Molecular Vision 2005; 11:922-8 ©2005 Molecular Vision Received 8 March 2005 | Accepted 29 October 2005 | Published 2 November 2005

Sequence variations in the retinal fascin FSCN2 gene in a Spanish population with autosomal dominant retinitis pigmentosa or macular degeneration

María José Gamundi,1 Imma Hernan,1 Miquel Maseras,2 Montserrat Baiget,3 Carmen Ayuso,4 Salud Borrego,5 Guillermo Antiñolo,5 José María Millán,6 Diana Valverde,7 Miguel Carballo1

1Servei de Laboratori, Laboratori de Biologia i Genètica Molecular, Hospital de Terrassa, Ctra. Torrebonica, Terrassa, Spain; 2Servei d’Oftalmologia de l’Hospital de Terrassa, Spain; 3Servei de Genètica Molecular, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 4Servicio de Genética, Fundación Jiménez Díaz, Madrid, Spain; 5Servicio de Genética y Diagnóstico Prenatal, Hospital Virgen del Rocío, Sevilla, Spain; 6Servicio de Genética, Hospital de la Fe, Valencia; 7Área de Genética, Facultad de Ciencias, Universidad de Vigo, La Coruña, Spain

Purpose: Only one mutation in the retinal fascin gene (FSCN2) has so far been associated with autosomal dominant retinitis pigmentosa (adRP) and macular dystrophy (adMD), in a Japanese population. Our study was designed to identify mutations in the FSCN2 gene among Spanish persons with adRP or adMD. Methods: Denaturing gradient gel electrophoresis and direct genomic sequencing were used to evaluate the complete coding region and flanking intronic sequences of the FSCN2 gene for mutations in 150 unrelated adRP and 15 adMD index patients, and in 50 sporadic cases of retinitis pigmentosa, together with 50 controls. Ophthalmic and electrophysi- ological examination of retinitis pigmentosa patients and their relatives was carried out according to pre-existing proto- cols. Results: Sixteen nucleotide substitutions were detected in the coding sequence of the index patients. Nine of these, His7Tyr, Ala122Thr, Ser126Phe, His138Tyr, Arg149Gln, Ala240Thr, Ala323Thr, Asn331His, and Phe367Leu are mis- sense mutations, one is a nonsense mutation (Lys302Stop), and six are silent mutations. Co-segregation of the mutations in the families showed no direct relation between mutation and disease. Conclusions: The photoreceptor-specific FSCN2 gene showed a relatively high number of sequence variations. The mutation 208delG in FSCN2, the only mutation so far associated with adRP or adMD, and which presumably causes a null allele, was not detected in these Spanish families. The nonsense mutation, Lys302Stop, detected in one adRP Spanish family is not the cause of the disease. These findings support the fact that the kind and frequency of the mutations depend on the ethnic population.

