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Loss-of-function mutations of FIG4 are responsible for neurological disorders in human and mouse that result from reduced abundance of the signaling lipid PI(3,5)P2. In contrast, loss-of-function mutations of the phosphoinositide kinase PIP4K2C result in elevated abundance of PI(3,5)P2. These opposing effects on PI(3,5)P2 suggested that we might be able to compensate for deficiency of FIG4 by reducing expression of PIP4K2C. To test this hypothesis in a whole animal model, we generated triallelic mice with genotype Fig 4−/−, Pip4k2c+/−; these mice are null for Fig 4 and haploinsufficient for Pip4k2c. The neonatal lethality of Fig 4 null mice in the C57BL/6J strain background was rescued by reduced expression of Pip4k2c. The lysosome enlargement characteristic of Fig 4 null cells was also reduced by heterozygous loss of Pip4k2c. The data demonstrate interaction between these two genes, and suggest that inhibition of the kinase PIPK4C2 could be a target for treatment of FIG4 deficiency disorders such as Charcot-Marie-Tooth Type 4J and Yunis-Varón Syndrome.

Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span. There is currently no treatment for CMT4J. Here, we present the results of preclinical studies testing a gene-therapy approach to restoring FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded adeno-associated virus serotype 9 (AAV9) to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at P1 or P4, mice survived at least 1 year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When mice were treated at P7 or P11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.

The lipid phosphatase gene FIG4 is responsible for Yunis-Varon syndrome and Charcot-Marie-Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents. Cultured fibroblasts exhibit enlarged vacuoles characteristic of FIG4 dysfunction. Two unrelated families segregate the same G > A variant in the +1 position of intron 21 in the homozygous state in one family and compound heterozygous in the other. This mutation in the splice donor site of exon 21 results in read-through from exon 20 into intron 20 and truncation of the final 115 C-terminal amino acids of FIG4, with retention of partial function. The observed CNS white matter disorder in these families is consistent with the myelination defects in the FIG4 null mouse and the known role of FIG4 in oligodendrocyte maturation. The families described here the expanded clinical spectrum of FIG4 deficiency to include leukoencephalopathy.

Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import–export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity. This article has an associated First Person interview with the first author of the paper.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper. ispartof: JOURNAL OF CELL SCIENCE vol:132 issue:5 ispartof: location:England status: published

The signaling lipid phosphatidylinositol 3,5-bisphosphate, PI(3,5)P(2), functions in vesicular trafficking through the endo-lysosomal compartment. Cellular levels of PI(3,5)P(2) are regulated by an enzyme complex comprised of the kinase PIKFYVE, the phosphatase FIG4, and the scaffold protein VAC14. Mutations of human FIG4 cause inherited disorders including Charcot-Marie-Tooth disease type 4J, polymicrogyria with epilepsy, and Yunis-Varón syndrome. Constitutive Fig4(−/−) mice exhibit intention tremor, spongiform degeneration of neural tissue, hypomyelination, and juvenile lethality. To determine whether PI(3,5)P(2) is required in the adult, we generated Fig4(flox/−); CAG-creER mice and carried out tamoxifen-induced gene ablation. Global ablation in adulthood leads to wasting, tremor, and motor impairment. Death follows within 2 months of tamoxifen treatment, demonstrating a life-long requirement for Fig4. Histological examinations of the sciatic nerve revealed profound Wallerian degeneration of myelinated fibers, but not C-fiber axons in Remak bundles. In optic nerve sections, myelinated fibers appear morphologically intact and carry compound action potentials at normal velocity and amplitude. However, when iKO mice are challenged with a chemical white matter lesion, repair of damaged CNS myelin is significantly delayed, demonstrating a novel role for Fig4 in remyelination. Thus, in the adult PNS Fig4 is required to protect myelinated axons from Wallerian degeneration. In the adult CNS, Fig4 is dispensable for fiber stability and nerve conduction, but is required for the timely repair of damaged white matter. The greater vulnerability of the PNS to Fig4 deficiency in the mouse is consistent with clinical observations in patients with Charcot-Marie-Tooth disease.

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux, and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects including embryonic lethality, neurodegeneration and overt inflammation. While being the most dramatic phenotype, the reasons for lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.Summary statementPIKfyve inhibition causes lysosomes to coalesce, resulting in fewer, enlarged lysosomes. We also show that TFEB-mediated lysosome biosynthesis does not contribute to swelling.

Mutations of FIG4 are responsible for Yunis-Varón syndrome, familial epilepsy with polymicrogyria, and Charcot-Marie-Tooth type 4J neuropathy (CMT4J). Although loss of the FIG4 phospholipid phosphatase consistently causes decreased PtdIns(3,5)P₂ levels, cell-specific sensitivity to partial loss of FIG4 function may differentiate FIG4-associated disorders. CMT4J is an autosomal recessive neuropathy characterized by severe demyelination and axonal loss in human, with both motor and sensory involvement. However, it is unclear whether FIG4 has cell autonomous roles in both motor neurons and Schwann cells, and how loss of FIG4/PtdIns(3,5)P₂-mediated functions contribute to the pathogenesis of CMT4J. Here, we report that mice with conditional inactivation of Fig4 in motor neurons display neuronal and axonal degeneration. In contrast, conditional inactivation of Fig4 in Schwann cells causes demyelination and defects in autophagy-mediated degradation. Moreover, Fig4-regulated endolysosomal trafficking in Schwann cells is essential for myelin biogenesis during development and for proper regeneration/remyelination after injury. Our data suggest that impaired endolysosomal trafficking in both motor neurons and Schwann cells contributes to CMT4J neuropathy.

In the PI(3,5)P2 biosynthetic complex, the lipid kinase PIKFYVE and the phosphatase FIG4 are bound to the dimeric scaffold protein VAC14, which is composed of multiple heat-repeat domains. Mutations of FIG4 result in the inherited disorders Charcot-Marie-Tooth disease type 4J, Yunis-Varon syndrome, and polymicrogyria with seizures. We here describe inherited variants of VAC14 in two unrelated children with sudden onset of a progressive neurological disorder and regression of developmental milestones. Both children developed impaired movement with dystonia, became nonambulatory and nonverbal, and exhibited striatal abnormalities on MRI. A diagnosis of Leigh syndrome was rejected due to normal lactate profiles. Exome sequencing identified biallelic variants of VAC14 that were inherited from unaffected heterozygous parents in both families. Proband 1 inherited a splice-site variant that results in skipping of exon 13, p.Ile459Profs(∗)4 (not reported in public databases), and the missense variant p.Trp424Leu (reported in the ExAC database in a single heterozygote). Proband 2 inherited two missense variants in the dimerization domain of VAC14, p.Ala582Ser and p.Ser583Leu, that have not been previously reported. Cultured skin fibroblasts exhibited the accumulation of vacuoles that is characteristic of PI(3,5)P2 deficiency. Vacuolization of fibroblasts was rescued by transfection of wild-type VAC14 cDNA. The similar age of onset and neurological decline in the two unrelated children define a recessive disorder resulting from compound heterozygosity for deleterious variants of VAC14.

