Mutation of Human Retinal Fascin Gene (FSCN2) Causes Autosomal Dominant Retinitis Pigmentosa

Abstract

purpose. To characterize the clinical features of 14 Japanese patients with autosomal dominant retinitis pigmentosa (ADRP) who were found to have a mutation in the FSCN2 gene.

methods. Mutation screening by single-strand conformation polymorphism (SSCP) was performed in 120 unrelated patients with ADRP, 200 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), and 100 patients with simplex RP (SRP). The DNA fragment that showed abnormal mobility on SSCP was sequenced. The clinical features of these patients were determined by visual acuity, slit lamp biomicroscopy, electroretinography, fluorescein angiography, and kinetic visual field testing.

results. A novel 208delG mutation in the FSCN2 gene was identified in 14 patients from four unrelated families with ADRP. The ophthalmic findings were typical of RP.

conclusions. The findings show that a 208delG mutation in the FSCN2 gene produces ADRP. This mutation was found in 3.3% of the patients with ADRP in Japan, which suggests that it may be relatively common in Japanese patients with ADRP.

Retinitis pigmentosa (RP) is a retinal disease that can have a dominant, a recessive, or an X-linked inheritance pattern. Genetic analyses of patients with RP have shown that approximately 30% of the patients with autosomal dominant RP (ADRP) have mutations of the rhodopsin gene or mutations in the RP1 gene. Among these mutations, the Pro23His mutation in the rhodopsin gene accounts for 12% of cases of ADRP. ¹ These percentages were determined in patients in the United States, but in Japan, only five mutations in the rhodopsin gene (Gly106Arg, Asn15Ser, Glu181Lys, Thr17Met, and Pro347Leu) and no RP1 mutations have been reported. ^{2 3 4 5 6 7} Our results from screening of the rhodopsin gene showed that no other mutation of the rhodopsin gene is present in 99% of Japanese patients with ADRP. Although 10 loci for ADRP, 1q, 3q, 6p, 7p, 7q, 8q, 14q, 17p, 17q, and 19q, have been reported, only four genes, the rhodopsin, peripherin/RDS, NRL, and RP1 genes, were identified. These findings strongly suggest that some other photoreceptor-specific gene may be the cause of RP in the Japanese population. 

The retinal fascin gene (FSCN2) is a newly identified photoreceptor-specific gene located on chromosome 17q25, which encodes 516 amino acids. ^{8 9} The fascin gene is associated with the assemblage of the actin-based structures of the connecting cilium plasma membrane and plays an important role in photoreceptor disc formation. ^{8 9 10 11} To date, only five polymorphic mutations in the FSCN2 gene have been reported, ⁹ and a disease-causing mutation in the FSCN2 gene has not been published. 

We report the presence of a 208delG mutation of the fascin gene in four unrelated Japanese families with ADRP and describe the clinical features of these patients. 

Materials and Methods

Subjects and Mutation Analysis

We screened genomic DNA samples isolated from 120 unrelated patients with ADRP, 200 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), and 100 patients with simplex RP (SRP) for mutations of the FSCN2 gene. We further screened 200 control chromosomes for mutations of this gene. 

Genomic DNA was isolated from leukocytes prepared from a sample of each patient’s blood (10–15 ml), by using a protocol previously described in detail. ² For the screenings, nine sets of oligonucleotide primer pairs were used from the genomic sequence of FSCN2. The primer sequences are given in Table 1 . PCR was performed in 50 μl of reaction mixture containing 250 ng genomic DNA, 20 μM of each primer, 200 μM of each dNTP, and 1.25 U Taq polymerase. The buffer contained 50 mM KCl, 10 mM TrisCl (pH 8.3), and 1.5 mM MgCl₂. 

The following steps were used for the PCR: an initial denaturation for 2 minutes at 94°C; 35 cycles of denaturation at 94°C for 1 minute; annealing at the exon-specific temperature for 1 minute; extension at 72°C for 2 minutes; and a final extension at 72°C for 10 minutes. Products of the PCR were analyzed by nonradioisotopic single-strand conformation polymorphism (SSCP). 

The amplified DNA fragment was then electrophoresed in 8% nondenaturing polyacrylamide gel containing 10% glycerol at 20 W for 8 hours at room temperature. After electrophoresis, DNA bands were visualized by silver staining. The mutation or polymorphism was observed by the presence of abnormal bands derived from a mutant allele. The DNA fragment that showed abnormal mobility on SSCP was then directly sequenced to identify the mutation in the FSCN2 gene on a sequencer (model 310; Perkin Elmer-Applied Biosystems, Foster City, CA). The product of the PCR amplification in exons 1d, 2, 3, and 5 were directly sequenced without SSCP. The PCR products were sequenced in the forward and reverse directions. 

The tenets of the Declaration of Helsinki were followed, and informed consent was obtained from all subjects who participated in the study. 

Clinical Examination

We examined 14 affected patients from four families (Fig. 1) . The ophthalmic examination included best corrected visual acuity, slit lamp biomicroscopy, kinetic visual field examination, fundus examination, fluorescein angiography (FA), and electroretinography (ERG). Ophthalmoscopic findings were recorded by color fundus photography. Kinetic visual field examination was performed on a Goldmann perimeter with V-4-e, I-4-e, I-3-e, and I-2-e isopters. The area of each isopter was expressed in steradians. ¹²  

The ERGs were recorded under controlled conditions that conformed to the standards of the International Society for Clinical Electrophysiology of Vision. ¹³ The ERGs were elicited by a single flash or 30-Hz flickering red light under light-adapted conditions for the cone-isolated responses. Rod-isolated responses were elicited by a dim blue flash under dark-adapted conditions (30 minutes in the dark). A bright white flash (20 J) in the dark-adapted state was used to elicit maximal mixed rod and cone responses. 

Results

DNA Analysis

The analysis of exon 1 of the FSCN2 gene showed abnormal band shifts in 14 patients from four unrelated families with ADRP. The subsequent nucleotide sequencing analysis disclosed an identical deletion of nucleotide G at cDNA position 208 that was designated as 208delG mutation of the FSCN2 gene (Fig. 2) . 

Fourteen affected members of four families were heterozygous for the 208delG mutation; nonaffected members did not have the mutation (Fig. 1) . The 208delG mutation was not detected in the chromosomes of 200 patients with ARRP, 100 patients with SRP, and 200 control subjects. Thus, the 208delG mutation cosegregated with the phenotype in the four pedigrees of Japanese families with ADRP. We also found two polymorphisms in the FSCN2 gene in two other patients with ADRP: ACG→ACA in codon 193 (Thr193Thr) and CCT→CCC in codon 228 (Pro228Pro), one of which (Pro288Pro) was the same polymorphism previously reported. ⁹  

Clinical Characteristics

The clinical characteristics of the 14 patients from four families associated with the 208delG mutation are summarized in Tables 2 and 3 . All the affected patients had had night blindness from childhood. The visual acuity of patients with 208delG mutation ranged from hand motion to 1.0 in all four families. Patients more than 40 years of age showed a marked decrease of the visual fields and visual acuity. 

In 11 patients, fundus examination disclosed bilateral pigmentary retinal degeneration and severe attenuation of the retinal arteries. Patient II-2 of family 4 showed pigmentary retinal degeneration associated with atrophic macular degeneration, and patient III-1 of the same family showed only attenuation of retinal vessels and a mottled appearance of the retinal pigment epithelium (RPE). The fundus of two patients (patient III-1 of family 2 and patient IV-1 of family 4), who were 10 and 4 years old, respectively, showed a mottled appearance of the RPE, attenuation of the retinal vessels, and absence of retinal pigmentary changes (Fig. 3) . These findings showed the early stage of retinal degeneration with the 208delG mutation. The natural course of the fundus changes can be estimated by examining the fundus of three members of family 2 whose ages extended over three generations (Fig. 3) . 

In six patients, Goldmann kinetic visual field testing showed severely constricted central visual fields with the V-4-e target, or in other cases, the patients could not even see the V-4-e target. Goldmann kinetic visual field testing of patient III-1 of family 4, whose only retinal abnormality was attenuation of the retinal vessels and mottled appearance of the RPE, demonstrated a constricted visual field for the I-4-e and I-2-e targets. Patient III-1 of family 2 showed constricted visual fields for the I-4-e, I-3-e, and I-2-e targets. 

FA disclosed hyperfluorescence from the posterior pole to the peripheral retina that corresponded with the mottled retina, suggesting atrophic changes in the RPE layer. Three patients (patients III-1 and III-3 of family 1 and patient II-2 of family 2) showed a combination of diffuse hyperfluorescence and patchy hypofluorescence. In addition, cystoid macula edema was observed in patient III-2 of family 3, and sharply demarcated macular degeneration was observed in patient II-2 of family 4. 

The results of the ERG recordings are presented in Table 3 , and those for the three members of family 2 are shown in Figure 4 . The scotopic, single-flash, standard-flash ERGs and 30-Hz flicker ERG were mildly reduced in patients III-1 of family 2 and III-1 of family 4, and the single-flash a- and b-waves of the ERGs were mildly reduced in patient IV-1 of family 4. The ERGs of the other patients were nonrecordable (Table 3) . 

Discussion

Fascin is a member of the family of actin-binding proteins. Five fascin genes from distant species, (i.e., urchin, Drosophila, Xenopus, mouse, and human) and two retinal fascins (human and bovine) have been identified. ^{8 9 10 14 15 16 17 18} They are highly conserved, actin-binding proteins, which supports the idea that fascin is a very important protein across species. Relevant to our study, the retinal fascin gene (FSCN2) is a newly identified photoreceptor-specific gene that encodes 516 amino acids and is located on chromosome 17q25. ⁹ Biochemical and morphologic studies have shown that retinal fascin also has actin-bundling activity. ¹¹ Retinal fascin is associated with the assemblage of the actin-based structures of the connecting cilium plasma membrane, which contains a cluster of F-actin and plays an important role in photoreceptor disc formation. ^{9 10 11} It is thus reasonable that mutations of the FSCN2 gene could alter the actin-binding and actin-bundling activities of the photoreceptor cells and lead to retinal dystrophies. 

RP17 is a novel locus for ADRP detected in two South African families. ^{8 19 20} Because the FSCN2 gene is located on 17q25, RP17 was considered to be a candidate gene for RP type 17 (RP17). However, Tubb et al. ⁹ reported that only five polymorphic mutations in the FSCN2 gene were found in two large RP17-carrying families, and no previous report of a disease-causing mutation in the FSCN2 gene had been published. 

We evaluated 120 Japanese patients with ADRP, 200 patients with ARRP, and 100 patients with SRP. Of note, molecular genetic analysis disclosed that 14 patients from four unrelated families had an identical 208delG mutation in the FSCN2 gene and that no mutation was detected in the patients with ARRP and SRP. 

This mutation resulted in a frame shift and a premature termination at codon 144, 359 bp downstream from the deletion. If translated, the mutated FSCN2 gene would not encode a functional protein. 

Fundus examination of three generations of family 2 disclosed the progression of retinal degeneration with increasing age (Fig. 3) . In the early stage, a 10-year-old patient showed a mottled appearance of the RPE and attenuation of the retinal vessels. In all families, affected individuals more than 40 years old showed marked retinal degeneration. 

For human fascin, Ser39 is very important in regulating actin binding, and this residue is also conserved in human retinal fascin. ¹¹ Thus, Ser39 is thought to play an important role, not only in human fascin but also in human retinal fascin. Because the 208delG mutation causes a frame shift and premature termination, patients with this mutation do not have Ser39 in the FSCN2 gene. Thus, these patients would be expected to lose the activity of actin binding and have a disorder of photoreceptor formation. 

We hypothesize that the 208delG mutation in the FSCN2 gene may be relatively common in Japanese patients with ADRP, because we have found this mutation in 3.3% of unrelated patients with ADRP in Japan, and there have been no reports about pathogenic mutations in the FSCN2 gene in RP type 17 of two families in other countries. ^{8 19 20} Additional families with ADRP, ARRP, and other retinal degenerations are being screened for this mutation to ascertain the phenotype–genotype correlation in the FSCN2 gene in the Japanese population. 