Retinitis pigmentosa (RP) is the name given to a group have resulted in the characterization of mutations in genes with of hereditary retinal degeneration diseases with a worldwide ubiquitous expression implicated in adRP, such as the pre- incidence of about 1 in 4000 individuals [1,2]. Clinical char- mRNA splicing factors PRPF8, PRPF31, PRPF3, and PAP-1 acteristics include night blindness, loss of the peripheral vi- [19-22] or the IMPDH1 and CAIV genes [23,24]. The clinical sual field, characteristic changes in the ocular fundus and de- and genetic heterogeneity of RP is demonstrated by the fact pression of the normal ocular electrophysiological responses that different mutations within genes associated with adRP [3,4]. Genetically, RP is heterogeneous and the disorder may can cause substantially different retinal degeneration pheno- be inherited through an autosomal dominant (adRP), autoso- types. For example, mutations within the RDS gene have been mal recessive (arRP), X-linked (XLRP) [5-7] or digenic trait associated with RP, different forms of the macular dystrophies [8]. Complex inheritance patterns such as tri-allelism [9] or or the cone-rod dystrophy that is also caused by some of the uniparental disomy [10,11] have been reported. Mutations mutations found in the CRX gene [25]. However, an extreme within seven genes (RHO, peripherin/RDS, ROM1, RP1, NRL, example of the clinical heterogeneity has recently been re- CRX, and FSCN2) that encode proteins specifically expressed ported in the FSCN2 gene; the only mutation so far found in in photoreceptor cells have been reported to cause adRP [12- FSCN2 associated with adRP (208delG) has recently been 18]. These proteins are involved with specific functions in the associated with autosomal dominant macular dystrophy retina, such as the visual transduction cycle, structural com- (adMD) in a Japanese population [18,26]. Similar heteroge- ponents of the rod, and cone photoreceptor cells or transcrip- neity was seen for the mutation 1147delA in the arrestin gene tion factors. Studies of genetic linkage and mutation detection in both Oguchi disease and arRP, also detected in Japanese patients and thus supporting the importance of ethnic varia- Correspondence to: Miguel Carballo, Hospital de Terrassa, Carretera tion [27]. de Torrebonica s/n, 08227 Terrassa, Spain; Phone: +34937312420; The photoreceptor-specific gene FSCN2, located on chro- FAX: +34937319045; e-mail: [email protected] mosome 17q25, encodes 516 amino acids [28]. Retinal fascin 922 Molecular Vision 2005; 11:922-8 ©2005 Molecular Vision is associated with the assembling of actin structures of the clamp consisting of a sequence of 40 CG nucleotides was in- connecting cilium plasma membrane, and it plays an impor- cluded in the 5' sequence of the forward or reverse primer to tant role in photoreceptor disc formation [28,29]. The human have a better resolution in the DGGE analysis. The running FSCN2 and actin ACTB genes lie within 200 kb of each other conditions of the DGGE are given in Table 1. Polymerase chain on chromosome 17q25, although they seem to show indepen- reaction (PCR) was performed in a 50 µl volume of buffer (20 dent gene regulation. Though possible linkage of FSCN2 to mM Tris-HCl pH 8.55, 16 mM (NH)2SO4, 1.5 mM MgCl2 150 the RP17 allele at distal 17q has been excluded [30], a muta- mg/ml BSA) containing 200-500 ng of human genomic DNA, tion in the FSCN2 gene associated with adRP and adMD has 25 pmol each primer, 10 nmol each deoxyribonucleoside triph- been found [18,26]. Analysis of this gene in western popula- osphate, and 1.5 units of Taq polymerase (Ecotaq, Ecogen, tions, however, is lacking. We screened for mutations in the Barcelona, Spain). Incubation was performed for 40 cycles retinal fascin FSCN2 gene in 150 adRP, 15 adMD families, consisting of 30 s (or 1 min) at 94 °C, 30 s (or 1 min) for and 50 sporadic cases of RP (SRP), together with 50 controls. annealing at different temperatures (Table 1) and 30 s (or 1 Interestingly, although we detected a relatively high sequence min) at 72 °C, followed by 1 min at 94 °C and 5 min at 72 °C. variation in FSCN2, none of the mutations detected seem to Electrophoresis of 8 µl of final PCR reaction volume was per- be directly causative of retinal disease. formed on a 1.5% agarose gel to test the amplification reac- tion. For DNA sequencing, PCR products were purified using METHODS Qiaquick columns (Qiagen). DNA sequencing was carried out Patients: The participants were all Caucasian from different using the OpenGene Automated DNA sequencing system from regions of Spain. We determined the pattern of disease inher- Visible Genetics and Thermo Sequenase Cy5.5 Dye Termina- itance of the patients from their family history and ophthalmic tor Cycle Sequencing Kit (Amersham Pharmacia Biotech, examination, which consisted of a funduscopic exam, visual Barcelona, Spain). The sequencing primers were the same as field, visual acuity, dark-adapted sensitivity and, in most cases, those used in PCR amplifications. electroretinograph analysis, all according to previously estab- lished protocols. Patients with autosomal dominant disease RESULTS usually had an affected parent or child. Prior to inclusion, all Screening for mutations in the flanking and coding sequences the patients and their relatives who were participating were of five exons of the FSCN2 gene was carried out by DGGE in informed about the aims of the study, which was approved by 150 adRP and 15 adMD index patients, 50 SRP patients, and all the participating institutions. Informed consent, which ad- 50 controls. We detected 16 sequence variations in the adRP hered to the tenets of the Declaration of Helsinki, was ob- index patients (Table 2). Nine of these mutations are missense tained from all the adults and from the children’s parents or mutations, one produces a premature stop codon and the other tutors. six are silent mutations in the coding region. Only one mis- Screening for mutations and sequencing conditions: Ge- sense mutation (His138Tyr), detected in FSCN2, occurred in nomic DNA was prepared from peripheral blood lymphocytes a residue conserved among other members of the fascin gene using QIAmp DNA Blood Mini Kit (Qiagen, Valencia, CA). family (Figure 1). Most of these sequence variations were ei- The coding region of the FSCN2 gene (NM_012418) was ther unique or had a low frequency in the population screened, amplified using primers located in the flanking intronic re- and should thus be considered as rare sequences or variations, gion (Table 1). Screening for mutations in FSCN2 was carried out by denaturing gradient gel electrophoresis (DGGE). A CG TABLE 2. SEQUENCE VARIATIONS IN THE FSCN2 GENE IN SPANISH PATIENTS OF RP

