IKBKAP Gene Research Articles

Phosphatidylserine increases IKBKAP levels in a humanized knock-in IKBKAP mouse model

Familial dysautonomia (FD) is a severe neurodegenerative genetic disorder restricted to the Ashkenazi Jewish population. The most common mutation in FD patients is a T-to-C transition at position 6 of intron 20 of the IKBKAP gene. This mutation causes aberrant skipping of exon 20 in a tissue-specific manner, leading to reduction of the IκB kinase complex-associated protein (IKAP) protein in the nervous system. We established a homozygous humanized mouse strain carrying human exon 20 and its two flanking introns; the 3' intron has the transition observed in the IKBKAP gene of FD patients. Although our FD humanized mouse does not display FD symptoms, the unique, tissue-specific splicing pattern of the IKBKAP in these mice allowed us to evaluate the effect of therapies on gene expression and exon 20 splicing. The FD mice were supplemented with phosphatidylserine (PS), a safe food supplement that increases mRNA and protein levels of IKBKAP in cell lines generated from FD patients. Here we demonstrated that PS treatment increases IKBAKP mRNA and IKAP protein levels in various tissues of FD mice without affecting exon 20 inclusion levels. We also observed that genes associated with transcription regulation and developmental processes were up-regulated in the cerebrum of PS-treated mice. Thus, PS holds promise for the treatment of FD.

IKAP expression levels modulate disease severity in a mouse model of familial dysautonomia

Hereditary sensory and autonomic neuropathies (HSANs) encompass a group of genetically inherited disorders characterized by sensory and autonomic dysfunctions. Familial dysautonomia (FD), also known as HSAN type III, is an autosomal recessive disorder that affects 1/3600 live births in the Ashkenazi Jewish population. The disease is caused by abnormal development and progressive degeneration of the sensory and autonomic nervous systems and is inevitably fatal, with only 50% of patients reaching the age of 40. FD is caused by a mutation in intron 20 of the Ikbkap gene that results in severe reduction in the expression of its encoded protein, inhibitor of kappaB kinase complex-associated protein (IKAP). Although the mutation that causes FD was identified in 2001, so far there is no appropriate animal model that recapitulates the disorder. Here, we report the generation and characterization of the first mouse models for FD that recapitulate the molecular and pathological features of the disease. Important for therapeutic interventions is also our finding that a slight increase in IKAP levels is enough to ameliorate the phenotype and increase the life span. Understanding the mechanisms underlying FD will provide insights for potential new therapeutic interventions not only for FD, but also for other peripheral neuropathies.

Crystal Structure of Elongator Subcomplex Elp4–6

Elongator is a multiprotein complex composed of two subcomplexes, Elp1-3 and Elp4-6. Elongator is highly conserved between yeast and humans and plays an important role in RNA polymerase II-mediated transcriptional elongation and many other processes, including cytoskeleton organization, exocytosis, and tRNA modification. Here, we determined the crystal structure of the Elp4-6 subcomplex of yeast. The overall structure of Elp4-6 revealed that Elp6 acts as a bridge to assemble Elp4 and Elp5. Detailed structural and sequence analyses revealed that each subunit in the Elp4-6 subcomplex forms a RecA-ATPase-like fold, although it lacks the key sequence signature of ATPases. Site-directed mutagenesis and biochemical analyses indicated that the Elp4-6 subcomplex can assemble into a hexameric ring-shaped structure in vitro and in vivo. Furthermore, GST pulldown assays showed that the ring-shaped assembly of the Elp4-6 subcomplex is important for its specific histone H3 binding. Our results may shed light on the substrate recognition and assembly of the holo-Elongator complex.

Nutraceutical‐mediated restoration of wild‐type levels of IKBKAP‐encoded IKAP protein in familial dysautonomia‐derived cells

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. 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 IΚB 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. 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.

IKAP/Elp1 is required in vivo for neurogenesis and neuronal survival, but not for neural crest migration.

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems.

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.

