Peters plus syndrome mutations affect the function and stability of human ß1,3-glucosyltransferase.

Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding ß1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.

Specific correction of a splice defect in brain by nutritional supplementation.

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.

Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia.

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 WT IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine whether 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/d for 28 d. An increase in WT IKBKAP mRNA expression in leukocytes was noted after 8 d in six of eight individuals; after 28 d, the mean increase compared with 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 seems 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 whether kinetin will prove therapeutic in FD patients.

Kinetin in familial dysautonomia carriers: implications for a new therapeutic strategy targeting mRNA splicing.

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.

Loss of mouse Ikbkap, a subunit of elongator, leads to transcriptional deficits and embryonic lethality that can be rescued by human IKBKAP.

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 mRNA in peripheral blood leukocytes: a molecular marker of gene expression and splicing in familial dysautonomia.

The common familial dysautonomia (FD) mutation results in tissue specific mis-splicing with reduced amount of wild-type (WT) IkappaB kinase associated protein gene (IKBKAP) mRNA and ELP1. ELP1 is a subunit of Elongator, formerly called the IkappaB kinase associated protein (IKAP) protein. We measured IKBKAP mRNA in peripheral blood leukocytes to determine whether FD subjects and carriers have characteristic levels. Estimated mean IKBKAP mRNA levels, measured by quantitative PCR and expressed as amount relative to the noncarrier average, were significantly different for the two groups when not adjusted for age and sex (p < 0.001): FD subjects 0.23, 95% confidence interval (CI) (0.19, 0.28); carriers 0.58, 95% CI (0.50, 0.68); or adjusted for age and sex (p < 0.001): FD subjects 0.21, 95% CI (0.16, 0.26); carriers 0.66, 95% CI (0.55, 0.79). Comparison of IKBKAP mRNA levels of the 22 FD subjects and their related carriers showed a strong correlation, providing evidence for genetic control of splicing efficiency. IKBKAP mRNA levels were not higher in those subjects using tocotrienols or epigallocatechin gallate. Levels of IKBKAP mRNA in peripheral blood leukocytes can be used to assess molecular response to therapies aimed at enhancing exon 20 inclusion and increasing cellular levels of ELP1/IKAP.

A humanized IKBKAP transgenic mouse models a tissue-specific human splicing defect.

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.

Therapeutic potential and mechanism of kinetin as a treatment for the human splicing disease familial dysautonomia.

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.

Fludrocortisone in patients with familial dysautonomia--assessing effect on clinical parameters and gene expression.

The common familial dysautonomia (FD) mutation causes a splicing defect that leads to production of both wild-type (WT) and mutant (MU) IKBKAP mRNA. Because drugs may alter splicing, seven drugs, fludrocortisone, midodrine, diazepam, albuterol, clonidine, caffeine, and dopamine were screened. Since only fludrocortisone negatively altered gene expression, we assessed fludrocortisone's efficacy in treating postural hypotension, and its effect on survival and secondary long-term FD problems. For 341 FD patients we obtained demographic data and clinical information from the last Center evaluation (most current or prior to death) including mean blood pressures (supine, 1 min erect and 5 min erect) and history regarding syncope and presyncope symptoms. For 175 fludrocortisone-treated patients, data from the evaluation prior to start of fludrocortisone and from the last Center evaluation were compared. The fludrocortisone-treated patient cohort was compared to the nontreated patient cohort with respect to overall survival and event-free survival for crisis frequency, worsening gait, frequent fractures, spine curvature, renal insufficiency, and pacemaker insertion. Overall survivals of patients on fludrocortisone alone, on fludrocortisone and midodrine, and on neither drug were compared. Cumulative survival was significantly higher in fludrocortisone-treated patients than in non-treated patients during the first decade. In subsequent decades, the addition of midodrine improved cumulative survival. Fludrocortisone significantly increased mean blood pressures and decreased dizziness and leg cramping, but not headaches or syncope. Fludrocortisone was associated with more long-term problems, which may reflect more symptomatic status associated with longer survival. Our data suggest that fludrocortisone has clinical efficacy despite negative in vitro observations on gene expression.

Rescue of a human mRNA splicing defect by the plant cytokinin kinetin.

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.

Identification of the first non-Jewish mutation in familial Dysautonomia.

Familial Dysautonomia is an autosomal recessive disease with a remarkably high carrier frequency in the Ashkenazi Jewish population. It has recently been estimated that as many as 1 in 27 Ashkenazi Jews is a carrier of FD. The FD gene has been identified as IKBKAP, and two disease-causing mutations have been identified. The most common mutation, which is present on 99.5% of all FD chromosomes, is an intronic splice site mutation that results in tissue-specific skipping of exon 20. The second mutation, R696P, is a missense mutation that has been identified in 4 unrelated patients heterozygous for the major splice mutation. Interestingly, despite the fact that FD is a recessive disease, normal mRNA and protein are expressed in patient cells. To date, the diagnosis of FD has been limited to individuals of Ashkenazi Jewish descent and identification of the gene has led to widespread diagnostic and carrier testing in this population. In this report, we describe the first non-Jewish IKBKAP mutation, a proline to leucine missense mutation in exon 26, P914L. This mutation is of particular significance because it was identified in a patient who lacks one of the cardinal diagnostic criteria for the disease-pure Ashkenazi Jewish ancestry. In light of this fact, the diagnostic criteria for FD must be expanded. Furthermore, in order to ensure carrier identification in all ethnicities, this mutation must now be considered when screening for FD.

Tissue-specific reduction in splicing efficiency of IKBKAP due to the major mutation associated with familial dysautonomia.

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.