adMD SRP TABLE 1. PRIMERS, AMPLIFICATION, AND DGGE CONDITIONS Nucleotide Amino acid adRP cases cases cases Controls Exon change change (n=150) (n=15) (n=50) (n=50) Fragment PCR annealing Percentage ------size temperature DGGE gradient 1A 98->C In promoter 51 (34.2%) 5 (33.3%) 20 (40%) 20 (40%) Exon Primers (5'-3') (bp) (°C)/time(s) denaturant 155 C->T His7Tyr 1 (0.7%) ------286 C->G Pro50Pro 1 (0.7%) 1A F: gcgggggccgtgagcactca 383 60/60 55-95% R: GC-clamp-caggaccaggaagcggcagt 1B 469 C->T Phe111Phe 4 (2.7%) 1 (6.7%) 2 (4%) 500 G->A Ala122Thr 1 (0.7%) 1B F: GC-clamp-cgctacctgtcggcagaagag 415 58/30 55-100% 513 C->T Ser126Phe 1 (0.7%) R: gctgtcacaggacttgaggcagta 548 C->T His138Tyr 1 (6.7%) 1 (2%) 572 G->A Val146Met 1 (2%) 1C F: GC-clamp-gacgccctcctcaccctcatc 426 58/30 55-90% 582 G->A Arg149Gln 1 (0.7%) R: ccccacggcctcctccctgaa 589 C->T Tyr151Tyr 2 (1.3%) 2 (13.3%) 1 (2%) 2 F: GC-clamp-ggagaggcgtgaggggcttc 262 58/30 40-70% R: ataggaggaagggatgtgtg 1C 854 G->A Ala240Thr 1 (6.7%) 874 T->C Pro246Pro 2 (1.3%) 3 F: GC-clamp-cacccccatctcctgtc 236 65/60 40-80% R: agtacctggcaggcagagtg 2 1040 A->T Lys302Stop 1 (0.7%) 1102 C->T His322His 1 (0.7%) 4 F: GC-clamp-gggaggggcagcgcagcaga 368 65/60 55-100% 1103 G->A Ala323Thr 19 (12.7%) 2 (13.3%) 3 (6%) 6 (12%) R: ctgccgtggctgcccgtgta 3 1127 A->C Asn331His 1 (0.7%) 5 F: ggggtggcagcgggcaggt 408 68/60 70-100% 1183 C->T Asn349Asn 2 (1.3%) 2 (13.3%) 2 (4%) R: GC-clamp-gggcctcctccacctccag 1235 T->C Phe367Leu 1 (0.7%) GC-clamp corresponds to the sequence 5'-CGC CCG CCG CGC CCC Sequence variations detected in the FSCN2 gene in Spanish patients. GCG CCC GGC CCG CCG CCC CCG CCC G-3'. 100% denaturant adRP is autosomal dominant retinitis pigmentosa, adMD is autoso- is 7 M urea, 40% v/v formamide in TAE buffer. Gradient denatur- mal dominant macular dystrophy, SRP is sporadic retinitis ation was permormed at 60 °C. pigmentosa. Controls are individuals from a control population. 923 Molecular Vision 2005; 11:922-8 ©2005 Molecular Vision

