Familial dysautonomia (FD; also known as "Riley-Day syndrome"), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

The defective splicing of pre-mRNA is a major cause of human disease. Exon skipping is a common result of splice mutations and has been reported in a wide variety of genetic disorders, yet the underlying mechanism is poorly understood. Often, such mutations are incompletely penetrant, and low levels of normal transcript and protein are maintained. Familial dysautonomia (FD) is caused by mutations in IKBKAP, and all cases described to date involve an intron 20 mutation that results in a unique pattern of tissue-specific exon skipping. Accurate splicing of the mutant IKBKAP allele is particularly inefficient in the nervous system. Here we show that treatment with the plant cytokinin kinetin alters splicing of IKBKAP. Kinetin significantly increases inclusion of exon 20 from the endogenous gene, as well as from an IKBKAP minigene. By contrast the drug does not enhance inclusion of alternatively spliced exon 31 in MYO5A. Benzyladenine, the most closely related cytokinin, showed a similar but less dramatic effect. Our findings reveal a remarkable impact on splicing fidelity by these small molecules, which therefore provide new tools for the dissection of mechanisms controlling tissue-specific pre-mRNA splicing. Further, kinetin should be explored as a treatment for increasing the level of normal IKAP in FD, and for other splicing disorders that may share a similar mechanism.

We recently identified a mutation in the I-kappa B 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), a devastating hereditary sensory and autonomic neuropathy, results from an intronic mutation in the IKBKAP gene that disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP) in the nervous system. To better understand the roles of IKAP in vivo, an Ikbkap knockout mouse model was created. Results from our study show that ablating Ikbkap leads to embryonic lethality, with no homozygous Ikbkap knockout (Ikbkap ؊/؊ ) embryos surviving beyond 12.5 days postcoitum. Morphological analyses of the Ikbkap ؊/؊ conceptus at different stages revealed abnormalities in both the visceral yolk sac and the embryo, including stunted extraembryonic blood vessel formation, delayed entry into midgastrulation, disoriented dorsal primitive neural alignment, and failure to establish the embryonic vascular system. Further, we demonstrate downregulation of several genes that are important for neurulation and vascular development in the Ikbkap ؊/؊ embryos and show that this correlates with a defect in transcriptional elongation-coupled histone acetylation. Finally, we show that the embryonic lethality resulting from Ikbkap ablation can be rescued by a human IKBKAP transgene. For the first time, we demonstrate that IKAP is crucial for both vascular and neural development during embryogenesis and that protein function is conserved between mouse and human.IKBKAP (encoding IB kinase-associated protein, also called Elongator protein 1) is the gene mutated in hereditary sensory and autonomic neuropathy type III, or familial dysautonomia (FD). All FD patients carry at least one copy of a splicing mutation in IKBKAP, which causes aberrant exon skipping and subsequent tissue-specific reduction of protein expression in FD patients (1,41,42). The IKBKAP gene is highly conserved across species, with the human and mouse proteins (IKAP and Ikap, respectively) sharing more than 80% amino acid homology (12). The IKBKAP protein, IKAP, was first reported to act as a scaffolding protein for the IB kinase complex (11). Recent studies, however, have shown that IKAP does not play a role in NF-B (nuclear factor B) signaling, but rather, it is a subunit of the human Elongator complex, which is important for efficient transcriptional elongation (19,28,36).FD (or Riley-Day syndrome) is one of the best known recessive hereditary neuropathies, with an extremely high carrier frequency in the Ashkenazi Jewish population, which ranges from 1 in 17 to 1 in 28 depending on the country of origin (29,33,42). Clinical characteristics of FD include diminished tear secretion, dysphagia, esophageal and gastric dysmotility, gastroesophageal reflux, spinal curvature, postural hypotension, blotching, excessive sweating, and decreased deep-tendon reflexes (2). Fatality in FD patients is high, and only half survive to 40 years of age. Clinical reports have shown that the failure of autonomic function is one of the major causes of death (21). To date, three FD-causing mutations have been identified in the IKBKAP gene: a...

Familial dysautonomia (FD) is a severe hereditary sensory and autonomic neuropathy, and all patients with FD have a splice mutation in the IKBKAP gene. The FD splice mutation results in variable, tissue-specific skipping of exon 20 in IKBKAP mRNA, which leads to reduced IKAP protein levels. The development of therapies for FD will require suitable mouse models for preclinical studies. In this study, we report the generation and characterization of a mouse model carrying the complete human IKBKAP locus with the FD IVS20+6T-->C splice mutation. We show that the mutant IKBKAP transgene is misspliced in this model in a tissue-specific manner that replicates the pattern seen in FD patient tissues. Creation of this humanized mouse is the first step toward development of a complex phenotypic model of FD. These transgenic mice are an ideal model system for testing the effectiveness of therapeutic agents that target the missplicing defect. Last, these mice will permit direct studies of tissue-specific splicing and the identification of regulatory factors that play a role in complex gene expression.

Mutations that affect the splicing of pre-mRNA are a major cause of human disease. Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a T to C transition at base pair 6 of IKBKAP intron 20. This mutation results in variable tissue-specific skipping of exon 20. Previously, we reported that the plant cytokinin kinetin dramatically increases exon 20 inclusion in RNA isolated from cultured FD cells. The goal of the current study was to investigate the nature of the FD splicing defect and the mechanism by which kinetin improves exon inclusion, as such knowledge will facilitate the development of future therapeutics aimed at regulating mRNA splicing. In this study, we demonstrate that treatment of FD lymphoblast cell lines with kinetin increases IKBKAP mRNA and IKAP protein to normal levels. Using a series of minigene constructs, we show that deletion of a region at the end of IKBKAP exon 20 disrupts the ability of kinetin to improve exon inclusion, pinpointing a kinetin responsive sequence element. We next performed a screen of endogenously expressed genes with multiple isoforms resulting from exon skipping events and show that kinetin's ability to improve exon inclusion is not limited to IKBKAP. Lastly, we highlight the potential of kinetin for the treatment of other human splicing disorders by showing correction of a splicing defect in neurofibromatosis.

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.

Familial dysautonomia (FD) is an autosomal recessive disorder characterized by developmental arrest in the sensory and autonomic nervous systems and by Ashkenazi Jewish ancestry. We previously had mapped the defective gene (DYS) to an 11-cM segment of chromosome 9q31-33, flanked by D9S53 and D9S105. By using 11 new polymorphic loci, we now have narrowed the location of DYS to <0.5 cM between the markers 43B1GAGT and 157A3. Two markers in this interval, 164D1 and D9S1677, show no recombination with the disease. Haplotype analysis confirmed this candidate region and revealed a major haplotype shared by 435 of 441 FD chromosomes, indicating a striking founder effect. Three other haplotypes, found on the remaining 6 FD chromosomes, might represent independent mutations. The frequency of the major FD haplotype in the Ashkenazim (5 in 324 control chromosomes) was consistent with the estimated DYS carrier frequency of 1 in 32, and none of the four haplotypes associated with FD was observed on 492 non-FD chromosomes from obligatory carriers. It is now possible to provide accurate genetic testing both for families with FD and for carriers, on the basis of close flanking markers and the capacity to identify >98% of FD chromosomes by their haplotype.