Terminal Osseous Dysplasia Is Caused by a Single Recurrent Mutation in the FLNA Gene
2010; Elsevier BV; Volume: 87; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2010.06.008
ISSN1537-6605
AutoresYu Sun, Rowida Almomani, Emmelien Aten, Jacopo Celli, Jaap van der Heijden, Hanka Venselaar, Stephen P. Robertson, Anna Baroncini, Brunella Franco, Lina Basel‐Vanagaite, Emiko Horii, Ricardo Drut, Yavuz Ariyürek, Johan T. den Dunnen, Martijn H. Breuning,
Tópico(s)Bone Metabolism and Diseases
ResumoTerminal osseous dysplasia (TOD) is an X-linked dominant male-lethal disease characterized by skeletal dysplasia of the limbs, pigmentary defects of the skin, and recurrent digital fibroma with onset in female infancy. After performing X-exome capture and sequencing, we identified a mutation at the last nucleotide of exon 31 of the FLNA gene as the most likely cause of the disease. The variant c.5217G>A was found in six unrelated cases (three families and three sporadic cases) and was not found in 400 control X chromosomes, pilot data from the 1000 Genomes Project, or the FLNA gene variant database. In the families, the variant segregated with the disease, and it was transmitted four times from a mildly affected mother to a more seriously affected daughter. We show that, because of nonrandom X chromosome inactivation, the mutant allele was not expressed in patient fibroblasts. RNA expression of the mutant allele was detected only in cultured fibroma cells obtained from 15-year-old surgically removed material. The variant activates a cryptic splice site, removing the last 48 nucleotides from exon 31. At the protein level, this results in a loss of 16 amino acids (p.Val1724_Thr1739del), predicted to remove a sequence at the surface of filamin repeat 15. Our data show that TOD is caused by this single recurrent mutation in the FLNA gene. Terminal osseous dysplasia (TOD) is an X-linked dominant male-lethal disease characterized by skeletal dysplasia of the limbs, pigmentary defects of the skin, and recurrent digital fibroma with onset in female infancy. After performing X-exome capture and sequencing, we identified a mutation at the last nucleotide of exon 31 of the FLNA gene as the most likely cause of the disease. The variant c.5217G>A was found in six unrelated cases (three families and three sporadic cases) and was not found in 400 control X chromosomes, pilot data from the 1000 Genomes Project, or the FLNA gene variant database. In the families, the variant segregated with the disease, and it was transmitted four times from a mildly affected mother to a more seriously affected daughter. We show that, because of nonrandom X chromosome inactivation, the mutant allele was not expressed in patient fibroblasts. RNA expression of the mutant allele was detected only in cultured fibroma cells obtained from 15-year-old surgically removed material. The variant activates a cryptic splice site, removing the last 48 nucleotides from exon 31. At the protein level, this results in a loss of 16 amino acids (p.Val1724_Thr1739del), predicted to remove a sequence at the surface of filamin repeat 15. Our data show that TOD is caused by this single recurrent mutation in the FLNA gene. Terminal osseous dysplasia (MIM 300244) is a rare condition, characterized by terminal skeletal dysplasia, pigmentary defects of the skin, and recurrent digital fibromata during infancy. It has been described as a male-lethal X-linked dominant disease in the previously reported families and cases.1Bacino C.A. Stockton D.W. Sierra R.A. Heilstedt H.A. Lewandowski R. Van den Veyver I.B. Terminal osseous dysplasia and pigmentary defects: clinical characterization of a novel male lethal X-linked syndrome.Am. J. Med. Genet. 2000; 94: 102-112Crossref PubMed Scopus (24) Google Scholar Linkage studies mapped the mutation to Xq27.3-q28.2Zhang W. Amir R. Stockton D.W. Van Den Veyver I.B. Bacino C.A. Zoghbi H.Y. Terminal osseous dysplasia with pigmentary defects maps to human chromosome Xq27.3-xqter.Am. J. Hum. Genet. 