Human fascin 1 MTANGTAEAVQIQFGLINCGNKYLTAEAFGFKVNASASSLKKKQIWTLEQ 50 Mouse fascin 1 MTANGTAEAVQIQFGLISCGNKYLTAEAFGFKVNASASSLKKKQIWTLEQ 50 Xenopus fascin MTSG------PLQLGLVNCNNKYLTAEAFGFKINASASSLKKKQVWSLEP 44 Human retinal fascin MPTNGLHQVLKIQFGLVNDTDRYLTAESFGFKVNASAPSLKRKQTWVLEP 50 Mutations in human retinal fascin MPTNGLYQVLKIQFGLVNDTDRYLTAESFGFKVNASAPSLKRKQTWVLEP 50 H7Y,P5OP Bovine retinal fascin MPTNGLHQVLKIQFGLVNDTDRYLTAESFGFKVNASAPSLKRKQMWVLEP 50 Sea urchin fascin MPAMN----LKYKFGLVNSAGRYLTAEKFGGKVNASGATLKARQVWILEQ 46

Human fascin 1 PPDEAGSAAVCLR-SHLGRYLAADKDGNVTC--EREVPGPDCRFLIVAHD 97 Mouse fascin 1 PPDEAGSAAVCLR-SHLGRYLAADKDGNVTC--EREVPDGDCRFLVVAHD 97 Xenopus fascin AGEDT--SAVLLR-SHLGRFLSADKDGKVSG--ESETAGPECRFLVSAQG 89 Human retinal fascin DPGQG--TAVLLRSSHLGRYLSAEEDGRVAC--EAEQPGRDCRFLVLPQP 96 Mutations in human retinal fascin DPGQG--TAVLLRSSHLGRYLSAEEDGRVAC--EAEQPGRDCRFLVLPQP 96 Bovine retinal fascin DPGEG--TAVLFRSSHLGRYLSAEEDGRVAC--EAERPGRDCRFLVLPQP 96 Sea urchin fascin EESST----ISYLKAPSGNFLSADKNGNVYCSVEDRTEDADTGFEIELQP 92

Human fascin 1 DGRWSLQSEAHRRYFGGTEDRLSCFAQTVS-PAEKWSVHIAMHPQVNIYS 146 Mouse fascin 1 DGRWSLQSEAHRRYFGGTEDRLSCFAQSVS-PAEKWSVHIAMHPQVNIYS 146 Xenopus fascin DGRWALQSEAYGRYFGGSEDRISCFSPSVS-PAEKWGVHLAMHPQFTLYS 138 Human retinal fascin DGRWVLRSEPHGRFFGGTEDQLSCFATAVS-PAELWTVHLAIHPQAHLLS 145 Mutations in human retinal fascin DGRWVLRSEPHGRFFGGTEDQLSCFTTAVF-PAELWTVHLAIYPQAHLLS 145 F111F, A122T, Bovine retinal fascin DGRWVLQSEPHGRFFGGTEDQLSCFATAIT-PAELWTVHLAIHPQAHLLS 145 S126F, H138Y Sea urchin fascin DGKWALKNVSHQRYLACNGEELICSESSTSNPSANWTVQLAIHPQVCMKN 142

