.
Genotyping was performed with 50 microsatellite markers from autosomes for the known arRP loci in family FR1 (Appendix 1). Then, genotyping and haplotyping analysis was performed with another six microsatellite markers - D1S237, D1S419, D1S556, D1S229, D1S227, and D1S2860 - around the USH2A gene. The fine mapping primer sequences were obtained from the Human Genome Database (GDB). Pedigree and haplotype maps were constructed using Cyrillic V. 2.0 software.
Mutation screening was performed in all four families using direct DNA sequence analysis. The coding region (exons 2–72) and the exon-intron boundaries of USH2A were amplified by PCR in the probands of the four families. The pairs of primers for exons 2–72 were used according to previously published (Table 1) articles [5,9,21]. For direct sequencing, amplicons were purified (Shenneng Bocai PCR purification kit; Shenneng, Shanghai, China). An automatic fluorescence DNA sequencer (ABI, Prism 373A; Perkin Elmer, Foster City, CA), used according to the manufacturer’s instructions, sequenced the purified PCR products in both the forward and reverse directions. Nucleotide sequences were compared with the published cDNA sequence of the USH2A gene (GenBankNM_206933.2). For USH2A, cDNA numbering +1 corresponds to A in the ATG translation initiation codon in RefSeq (AY481573.1).
Variations (c.2802T>G, c.8232G>C, c.3788G>A, and c.14403C>G) found in the sequencing were confirmed with the restriction endonucleases Hinc II (TaKaRa, Dalian, China), HpyCH4V, BsaI, and SpeI (New England Biolabs, Ipswich, MA), respectively, which were used in all available family members and in the100 normal controls.
To validate the variations (c. 1876C>T and c.7123delG) found in the sequencing, a single strand conformation polymorphism (SSCP) analysis was performed in all available family members and in the 100 normal controls. As the PCR fragments used in SSCP analysis were between 150 and 300 bp, two pairs of specific primers were designed for detecting mutations in exon 11 and exon 38 (Table 2).
To confirm the variation (c.6249delT) found in the sequencing, a high-resolution melt curve analysis (HRM) was performed in the available family members and in the 100 normal controls. Primer sequences were designed to obtain the best HRM performance, avoiding hairpin and primer–dimer formation as much as possible, and keeping the amplicon length under 250 base pairs. One pair of specific primers was designed for detecting a mutation in exon 32 (Table 2). The 10 μl reaction mixture consisted of 5 μl SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA), 1 μl genomic DNA (10–150 ng/μl), 0.5 μl forward primer (10 pmol/μl), 0.5 μl reverse primer (10 pmol/μl), and 3 μl double distilled water. PCR cycling and an HRM analysis were performed on the Rotor-Gene 6000TM (Corbett Research, Mortlake, NSW, Australia) .
Garnier-Osguthorpe-Robson (GOR) software was used to predict the effect of the mutation on the secondary structure of USH2A . This method infers the secondary structure of a sequence by calculating the probability for each of the four structure classes (helix, sheet, turn, and loop), based on the central residue and its neighbors from the calculated matrices .
This study identified one Chinese family, consisting of four patients and one unaffected relative, diagnosed with non-syndromic RP, and three unrelated Chinese families, including four patients and six unaffected relatives diagnosed with USH2. The inheritance pattern in the families was autosomal recessive (Figure 1). All the patients had experienced night blindness and vision acuity impairment. The patients with USH2 had hearing impairment in early childhood. Ophthalmoscopic examination demonstrated attenuation of the retinal vessels, bone-spicule pigmentation in the fundus, and waxy pallor of the optic nerve head (Figure 2). The wave amplitudes of the ERG of the probands were indistinguishable from the baseline. Audiometric tests indicated moderate to severe sensorineural hearing impairment in the patients with USH2; in contrast, the results from the patients with non-syndromic arRP were normal. Vestibular functions of all the patients were normal. The detailed clinical information for each family’s proband is summarized in Table 3.
Family FR1 was genotyped with 50 polymorphic markers around the known arRP loci. The mapping results excluded the other known arRP loci with the exception of the USH2A. Further genotyping and haplotyping analysis for the six markers (D1S237, D1S419, D1S556, D1S229, D1S227, and D1S2860) suggested that the USH2A gene might be the disease-causing gene in this family (Figure 1).
Sequencing of the USH2A gene revealed 17 sequence variants in this study, eight of which were pathogenic mutations (Table 4). All eight pathogenic mutations were heterozygous; seven of them were first detected in the current study (Figure 3 and Table 4). Using RFLP, SSCP, or HRM analysis, the eight mutations co-segregated with the RP/USH2 phenotype, respectively (Figure 4, Figure 5, Figure 6). Analyses did not detect the other seven mutations in the 100 normal controls, with the exception of p.C934W, which was identified in its heterozygous state in two individuals among the 100 normal controls (Table 4).
