Tissue-Specific Reduction in Splicing Efficiency of IKBKAP Due to the Major Mutation Associated with Familial Dysautonomia
2003; Elsevier BV; Volume: 72; Issue: 3 Linguagem: Inglês
10.1086/368263
ISSN1537-6605
AutoresMath P. Cuajungco, Maire Leyne, James Mull, Sandra Gill, Lu W, David Zagzag, Felicia B. Axelrod, Channa Maayan, James F. Gusella, Susan A. Slaugenhaupt,
Tópico(s)Nuclear Structure and Function
ResumoWe recently identified a mutation in the I-κB kinase associated protein (IKBKAP) gene as the major cause of familial dysautonomia (FD), a recessive sensory and autonomic neuropathy. This alteration, located at base pair 6 of the intron 20 donor splice site, is present on >99.5% of FD chromosomes and results in tissue-specific skipping of exon 20. A second FD mutation, a missense change in exon 19 (R696P), was seen in only four patients heterozygous for the major mutation. Here, we have further characterized the consequences of the major mutation by examining the ratio of wild-type to mutant (WT:MU) IKBKAP transcript in EBV-transformed lymphoblast lines, primary fibroblasts, freshly collected blood samples, and postmortem tissues from patients with FD. We consistently found that WT IKBKAP transcripts were present, albeit to varying extents, in all cell lines, blood, and postmortem FD tissues. Further, a corresponding decrease in the level of WT protein is seen in FD cell lines and tissues. The WT:MU ratio in cultured lymphoblasts varied with growth phase but not with serum concentration or inclusion of antibiotics. Using both densitometry and real-time quantitative polymerase chain reaction, we found that relative WT:MU IKBKAP RNA levels were highest in cultured patient lymphoblasts and lowest in postmortem central and peripheral nervous tissues. These observations suggest that the relative inefficiency of WT IKBKAP mRNA production from the mutant alleles in the nervous system underlies the selective degeneration of sensory and autonomic neurons in FD.Therefore, exploration of methods to increase the WT:MU IKBKAP transcript ratio in the nervous system offers a promising approach for developing an effective therapy for patients with FD. We recently identified a mutation in the I-κB kinase associated protein (IKBKAP) gene as the major cause of familial dysautonomia (FD), a recessive sensory and autonomic neuropathy. This alteration, located at base pair 6 of the intron 20 donor splice site, is present on >99.5% of FD chromosomes and results in tissue-specific skipping of exon 20. A second FD mutation, a missense change in exon 19 (R696P), was seen in only four patients heterozygous for the major mutation. Here, we have further characterized the consequences of the major mutation by examining the ratio of wild-type to mutant (WT:MU) IKBKAP transcript in EBV-transformed lymphoblast lines, primary fibroblasts, freshly collected blood samples, and postmortem tissues from patients with FD. We consistently found that WT IKBKAP transcripts were present, albeit to varying extents, in all cell lines, blood, and postmortem FD tissues. Further, a corresponding decrease in the level of WT protein is seen in FD cell lines and tissues. The WT:MU ratio in cultured lymphoblasts varied with growth phase but not with serum concentration or inclusion of antibiotics. Using both densitometry and real-time quantitative polymerase chain reaction, we found that relative WT:MU IKBKAP RNA levels were highest in cultured patient lymphoblasts and lowest in postmortem central and peripheral nervous tissues. These observations suggest that the relative inefficiency of WT IKBKAP mRNA production from the mutant alleles in the nervous system underlies the selective degeneration of sensory and autonomic neurons in FD.Therefore, exploration of methods to increase the WT:MU IKBKAP transcript ratio in the nervous system offers a promising approach for developing an effective therapy for patients with FD. Familial dysautonomia (FD, Riley-Day syndrome, hereditary sensory, and autonomic neuropathy type III [MIM 223900]) is an autosomal recessive disease that affects the sensory and autonomic nervous systems. FD has a remarkably high carrier frequency of 1 in 32 in the Ashkenazi Jewish population (Dong et al. Dong et al., 2002Dong J Edelmann L Bajwa AM Kornreich R Desnick RJ Familial dysautonomia: detection of the IKBKAP IVS20+6T→C and R696P mutations and frequencies among Ashkenazi Jews.Am J Med Genet. 