Human fascin 1 VTRKRYAHLSARP--ADEIAVDRDVPWGVDSLITLAFQDQ-RYSVQTADH 193 Mouse fascin 1 VTRKRYAHLSARP--ADEIAVDRDVPWGVDSLITLAFQDQ-RYSVQTSDH 193 Xenopus fascin VTRKRYAHLSAN---GDELSVERDVPWGVDSLITLIFQDN-RYSIQTPDH 184 Human retinal fascin VSRRRYVHLCPR---EDEMAADGDKPWGVDALLTLIFRSR-RYCLKSCDS 191 Mutations in human retinal fascin VSRNRYVHLCPR---EDEMAADGDKPWGVDALLTLIFRSR-RYCLKSCDS 191 R149N, Y151Y Bovine retinal fascin VSRRRYAHLCPQ---EDEIAADSNTPWGVDALVTLIFQNR-QYCLKSCDS 191 Sea urchin fascin VQHQRYAHLKTSEEGEDSVVVDELVPWGADSTLTLVYLGKGKYGLEAFNG 192

Human fascin 1 RFLRHDGRLVARPEPATGYTLEFRSGKVAFRDCEGRYLAPSGPSGTLKAG 243 Mouse fascin 1 RFLRHDGRLVARPEPATGFTLEFRSGKVAFRDCEGRYLAPSGPSGTLKAG 243 Xenopus fascin RLLASDGSLRDGPGPDTGYTLDISSGKVAFRANDGRYLTSSGPSGTMKAG 234 Human retinal fascin RYLRSDGRLVWEPEPRACYTLEFKAGKLAFKDCDGHYLAPVGPAGTLKAG 241 Mutations in human retinal fascin RYLRSDGRLVWEPEPRACYTLEFKAGKLAFKDCDGHYLAPVGPAGTLKTG 241 A240T Bovine retinal fascin RYLRSDGRLVWEPEPRARYTLEFKAGKLAFKDCDGHYLAPVGPAGTLRAG 241 Sea urchin fascin KFVQTDGQLAGTANEQTQFTLIFTSGHLVLRDNNGRHLGVDSGTRVLKSS 242

Human fascin 1 KATKVGKDELFALEQSCAQVVLQAANERNVSTRQGMDLSAN---QDEETD 290 Mouse fascin 1 KATKVGKDELFALEQSCAQVVLQAANERNVSTRQGMDLSAN---QDEETD 290 Xenopus fascin KNSKAGRDELFVLERSCPQVVLTAGNGRNVSTRQGIDLSAN---QDEESD 281 Human retinal fascin RNTRPGKDELFDLEESHPQVVLVAANHRYVSVRQGVNVSAN---QDDELD 288 Mutations in human retinal fascin RNTRPGKDELFDLEESHPQVVLVAANHRYVSVRQGVNVSAN---QDDELD 288 P246P Bovine retinal fascin RNTRPGKDELFDLEESHPQVVLVAANHRYVSVRQGVNVSAN---QDDELD 288 Sea urchin fascin K-PGLTKANYFILEDSCPQGAFEFG-GKYASLKQGEDVSFKLLVDEDIED 290

Human fascin 1 QETFQLEIDRDTKKCA------FRTHTGKYWTLTATGGVQSTASSKNASC 334 Mouse fascin 1 QETFQLEIDRDTRKCA------FRTHTGKYWTLTATGGVQSTASSKNASC 334 Xenopus fascin QETFQLEINKETKMCA------FRTHTGKYWTLSNNGGIQASASTLNNSC 325 Human retinal fascin HETFLMQIDQETKKCT------FYSSTGGYWTLVTHGGIHATATQVSANT 332 K302X, H322H, Mutations in human retinal fascin HETFLMQIDQETKXCT------FYSSTGGYWTLVTHGGIHTTATQVSAHT 332 A323T, N331H Bovine retinal fascin HETFLMQIDQETKKCT------FYSSTGGYWTLVTHGGIQATATQVSENT 332 Sea urchin fascin TETFQLEFVETDKYAIRVCDPKKNSRDAKFWKTVAAGIQANGNSKDQTDC 340

