Familial dysautonomia (FD)

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy which results from poor development and progressive degeneration of the nervous system. The mutation responsible for FD was found at the 5’ss of intron 20 of the IKBKAP gene, encoding the IκB kinase complex-associated protein (IKAP). The mutation results in exon 20 shifting from constitutive inclusion in normal individuals to alternative splicing in the patients, causing a frameshift and a stop codon in the skipped isoform, which leads to reduced IKAP expression. From our current knowledge of FD we can presume that the key for effective therapy of FD is increasing the amount of the normal, functional IKAP protein by restoring normal IKAP mRNA splicing. We screened a variety of substances regarding their effect on the splicing pattern of the IKAP mRNA in FD cells. A food supplement based on Serine - Phosphatidylserine (PS) which is produced by Enzymotec under Sharp•PS® brand (http://www.enzymotec.com/) showed a significant increase in the levels of IKAP mRNA. PS was added to FD cell lines (fibroblast cell lines, derived from the appendix of FD patients, which was immortalized in our lab) at different concentrations and for different time periods. In addition, we conducted a long term treatment of PS with an optimal concentration of 100µg/ml PS, which gave the optimal effect on the inclusion level of exon 20 (found by perliminary experiments). Samples taken after 3-days, 1-week and 2-week, showed a great increase in the amount of wt IKAP mRNA as shown by RT-PCR and Real-Time PCR analysis in Figure 1. The results indicate that a long-term PS treatment significantly raises its effect on the wt IKAP mRNA level. Our results also showed increase in IKAP protein level as a result of PS treatment.

Figure : PS significantly raises IKAP mRNA level on a long term treatment. PS was added to FDB cell line at a concentrations 100µg/ml. Every two days the cellsí medium was replaced and new fresh PS was added. RNA was extracted 3-day, 1-week and 2-week following the initial addition of PS. Left side - Real-Time PCR analysis of the level of exon 20 inclusion isoform (wt). Each relative quantity represents normalization to untreated control cells harvested on the same day. Right side - RT-PCR analysis of the splicing of the endogenous IKAP gene in FD cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced.