570 Mol. Nutr. Food Res. 2012, 56, 570–579 DOI 10.1002/mnfr.201100670 RESEARCH ARTICLE Nutraceutical-mediated restoration of wild-type levels of IKBKAP-encoded IKAP protein in familial dysautonomia-derived cells Sylvia L. Anderson 1 , Bo Liu 1 , Jinsong Qiu 1,2 , Andrea J. Sturm 1 , Jamie A. Schwartz 3 , Austin J. Peters 1 , Kerry A. Sullivan 1 and Berish Y. Rubin 1 1 Laboratory for Familial Dysautonomia Research, Department of Biological Sciences, Fordham University, Bronx, NY, USA 2 Department of Cellular & Molecular Medicine, University of California (San Diego), La Jolla, CA, USA 3 Department of Surgery, St. Luke’s-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, NY, USA Scope: The reported ability to modulate the production of the wild-type transcript in cells bearing the splice-altering familial dysautonomia (FD)-causing mutation in the IKBKAP gene prompted an evaluation of the impact of commonly consumed nutraceuticals on the splicing of this transcript. Methods and results: Screening efforts revealed the ability of the isoflavones, genistein, and daidzein, to impact splicing and increase the production of the wild-type, exon-20-containing, transcript, and the full-length IKBKAP-encoded IKB kinase complex associated protein (IKAP) in FD-derived cells. Genistein was also found to impact splicing in neuronal cells, a cell type profoundly impacted by FD. The simultaneous exposure of FD-derived cells to genistein and epigallocatechin gallate (EGCG) resulted in the almost exclusive production of the exon-20- containing transcript and the production of wild-type amounts of IKAP protein. Conclusion: This study represents the first demonstration that the isoflavones, genistein and daidzein, possess splice-altering capabilities and that simultaneous treatment with genistein and EGCG reverses the splice-altering impact of the FD-causing mutation. These findings support the clinical evaluation of the therapeutic impact of the combined administration of these two commonly consumed nutraceuticals on this patient population and suggest a broader evaluation of the impact of these nutraceuticals on the in vivo RNA splicing process. Keywords: Alternative splicing / Familial dysautonomia / Genistein / Isoflavone / Nutraceutical Received: October 5, 2011 Revised: November 10, 2011 Accepted: December 8, 2011 1 Introduction Familial dysautonomia (FD) is an autosomal recessive dis- order that impacts the development and survival of sensory, sympathetic, and some parasympathetic neurons [1, 2]. Indi- Correspondence: Professor Berish Y. Rubin, Department of Bio- logical Sciences, Fordham University, 441 E. Fordham Rd., Bronx, NY 10458, USA E-mail: rubin@fordham.edu Fax: +1-718-817-2792 Abbreviations: EGCG, epigallocatechin gallate; FD, familial dysautonomia; IKAP, IKB kinase complex associated protein; IK- BKAP, inhibitor of kappa light polypeptide gene enhancer in B- cells, kinase complex-associated protein viduals with FD exhibit decreased sensitivity to pain and tem- perature, cardiovascular instability, recurrent pneumonias, vomiting/dysautonomic crises, gastrointestinal dysfunction, and defective lacrimation [1–4]. FD is a life-threatening dis- order with a high mortality rate. Two FD-causing mutations have been identified in individuals of Ashkenazi Jewish de- scent [5, 6]. The more common FD-causing mutation is a C→T transition in the sixth position of the 5 ′ -splice donor site of intron 20 (termed as IVS20 +6T→C ) of the IKBKAP gene. This mutation alters the splicing process, resulting in the al- most exclusive production of an exon-20-lacking transcript. Translation of this transcript produces a truncated nonfunc- tional protein. The IKBKAP-encoded IKB (Ikappa B)-kinase-complex- associated protein (IKAP), initially named on the basis of C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com