(Investigative Ophthalmology and Visual Science. 2007;48:530-533.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI: 10.1167/iovs.06-0669
The 208delG Mutation in FSCN2 Does Not Associate with Retinal Degeneration in Chinese Individuals
Qingjiong Zhang,
Shiqiang Li,
Xueshan Xiao,
Xiaoyun Jia, and
Xiangming Guo
From the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou China.
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Abstract
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PURPOSE. The 208delG (c.72delG, p.Thr25GlnfsX120) mutation in the FSCN2 gene was reported to cause autosomal dominant retinitis pigmentosa (ADRP) and autosomal dominant macular degeneration (ADMD). The purpose of this study was to detect the 208delG mutation in Chinese individuals, with or without hereditary retinal degeneration.
METHODS. DNA fragments encompassing the 208delG mutation were amplified by polymerase chain reaction (PCR). The amplicons were analyzed by sequencing or/and heteroduplex- single-strand conformational polymorphism (SSCP) analysis. An ophthalmic evaluation was conducted in those individuals with the 208delG mutation.
RESULTS. The 208delG mutation was detected in 8 of 242 unrelated probands: 175 with retinitis pigmentosa (RP), 20 with Leber congenital amaurosis (LCA), and 47 with cone–rod dystrophy (CORD). Of the eight, the retinal diseases were RP in six probands, LCA in one proband, and CORD in one proband. The disease was transmitted as an autosomal dominant (one family), autosomal recessive (two families), or sporadic (five families) trait. The mutation did not cosegregate with retinal degeneration in three families, whereas five normal family members also had the mutation. In addition, this mutation was also detected in 13 of 521 unrelated control subjects.
CONCLUSIONS. The 208delG mutation in FSCN2 is not associated with hereditary retinal degeneration in the Chinese individuals examined, which contradicts the original report about mutation in FSCN2 as a cause of ADRP and ADMD. This finding reminds us that great care is needed in making mutation–disease associations.
Hereditary retinal degeneration is a group of severe disorders affecting vision, which can be transmitted as an autosomal dominant, autosomal recessive, or X-linked trait. Several loci or genes responsible for retinal degeneration have been reported (RetNet, http://www.sph.uth.tmc.edu/Retnet/ provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX).
FSCN2 (OMIM 607643; Online Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov/Omim/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), mapped to 17q25, encodes a photoreceptor-specific fascin that belongs to the family of actin-binding proteins. This protein most likely assembles the actin microfilaments associated with photoreceptor discs.1 A novel locus (RP17) for ADRP has been mapped to 17q25.2 FSCN2 is therefore considered to be a good candidate for RP17 but has been excluded as a causative gene. Subsequently, genomic sequences of FSCN2 in 420 unrelated Japanese patients with retinitis pigmentosa were screened. A single mutation, previously designated as 208delG, now c.72delG, p.Thr25GlnfsX120 according to the current recommendation of the Human Genome Variation Society (HGVS; http://www.hgvs.org/mutnomen/ St.Vincent’s Hospital Melbourne, Fitzroy VIC, Australia), was identified in four unrelated Japanese families with ADRP.3 This mutation was further detected in 2 of 54 unrelated Japanese families with autosomal dominant cone–rod dystrophy or macular degeneration.4
It would be interesting to know whether the 208delG in FSCN2 represents a mutation hot spot or a mutation founder in certain populations. In light of the close ethnic relation of Chinese and Japanese, evaluation of the 208delG mutation in Chinese patients with retinitis pigmentosa (RP) or other related diseases would be logical. We analyzed the 208delG mutation in 242 unrelated patients with retinal degeneration (including 175 with RP, 47 with CORD [cone–rod dystrophy], and 20 with LCA [Leber congenital amaurosis]) and 521 unrelated control subjects. Surprisingly, the 208delG mutation was detected in 8 of the 242 patients with retinal degeneration, but it did not cosegregate with retinal degeneration in three Chinese families. In addition, this mutation was detected in 13 of 521 unrelated individuals, who do not have any type of hereditary retinal degeneration.