2002; 110: 253-257Crossref PubMed Scopus (50) Google Scholar). In FD, there is progressive depletion of unmyelinated sensory and autonomic neurons (Pearson and Pytel Pearson and Pytel, 1978aPearson J Pytel BA Quantitative studies of ciliary and sphenopalatine ganglia in familial dysautonomia.J Neurol Sci. 1978a; 39: 123-130Abstract Full Text PDF PubMed Scopus (53) Google Scholar, Pearson and Pytel, 1978bPearson J Pytel BA Quantitative studies of sympathetic ganglia and spinal cord intermedio-lateral gray columns in familial dysautonomia.J Neurol Sci. 1978b; 39: 47-59Abstract Full Text PDF PubMed Scopus (103) Google Scholar; Pearson et al. 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Riley et al., 1949Riley CM Day RL Greely D Langford WS Central autonomic dysfunction with defective lacrimation.Pediatrics. 1949; 3: 468-477PubMed Google Scholar; Axelrod et al. Axelrod et al., 1974Axelrod FB Nachtigal R Dancis J Familial dysautonomia: diagnosis, pathogenesis and management.Adv Pediatr. 1974; 21: 75-96PubMed Google Scholar; Axelrod Axelrod, 1984Axelrod FB Familial dysautonomia and other congenital and sensory autonomic neuropathies.in: Blake IB Cell and molecular biology of neuronal development. Plenum Press, New York1984: 331-340Crossref Google Scholar). Recently, we and others identified a single noncoding mutation in the I-κB kinase–associated protein (IKBKAP) gene that accounts for >99.5% of all cases of FD (Anderson et al. Anderson et al., 2001Anderson SL Coli R Daly IW Kichula EA Rork MJ Volpi SA Ekstein J Rubin BY Familial dysautonomia is caused by mutations of the IKAP gene.Am J Hum Genet. 2001; 68: 753-758Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar; Slaugenhaupt et al. Slaugenhaupt et al., 2001Slaugenhaupt SA Blumenfeld A Gill SP Leyne M Mull J Cuajungco MP Liebert CB Chadwick B Idelson M Reznik L Robbins CM Makalowska I Brownstein MJ Krappmann D Scheidereit C Maayan C Axelrod FB Gusella JF Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.Am J Hum Genet. 2001; 68: 598-605Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). This major IKBKAP mutation is a single-base change at base pair 6 of the donor splice site of intron 20 (IVS20+6T→C). We found that this mutation causes a decrease in splicing efficiency with sporadic skipping of exon 20, thereby reducing the level of WT IKBKAP mRNA in patient cells (Slaugenhaupt et al. Slaugenhaupt et al., 2001Slaugenhaupt SA Blumenfeld A Gill SP Leyne M Mull J Cuajungco MP Liebert CB Chadwick B Idelson M Reznik L Robbins CM Makalowska I Brownstein MJ Krappmann D Scheidereit C Maayan C Axelrod FB Gusella JF Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.Am J Hum Genet. 2001; 68: 598-605Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). By contrast, data presented by Anderson et al. (Anderson et al., 2001Anderson SL Coli R Daly IW Kichula EA Rork MJ Volpi SA Ekstein J Rubin BY Familial dysautonomia is caused by mutations of the IKAP gene.Am J Hum Genet. 2001; 68: 753-758Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) suggested complete elimination of WT IKBKAP mRNA in lymphoblast cells of patients with FD. The only other mutation found in patients with FD to date is a rare missense alteration (R696P), existing only in four patients heterozygous for the major splicing mutation. It is located in exon 19 and is predicted to disrupt a phosphorylation site in I-κB–associated protein (IKAP), the IKBKAP gene product. IKAP is a conserved protein found in mouse, fruitfly, mustard plant, nematode, and yeast (Cuajungco et al. Cuajungco et al., 2001Cuajungco MP Leyne M Mull J Gill SP Gusella JF Slaugenhaupt SA Cloning, characterization, and genomic structure of the mouse Ikbkap gene.DNA Cell Biol. 2001; 20: 579-586Crossref PubMed Scopus (22) Google Scholar). It was initially reported as a scaffold protein for the I-κB kinase (IKK) complex (Cohen et al. Cohen et al., 1998Cohen L Henzel WJ Baeuerle PA IKAP is a scaffold protein of the Iκ-B kinase complex.Nature. 