Filamin A Is Mutated in X-Linked Chronic Idiopathic Intestinal Pseudo-Obstruction with Central Nervous System Involvement
2007; Elsevier BV; Volume: 80; Issue: 4 Linguagem: Inglês
10.1086/513321
ISSN1537-6605
AutoresAnnagiusi Gargiulo, Renata Auricchio, Maria Vittoria Barone, Gabriella Cotugno, William Reardon, Peter J. Milla, Andrea Ballabio, Alfredo Ciccodicola, Alberto Auricchio,
Tópico(s)Child Nutrition and Feeding Issues
ResumoWe have previously reported that an X-linked recessive form of chronic idiopathic intestinal pseudo-obstruction (CIIPX) maps to Xq28. To select candidate genes for the disease, we analyzed the expression in murine fetal brain and intestine of 56 genes from the critical region. We selected and sequenced seven genes and found that one affected male from a large CIIPX-affected kindred bears a 2-bp deletion in exon 2 of the FLNA gene that is present at the heterozygous state in the carrier females of the family. The frameshift mutation is located between two close methionines at the filamin N terminus and is predicted to produce a protein truncated shortly after the first predicted methionine. Loss-of-function FLNA mutations have been associated with X-linked dominant nodular ventricular heterotopia (PVNH), a central nervous system (CNS) migration defect that presents with seizures in females and lethality in males. Notably, the affected male bearing the FLNA deletion had signs of CNS involvement and potentially has PVNH. To understand how the severe frameshift mutation we found can explain the CIIPX phenotype and its X-linked recessive inheritance, we transiently expressed both the wild- type and mutant filamin in cell culture and found that filamin translation can start from either of the two initial methionines in these conditions. Therefore, translation of a normal shorter filamin can occur in vitro from the second methionine downstream of the 2-bp insertion we found. We confirmed this, demonstrating that the filamin protein is present in the patient's lymphoblastoid cell line that shows abnormal cytoskeletal actin organization compared with normal lymphoblasts. We conclude that the filamin N terminal region between the initial two methionines is crucial for proper enteric neuron development. We have previously reported that an X-linked recessive form of chronic idiopathic intestinal pseudo-obstruction (CIIPX) maps to Xq28. To select candidate genes for the disease, we analyzed the expression in murine fetal brain and intestine of 56 genes from the critical region. We selected and sequenced seven genes and found that one affected male from a large CIIPX-affected kindred bears a 2-bp deletion in exon 2 of the FLNA gene that is present at the heterozygous state in the carrier females of the family. The frameshift mutation is located between two close methionines at the filamin N terminus and is predicted to produce a protein truncated shortly after the first predicted methionine. Loss-of-function FLNA mutations have been associated with X-linked dominant nodular ventricular heterotopia (PVNH), a central nervous system (CNS) migration defect that presents with seizures in females and lethality in males. Notably, the affected male bearing the FLNA deletion had signs of CNS involvement and potentially has PVNH. To understand how the severe frameshift mutation we found can explain the CIIPX phenotype and its X-linked recessive inheritance, we transiently expressed both the wild- type and mutant filamin in cell culture and found that filamin translation can start from either of the two initial methionines in these conditions. Therefore, translation of a normal shorter filamin can occur in vitro from the second methionine downstream of the 2-bp insertion we found. We confirmed this, demonstrating that the filamin protein is present in the patient's lymphoblastoid cell line that shows abnormal cytoskeletal actin organization compared with normal lymphoblasts. We conclude that the filamin N terminal region between the initial two methionines is crucial for proper enteric neuron development. Chronic idiopathic intestinal pseudo-obstruction (CIIP [MIM %300048]) is a clinical syndrome caused by a heterogeneous group of enteric neuromuscular diseases that result