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Methods
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Patient Samples and Pedigrees
Patients with RP, CORD, or LCA were ascertained from our Pediatric and Genetic Clinic, Zhongshan Ophthalmic Center as part of our 863-project to identify genes responsible for genetic eye diseases. This study was approved by IRB of the Zhongshan Ophthalmic Center and adhered to the tenets of the Declaration of Helsinki and the Guidance of Sample Collection of Human Genetic Diseases (863-Plan) by the Ministry of Public Health of China. Informed consent was obtained from the participating individuals or their guardians before the study. Medical and ophthalmic histories were obtained, and ophthalmic examination (by QZ and XG) included visual acuity, slit lamp and funduscopic examinations. Electroretinogram (ERG) responses were recorded in selected probands consistent with ISCEV standards.5
Detection of the 208delG Mutation in FSCN2
DNA fragments encompassing the 208delG mutation in FSCN2 (human genome build 35.1, NC_000017 region between nucleotides 7110017 and 77114632 for genomic DNA, NM_012418 for mRNA, NP_036550 for protein; http://www.ncbi.nlm.nih.gov/ provided by the National Center for Biotechnology Information [NCBI], Bethesda, MD) were amplified by polymerase chain reaction with two pairs of primers as follows: (1) for DNA sequencing: FSCN2-1AF (forward) 5'-CCCCGCCCGCCCTCTGCT-3', FSCN2-R (reverse) 5'-CACGGCCCGGCTGCTCTGC-3'; (2) for heteroduplex-SSCP (single-strand conformational polymorphism) analysis: FSCN2-HAF 5'-CCCGGCCAGCCTGAAGATGC-3', FSCN2-HAR 5'-CACAGCCGTGCCTTGTCCT-3'. The DNA sequences in 96 patients with RP were identified (BigDye Terminator cycle sequencing kit ver. 3.1; Applied Biosystems [ABI], Foster City, CA, and the 3100 Genetic Analyzer; ABI). The DNA sequences in additional patients with RP, CORD, or LCA as well as control subjects were screened by heteroduplex-SSCP analysis, as we have described elsewhere.6 Genomic DNA from each individual suspected with the 208delG mutation on heteroduplex-SSCP analysis was further examined by direct sequencing. For patients with the 208delG mutation, the FSCN2 gene in available family members was analyzed.
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Results
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In initial sequenceanalysis of 96 probands with RP, heterozygous 208delG mutation in FSCN2 was detected in 3 probands (Fig. 1) . Heteroduplex-SSCP analysis of an additional 146 probands with RP, CORD, or LCA disclosed another five probands with the same band pattern as those with the 208delG mutation. Sequence analysis of these five probands revealed a heterozygous 208delG mutation.
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FIGURE 1. Sequence chromatograms around the 208delG mutation. Both forward and reverse sequences were shown. Wild: normal sequence. Family A IV:4: individual IV:4 with LCA from family A had the 208delG mutation. C1: a normal control subject also had the 208delG mutation. Arrow: the site where a normal sequence overlaps with the shifted mutant sequence due to the 208delG mutation.
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In total, the heterozygous 208delG (c.72delG, p.Thr25GlnfsX120) mutation was detected in 8 of 242 unrelated probands with RP (n = 175), LCA (n = 20), or CORD (n = 47). Of the eight probands, the retinal diseases were RP in six, LCA in one, and CORD in one (Table 1) . In the eight probands with the mutation, the disease was transmitted as an autosomal dominant (one family), autosomal recessive (two families), or sporadic (five families) trait (Fig. 2) .
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FIGURE 2. The 208delG mutation was identified in five families with retinal degeneration. Arrow: proband in each family. Filled symbols: individuals affected with retinal degeneration. ++, normal sequence around the 208delG region. + –, presence of the heterozygous 208delG mutation.
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The mutation did not cosegregate with the disease in three families in which five unaffected family members also had the mutation (Fig. 2) . In family-A with LCA, the 208delG mutation was detected in only one (Fig. 2 , IV:4) of the six affected individuals. This mutation was also present in three unaffected individuals of family A: II:5, II:6, and III:9. In family-B, the 208delG mutation was identified in an unaffected mother and her son who had CORD. In family C, the 208delG mutation was found in a patient with RP and in his elder sister, without any sign of retinal degeneration. Ophthalmoscopic observation revealed a normal fundus in all five unaffected family members in families A, B, and C (Supplementary Fig. S1, online at http://www.iovs.org/cgi/content/full/48/2/530/DC1). Electroretinograms demonstrated normal retinal rod–cone function in three unaffected family members with the 208delG mutation (Fig. 3) . As these five unaffected family members were older than the corresponding proband in each family and were more than 40 years old except one, who was 20 years old, a normal ocular phenotype was unlikely due to the late expression of the retinal diseases (Table 2) .