Human fascin 1 YFDIEWRDRRITLRASNGKFVTSKKNGQLAASVETAGDSELFLMKLINRP 384 Mouse fascin 1 YFDIEWCDRRITLRASNGKFVTAKKNGQLAASVETAGDSELFLMKLINRP 384 Xenopus fascin YFDIEWCDRRITLKGANGKFVTSKKNGQLAASVETAGDSELFLMKLINRP 375 Human retinal fascin MFEMEWRGRRVALKASNGRYVCMKKNGQLAAISDFVGKDEEFTLKLINRP 382 Mutations in human retinal fascin MFEMEWRGRRVALKASNGRYVCMKKNGQLAAISDLVGKDEEFTLKLINRP 382 N349N, F367L Bovine retinal fascin MFEMEWRGRRVALKASNGRYVCMKKNGQLAAISDFVGEDEEFTLKLINRP 382 Sea urchin fascin QFSVEYNGNDMHVRAPGGKYVSVRDNGHLFLQDSPKD----FIFRLLNRP 386

Human fascin 1 IIVFRGEHGFIGCRKVTGTLDANRSSYDVFQLEFNDGAYNIKDSTGKYWT 434 Mouse fascin 1 IIVFRGEHGFIGCRKVTGTLDANRSSYDVFQLEFNDGAYNIKDSTGKYWT 434 Xenopus fascin LIVFRGEHGFIGCRKMTGTLDSNRSIYDVFELEFNDGAYSLKDSTGKYWT 425 Human retinal fascin ILVLRGLDGFVCHHRGSNQLDTNRSVYDVFHLSFSDGAYRIRGRDGGFWY 432 Mutations in human retinal fascin ILVLRGLDGFVCHHRGSNQLDTNRSVYDVFHLSFSDGAYRIRGRDGGFWY 432 Bovine retinal fascin ILVLRGLDGFVCHRRGSNQLDTNRSVYDVFHLSFSDGAYQIRGRGGGFWH 432 Sea urchin fascin KLVLKCPHGFVGMKEGKAEVACNRSNFDVFTVTYKEGGYTIQDSCGKYWS 436

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Human fascin 1 VGSDSAVTSSGDTPVDFFFEFCDYNKVAIKVG--GRYLKGDHAGVLKASA 482 Mouse fascin 1 VGSDSSVTSSSDTPVDFFFEFCDYNKVALKVG--GRYLKGDHAGVLKACA 482 Xenopus fascin VGSDMSVTSSSPTRVDFYFEFCDYNKVAIKVN--GLYLKGDHAGVLKANA 473 Human retinal fascin TGSHGSVCSDGERAEDFVFEFRERGRLAIRARS-GKYLRGGASGLLRADA 481 Mutations in human retinal fascin TGSHGSVCSDGERAEDFVFEFRERGRLAIRARS-GKYLRGGASGLLRADA 481 Bovine retinal fascin TGSHGSVCSDGERAEDFLFEFRERGRLAIRARS-GKYLRGGASGLLRADA 481 Sea urchin fascin CD-DSSRIVLGEAAGTFFFEFHELSKFAIRAESNGMLIKGEQSGLFTANG 485

Human fascin 1 ETVDPASLWEY 493 Mouse fascin 1 ETIDPASLWEY 493 Xenopus fascin ETIDSSTLWEY 484 Human retinal fascin DAPAGTALWEY 492 Mutations in human retinal fascin DAPAGTALWEY 492 Bovine retinal fascin DAPAGVALWEY 492 Sea urchin fascin SEVSKDTLWEF 496