2000; 66: 1461-1464Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar However, no disease-causing gene had been discovered. In the present study, we examined terminal osseous dysplasia (TOD) in three families and three sporadic case individuals (patients 1, 2, and 3 described by Horii,3Horii E. Sugiura Y. Nakamura R. A syndrome of digital fibromas, facial pigmentary dysplasia, and metacarpal and metatarsal disorganization.Am. J. Med. Genet. 1998; 80: 1-5Crossref PubMed Scopus (18) Google Scholar Drut,4Drut R. Pedemonte L. Rositto A. Noninclusion-body infantile digital fibromatosis: a lesion heralding terminal osseous dysplasia and pigmentary defects syndrome.Int. J. Surg. Pathol. 2005; 13: 181-184Crossref PubMed Scopus (16) Google Scholar and Breuning5Breuning M.H. Oranje A.P. Langemeijer R.A. Hovius S.E. Diepstraten A.F. den Hollander J.C. Baumgartner N. Dwek J.R. Sommer A. Toriello H. Recurrent digital fibroma, focal dermal hypoplasia, and limb malformations.Am. J. Med. Genet. 2000; 94: 91-101Crossref PubMed Scopus (23) Google Scholar). The Dutch family (Figure 1A , family 1) and Italian family (Figure 1A, family 2) have been described before (Breuning5Breuning M.H. Oranje A.P. Langemeijer R.A. Hovius S.E. Diepstraten A.F. den Hollander J.C. Baumgartner N. Dwek J.R. Sommer A. Toriello H. Recurrent digital fibroma, focal dermal hypoplasia, and limb malformations.Am. J. Med. Genet. 2000; 94: 91-101Crossref PubMed Scopus (23) Google Scholar and Baroncini6Baroncini A. Castelluccio P. Morleo M. Soli F. Franco B. Terminal osseous dysplasia with pigmentary defects: clinical description of a new family.Am. J. Med. Genet. A. 2007; 143: 51-57Crossref PubMed Scopus (10) Google Scholar). The third family (Figure 1A, family 3) has not been reported before and is nonconsanguineous and of Israeli Arab origin. All patients, a mother and her two daughters, have normal cognitive development. The mother (3I:2) suffers from chronic mild obstructive lung disease and vitamin B12 deficiency. Since her childhood, she has had multiple minor surgeries to remove small skin lesions from her hands and legs. On clinical examination at the age of 25 yrs, her head circumference was 54 cm (25%–50%), her height was 170 cm (75%–90%), and her arm span was 171 cm. Her right hand showed brachydactyly of digit III-V, a short fingernail on digitus IV, and lateral deviation of the fifth digit. On her left hand, there was lateral deviation of the fourth digit, with a small lesion on the lateral aspect of the distal phalanx, and clinodactyly of the fifth digit (Figure 1B). Her right foot showed a short and highly implanted fourth digit. There was bilateral widening of the distal portion of the second–fifth digits. She had no gingival extra frenulum and no pterygium. A skeletal X-ray survey revealed unilateral flattening of her vertebral bodies at L1-L3, secondary right scoliosis, and wedging of her L1 vertebral body. Her daughter (3II:4) underwent surgery at 2 mo of age to remove small skin lesions from her hands, feet, and gingiva. On clinical examination at the age of 3 yrs, she had a head circumference of 48 cm (25%–50%), a height of 85 cm (< 3%), and a weight of 11.1 kg (< 3%). She showed hypertelorism—interpupillary distance of 5.4cm (> 97%), a right epicanthal fold, a normal palate, an upper and lower accessory frenulum (Figure 1C), a short neck, and a short thorax. Despite earlier surgery, she had bilateral skin lesions on her second and fifth digits and bilateral clinodactyly of the fifth digit (Figure 1D). Her feet showed a lesion in her third toes and thickening of the nail of the fifth toes bilaterally. A skeletal X-ray survey revealed bilateral lytic lesions in the proximal humerus and the proximal femur, as well as multiple soft-tissue lesions in her feet and hands. The youngest daughter (3II:5) was born with multiple lesions on her hands and feet, including bilateral camptodactyly of the third digit, and bilateral overriding of the fourth toe. Echocardiogram at birth showed persistent foramen ovale. On clinical examination at the age of 6 mo, her head circumference was 42 cm (25%–50%), her height was 58.8 cm (< 3%), and her weight was 5.1 kg (< 3%). She has