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FIGURE 3. Electroretinogram recording of four individuals with the 208delG mutation. Individual family A-IV4, affected with LCA, had no appreciable rod and cone responses. The other three were unaffected family members from families A and C, who had normal rod and cone responses.
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In addition, the 208delG mutation was detected in 13 of 521 unrelated control subjects (Tables 1 2 ; Fig. 4 ), including 9 of 329 normal control subjects, and 4 of 192 individuals with Leber hereditary optic neuropathy (LHON) who harbored one of the three common mtDNA primary mutations for LHON.7 None of the 13 control individuals had night blindness. Signs for retinal degeneration, such as attenuation of retinal vessels and pigment deposits on fundus, were not present in these individuals. Clinical information on the 26 individuals with the 208delG mutation, including 8 patients with retinal degeneration, 5 unaffected family members, and 13 unrelated control subjects is shown in Table 2 .
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FIGURE 4. Results of heteroduplex SSCP analysis. The same band patterns were observed in samples with the heterozygous 208delG mutation, including 8 samples from probands with retinal degeneration (lanes 4–11, families A–H) as well as 13 from unrelated control subjects (lanes 15–27). These samples had additional bands in the SSCP (top) and heteroduplex (bottom) regions, compared with samples without the mutation (lanes 1–3 and 12–14 from probands with RP, and lanes 28–30 from unrelated control subjects).
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Discussion
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In this study, the 208delG mutation in FSCN2 was detected in 8 of 242 patients with retinal degeneration. The mutation in three families is apparently not associated with the disease, in that five normal family members also had the mutation. The mutation was also detected in 13 of 521 control individuals.
Previously, the 208delG mutation in FSCN2 was detected in only six Japanese families: four with ADRP and two with ADMD.3 4 Subsequently, a mouse model involving targeted disruption of the FSCN2 gene was constructed by replacing exon 1 of FSCN2 with the cDNA of a green fluorescent protein. It was suggested that haploinsufficiency of the FSCN2 gene results in retinopathy in the FSCN2 knockout mice.8 Unfortunately, the phenotype of mice with homozygous knockout of FSCN2 has not been reported.
FSCN2 was excluded as a candidate gene for RP17 mapped to this region.1 2 The 208delG mutation was not detected in 458 families with retinal degeneration from other ethnic groups so far reported.9 10 11 This mutation was not detected in 215 Spanish probands with ADRP (200 cases) or ADMD (15 cases).9 It was not detected in 43 Italian families with ADRP10 or in 200 U.S. families with ADRP in a recent study.11 In addition, no other mutation, other than 208delG, has been identified in the FSCN2 gene of patients with retinal degeneration.3 4 9 10
It is unusual that a gene is a responsible for disease in one ethnic group but not in many others, if a reasonable number of cases have been studied. It is highly unusual that the same mutation can cause both rod–cone and cone–rod retinal degeneration, although different mutations in the same gene have been reported to cause both types of retinal degeneration. In this case, careful and extensive re-evaluation of a larger number of control subjects and unaffected family members is of the first priority. It is almost impossible to claim a disease-causing mutation if it is equally distributed in normal individuals and in patients. Our results indicate that the 208delG mutation was not associated with RP, CORD, and LCA in the Chinese population studied. Further studies in other populations are needed to clarify the different findings in Japanese and Chinese populations. If our result is supported by further studies, it is advised that care be taken in correlating a mutation with a disease until confirmed by multiple findings.
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Acknowledgements
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The authors thank all patients and family members for their participation.
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Footnotes
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Supported by Grant Z19-01-04-02 from the 863 Plan of China; Grant 010765 from the Guangdong Natural Science Foundation; Grant 6041 from the Fok Ying Tung Education Foundation; and Grant Q02028 from the Guangdong Education Bureau for Excellent Talent.
Submitted for publication June 16, 2006; revised September 28, 2006; accepted December 15, 2006.
Disclosure: Q. Zhang, None; S. Li, None; X. Xiao, None; X. Jia, None; X. Guo, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Qingjiong Zhang, Ophthalmic Genetics and Molecular Biology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xianlie Road, Guangzhou 510060, China; qingjiongzhang{at}yahoo.com.
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Abstract
Methods