Deletion of Exon 20 of the Familial Dysautonomia Gene Ikbkap in Mice Causes Developmental Delay, Cardiovascular Defects, and Early Embryonic Lethality

Familial Dysautonomia (FD) is an autosomal recessive disorder that affects 1/3,600 live births in the Ashkenazi Jewish population, and leads to death before the age of 40. The disease is characterized by abnormal development and progressive degeneration of the sensory and autonomic nervous system. A single base pair substitution in intron 20 of the Ikbkap gene accounts for 98% of FD cases, and results in the expression of low levels of the full-length mRNA with simultaneous expression of an aberrantly spliced mRNA in which exon 20 is missing. To date, there is no animal model for the disease, and the essential cellular functions of IKAP - the protein encoded by Ikbkap - remain unknown. To better understand the normal function of IKAP and in an effort to generate a mouse model for FD, we have targeted the mouse Ikbkap gene by homologous recombination. We created two distinct alleles that result in either loss of Ikbkap expression, or expression of an mRNA lacking only exon 20. Homozygosity for either mutation leads to developmental delay, cardiovascular and brain malformations, accompanied with early embryonic lethality. Our analyses indicate that IKAP is essential for expression of specific genes involved in cardiac morphogenesis, and that cardiac failure is the likely cause of abnormal vascular development and embryonic lethality. Our results also indicate that deletion of exon 20 abolishes gene function. This implies that the truncated IKAP protein expressed in FD patients does not retain any significant biological function.

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.

Effects of IKAP/hELP1 Deficiency on Gene Expression in Differentiating Neuroblastoma Cells: Implications for Familial Dysautonomia

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype.

Phosphatidylserine Increases IKBKAP Levels in Familial Dysautonomia Cells

Familial Dysautonomia (FD) is an autosomal recessive congenital neuropathy that results from abnormal development and progressive degeneration of the sensory and autonomic nervous system. The mutation observed in almost all FD patients is a point mutation at position 6 of intron 20 of the IKBKAP gene; this gene encodes the IκB kinase complex-associated protein (IKAP). The mutation results in a tissue-specific splicing defect: Exon 20 is skipped, leading to reduced IKAP protein expression. Here we show that phosphatidylserine (PS), an FDA-approved food supplement, increased IKAP mRNA levels in cells derived from FD patients. Long-term treatment with PS led to a significant increase in IKAP protein levels in these cells. A conjugate of PS and an omega-3 fatty acid also increased IKAP mRNA levels. Furthermore, PS treatment released FD cells from cell cycle arrest and up-regulated a significant number of genes involved in cell cycle regulation. Our results suggest that PS has potential for use as a therapeutic agent for FD. Understanding its mechanism of action may reveal the mechanism underlying the FD disease.

Olfactory Stem Cells, a New Cellular Model for Studying Molecular Mechanisms Underlying Familial Dysautonomia

BackgroundFamilial dysautonomia (FD) is a hereditary neuropathy caused by mutations in the IKBKAP gene, the most common of which results in variable tissue-specific mRNA splicing with skipping of exon 20. Defective splicing is especially severe in nervous tissue, leading to incomplete development and progressive degeneration of sensory and autonomic neurons. The specificity of neuron loss in FD is poorly understood due to the lack of an appropriate model system. To better understand and modelize the molecular mechanisms of IKBKAP mRNA splicing, we collected human olfactory ecto-mesenchymal stem cells (hOE-MSC) from FD patients. hOE-MSCs have a pluripotent ability to differentiate into various cell lineages, including neurons and glial cells.Methodology/Principal FindingsWe confirmed IKBKAP mRNA alternative splicing in FD hOE-MSCs and identified 2 novel spliced isoforms also present in control cells. We observed a significant lower expression of both IKBKAP transcript and IKAP/hELP1 protein in FD cells resulting from the degradation of the transcript isoform skipping exon 20. We localized IKAP/hELP1 in different cell compartments, including the nucleus, which supports multiple roles for that protein. We also investigated cellular pathways altered in FD, at the genome-wide level, and confirmed that cell migration and cytoskeleton reorganization were among the processes altered in FD. Indeed, FD hOE-MSCs exhibit impaired migration compared to control cells. Moreover, we showed that kinetin improved exon 20 inclusion and restores a normal level of IKAP/hELP1 in FD hOE-MSCs. Furthermore, we were able to modify the IKBKAP splicing ratio in FD hOE-MSCs, increasing or reducing the WT (exon 20 inclusion):MU (exon 20 skipping) ratio respectively, either by producing free-floating spheres, or by inducing cells into neural differentiation.Conclusions/SignificancehOE-MSCs isolated from FD patients represent a new approach for modeling FD to better understand genetic expression and possible therapeutic approaches. This model could also be applied to other neurological genetic diseases.