mild hypertelorism, three brownish pigmented spots of different sizes (3 mm to 1.5 cm) in her right temporal groove, mild retrognathia, a right upper accessory frenulum, a cleft palate, a short neck, and a short thorax. She has a bilateral axillary pterygium (Figure 1E), which is more severe on the right side. Bilaterally, there is limited extension of her elbows, with normal supination and pronation of her hands. In her right hand (Figure 1F), she had multiple skin lesions on her second–fifth digits, clinodactyly and lateral deviation of her second and third digits, and a narrow fifth digit with an absent distal crease. Her left hand showed skin lesions on her second-fourth digits. Her second digit was narrow and laterally deviated. There was camptodactyly of the third-fifth digits, brachydactyly and clinodactyly of the fifth digit, and absence of a distal crease. In her feet, she had bilateral plantar pits. The right foot has distal broadening of the second-fifth toe and brachydactyly of the second and third toe accompanied by syndactyly. There was overriding of the third and fourth toe. On her left foot, the second-fifth toes were distally broad. She had a overlapping of the second and fourth toes over her third toe, brachydactyly of the third toe that was proximally implanted. A skeletal X-ray survey revealed bilateral lytic lesions of the proximal humerus, lytic lesions of the left proximal femur, and multiple soft-tissue lesions. She had underdeveloped tarsal bones in her feet. The phenotypes from different patients are summarized in Table 1.Table 1Clinical Features of the Patients Studied in This Report1II:41III:62II:42III:53I:23II:43 II:5Patient 1Patient 2Patient 3OriginDutchDutchItalianItalianIsraeli ArabIsraeli ArabIsraeli ArabJapaneseArgentinianDutchAge at Onset1 mo3 mo7 mo2 mobirth3 mo4 moPigmentary Skin AnomaliesFace++−+++++Scalp−−+−FibromatosisDigital fibromas++−+++++++Limbs and Skeletal SystemSynadactyly−−−+++−−−Brachydactyly+−++++Clinodactyly−++++Camptodactyly++Metacarpal disorganization++−++++Metatarsal disorganization++−+++++Limb long bones anomalies−+−+−+++++Articular abnormalities++−++++Facial FeaturesCleft palate−−−−−+−−Upslanting palpebral fissures−−++Hypertelorism/Telecanthus+−−+++Epicanthic folds−−+++Coloboma of Iris−+−−−−−Flat/depressed nasal tip−+−−+−Thick lips/Prominent+−+Lower LipPapillomata−−−−Multiple frenula++−Preauricular pits and tags+− Open table in a new tab DNA of patients and family members were extracted from peripheral blood (families 1, 2, and 3), buccal cells (patient 1), or paraffin-embedded tissue (patients 2 and 3). Two probands (1II:4 and 2III:5) of the Dutch and the Italian families were tested with the X-exome target-enrichment methodology (SureSelect, Agilent) and next-generation sequencing (Illumina Genome Analyzer II). The methods used for sequence capture, enrichment, and elution followed instructions and protocols provided by the manufacturers (SureSelect, Agilent) with a little modification. In brief, 500 ng of DNA was fragmented (Bioruptor, Diagenode) according to manufacturer's instructions to yield fragments from 200 to 300 bp. Paired-end adaptor oligonucleotides from Illumina were added to both ends. The DNA-adaptor-ligated fragments were then hybridized to 250 ng of SureSelect X chromosome oligo capture library (SureSelect, Agilent) for 14 hr. After hybridization, washing, and elution, the elute was amplified to create sufficient DNA template for downstream applications. The eluted-enriched DNA fragments were sequenced with the Illumina technology platform. We prepared the paired-end flow cell on the supplied cluster station, following the instructions of the manufacturer. The reads were aligned to the reference human genome (hg 18, NCBI build 36.2) by Bowtie7Langmead B. Trapnell C. Pop M. Salzberg S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biol. 2009; 10: R25Crossref PubMed Scopus (13292) Google Scholar (Table S1, available online). Substitution-variant calling was performed by searching for positions where a variant nucleotide was present in more than 30% of the reads. After