in a severe abnormality of gastrointestinal motility.1Milla PJ Intestinal pseudo-obstruction in children. Wrightson Biomedical, Petersfield, United Kingdom and Bristol, PA1994Google Scholar CIIP may present at any age and is diagnosed by radiological, surgical, or manometric evidence of abnormal bowel motility causing intestinal obstruction in the absence of any mechanical occlusion.1Milla PJ Intestinal pseudo-obstruction in children. Wrightson Biomedical, Petersfield, United Kingdom and Bristol, PA1994Google Scholar CIIP is an uncommon, often fatal, syndrome in infancy, which may occur because of primary intrinsic visceral neuromuscular disorders or secondary to a variety of conditions, such as drug toxicity, ischemia, inflammatory or autoimmune diseases, myopathies, or viral infections (e.g., Epstein-Barr or cytomegalovirus [CMV]).1Milla PJ Intestinal pseudo-obstruction in children. Wrightson Biomedical, Petersfield, United Kingdom and Bristol, PA1994Google Scholar Primary forms of CIIP are neurogenic (28%), myopathic (36%), or unclassifiable (36%).2Staiano A Tozzi A Auricchio A Aetiology and assessment of constipation in children.in: Barbara L Corindolesi R Gizzi G Stanghellini V Chronic constipation. Saunders Company, Philadelphia1996: 153-168Google Scholar Neuropathic abnormalities of enteric innervation cause neurogenic CIIP and may be quantitative (hypo-, hyper-, and aganglionosis) or qualitative in nature.2Staiano A Tozzi A Auricchio A Aetiology and assessment of constipation in children.in: Barbara L Corindolesi R Gizzi G Stanghellini V Chronic constipation. Saunders Company, Philadelphia1996: 153-168Google Scholar In Hirschsprung disease (HSCR [MIM #142623]), the most common neuronal CIIP, lack of migration of enteric ganglion cells during development results in aganglionosis along gastrointestinal segments of variable length.2Staiano A Tozzi A Auricchio A Aetiology and assessment of constipation in children.in: Barbara L Corindolesi R Gizzi G Stanghellini V Chronic constipation. Saunders Company, Philadelphia1996: 153-168Google Scholar In other cases, migration of enteric neurons is not affected, but enteric ganglia and nerve fibers show qualitative abnormalities, suggesting the presence of a differentiation defect. This may be the case for the patients described by Tanner3Tanner MS Smith B Lloyd JK Functional intestinal obstruction due to deficiency of argyrophil neurones in the myenteric plexus: familial syndrome presenting with short small bowel, malrotation, and pyloric hypertrophy.Arch Dis Child. 1976; 51: 837-841Crossref PubMed Scopus (74) Google Scholar—affected by a short intestine, malrotation, and hypertrophic pyloric stenosis—who were reported to have absence of argyrophilic neurons in the myenteric plexus.3Tanner MS Smith B Lloyd JK Functional intestinal obstruction due to deficiency of argyrophil neurones in the myenteric plexus: familial syndrome presenting with short small bowel, malrotation, and pyloric hypertrophy.Arch Dis Child. 1976; 51: 837-841Crossref PubMed Scopus (74) Google Scholar The condition was thought to be inherited as an autosomal recessive trait.3Tanner MS Smith B Lloyd JK Functional intestinal obstruction due to deficiency of argyrophil neurones in the myenteric plexus: familial syndrome presenting with short small bowel, malrotation, and pyloric hypertrophy.Arch Dis Child. 1976; 51: 837-841Crossref PubMed Scopus (74) Google Scholar More recently, we reported an Italian family with 10 affected males in 4 generations, all related through healthy females; of the 10, 9 died in the first months of life. Two affected subjects (IV-1 and IV-5) in the last generation (fig. 1A) had severe CIIP associated with a short small intestine, malrotation, and hypertrophic pyloric stenosis.4Auricchio A Brancolini V Casari G Milla PJ Smith VV Devoto M Ballabio A The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28.Am J Hum Genet. 1996; 58: 743-748PubMed Google Scholar Subject IV-1 survived repeated surgery and is still alive. Histological analysis of full-thickness ileal and colonic biopsies of samples from the two subjects evidenced abnormal neurons in the myenteric and submucosal plexuses.4Auricchio A Brancolini V Casari G Milla PJ Smith VV Devoto M Ballabio A The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28.Am J Hum Genet. 