IKAP/hELP1 down-regulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor, and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP – down - regulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development.

Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs

SUMMARYThe isolation of human induced pluripotent stem cells (iPSCs)1-3 offers a novel strategy for modeling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs4-7. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy caused by a point mutation in IKBKAP8 involved in transcriptional elongation9. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood due to the lack of an appropriate model system.Here we report the derivation of patient specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC derived lineages demonstrates tissue specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell based assays revealing marked defects in neurogenic differentiation and migration behavior. Finally, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining novel insights into human disease pathogenesis and treatment.

IKAP localizes to membrane ruffles with filamin A and regulates actin cytoskeleton organization and cell migration

Loss-of-function mutations in the IKBKAP gene, which encodes IKAP (ELP1), cause familial dysautonomia (FD), with defective neuronal development and maintenance. Molecular mechanisms leading to FD are poorly understood. We demonstrate that various RNA-interference-based depletions of IKAP lead to defective adhesion and migration in several cell types, including rat primary neurons. The defects could be rescued by reintroduction of wild-type IKAP but not by FD-IKAP, a truncated form of IKAP constructed according to the mutation found in the majority of FD patients. Cytosolic IKAP co-purified with proteins involved in cell migration, including filamin A, which is also involved in neuronal migration. Immunostaining of IKAP and filamin A revealed a distinct co-localization of these two proteins in membrane ruffles. Depletion of IKAP resulted in a significant decrease in filamin A localization in membrane ruffles and defective actin cytoskeleton organization, which both could be rescued by the expression of wild-type IKAP but not by FD-IKAP. No downregulation in the protein levels of paxillin or beclin 1, which were recently described as specific transcriptional targets of IKAP, was detected. These results provide evidence for the role of the cytosolic interactions of IKAP in cell adhesion and migration, and support the notion that cell-motility deficiencies could contribute to FD.

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.

Weak definition ofIKBKAPexon 20 leads to aberrant splicing in familial dysautonomia

Splicing mutations that lead to devastating genetic diseases are often located in nonconserved or weakly conserved sequences that normally do not affect splicing. Thus, the underlying reason for the splicing defect is not immediately obvious. An example of this phenomenon is observed in the neurodevelopmental disease familial dysautonomia (FD), which is caused by a single-base change in the 5' splice site (5'ss) of intron 20 in the IKBKAP gene (c.2204+6T>C). This mutation, which is in the sixth position of the intron and results in exon 20 skipping, has no phenotype in many other introns. To determine why the position 6 mutation causes aberrant splicing only in certain cases, we first used an in silico approach to identify potential sequences involved in exon 20 skipping. Computational analyses of the exon 20 5'ss itself predicted that this nine-nucleotide splicing signal, even when it contains the T>C mutation, is not sufficiently weak to explain the FD phenotype. However, the computational analysis predicted that both the upstream 3' splice site (3'ss) and exon 20 contain weak splicing signals, indicating that the FD 5'ss, together with the surrounding splicing signals, are not adequate for defining exon 20. These in silico predictions were corroborated using IKBKAP minigenes in a new rapid and simple in vitro coupled RNA polymerase (RNAP) II transcription/splicing assay. Finally, the weak splicing signals that flank the T>C mutation were validated as the underlying cause of familial dysautonomia in vivo using transient transfection assays. Together, our study demonstrates the general utility of combining in silico data with an in vitro RNAP II transcription/splicing system for rapidly identifying critical sequences that underlie the numerous splicing diseases caused by otherwise silent mutations.