removing substitutions present with high frequency in dbSNP, the variants located in the previously identified TOD linkage interval, Xq27.3-q28, were listed in Table 2. From these variants, c.5217G>A, the only variant shared by the two patients, in the FLNA gene was selected for further study for the following reasons: (1) c.5217G>A, the last nucleotide of exon 31, was predicted to affect splicing by Human Splicing Finder.8Desmet F.O. Hamroun D. Lalande M. Collod-Béroud G. Claustres M. Béroud C. Human Splicing Finder: an online bioinformatics tool to predict splicing signals.Nucleic Acids Res. 2009; 37: e67Crossref PubMed Scopus (1702) Google Scholar The score of the splicing donor site dropped from 91.2 to 80.63, indicating that the wild-type site may not function as usual. (2) Mutations in FLNA have been reported to be involved in diseases showing a partial phenotypic overlap with TOD.9Robertson S.P. Filamin A: phenotypic diversity.Curr. Opin. Genet. Dev. 2005; 15: 301-307Crossref PubMed Scopus (153) Google ScholarTable 2List of all Exonic Variants with Low Frequency in the European Population in Xq27.3-Xq28HGVS NameGenePredicted FunctionPredicted Protein Change1II:42III:5NM_002025.2:c.1653A>GAFF2silentp.(=)−+NM_001183.4:c.∗461A>CATP6AP13′ UTRp.(=)−+NM_001009932.1:c.364G>ADNASE1L1silentp.(=)+−NM_001110556.1:c.5217G>AFLNAsilentp.(=)++NM_001110556.1:c.5814C>TFLNAsilentp.(=)−+rs2070825, high frequency in a group of multiple populationsNM_001110556.1:c.5850T>CFLNAsilentp.(=)+−does not segregate with phenotypeNM_004961.3:c.186G>AGABREsilentp.(=)+−NM_005342.2:c.166G>CHMGB3missensep.(Glu56Gln)−+NM_005367.4:c.888A>GMAGEA12silentp.(=)−+NM_005362.3:c.455G>TMAGEA6missensep.(Ser152Ile)+−repetitive regionNM_005365.4:c.92C>AMAGEA9missensep.(Pro31His)+−repetitive regionNM_001170944.1:c.468C>TPNMA6Bsilentp.(=)+−NM_005629.3:c.324A>GSLC6A8silentp.(=)−+NM_032539.2:c.1002T>CSLITRK2silentp.(=)−+NM_032539.2:c.309G>ASLITRK2silentp.(=)−+NM_001009615.1:c.240C>ASPANXN2silentp.(=)+−NM_014370.2:c.1014G>ASRPK3silentp.(=)+−NM_006280.1:c.430G>ASSR4missensep.(Gly144Arg)+−All of the HGVS numbers were generated with the use of the longest isoforms if multiple transcripts existed. Open table in a new tab All of the HGVS numbers were generated with the use of the longest isoforms if multiple transcripts existed. Sanger sequencing results confirmed the presence of c.5217G>A (Figure 2A ) and c.5850T>C (Figure 2B) in all affected cases (1II:4 and 1III:6) in family 1, as well as c.5686+84A>G found in an intron but not in an unaffected individual (1I:2). Further evidence came from the analysis of the Italian family, in whom affected cases (2II:4 and 2III:5) carry exactly the same variant, c.5217G>A, together with another exonic variant, c.5814C>T. Unfortunately, we did not have access to material from both parents and therefore could not determine whether the mutations occurred de novo. Notably, families 1 and 2 had two distinct variants adjacent to the c.5217G>A mutation, making a close and common ancestor highly unlikely. Finally, upon analysis of a third TOD family and three unrelated sporadic cases, we identified exactly the same c.5217G>A variant again in all patients, but not in unaffected family members (1I:2, 3I:1, and 3II:3). Variant c.5217G>A affects the last nucleotide of exon 31 of the FLNA gene (Figure 2C). At the protein level, it is not predicted to change the encoded amino acid, but as the last nucleotide of an exon, it may affect splicing.10Agarwal N. Kutlar F. Mojica-Henshaw M.P. Ou C.N. Gaikwad A. Reading N.S. Bailey L. Kutlar A. Prchal J.T. Missense mutation of the last nucleotide of exon 1 (G->C) of beta globin gene not only leads to undetectable mutant peptide and transcript but also interferes with the expression of wild allele.Haematologica. 2007; 92: 1715-1716Crossref PubMed Scopus (10) Google Scholar, 11Yamada K. Fukao T. Zhang G. Sakurai S. Ruiter J.P. Wanders R.J. Kondo N. Single-base substitution at the last nucleotide of exon 6 (c.671G>A), resulting in the skipping of exon 6, and exons 6 and 7 in human succinyl-CoA:3-ketoacid CoA transferase (SCOT) gene.Mol. Genet. Metab. 