 Supported in part by a grant from the Research Committee on Chorioretinal Degenerations and Optic Atrophy, the Ministry of Health, Labour and Welfare of the Japanese Government, Tokyo, Japan (MT); and Grants-in-Aid A-2-10307041 and 12357010 for Scientific Research from the Ministry of Education, Science, Sports and Culture of the Japanese Government, Tokyo, Japan (MT).

 Submitted for publication October 10, 2000; revised March 13, 2001; accepted April 6, 2001.

 Commercial relationships policy: N.

 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.

 

Table 1.

 

FSCN2 Gene Primers Used for Mutation Screening

EXON	Forward Primer	Reverse Primer	Length	Annealing Temperature
1a	5′-GGCCAGCCTGAAGATGCC-3′	5′-CTCTTCTGCCGACAGGTAGC-3′	232 bp	61
1b	5′-TCCGCAGCAGCCACCT-3′	5′-TCGGTGCCTCCGAAGA-3′	166 bp	63
1c	5′-TCTTCGGAGGCACCGA-3′	5′-AGGACTTGAGGCAGTACCGT-3′	237 bp	62
1d	5′-GCAGACGGAGACAAGCC-3′	5′-TCAGGAGGTCGCCACCT-3′	370 bp	62
2	5′-GGTCTCTGAGAGGTGCCTTC-3′	5′-GCACTCACACTTGTGTGGCT-3′	317 bp	60
3	5′-GATTGCCGTAGCAGCTCAGT-3′	5′-TCCAGCTCTTGGTGGAGATG-3′	398 bp	62
4a	5′-CACATGAGGCAATGGCA-3′	5′-CAGGTGGAAGACGTCGTAGA-3′	263 bp	62
4b	5′-AACCAGCTGGACACCAA-3′	5′-ACTCGAAGACGAAGTCCTCG-3′	245 bp	63
5	5′-TACCGGATCCGAGGTGCG-3′	5′-CCTCCACCTCCAGCTGCAG-3′	408 bp	63

Figure 1.

 

Pedigrees of four Japanese families with the 208delG mutation. M, 208delG mutation; +, wild-type sequence of the FSCN2 gene.

Figure 2.

 

Sequence analysis of exon 1 in patient II-4 of family 1 showing the heterozygous 208delG mutation (arrow).

Table 2.

 