1998; 395: 292-297Crossref PubMed Scopus (264) Google Scholar), but this function was subsequently contested by Krappmann et al. (Krappmann et al., 2000Krappmann D Hatada EH Tegethoff S Li J Klippel A Giese K Baeuerle PA Scheidereit C The Ik-B kinase (IKK) complex tripartite and contains IKKγ but not IKAP as a regular component.J Biol Chem. 2000; 275: 29779-29787Crossref PubMed Scopus (103) Google Scholar), who reported that IKAP was, in fact, a member of a different cellular complex. IKAP is now recognized as the largest member of the three-subunit protein complex called “core Elongator,” which was originally identified in yeast and, more recently, in humans (Otero et al. Otero et al., 1999Otero G Fellows J Li Y de Bizemont T Dirac AM Gustafsson CM Erdjument-Bromage H Tempst P Svejstrup JQ Elongator, a multisubunit component of a novel RNA polymerase II holoenzyme for transcriptional elongation.Mol Cell. 1999; 3: 109-118Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar; Wittschieben et al. Wittschieben et al., 1999Wittschieben BO Otero G de Bizemont T Fellows J Erdjument-Bromage H Ohba R Li Y Allis CD Tempst P Svejstrup JQ A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme.Mol Cell. 1999; 4: 123-128Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar; Fellows et al. 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Studies in yeast and human have revealed that core Elongator interacts with another three-subunit protein to form the six-subunit holo-Elongator complex (Krogan and Greenblatt Krogan and Greenblatt, 2001Krogan NJ Greenblatt JF Characterization of a six-subunit holo-elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae..Mol Cell Biol. 2001; 21: 8203-8212Crossref PubMed Scopus (127) Google Scholar; Li et al. Li et al., 2001Li Y Takagi Y Jiang Y Tokunaga M Erdjument-Bromage H Tempst P Kornberg RD A multiprotein complex that interacts with RNA polymerase II elongator.J Biol Chem. 2001; 276: 29628-29631Crossref PubMed Scopus (57) Google Scholar; Winkler et al. 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Otero et al., 1999Otero G Fellows J Li Y de Bizemont T Dirac AM Gustafsson CM Erdjument-Bromage H Tempst P Svejstrup JQ Elongator, a multisubunit component of a novel RNA polymerase II holoenzyme for transcriptional elongation.Mol Cell. 1999; 3: 109-118Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar; Winkler et al. Winkler et al., 2001Winkler GS Petrakis TG Ethelberg S Tokunaga M Erdjument-Bromage H Tempst P Svejstrup JQ RNA polymerase II elongator holoenzyme is composed of two discrete subcomplexes.J Biol Chem. 2001; 276: 32743-32749Crossref PubMed Scopus (132) Google Scholar). Using purified human core Elongator complex from HeLa cell extracts, Kim and colleagues (2002) demonstrated that core Elongator indeed facilitates transcription by RNA polymerase II in a chromatin- and acetyl-coA-dependent manner. However, recent findings in yeast have called previous observations into question. For example, when chromatin immunoprecipitation assay was used, no evidence was observed to suggest that yeast Elongator is directly involved in transcription elongation in vivo (Pokholok et al. Pokholok et al., 2002Pokholok DK Hannett NM Young RA Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo.Mol Cell. 2002; 9: 799-809Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) or in vitro (Krogan et al. Krogan et al., 2002Krogan NJ, Kim M, Ahn SH, Zhong G, Kobor MS, Cagney G, Emili A, Shilatifard A, Buratowski S, Greenblatt JF (2002) RNA polymerase II elongation factors of Saccharomyces cer-evisiae: a targeted proteomics approach. Mol Cell Biol 22: 6979-6992Google Scholar). Likewise, using tandem affinity purification, Krogan et al. (Krogan et al., 2002Krogan NJ, Kim M, Ahn SH, Zhong G, Kobor MS, Cagney G, Emili A, Shilatifard A, Buratowski S, Greenblatt JF (2002) RNA polymerase II elongation factors