Figure 1. A multiple sequence alignment of fascins. Hyphens indicate gaps of residues inserted by the Clustal analysis program ClustalW. Colors are related to the nature of the residues: red indicates small, hydrophobic, and aromatic amino acids; blue indicates acidic residues; pink denotes basic residues; green denotes hydroxyl, amino, and basic residues; others in gray color. Labels to the right of the sequence numbers denote the amino acid substitutions found in a Spanish population that are shown in purple in the sequence. except the variation in the promoter region (34%) and the this family we also detected the mutation Gly182Ser within Ala323Thr polymorphism, which were also found in the con- the rhodopsin (RHO) gene. This mutation in RHO co-segre- trol population (Table 2). gated with the disease in this family (Figure 2), and it has We checked co-segregation of all these sequence varia- been reported to cause adRP in other families. tions with adRP and adDM in the families in this study. We considered a major criterion of non co-segregation to be the DISCUSSION absence of the mutation in one or more patients in the family. The fascins are a highly conserved family of actin-bundling All the families proved informative for this criterion. Even proteins identified across a wide range of species [31]. Fascin the family carrying the nonsense Lys302Stop mutation in the expression has been shown to be specific for distinct cell types, FSCN2 gene lacked co-segregation (Figure 2). However, in including neurons, macrophages, dendritic, and other cells.

Figure 2. Analysis of family adRP111. Pedigree and direct ge- nomic sequencing of a Spanish fam- ily carrying a mutation in the FSCN2 and RHO genes. A: Pedigree of the family. The shaded circles (females) and shaded boxes (males) represent the affected members and the un- shaded boxes and circles denote the unaffected members in the family. All the affected members are carri- ers of the mutation in the RHO gene. Heterozygous carriers of the FSCN2 mutation are represented by (+) while (-) represents a wild-type al- lele. B: Chromatogram of direct ge- nomic sequencing of members of the family. The mutation and a wild- type control of FSCN2 and RHO are shown.