2007; 90: 291-297Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 12Kuivaniemi H. Tromp G. Bergfeld W.F. Kay M. Helm T.N. Ehlers-Danlos syndrome type IV: a single base substitution of the last nucleotide of exon 34 in COL3A1 leads to exon skipping.J. Invest. Dermatol. 1995; 105: 352-356Crossref PubMed Scopus (32) Google Scholar RNA was isolated from cultured fibroblasts of arm skin from 1III:6, removed during a recent orthopaedic procedure under general anesthesia with informed consent. Cells were cultured in standard medium for human fibroblasts (Dulbecco's modified Eagle's medium with 10% FBS, 1% penicillin/streptomycin, 1% glucose, 1% glutamax) with 5% CO2 in 37°C. RNA was extracted with the RNeasy Mini Kit (QIAGEN). cDNA was synthesized from 500 ng of total RNA by RevertAid RNaseH-M-MuLV reverse transcriptase in a total volume of 20 μl according to the protocol provided by the supplier (MBI-Fermentas). Target regions were amplified by RT-PCR with the use of the primers listed in Table S2. The products were evaluated with the Bioanalyzer 2100 DNA chip 1000 (Agilent), according to the manufacturer's instructions. RNA from patient fibroblasts showed only normal transcripts, both transcripts 1 (NM_01456) and 2 (NM_001110556) differing by insertion of the 24 bp exon 30 in transcript 2. Although transcript 1 has been reported as the predominant transcript in controls,13Maestrini E. Patrosso C. Mancini M. Rivella S. Rocchi M. Repetto M. Villa A. Frattini A. Zoppè M. Vezzoni P. et al.Mapping of two genes encoding isoforms of the actin binding protein ABP-280, a dystrophin like protein, to Xq28 and to chromosome 7.Hum. Mol. Genet. 1993; 2: 761-766Crossref PubMed Scopus (67) Google Scholar we detected about equal expression levels in controls (Figure 3B , lanes 2–4 and 8) and higher expression of transcript 2 in patient fibroblasts (Figure 3B, lane 1). Both bands were isolated from the agarose gel by the Qiaquick Gel Extraction Kit (QIAGEN) and analyzed by Sanger sequencing. Interestingly, we detected no expression of the mutant allele. This could be due to nonsense-mediated decay 14Holbrook J.A. Neu-Yilik G. Hentze M.W. Kulozik A.E. Nonsense-mediated decay approaches the clinic.Nat. Genet. 2004; 36: 801-808Crossref PubMed Scopus (470) Google Scholar and/or skewed X chromosome inactivation (XCI). To test the first possibility, the fibroblasts were treated with cycloheximide15Kim C.E. Gallagher P.M. Guttormsen A.B. Refsum H. Ueland P.M. Ose L. Folling I. Whitehead A.S. Tsai M.Y. Kruger W.D. Functional modeling of vitamin responsiveness in yeast: a common pyridoxine-responsive cystathionine beta-synthase mutation in homocystinuria.Hum. Mol. Genet. 1997; 6: 2213-2221Crossref PubMed Google Scholar for 4.5 hr followed by RNA analysis using the same procedures as those for RNA from untreated cells. The mutant allele was still absent in RNA from cycloheximide treated cells. XCI was analyzed with the Androgen Receptor (AR) assay.16Kubota T. Nonoyama S. Tonoki H. Masuno M. Imaizumi K. Kojima M. Wakui K. Shimadzu M. Fukushima Y. A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR.Hum. Genet. 1999; 104: 49-55Crossref PubMed Scopus (139) Google Scholar The assay showed random XCI in 1I:2 versus 100% XCI of the mutant chromosome in patient 1II:4 (patient 1III:6 was uninformative), indicating that the mutant allele was inactivated. Fifteen years ago, at the age of 1 yr, patient 1III:6 had fibroma tissue from the fifth digits of both hands and the fifth toe of the left foot surgically removed and stored in liquid nitrogen. We cultured these cells and analyzed RNA. In the fibroma cells, we observed two sets of two bands (Figure 3B, lanes 5–7), indicating altered splicing. One set had the same length as that observed in normal fibroblasts (Figure 3A, transcripts 1 and 2), and the other set was shorter (Figure 3A, transcripts 3 and 4, faint from RNA of a tumor in left fifth finger and toe; Figure 3B, lanes 6 and 7). Note that the fibroma always contains a mixture of tumor and normal stroma cells. Sequence analysis showed a deletion removing the last 48 nucleotides of exon 31 (Figure 3C), resulting in a deletion of 16 amino acids. To facilitate clinical diagnostics of FLNA gene mutations, we have established a web-based FLNA gene variant database using the LOVD software.17Fokkema I.F. den Dunnen J.T. Taschner P.E. LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach.Hum. Mutat. 