of Saccharomyces cer-evisiae: a targeted proteomics approach. Mol Cell Biol 22: 6979-6992Google Scholar) failed to copurify yeast Elongator with RNA polymerase II, even at microgram quantities. It is interesting that immunofluorescence localization studies show that each core Elongator subunit, including IKAP, is primarily detected in the cytoplasm of human (Krappmann et al. Krappmann et al., 2000Krappmann D Hatada EH Tegethoff S Li J Klippel A Giese K Baeuerle PA Scheidereit C The Ik-B kinase (IKK) complex tripartite and contains IKKγ but not IKAP as a regular component.J Biol Chem. 2000; 275: 29779-29787Crossref PubMed Scopus (103) Google Scholar; Hawkes et al. 2002; Holmberg et al. 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(Holmberg et al., 2002Holmberg C Katz S Lerdrup M Herdegen T Jaattela M Aronheim A Kallunki T A novel specific role for I-κ B kinase complex-associated protein in cytosolic stress signaling.J Biol Chem. 2002; 277: 31918-31928Crossref PubMed Scopus (94) Google Scholar) reported a novel scaffolding role for IKAP in the c-Jun N-terminal kinase (JNK) signal transduction pathway, owing to its discerned strong physical interaction with JNK and enhancement of JNK activation. These studies suggest that IKAP may indeed have multiple roles in the cell, and it remains to be determined how a decrease in cellular IKBKAP causes the FD phenotype and why this phenotype displays tissue specificity. The regulation of gene splicing (reviewed by Nissim-Rafinia and Kerem [Nissim-Rafinia and Kerem, 2002Nissim-Rafinia M Kerem B Splicing regulation as a potential genetic modifier.Trends Genet. 2002; 18: 123-127Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar]) can potentially play an important role in inherited disorders, influencing phenotypic parameters such as organ specificity, disease severity, and age at onset. Splicing mutations that result in exon skipping have been reported for many disorders, including adrenoleukodystrophy, ataxia telangiectasia, Marfan syndrome, cystic fibrosis, retinoblastoma, tuberous sclerosis, and neurofibromatosis (Mayer et al. Mayer et al., 2000Mayer K Ballhausen W Leistner W Rott H Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences.Biochim Biophys Acta. 2000; 1502: 495-507Crossref PubMed Scopus (38) Google Scholar; Pagani et al. 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The remarkable features of FD are that a single splicing mutation is responsible for virtually all cases of the disease and that, in our hands, homozygous mutant cultured cells are capable of producing substantial amounts of WT IKBKAP mRNA and protein (Slaugenhaupt et al. Slaugenhaupt et al., 2001Slaugenhaupt SA Blumenfeld A Gill SP Leyne M Mull J Cuajungco MP Liebert CB Chadwick B Idelson M Reznik L Robbins CM Makalowska I Brownstein MJ Krappmann D Scheidereit C Maayan C Axelrod FB Gusella JF Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.Am J Hum Genet. 2001; 68: 598-605Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). These observations suggest that manipulation of splicing mechanisms could offer a route to treating FD and other human disorders involving splicing defects. In view of the differences in the reported effects of the major FD mutation on splicing of exon 20 (Anderson et al. Anderson et al., 2001Anderson SL Coli R Daly IW Kichula EA Rork MJ Volpi SA Ekstein J Rubin BY Familial dysautonomia is caused by mutations of the IKAP gene.Am J Hum Genet. 2001; 68: 753-758Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar; Slaugenhaupt et al. Slaugenhaupt et al., 2001Slaugenhaupt SA Blumenfeld A Gill SP Leyne M Mull J Cuajungco MP Liebert CB Chadwick B Idelson M Reznik L Robbins CM Makalowska I Brownstein MJ Krappmann D Scheidereit C Maayan C Axelrod FB Gusella JF Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.Am J Hum Genet. 