1996; 58: 743-748PubMed Google Scholar Like the patients described by Tanner et al.,3Tanner MS Smith B Lloyd JK Functional intestinal obstruction due to deficiency of argyrophil neurones in the myenteric plexus: familial syndrome presenting with short small bowel, malrotation, and pyloric hypertrophy.Arch Dis Child. 1976; 51: 837-841Crossref PubMed Scopus (74) Google Scholar there were abnormalities of argyrophilic neurons. In our study family, the condition appeared to be inherited with a clear X-linked recessive pattern—that is, X-linked chronic idiopathic intestinal pseudo-obstruction (CIIPX)—and we mapped the disease locus to Xq28 between DXS15 and DXYS154.4Auricchio A Brancolini V Casari G Milla PJ Smith VV Devoto M Ballabio A The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28.Am J Hum Genet. 1996; 58: 743-748PubMed Google Scholar There was an additional report by FitzPatrick et al.5FitzPatrick DR Strain L Thomas AE Barr DG Todd A Smith NM Scobie WG Neurogenic chronic idiopathic intestinal pseudo-obstruction, patent ductus arteriosus, and thrombocytopenia segregating as an X linked recessive disorder.J Med Genet. 1997; 34: 666-669Crossref PubMed Scopus (24) Google Scholar about neurogenic CIIP with patent ductus arteriosus and large platelet thrombocytopenia, apparently inherited in an X-linked recessive manner. By April 2003, 63 genes were annotated in the CIIPX critical region (Human Genome Browser Gateway). We analyzed their expression in fetal murine gut and brain, to select candidate CIIPX genes, and we found a frameshift mutation in the filamin A gene (FLNA [MIM +300017]) that was associated with CIIPX in the Italian kindred. The index patient (IV-1), second cousin to subject IV-5, is a male and the first offspring of healthy, nonconsanguineous parents (fig. 1A). He was born at term after an uneventful pregnancy. On the 3rd d of life, he presented with bilious vomiting, and laparatomy showed a short small bowel with intestinal malrotation, pyloric hypertrophy, and an ileal volvulus. Fifteen days later, he required additional surgery for intestinal obstruction, and an ileostomy was raised. Histological examination of full-thickness ileal and colonic biopsies showed abnormal argyrophilic neurons in the myenteric plexus, as well as nerve fibers in the lamina propria in the colon.4Auricchio A Brancolini V Casari G Milla PJ Smith VV Devoto M Ballabio A The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28.Am J Hum Genet. 1996; 58: 743-748PubMed Google Scholar Findings similar to the myenteric plexus were present in the submucosal plexus. At age 3 mo, continuity of his bowel was restored with an ileoileal anastomosis, but the bowel once again became obstructed. Manometric studies suggested a neuropathic disorder of his gut. A further ileostomy was raised, and full-thickness material was taken for further histology. At age 3 years, a Lester-type ileorectal anastomosis was performed to restore bowel continuity. In addition to the severe CIIP, by age 7 years, an asymmetrical spastic diplegia with impairment of fine finger movements also became apparent. Magnetic resonance imaging (MRI) of his brain showed an abnormal intermediate signal in the peritrigonal white matter. The patient and two carrier females in the family declined to undergo additional brain MRI after the identification of the FLNA mutation. The patient required lengthening operations for both Achilles tendons. He also had seizures: the first time after surgery during his 1st mo of life, then again at ages 8 and 18 years. Since the last episode, he has received carbamazepine treatment. He continues to require supplemental parenteral nutrition to maintain good health. The second patient (IV-5) was a male, the third offspring of healthy, unrelated parents (fig. 1A). His older brother and sister have no gastrointestinal disorders and are completely well. He was born at term, weighed 4.2 kg, and measured 52 cm in length. On his 1st d of life, he had abdominal distension and bilious vomiting. Laparotomy demonstrated a small-intestinal malrotation with CIIP. Because of ongoing subocclusive symptoms, two intestinal