IKBKAP mRNA in Peripheral Blood Leukocytes: A Molecular Marker of Gene Expression and Splicing in Familial Dysautonomia

Simson, Gabrielle Gold‐von; Leyne, Maire; Mull, James; Rolnitzky, Linda; Goldberg, Judith D.; Berlin, Dena; Axelrod, Felicia B.; Slaugenhaupt, Susan A.

doi:10.1203/pdr.0b013e31815ef74b

Cited by 10 publications

(7 citation statements)

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“…This is the first demonstration of in vivo modification of IKBKAP splicing. The measurement of exon 20 inclusion as a direct marker for splicing alteration has been proven (9,12,13). The misspliced message is targeted for NMD, however, our previous molecular studies have shown that blocking NMD does not alter the effect of kinetin, which targets mRNA splicing directly due to the presence of a specific "kinetin response element" at the end of IKBKAP exon 20 (12,13).…”

Section: Discussionmentioning

confidence: 99%

“…The alternative splicing of IKBKAP in FD is also tissue specific, and the most profound aberrant splicing (that leads to overproduction of mutant IKBKAP mRNA and low levels of functional protein) occurs in neuronal tissue (5)(6)(7)(8). Although carriers seem to be clinically asymptomatic, they demonstrate some degree of reduced splicing efficiency (9), and may have subclinical neurophysiologic manifestations that have not yet been described.…”

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confidence: 99%

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Kinetin in Familial Dysautonomia Carriers: Implications for a New Therapeutic Strategy Targeting mRNA Splicing

et al. 2009

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Familial dysautonomia (FD) is caused by an intronic splice mutation in the IkappaB kinase-associated protein gene (IKBKAP) that leads to partial skipping of exon 20 and tissue-specific reduction of IkappaB kinase-associated protein/elongator protein 1 (IKAP/ELP-1 protein). Kinetin increases IKBKAP mRNA and protein expression in FD cell lines. To determine whether oral kinetin alters IKBKAP splicing in vivo, we administered kinetin to 29 healthy carriers of the major FD mutation for 8 d. Adverse effects, kinetin, and IKBKAP mRNA levels were monitored. In the highest dosing cohorts (23.5 mg/kg/d), the target plasma kinetin level was achieved in 91% of subjects at 2 h. After 8 d, IKBKAP mRNA expression in leukocytes increased as kinetin levels increased. There is a linear association between log plasma kinetin level and corresponding log change from baseline in IKBKAP mRNA expression that allows estimation of IKBKAP mRNA levels because of kinetin ingestion. Adverse effects were transient and mild. This is the first report of in vivo IKBKAP splicing modification and strongly suggests kinetin's therapeutic potential in FD and perhaps in other splicing disorders. Furthermore, our findings support our hypothesis that treatments, which target a particular splicing mutation, can be successfully developed.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Kinetin in Familial Dysautonomia Carriers: Implications for a New Therapeutic Strategy Targeting mRNA Splicing

et al. 2009

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“…As previously described (13, 14), RNA extraction and quantification were performed and the relative amount of wild-type IKBKAP was measured using semi quantitative RT-PCR. Briefly, the relative amount of spliced transcripts were determined using an Alpha 2000 Imager Analyzer (BioRad, Hercules, CA) and Image Quant QL (Amersham, Piscataway, NJ) software, using the integrated density value for each band.…”

Section: Methodsmentioning

confidence: 99%

Kinetin Improves IKBKAP mRNA Splicing in Patients With Familial Dysautonomia

et al. 2011

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Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/day for 28 days. An increase in wild-type IKBKAP mRNA expression in leukocytes was noted after eight days in six of eight individuals; after 28 days the mean increase as compared to baseline was significant (p=0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients, but also that effect appears to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine if kinetin will prove therapeutic in FD patients.

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“…Interestingly, they were also able to show that kinetin treatment led to a higher percentage of differentiating neurons and an increase in expression of key peripheral neuronal genes, demonstrating positive down-stream effects of increased IKAP production. Further, our clinical studies have shown that oral administration of kinetin to adult FD carriers improves IKBKAP splicing in blood and that these changes in IKBKAP splicing are dose dependent (37,43). Kinetin activity has further been confirmed in a new model system where it improved exon 20 inclusion and restored IKAP levels in olfactory ecto-mesenchymal stem cells derived from FD patients (44).…”

Section: Discussionmentioning

confidence: 85%

Specific correction of a splice defect in brain by nutritional supplementation

Shetty

Gallagher

Chen

et al. 2011

Human Molecular Genetics

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Recent studies emphasize the importance of mRNA splicing in human genetic disease, as 20-30% of all disease-causing mutations are predicted to result in mRNA splicing defects. The plasticity of the mRNA splicing reaction has made these mutations attractive candidates for the development of therapeutics. Familial dysautonomia (FD) is a severe neurodegenerative disorder, and all patients have an intronic IVS20+6T>C splice site mutation in the IKBKAP gene, which results in tissue-specific skipping of exon 20 and a corresponding reduction in ikappaB kinase complex associated protein (IKAP) levels. We created transgenic mouse lines using a human IKBKAP bacterial artificial chromosome (BAC) into which we inserted the IKBKAP splice mutation (FD BAC) and have shown that the transgenic mice exhibit the same tissue-specific aberrant splicing patterns as seen in FD patients. We have previously demonstrated that the plant cytokinin kinetin can significantly improve the production of wild-type IKBKAP transcripts in FD lymphoblast cell lines by improving exon inclusion. In this study, we tested the ability of kinetin to alter IKBKAP splicing in the transgenic mice carrying the FD BAC and show that it corrects IKBKAP splicing in all major tissues assayed, including the brain. The amount of wild-type IKBKAP mRNA and IKAP protein was significantly higher in the kinetin-treated mice. These exciting results prove that treatment of FD, as well as other mechanistically related splicing disorders, with kinetin holds great promise as a potential therapeutic aimed at increasing normal protein levels, which may, in turn, slow disease progression.

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IKBKAP mRNA in Peripheral Blood Leukocytes: A Molecular Marker of Gene Expression and Splicing in Familial Dysautonomia

Cited by 10 publications

References 15 publications

Kinetin in Familial Dysautonomia Carriers: Implications for a New Therapeutic Strategy Targeting mRNA Splicing

Kinetin in Familial Dysautonomia Carriers: Implications for a New Therapeutic Strategy Targeting mRNA Splicing

Kinetin Improves IKBKAP mRNA Splicing in Patients With Familial Dysautonomia

Specific correction of a splice defect in brain by nutritional supplementation

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