2001; 68: 598-605Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar), we have characterized the defective IKBKAP splicing using both densitometry and real-time quantitative PCR assays in patient cell lines, freshly collected blood, and FD postmortem tissues. We collected patient blood samples through the Dysautonomia Diagnostic and Treatment Center at New York University Medical Center, with approval from by the Institutional Review Boards of New York University Medical Center, Massachusetts General Hospital, and Harvard Medical School. For each patient, total RNA was extracted, and a lymphoblast cell line was initiated. We also used previously generated patient lymphoblast and fibroblast lines, as well as additional FD cell lines, purchased from the Coriell Mutant Cell Repository, for comparative experiments. All cultured cells were propagated in a standard 37°C incubator at 85% humidity and 5% CO2, with lymphoblast lines grown in RPMI-1640 and primary fibroblasts in Minimum Essential Medium with Earle’s balanced salts. Both media were supplemented with 2 mM L-glutamine and either 10% (GUS lines), 15% (GM lines), or 20% (MIN lines) fetal bovine serum (FBS) (Invitrogen). In some experiments, lymphoblast lines were grown with or without 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Postmortem tissues were obtained from the Pathology Department at New York University Medical Center and the University of Miami Brain and Tissue Bank for Developmental Disorder. Total RNA was extracted using Tri-Reagent for all tissues and cultured cell samples, or Tri-Reagent BD for blood (Molecular Research Center), and quantitated by UV absorbance. Reverse-transcription reactions using oligo-dT15/random hexamer primers (Promega), Superscript II reverse transcriptase (200 U/μL; Invitrogen) and RNase-inhibitor (80 U/μL; Roche) were performed as described (Cuajungco et al. Cuajungco et al., 2001Cuajungco MP Leyne M Mull J Gill SP Gusella JF Slaugenhaupt SA Cloning, characterization, and genomic structure of the mouse Ikbkap gene.DNA Cell Biol. 2001; 20: 579-586Crossref PubMed Scopus (22) Google Scholar). PCR was done on all samples to evaluate both WT and MU transcripts (fig. 1). The PCR products were fractionated by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Each gel band was assessed using an Alpha 2000 Image Analyzer and software coupled with automatic background subtraction (Alpha Innotech). Subsequent analysis of the integrated density value (IDV) to calculate the WT:MU transcript ratio enabled us to obtain the relative differences in expression within each patient tissue or cell samples. To validate our densitometry findings, we used real-time quantitative PCR (QPCR). For QPCR, the 18S ribosomal RNA (Applied Biosystems) was used as an internal normalization control to correct for variations in input RNA amount or reverse transcription efficiency. The 18S rRNA is known for its robustness and stable expression across different tissues and cell lines (Schmittgen and Zakrajsek Schmittgen and Zakrajsek, 2000Schmittgen TD Zakrajsek BA Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.J Biochem Biophys Methods. 2000; 46: 69-81Crossref PubMed Scopus (965) Google Scholar; Goidin et al. Goidin et al., 2001Goidin D Mamessier A Staquet MJ Schmitt D Berthier-Vergnes O Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and β-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations.Anal Biochem. 2001; 295: 17-21Crossref PubMed Scopus (322) Google Scholar). We used a One-Step RT-Platinum Taq kit (Invitrogen), optimized to 5 mM Mg++, with 500 nM forward and reverse primers, 400 nM TaqMan probe, and 200 ng of total RNA sample. QPCR was done on a BioRad thermal ICycler with the following PCR program: 1 cycle at 50°C for 15 min; 1 cycle at 95°C for 5 min; and 45 cycles at 95°C for 15 sec, and at 59 °C for 1 min. All reactions were carried out in triplicate in a 96-well plate format, with a final volume of 50 μL. Trials for each WT, MU, or 18S assay included four duplicated tissue samples and “no RT” controls. For relative gene-expression analysis, we used the standard curve method (generated from the same serial dilutions of MIN9741 lymphoblast sample used in every plate run) to correct for minor differences in PCR efficiency (Heid et al. Heid et al., 1996Heid CA Stevens J Livak KJ Williams PM Real time quantitative PCR.Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (4808) Google Scholar; Winer et al. Winer et al., 1999Winer J Jung CK Shackel I Williams PM Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.Anal Biochem. 1999; 270: 41-49Crossref PubMed Scopus (1171) Google Scholar; Giulietti et al. Giulietti et al., 2001Giulietti A Overbergh L Valckx D Decallonne B Bouillon R Mathieu C An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.Methods. 2001; 25: 386-401Crossref PubMed Scopus (1046) Google Scholar). QPCR values were expressed relative to an FD spleen sample that showed virtually equal WT and MU IKBKAP expression and was included as a calibrator in every experimental run. For protein analysis, available FD postmortem tissues and cell lines were homogenized and total protein quantitated by use of bicinchoninic assay (Pierce). For each sample, 400 μg of total protein was run on a 6% denaturing polyacrylamide gel and Western blotted. The blots were probed using an anti-IKAP C-terminal polyclonal antibody (a kind gift from Dr. Jesper Svejstrup and Dr. Claus Schedereit) and the protein detected by ECL (Amersham), as described elsewhere (Slaugenhaupt et al. Slaugenhaupt et al., 2001Slaugenhaupt SA Blumenfeld A Gill SP Leyne M Mull J Cuajungco MP Liebert CB Chadwick B Idelson M Reznik L Robbins CM Makalowska I Brownstein MJ Krappmann D Scheidereit C Maayan C Axelrod FB Gusella JF Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.Am J Hum Genet. 2001; 68: 598-605Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). The polyvinylidene fluoride membrane was stained for total protein, using 0.2% Ponceau-S (Sigma) to confirm equal loading and transfer. Our original report (Slaugenhaupt et al. Slaugenhaupt et al., 2001Slaugenhaupt SA Blumenfeld A Gill SP Leyne M Mull J Cuajungco MP Liebert CB Chadwick B Idelson M Reznik L Robbins CM Makalowska I Brownstein MJ Krappmann D Scheidereit C Maayan C Axelrod FB Gusella JF Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.Am J Hum Genet. 2001; 68: 598-605Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar) suggested that lymphoblast cell lines from patients with FD were capable of expressing WT IKBKAP transcript from the mutant FD allele. These data contrasted with the absence of WT transcript in FD lymphoblast cells in the report of Anderson et al. (Anderson et al., 2001Anderson SL Coli R Daly IW Kichula EA Rork MJ Volpi SA Ekstein J Rubin BY Familial dysautonomia is caused by mutations of the IKAP gene.Am J Hum Genet. 2001; 68: 753-758Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). To address this discrepancy, we obtained the GM5106 FD cell line used by Anderson et al. from the Coriell Mutant Cell Repository and compared it with a frozen GM5106 stock already present in our laboratory, as well as with independent lymphoblast cell lines from other patients homozygous for the major mutation. We employed densitometry as a rapid assay to characterize the relative WT:MU transcript ratio of 81 FD lymphoblast cell lines. Of these samples, we consistently found significant WT IKBKAP mRNA in all FD cell lines examined (fig. 2A), including that of the patient sample tested by Anderson et al. (Anderson et al., 2001Anderson SL Coli R Daly IW Kichula EA Rork MJ Volpi SA Ekstein J Rubin BY Familial dysautonomia is caused by mutations of the IKAP gene.Am J Hum Genet. 2001; 68: 753-758Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) (see lane 6 and lane 7 in fig. 2A). To assess whether the efficiency of accurate IKBKAP splicing might vary over time, we used four independent lymphoblast cell lines (MIN9741, MIN9744, MIN9745, and MIN9754) from patients with FD who were homozygous for the major mutation. The cells were cultured and assayed weekly for 14 wk (fed every 3 d and harvested on the 7th d at saturation density of ∼20 million cells). The cell lines did not differ significantly in their overall average WT:MU IDV ratio for the entire culture period (table 1). However, the ratio did show significant fluctuation (P<.0001), over a twofold
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