Tocotrienols reverse IKAP and monoamine oxidase deficiencies in familial dysautonomia

Familial dysautonomia (FD), a recessive neurodegenerative disease, is caused by mutations in the IKBKAP gene that result in the production of nonfunctional IKAP protein. Manifestations of FD include autonomic crises characterized by hypertension, tachycardia, diaphoresis, and vomiting. Elevated plasma levels of norepinephrine (NE) and dopamine observed during autonomic crises and an exaggerated hypertensive response to low doses of NE prompted an examination of monoamine oxidase (MAO) levels, key isoenzymes responsible for degrading biogenic and dietary monoamines, in individuals with FD. Fetal tissue homozygous for the common FD-causing mutation and peripheral blood cells of individuals with FD have reduced MAO A mRNA levels. FD-derived cells, stimulated with tocotrienols or EGCG to produce increased levels of functional IKAP, express increased amounts of MAO A mRNA transcript and protein. Administration of tocotrienol to individuals with FD results in increased expression of both functional IKAP and MAO A transcripts in their peripheral blood cells. These findings provide new insight into the pathophysiology of FD and demonstrate the value of therapeutic approaches designed to elevate cellular levels of functional IKAP and MAO A.

Of splice and men: what does the distribution of IKAP mRNA in the rat tell us about the pathogenesis of familial dysautonomia?

Familial dysautonomia (FD) is the best-known and most common member of a group of congenital sensory/autonomic neuropathies characterized by widespread sensory and variable autonomic dysfunction. As opposed to the sensory/motor neuropathies, little is known about the causes of neuronal dysfunction and loss in the sensory/autonomic neuropathies. FD involves progressive neuronal degeneration, has a broad impact on the operation of many of the body’s systems, and leads to a markedly reduced quality of life and premature death. In 2001, we identified two mutations in the IKBKAP gene that result in FD. IKBKAP encodes IKAP, a member of the putative human holo-Elongator complex, which may facilitate transcription by RNA polymerase II. Whether or not the Elongator plays this role is moot. The FD mutation found on >99.5% of FD chromosomes does not cause complete loss of function. Instead, it results in a tissue-specific decrease in splicing efficiency of the IKBKAP transcript; cells from patients retain some capacity to produce normal mRNA and protein. To better understand the relationship between the genotype of FD patients and their phenotype, we have used in situ hybridization histochemistry to map the IKAP mRNA in sections of whole rat embryos. The mRNA is widely distributed. Highest levels are in the nervous system, but substantial amounts are also present in peripheral organs.

Tocotrienols induce IKBKAP expression: a possible therapy for familial dysautonomia

Familial dysautonomia (FD), a neurodegenerative genetic disorder primarily affecting individuals of Ashkenazi Jewish descent, is caused by mutations in the IKBKAP gene which encodes the IκB kinase complex-associated protein (IKAP). The more common or major mutation causes aberrant splicing, resulting in a truncated form of IKAP. Tissues from individuals homozygous for the major mutation contain both mutant and wild-type IKAP transcripts. The apparent leaky nature of this mutation prompted a search for agents capable of elevating the level of expression of the wild-type IKAP transcript. We report the ability of tocotrienols, members of the vitamin E family, to increase transcription of IKAP mRNA in FD-derived cells, with corresponding increases in the correctly spliced transcript and normal protein. These findings suggest that in vivo supplementation with tocotrienols may elevate IKBKAP gene expression and in turn increase the amount of functional IKAP protein produced in FD patients.

Preimplantation genetic diagnosis for familial dysautonomia

Familial dysautonomia (FD) is the most common congenital sensory neuropathy in Ashkenazi Jews, caused by a single major mutation in the IKBKAP gene. Effective management for this severe debilitating disease is still not available, making preimplantation genetic diagnosis (PGD) a useful option for at-risk couples to establish an FD free pregnancy from the outset. PGD was performed for a couple with a previous affected child with FD, using first and second polar body testing to preselect mutation-free oocytes, based on mutation analysis with simultaneous testing of two closely linked markers, D9S58 and D9S1677. Of 15 tested oocytes, 11 carried information about both polar bodies' genotype, of which seven were predicted to be free of the FD gene. Three embryos resulting from these oocytes were transferred back to the patient, resulting in a triplet pregnancy and the birth of three unaffected children confirmed to be free of FD. This is the first PGD for FD, providing an alternative for those at-risk couples who cannot accept prenatal diagnosis and termination of pregnancy as an option for avoiding FD.