Clinical Characteristics of Patients with the 208delG Mutation in the FSCN2 Gene

Patient/Age (y)	Visual Acuity	Manifest Refraction	Goldmann Perimetry Results	Fundus Findings	Fluorescein Angiography
Family 1
II-4/81	0.1 OD	Pseudophakia	No data	BP, AN, atrophy, OA	No data
	0.1 OS	Pseudophakia		BP, AN, atrophy, OA
III-1/51	HM OD	Pseudophakia	NS	BP, AN, atrophy, OA	DHyperF, patchyHypoF
	0.01 OS	Pseudophakia	0.003 (V4e)	BP, AN, atrophy, OA
III-3/57	HM OD	Pseudophakia	NS	BP, AN, atrophy, OA	DHyperF, patchyHypoF
	0.01 OS	Pseudophakia	0.019 (V4e)	BP, AN, atrophy
III-5/49	0.5 OD	−1.0 sphere	No data	BP, AN, atrophy	No data
	0.5 OS	−0.5 sphere		BP, AN, atrophy
Family 2
I-2/73	0.15 OD	−8.0+ 1.0× 58	0.025 (V4e)	BP, AN, atrophy	No data
	HM OS	−9.0+ 1.0× 153	NS	BP, AN, atrophy
II-2/40	HM OD	−1.5+ 1.25× 97	NS	BP, AN, atrophy, OA	DHyperF, patchy HypoF
	HM OS	−1.0 sphere	NS	BP, AN, atrophy, OA
III-1/10	0.04 OD	−13.0+ 3.0× 90	3.326 (V4e), 1.361 (I4e), 0.041 (I3e)	AN, mottled RPE	DHyperF
	0.8 OS	−8.25+ 1.25× 120	3.42 (V4e), 1.768 (I4e), 0.702 (I3e), 0.087 (I2e)	AN, mottled RPE
Family 3
I-2/74	HM OD	Pseudophakia	NS	BP, AN, atrophy, OA	No data
	HM OS	Pseudophakia	NS	BP, AN, atrophy, OA
II-1/52	0.2 OD	Pseudophakia	0.049 (V4e)	BP, AN, atrophy	DHyperF
	0.4 OS	Pseudophakia	0.047 (V4e)	BP, AN, atrophy
III-2/29	1.0 OD	−1.25+ 0.75× 110	0.262 (V4e), 0.046 (I4e), 0.009 (I2e)	BP, AN, atrophy	DHyperF, CME
	0.5 OS	−2.0 sphere	0.143 (V4e), 0.031 (I4e), 0.001 (I2e)	BP, AN, atrophy, ME
Family 4
II-2/70	HM OD	Pseudophakia	0.234 (V4e)	BP, AN, atrophy, CRA in MA	DHyperF, sharply demarcated CRA
	HM OS	Pseudophakia	0.074 (V4e)	BP, AN, atrophy, CRA in MA
II-3/68	HM OD	Pseudophakia	No data	BP, AN, atrophy	No data
	HM OS	Pseudophakia	No data	BP, AN, atrophy
III-1/32	1.0 OD	−3.5+ 1.0× 30	3.271 (V4e), 1.986 (I4e), 0.073 (I2e)	AN, mottled RPE	No data
	1.0 OS	−3.0 sphere	2.592 (V4e), 1.437 (I4e), 0.037 (I2e)	AN, mottled RPE
IV-1/3	0.2 OD	−6.5+ 1.5× 100	No data	AN, mottled RPE	No data
	0.4 OS	−6.0+ 1.5× 90	No data	AN, mottled RPE

 HM, hand motion; NS, not seen; BP, bone spicule–like pigmentation; OA, optic atrophy; CRA, chorioretinal atrophy; ME, macular edema; CME, cystoid macular edema; AN, attenuation of retinal vessels; DhyperF, diffuse hyperfluorescence; Patchy hypoF, patchy hypofluorescence; MA, macula.

Table 3.

 

ERG Results

Patient	Standard-Flash							Scotopic			Photopic							30-Hz Flicker
		OD			OS			OD	OS	OD			OS			OD	OS
		a Wave	b Wave	a Wave	b Wave	a Wave	b Wave	a Wave	b Wave	a Wave	b Wave
Family 1
II-4	Nonrecordable			Nonrecordable			NP			NP			NP			NP	NP
III-1	Nonrecordable			Nonrecordable			NP			NP			NP			NP	NP
III-3	Nonrecordable			Nonrecordable			NP			NP			NP			NP	NP
III-5	NP			NP			NP			NP			NP			NP	NP
Family 2
I-2	Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable
II-2	Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable
III-1	29.9	29	43.2	40.2	21.7	40.7	43.7	45.3	77	68	32.2	34.7
Family 3
I-2	Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable
II-1	Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable
III-2	Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable
Family 4
II-2	Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable
II-3	Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable			Nonrecordable
III-1	73.6	61.4	76.5	69.3	Normal	Normal	97.5	80.3	77.6	77.9	55.1	60.2
IV-1	46.5	55.7	43.2	64.2	NP			NP			NP			NP

 Percentage of mean normal amplitude of results from participating ERG laboratories. NP, not performed.

Figure 3.

 

Fundus photographs of the affected members of three generations of family 2. Patients I-2 and II-2 showed advanced, severe retinal degeneration, including attenuated retinal vessels, bone spicule deposits, and atrophy of the macula. Patient III-1 showed a mottled appearance of the RPE and attenuation of retinal vessels without retinal pigmentation.

Figure 4.

 

ERGs of patients I-2 and II-2 of family 2 showing nondetectable rod and cone responses in scotopic, photopic, standard-flash, and 30-Hz flicker ERGs. Patient III-1 of family 2 showed mildly reduced amplitudes in all ERGs.