Four different combinations of heterozygous mutations were detected in the four families. In family FR1 (non-syndromic arRP), two missense mutations, c.2802T>G (p.C934W) and c.8232G>C (p.W2744C), were detected in different alleles of patient 077006 (Figure 1, Figure 3, Figure 4). Using the GOR method, the results for secondary structure prediction suggested that p.C934W replaced two β sheets “E” with two coils “C” at amino acids 935 and 940, respectively. Mutation p.W2744C substituted a β sheet “E” and two turn sheets “T” for three coils “C” at amino acid 2745, 2747, and 2748, respectively (Figure 7). For the three USH2 families (F6, F7, and F8), one allele carried nonsense mutations, c.1876C>T (p.R626X), c.3788G>A (p.W1263X), and c.14403C>G (p. Y4801X), respectively, while the other allele harbored deletion mutations c.6249delT (p. I2084fs), c.9492_9498del TGATGAT (p. D3165fs), and c.7123delG (p. G2375fs), respectively (Figure 1, Figure 3, Figure 4, Figure 5, Figure 6).
In addition to the eight pathogenic mutations detected in this study, nine nonpathogenic sequence variants were also identified. Table 5 summarizes these variants based on their nature and frequency.
This study detected eight different mutations of the USH2A gene isoform b in one non-syndromic arRP family and in three USH2 families. Scandinavian, French, European, and Canadian studies [12,14,16,24-26] previously reported the nonsense mutation p.R626X. The remaining seven mutations were first identified in this study.
Rivolta et al. first reported that about 4.5% of 225 patients from North America with non-syndromic recessive RP carried the missense mutation p.C759F . Then, Bernal et al. found that there was a similar detecting frequency (4.6%) for p.C759F in Spanish patients .Two novel missense mutations, p.C934W and p.W2744C, were found in family FR1. Although p.C934W was identified (in a heterozygous state) in two individuals among the 100 normal controls, both mutations have been classified as deleterious-effect missense mutations with several lines of evidence. Both mutations co-segregated with the phenotype of family FR1 and both residues (C934 and W2744), located in the 8th Lam EGF domains and in the 14th FN3 repeat of the usherin, respectively, were highly conserved in different species (Figure 8). The results of GOR suggested that p.C934W and p.W2744C lead to secondary structure changes around residues 934 and 2744, which might interfere with the correct folding of the usherin (Figure 7). As the results of audiometric tests for the patients from family FR1 were normal, the two compound missense mutations might be responsible for RP without hearing loss.
Three different compound heterozygous mutations were identified in three families (F6, F7, and F8) with USH2 and all six mutations directly or indirectly resulted in premature termination of the USH2A translation. This is consistent with Dreyer et al.  previous observation that patients carrying compound heterozygous mutations (either two truncating or one truncating combined with one missense) in exon 22–72 presented the Usher type II phenotype. In contrast to the patients from the three USH2 families, the patients in FR1 carried two missense mutations. A recent study in a cohort of 272 Spanish patients with non-syndromic RP resulted in the identification of two mutant alleles of the USH2A gene in nine patients, with seven of them carrying either homozygous missense mutations or two heterozygous missense mutations . In a large Chinese family, four patients carrying one truncating combined with one missense mutation (p.G1734R) exhibited RP with hearing loss, while the only person harboring the homozygous misense mutation (p.G1734R) presented RP without hearing loss . However, this phenomenon was not observed in one Israeli family with three non-syndromic RP patients carrying one missense mutation and one truncating mutation .
As in our previous study , with the exception of one mutation (p.R626X), the other mutations identified in the current study were novel and were spread relatively evenly along the USH2A gene (Figure 9). These results indicate that the mutation spectrum for the USH2A gene among Chinese or Asian patients differs from the mutation spectrum among European Caucasians. The common mutations, p. E767fs for USH2 and p.C759F for arRP in Caucasians, are not detected in Chinese and Japanese patients [12-14,16,18,19,21,27-29].
In conclusion, our results further support previous indications that the mutations of the USH2A gene are also responsible for non-syndromic RP in Chinese patients. The mutation spectrum among Chinese patients appears to differ from that among European Caucasians.
Appendix 1. 50 markers used in the known arRP genotyping.
We thank the patients and their families for participation in this study. The study was supported by the Beijing National Science Foundation (No. 07G0069 and No. 07G0069). The funding organization had no role in the design or conduct of this research.