resections were performed during the following weeks. He required total parenteral nutrition and an additional operation with ileoileal anastomosis. Intestinal histology of the ileum showed abnormalities of the myenteric and submucosal neurons similar to those of subject IV-1. Subject IV-5 died shortly after surgery, at age 8 mo. Genomic DNA was isolated from peripheral–whole-blood lymphocytes and from paraffin-embedded sections by use of standard protocols (Qiagen Italy) or as described elsewhere.4Auricchio A Brancolini V Casari G Milla PJ Smith VV Devoto M Ballabio A The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28.Am J Hum Genet. 1996; 58: 743-748PubMed Google Scholar The complete coding sequence of seven genes selected for direct sequencing, including the exon-intron boundaries, was amplified by PCR with Taq Gold DNA Polymerase (Roche). These genes are (from X chromosome centromere to telomere): ZNF275, ATP2B3 (MIM *300014), DUSP9 (MIM *300134), SLC6A8 (MIM *300036), ABCD1 (MIM *300371), L1CAM (MIM *308840), and FLNA. PCR was performed as follows: 35 cycles with 50 ng of genomic DNA at 94°C for 1 min, at the appropriate primer annealing temperature for 1 min, and at 72°C for 1 min. Primers and reaction conditions for PCR amplification are available on request. Amplicons were screened for mutations by direct sequencing with an ABI PRISM Big Dye terminator cycle sequencing kit, and the reactions were analyzed with an ABI PRISM 3100 genetic analyzer (Applied Biosystems). The sequenced exon and intron-exon boundaries were compared with consensus sequences obtained from the National Center for Biotechnology Information Database with use of standard software for DNA sequencing analysis (Autoassembler v. 2.1 [Applied Biosystems]). Sixty-six females and 32 males from the general Italian population were used as controls, for a total of 164 X chromosomes. Total RNA from C57BL/6 mice (Harlan Italy), from embryonic day–18 (E18) mouse brain or gut, from 5-wk-old mouse gut, and from V-1 individual lymphoblastoid cells was extracted using the Trizol reagent (Invitrogen Italy) according to the manufacturer's instructions. After DNAse treatment, 5 μg of total RNA was used as a template for cDNA synthesis, with use of random primers and SuperScript III (Invitrogen Italy). Subsequently, PCR amplification was performed using oligonucleotides specific for each of the 63 genes included in the CIIPX critical region and was designed in the region of homology between human and murine RNAs with use of the Oligo 4.0 software (National Biosciences). PCR was performed using Taq Gold DNA polymerase (Roche), with 35 cycles at 94°C for 1 min, at the appropriate primer annealing temperature for 1 min, and at 72°C for 1 min. Primer sequences and reaction conditions for each gene are available on request. We generated two plasmids that express the first N-terminal 124 aa of FLNA by fusing to a hemoagglutinin tag (HA) under the transcriptional control of the ubiquitous CMV promoter. In plasmid 1, the wild-type FLNA sequence was included, whereas, in plasmid 2, the FLNA sequence corresponding to c.65-66delAC was introduced. To do so, FLNA exon 2 sequence from the initial ATG codon to genome position 3688 was amplified from control and the patient's genomic DNA, with use of primers NotI, 5′-ATG AGT AGC TCC CAC TCT CGG GCG GGC CAG-3′, and BamHI-TTA-HA epitope tag, 5′-GAT GGA CAC CAG TTT GAT GCT CTC GCG GT-3′. The amplification was performed with Pfu DNA Polymerase (Promega Italy). The PCR product was inserted into PCR 2.1-TOPO plasmid (Invitrogen) and then was digested with NotI and BamHI restriction enzymes. The corresponding fragments from control and patient were then cloned into pAAV2.1-CMV-EGFP6Auricchio A Kobinger G Anand V Hildinger M O'Connor E Maguire AM Wilson JM Bennett J Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.Hum Mol Genet. 