925 Molecular Vision 2005; 11:922-8 ©2005 Molecular Vision

Fascins are thought to play a specialized role in forming or FSCN2 gene. Furthermore, clinical examination of the patients stabilizing dynamic cell extensions. The retina-specific fascin carrying both mutations in the rhodopsin and FSCN2 genes has been identified in bovine [32] and human [28] retinas. showed no significant differences in phenotype compared with The human retinal fascin FSCN2 gene is located on chromo- the carriers of the mutation in the rhodopsin gene only. some 17q (17q25-qter), with close physical linkage to the cy- Because neither the mutation 208delG nor Lys302Stop toplasmic actin ACTG1 gene. Interestingly, the human FSCN1 are located in the final exon of the FSCN2 gene, it is unlikely gene shows a similar close linkage with the actin ACTB gene that their products could be translated stably [33]. Both muta- at 7p22, suggesting that these two fascin/actin genes derive tions probably lead to the loss of a functional allele in the from a single chromosome duplication event. carriers. However, while the mutation 208delG causes retin- The fascin FSCN2 gene is a retina-specific gene. The pro- opathy in a Japanese population, Lys302Stop is unlikely to be moter region of the human FSCN2 gene contains potential the cause of the disease in the Spanish family. Recently, tar- binding sites for retina-specific transcription factors. Thus, a geted disruption of the FSCN2 gene in a mouse model has potential retinoic acid response element (RARE) and consen- been reported to produce retinopathy [34]. Furthermore, the sus sequences for the retina-specific CRX and NRL transcrip- generation of one mouse line of homozygous 208delG showed tion factors are observed in the flanking 5' sequences of the no retinal expression of FSCN2 while heterozygous murine FSCN2 gene. Although the localization of FSCN2 to distal carriers of the mutation showed progressive photoreceptor de- 17q is where retinitis pigmentosa 17 (RP17) was mapped [30], generation [34]. These findings indicate a pathogenic mecha- linkage analysis studies in two large RP17 families have shown nism of haploinsufficiency for the mutation 208delG. Whether that FSCN2 and RP17 are not linked [28]. However, FSCN2 the mutation Lys302Stop also produces haploinsufficency in is a candidate gene for RP and screening for mutations has a mouse model leading to retinal degeneration remains to be been carried out in a Japanese population. One mutation, established. It is well known that truncated mutations located 208delG in FSCN2, was detected in four unrelated families in exon 4 near the 5' region of RP1 cause adRP [35], and our (14 patients) with adRP [18]. This mutation was found in 3.3% results (data not shown). However, in a Chinese family a trun- of adRP patients in Japan, and it was suggested that it might cated mutation detected in the C-terminal region of RP1 did be relatively common in Japanese patients with adRP. The not cause the disease [36]. A similar situation is seen with the identical 208delG mutation has recently been found in four nonsense mutations of FSCN2, with the difference that the patients from two adMD Japanese families [26], showing the mutations in RP1 are located in the last exon of the gene, with remarkable clinical heterogeneity of this FSCN2 mutation. No a possible stable translation product, and a deleterious effect other sequence variation in FSCN2 has been reported in asso- of such mutants is not ruled out, although haploinsufficiency ciation with retinal disease. However, our analysis of the has been postulated as the most probable pathogenic mecha- FSCN2 gene in a Spanish population detected 14 sequence nism for these mutations [37]. variations in the coding sequence and two polymorphisms, In summary, our report of the mutational analysis of the one in the promoter region (34%) and a second (13%), FSCN2 gene in a Spanish population suggests similar analy- Ala323Thr, in the coding sequence. One of the mutations, ses in other populations to clearly associate mutations in the His138Tyr, corresponds to a conserved residue in the retinal FSCN2 gene with retinal degeneration. Compared with the fascin of different species. This His138Tyr mutation in the other known genes associated with adRP, in our population conserved fascin residue was detected in a patient with macu- the FSCN2 gene shows a relatively high proportion of sequence lar degeneration. However, this mutation was also observed variation that is not correlated with retinal degeneration dis- in the patient’s unaffected father but was absent in one af- ease. This variation, together with the clinical heterogeneity fected brother. We detected the His138Tyr mutation in a sim- shown for the 208delG mutation in a Japanese population, plex case of RP. The rest of the missense mutations were also suggests that the frequency and kind of mutations depend on analyzed in the families, but none of the mutations could be ethnic populations and raises the possibility that unknown directly associated with the disease, because one or more pa- genetic factors may be linked to FSCN2 and modulate its tients in the family were not carriers of the mutation (data not mutant expression in retinal degeneration. Further studies of shown). phenotype and genotype correlation in persons with adRP and We detected a nonsense mutation, 904A->T, in the FSCN2 adMD carrying mutations in the FSCN2 gene will be neces- gene, which introduces a premature stop codon (Lys302Stop), sary to clarify the situation. presumably producing a truncated protein. If the translation product of this mutant allele is stable, nearly half the C-termi- ACKNOWLEDGEMENTS nal lacks the encoded protein. While the Lys302Stop muta- We thank the families for their participation in this research tion does not seem to be causative of adRP, as seen from its and Ian Johnstone for editing this manuscript. This work was co-segregation in the family (Figure 2), we nevertheless de- partially supported by grants from the Fondo de tected in this family an additional mutation (Gly182Ser) in Investigaciones Sanitarias (01/0081-01, FIS G03/018), ONCE the RHO gene, which has been previously reported in asso- and Fundación ONCE. ciation with adRP and that co-segregates with RP (Figure 2). Thus, the cause of the disease in the family in question is prob- REFERENCES ably this rhodopsin mutation rather than the mutation in the 1. Humphries P, Kenna P, Farrar GJ. On the molecular genetics of 926 Molecular Vision 2005; 11:922-8 ©2005 Molecular Vision

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