2005; 26: 63-68Crossref PubMed Scopus (208) Google Scholar In this publicly available database, we have collected all variants reported in the literature thus far (83 in total; see FLNA mutation database), including the variants described here. The c.5217G>A variant detected in TOD patients has not been described before; it is listed neither in dbSNP nor in the pilot study 1 of the 1000 Genomes Project. Finally, over 400 chromosomes have been sequenced and the mutant allele was not found (data not shown). Mutations in FLNA have been reported to cause a wide range of developmental malformations in the brain, bones, limbs, heart,18Kyndt F. Gueffet J.P. Probst V. Jaafar P. Legendre A. Le Bouffant F. Toquet C. Roy E. McGregor L. 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Genet. 2003; 33: 487-491Crossref PubMed Scopus (315) Google Scholar, 27Robertson S.P. Thompson S. Morgan T. Holder-Espinasse M. Martinot-Duquenoy V. Wilkie A.O. Manouvrier-Hanu S. Postzygotic mutation and germline mosaicism in the otopalatodigital syndrome spectrum disorders.Eur. J. Hum. Genet. 2006; 14: 549-554Crossref PubMed Scopus (26) Google Scholar Although each of the OPD spectrum disorders are characterized by specific clinical symptoms, there clearly is a clinical overlap with TOD, including a generalized bone dysplasia that includes craniofacial anomalies and anomalies in digits and long bones.9Robertson S.P. Filamin A: phenotypic diversity.Curr. Opin. Genet. Dev. 2005; 15: 301-307Crossref PubMed Scopus (153) Google Scholar, 32Robertson S.P. Molecular pathology of filamin A: diverse phenotypes, many functions.Clin. Dysmorphol. 2004; 13: 123-131Crossref PubMed Scopus (24) Google Scholar Interestingly, the most conspicuous symptoms of TOD patients are skeletal dysplasia of the limbs and recurrent digital fibroma, suggesting a significant role of the FLNA mutation in the TOD phenotype. The FLNA gene encodes a cytoskeletal protein, filamin A, which crosslinks actin filaments into an orthogonal network and links these to the cell membrane. Within the cytoskeleton, filamin A also mediates functions relating to cell signaling, transcription, and development.33Zhou A.X. Hartwig J.H. Akyürek L.M. Filamins in cell signaling, transcription and organ development.Trends Cell Biol. 2010; 20: 113-123Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar Filamin A consists of two calponin homology sequences (CH1 and CH2) at the N terminus and connects with 24 immunoglobin-like filamin repeats, divided by two hinges, one between repeats 15 and 16 and one between repeats 23 and 24. To check the stability of filamin A in patient cells, protein was extracted from both fibroblast and fibroma cells. Immunoblot was performed with the use of mouse human filamin A monoclonal antibody, MAB1680, from Millipore. No difference in molecular weight or quantity was observed. The difference of 18 amino acids was likely too small to be distinguished by immunoblot. The c.5217G>A mutation is located in a highly conserved position at the DNA level, across a wide range of vertebrate and invertebrate species except rodent, and found in all ten affected patients from six different unrelated families. In addition, the mutation introduced abnormal splicing in fibroma cells. At the protein level, c.5217G>A encodes the second-to-last amino acid of repeat 15, which is immediately adjacent to hinge 1. Recent studies demonstrated repeats 9–15 contain an F-actin binding domain necessary for high avidity F-actin binding.34Nakamura F. Osborn T.M. Hartemink C.A. Hartwig J.H. Stossel T.P. Structural basis of filamin A functions.J. Cell Biol. 2007; 179: 1011-1025Crossref PubMed Scopus (193) Google Scholar Hinge 1 plays an important
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