2001; 10: 3075-3081Crossref PubMed Scopus (302) Google Scholar by removing the EGFP-coding sequence, to obtain plasmid 1 and plasmid 2, respectively. Human embryonic kidney (HEK) 293 cells were plated in 6-well plates containing 3×105 cells/well. After 24 h, the cells were separately transfected with 2 μg/well of plasmid 1 and plasmid 2 with use of FUGENE 6 (Roche), according to the manufacturer's instruction. Transfection with pAAV2.1-CMV-EGFP was used as a control. After 48 h, proteins from transfected cells were extracted with Lysis buffer (50 mM NaCl, 25 mM Tris [pH 8.0], 0.5% NP40, 0.1% SDS, 1 mM protease inhibitors [Roche], and 1 mM phenylmethylsulphonyl fluoride) and were kept on ice for 20 min. Samples were spun at 13,000 rpm for 20 min, and supernatants were collected. Protein concentrations were determined with the Bio-Rad dye protein assay. The proteins were denatured by heating at 98°C for 3 min and were separated on a 16% SDS-polyacrylamide gel electrophoresis with 4% stacking gel in 1× Tris-glycine buffer (0.025 M Tris, 0.192 M glycine, and 0.1% SDS [pH 8.3]) in a miniprotean cell (Bio-Rad) at 130 volts for 2 h. The separated proteins were electrotransferred onto a polyvinylidene fluoride (PVDF) membrane with transfer buffer (25 mM Tris base, 0.2 M glycine, and 20% methanol [pH 8.5]) in a minitransfer cell (Bio-Rad) at 100 volts at 4°C for 1 h. Membranes were incubated at room temperature for 1.5 h in blocking buffer containing 1× TBS and 0.05% Tween 20 with 5% dried nonfat milk and then were probed with an anti-HA antibody (Sigma-Aldrich) for 2 h at room temperature. This was followed by incubation with a peroxidase-conjugated secondary anti-rabbit IgG for 1 h at room temperature. Signals were detected by chemoluminescence with the Pico Enhanced Chemiluminescence Kit (Pierce Chemical). A prestained molecular-weight ladder (Fermentas) was used to determine protein size. Lymphoblasts from patient IV-1 and from two control individuals were grown in Roswell Park Memorial Institute PMI 1640 plus medium with 20% fetal bovine serum (FBS) in 5% CO2 at 37°C, were harvested, precipitated, and lysed with Lysis buffer (50 mM NaCl, 25 mM Tris [pH 8.0], 0.5% NP40, 0.1% SDS, 1 mM protease inhibitors [Roche], and 1 mM PMSF) on ice for 20 min. Samples were spun at 13,000 rpm for 20 min, and supernatants were collected. Protein concentrations were determined with the Bio-Rad dye protein assay. The proteins were denatured by heating to 98°C for 3 min and then were separated on a 7% SDS-polyacrylamide gel electrophoresis with 4% stacking gel in 1× Tris-glycine buffer (0.025 M Tris, 0.192 M glycine, and 0.1% SDS [pH 8.3]) in a miniprotean cell (Bio-Rad). The separated proteins were electrotransferred onto PVDF membrane with a transfer buffer (25 mM Tris base, 0.2 M glycine, and 20% methanol [pH 8.5]) in a minitransfer cell (Bio-Rad) at 4°C for 1 h. Membranes were incubated overnight in blocking buffer containing 1× TBS and 0.1% Tween 20 with 5% dried nonfat milk and then were probed with anti–filamin A antibody (Cell Signaling) for 3 h at room temperature. This was followed by incubation with the peroxidase-conjugated secondary anti-rabbit IgG for 1 h at room temperature. Signals were detected by chemoluminescence through use of the Pico Enhanced Chemiluminescence Kit (Pierce Chemical). A prestained molecular-weight ladder (Fermentas) was used to determine protein size. Lymphoblasts were obtained from two unaffected subjects and from patient IV-1. Cells were cultured in serum-free RPMI 1640 medium plus 20% FBS (Invitrogen). Cells (∼3,000) were centrifuged with Cytospin Centrifuge for Cells Suspensions (Shandon Cytospin 3 Cytocentrifuge [Global Medical Instrumentation]) at 800 rpm for 3 min and were transferred to the corresponding glass slides. Cells were fixed with 3% paraformaldehyde (Sigma) for 10 min at room temperature. After one wash with 1× PBS (Invitrogen), Triton 0.2% (Bio-Rad) was applied to the slides, which were then incubated at room temperature for 5 min. After another wash with 1× PBS, slides were incubated for 45 min in the dark with phalloidin-Texred (Sigma) (diluted 1:500 in 1× PBS), with mouse antibodies directed to γ-tubulin (Sigma) (diluted 1:50 in 1× PBS), or with rabbit antibodies to filamin A (Cell Signalling) (diluted 1:250 in 1× PBS). Fluorochromes can directly bind the phalloidin, which in turn links F-actin, revealing F-actin without antibodies. Secondary anti-mouse and anti-rabbit antibodies (Invitrogen) conjugated to rhodamine were used at concentrations of 1:1000 and 1:200 dilutions, respectively, in 1× PBS. Slides were covered with Mowiol gel (Calbiochem) and then were analyzed by fluorescence microscopy (Laser Scanner Microscopy 510 Zeiss). To identify the gene mutated in CIIPX, we analyzed, by reverse transcriptase, the expression pattern of the genes from the CIIPX critical region on Xq28 in c57/BL6 fetal (E18) brain and gut. Of the 63 genes located in the CIIPX critical region, 56 are homologous in mouse and human and therefore were analyzed. We found that 7 of 56 genes are expressed only in intestine, 13 are expressed only in brain, and 28 are expressed in both intestine and brain. Eight genes are not expressed in either tissue but are expressed in lymphoblastoid cell lines (data not shown). We selected seven genes (ZNF275, ATP2B3, DUSP9, SLC6A8,ABCD1, L1CAM, and FLNA) from those expressed in both tissues for direct sequencing analysis of exon and intron-exon boundaries. ABCD1, L1CAM, SLC6A8, and FLNA were selected as candidate genes because of their involvement in already-known inherited disorders of the nervous system7Sarde CO Mosser J Kioschis P Kretz C Vicaire S Aubourg P Poustka A Mandel JL Genomic organization of the adrenoleukodystrophy gene.Genomics. 1994; 22: 13-20Crossref PubMed Scopus (78) Google Scholar, 8Jouet M Rosenthal A Armstrong G MacFarlane J Stevenson R Paterson J Metzenberg A Ionasescu V Temple K Kenwrick S X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from mutations in the L1 gene.Nat Genet. 1994; 7: 402-407Crossref PubMed Scopus (354) Google Scholar, 9Hahn KA Salomons GS Tackels-Horne D Wood TC Taylor HA Schroer RJ Lubs HA Jakobs C Olson RL Holden KR et al.X-linked mental retardation with seizures and carrier manifestations is caused by a mutation in the creatine-transporter gene (SLC6A8) located in Xq28.Am J Hum Genet. 2002; 70: 1349-1356Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 10Robertson SP Twigg SR Sutherland-Smith AJ Biancalana V Gorlin RJ Horn D Kenwrick SJ Kim CA Morava E Newbury-Ecob R et al.Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans.Nat Genet. 2003; 33: 487-491Crossref PubMed Scopus (315) Google Scholar; ATP2B3 is a plasma-membrane protein possibly involved in the regulation of physiological ions homeostasis11Brown BJ Hilfiker H DeMarco SJ Zacharias DA Greenwood TM Guerini D Strehler EE Primary structure of human plasma membrane Ca2+-ATPase isoform 3.Biochim Biophys Acta. 1996; 1283: 10-13Crossref PubMed Scopus (18) Google Scholar; DUSP9 was selected because of its protein's interactions with members of the extracellular signal-regulated kinase family of mitogen-activated protein kinases, its possible involvement in regulation of gene expression in neurons of neuroenteric system, and its contribution to pain caused by inflammation12Ji RR Befort K Brenner GJ Woolf CJ ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity.J Neurosci. 2002; 22: 478-485Crossref PubMed Google Scholar; finally, ZNF275, because of its protein's high similarity to a Zinc-finger protein, was selected as a possible uncharacterized novel putative transcriptional activator.13Mallon AM Platzer M Bate R Gloeckner G Botcherby MR Nordsiek G Strivens MA Kioschis P Dangel A Cunningham D et al.Comparative genome sequence analysis of the Bpa/Str region in mouse and man.Genome Res. 2000; 10: 758-775Crossref PubMed Scopus (44) Google Scholar We found that the index patient (IV-1) bears a 2-bp deletion in exon 2 of FLNA (c.65-66delAC). Segregation analysis of the FLNA mutation confirms that all obligate carriers, by pedigree or established by linkage analysis,4Auricchio A Brancolini V Casari G Milla PJ Smith VV Devoto M Ballabio A The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28.Am J Hum Genet. 1996; 58: 743-748PubMed Google Scholar are heterozygous for the 2-bp deletion (fig. 1B). The mutation is absent in 164 control X chromosomes. Recently, a wide spectrum of developmental anomalies has been shown to be caused by mutations in FLNA.14Robertson SP Filamin A: phenotypic diversity.Curr Opin Genet Dev. 2005; 15: 301-307Crossref PubMed Scopus (153) Google Scholar Null mutations in the FLNA gene result in bilateral periventricular nodular heterotopia (PVNH [MIM #300049]), an X-linked dominant neuronal migration disorder clinically characterized by seizures.15Fox JW Lamperti ED Eksioglu YZ Hong SE Feng Y Graham DA Scheffer IE Dobyns WB Hirsch BA Radtke RA et al.Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia.Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar The association between a potential severe loss-of-function FLNA mutation and an X-linked recessive disease involving enteric neuron development prompted us to investigate the effect of FLNA c.65-66delAC at the protein level. The c.65-66delAC deletion found in patients with CIIPX is predicted to cause a frameshift and a premature stop codon at filamin A amino acid position 103 (fig. 2A). The predicted mutant protein retains the initial 22 aa identical to wild-type filamin A, followed by 81 different aa before a premature stop codon. This is the most severe FLNA loss-of-function mutation described to date,16Parrini E Ramazzotti A Dobyns WB Mei D Moro F Veggiotti P Marini C Brilstra EH Dalla Bernardina B Goodwin L et al.Periventricular heterotopia: phenotypic heterogeneity and correlation with filamin A mutations.Brain. 2006; 129: 1892-1906Crossref PubMed Scopus (243) Google Scholar predicted to cause lethality in males and PVNH phenotypes in females. Interestingly, the c.65-66delAC deletion is located 22 codons downstream of the initial FLNA ATG (Met1) (fig. 2A) and 5 codons upstream of a second ATG (Met2) (fig. 2A). If filamin A translation occurs from either methionine and results in the synthesis of two proteins differing in 27 aa at the NH2 terminus, the c.65-66delAC deletion would affect only the translation of the longer form of filamin A. To determine whether this is the case, we transiently transfected HEK 293 cells with two different eukaryotic expression plasmids containing the CMV promoter, as shown in figure 2B. In both plasmids, the FLNA-coding sequence from the first methionine to amino acid residue 124 (corresponding to the end of exon 2) was fused to an HA peptide at its 3′ end, resulting in peptides of 124 aa of predicted length if translation occurs from Met1 or of 96 aa if it occurs from Met2. Whereas the wild-type FLNA sequence was included in plasmid 1, that bearing the c.65-66delAC deletion was present in plasmid 2. As shown in figure 2C, cells transfected with plasmid 1 express both filamin A forms, whereas those transfected with plasmid 2 express only the shorter form of the protein, albeit at lower levels, possibly because of nonsense-mediated decay. This suggests that (1) filamin A translation can start from either of its two initial methionines, (2) translation of the shorter filamin A form alone occurs because of the c.65-66delAC deletion. To confirm this, we analyzed endogenous filamin A expression in control and patient IV-1 lymphoblasts. Western blot with anti–filamin A antibodies shows that filamin A expression in the patient's lymphoblasts is similar to expression in control lymphoblasts (fig. 3A). Although, because of filamin A's high molecular weight (280 kDa), we cannot discriminate whether the protein present in the patient's lymphoblasts is of lower molecular weight than that in control lymphoblasts, these data confirm that c.65-66delAC results in filamin A translation despite the early frameshift. Finally, we investigated the impact of c.65-66delAC on cytoskeletal organization in the patient's lymphoblasts. Whereas actin immunofluorescence staining with phalloidin-Texred is predominantly diffused in the cytoplasm in lymphoblasts from healthy control individuals, in the cells derived from the patient, actin appears mainly concentrated in large dots localized on the centrosome side of the cells (fig. 4; the percentage of cells containing phalloidin dots in patient vs. control lymphoblasts was 78% vs. 9.4%). We confirmed, by immunofluorescence, the presence of filamin A in the patient's lymphoblasts and that it has a distribution similar to that of actin (fig. 5).Figure 3Western blot analysis of filamin A expression in patient IV-1 lymphoblasts. The amount of total protein loaded for each individual is shown above the lanes. Protein molecular weight is shown on the left. The primary antibodies used are shown on the right.View Large Image Figure
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