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis. DOI: http://dx.doi.org/10.7554/eLife.13023.001, eLife digest Neurons communicate with each other through long cable-like extensions called axons. An insulating sheath called myelin (or white matter) surrounds each axon, and allows electrical impulses to travel more quickly. Cells in the brain called oligodendrocytes produce myelin. If the myelin sheath is not properly formed during development, or is damaged by injury or disease, the consequences can include paralysis, impaired thought, and loss of vision. Oligodendrocytes have complex shapes, and each can generate myelin for as many as 50 axons. Oligodendrocytes produce the building blocks of myelin inside their cell bodies, by following instructions encoded by genes within the nucleus. However, the signals that regulate the trafficking of these components to the myelin sheath are poorly understood. Mironova et al. set out to determine whether signaling molecules called phosphoinositides help oligodendrocytes to mature and move myelin building blocks from the cell bodies to remote contact points with axons. Genetic techniques were used to manipulate an enzyme complex in mice that controls the production and turnover of a phosphoinositide called PI(3,5)P2. Mironova et al. found that reducing the levels of PI(3,5)P2 in oligodendrocytes caused the trafficking of certain myelin building blocks to stall. Key myelin components instead accumulated inside bubble-like structures near the oligodendrocyte’s cell body. This showed that PI(3,5)P2 in oligodendrocytes is essential for generating myelin. Further experiments then revealed that reducing PI(3,5)P2 in the neurons themselves indirectly prevented the oligodendrocytes from maturing. This suggests that PI(3,5)P2 also takes part in communication between axons and oligodendrocytes during development of the myelin sheath. A key next step will be to identify the regulatory mechanisms that control the production of PI(3,5)P2 in oligodendrocytes and neurons. Future studies could also explore what PI(3,5)P2 acts upon inside the axons, and which signaling molecules support the maturation of oligodendrocytes. Finally, it remains unclear whether PI(3,5)P2signaling is also required for stabilizing mature myelin, and for repairing myelin after injury in the adult brain. Further work could therefore address these questions as well. DOI: http://dx.doi.org/10.7554/eLife.13023.002

FIG4 is a ubiquitously expressed phosphatase that, in complex with FAB1/PIKFYVE and VAC14, regulates the biosynthesis of the signaling lipid PI(3,5)P(2). Null mutation of Fig4 in the mouse results in spongiform degeneration of brain and peripheral ganglia, defective myelination and juvenile lethality. Partial loss-of-function of human FIG4 results in a severe form of Charcot-Marie-Tooth neuropathy. Neurons from null mice contain enlarged vacuoles derived from the endosome/lysosome pathway, and astrocytes accumulate proteins involved in autophagy. Other cellular defects include astrogliosis and microgliosis. To distinguish the contributions of neurons and glia to spongiform degeneration in the Fig4 null mouse, we expressed Fig4 under the control of the neuron-specific enolase promoter and the astrocyte-specific glial fibrillary acidic protein promoter in transgenic mice. Neuronal expression of Fig4 was sufficient to rescue cellular and neurological phenotypes including spongiform degeneration, gliosis and juvenile lethality. In contrast, expression of Fig4 in astrocytes prevented accumulation of autophagy markers and microgliosis but did not prevent spongiform degeneration or lethality. To confirm the neuronal origin of spongiform degeneration, we generated a floxed allele of Fig4 and crossed it with mice expressing the Cre recombinase from the neuron-specific synapsin promoter. Mice with conditional inactivation of Fig4 in neurons developed spongiform degeneration and the full spectrum of neurological abnormalities. The data demonstrate that expression of Fig4 in neurons is necessary and sufficient to prevent spongiform degeneration. Therapy for patients with FIG4 deficiency will therefore require correction of the deficiency in neurons.

Charcot-Marie-Tooth disease is a genetically heterogeneous group of motor and sensory neuropathies associated with mutations in more than 30 genes. Charcot-Marie-Tooth disease type 4J (OMIM 611228) is a recessive, potentially severe form of the disease caused by mutations of the lipid phosphatase FIG4. We provide a more complete view of the features of this disorder by describing 11 previously unreported patients with Charcot-Marie-Tooth disease type 4J. Three patients were identified from a small cohort selected for screening because of their early onset disease and progressive proximal as well as distal weakness. Eight patients were identified by large-scale exon sequencing of an unselected group of 4000 patients with Charcot-Marie-Tooth disease. In addition, 34 new FIG4 variants were detected. Ten of the new CMT4J cases have the compound heterozygous genotype FIG4(I41T/null) described in the original four families, while one has the novel genotype FIG4(L17P/nul)(l). The population frequency of the I41T allele was found to be 0.001 by genotyping 5769 Northern European controls. Thirty four new variants of FIG4 were identified. The severity of Charcot-Marie-Tooth disease type 4J ranges from mild clinical signs to severe disability requiring the use of a wheelchair. Both mild and severe forms have been seen in patients with the same genotype. The results demonstrate that Charcot-Marie-Tooth disease type 4J is characterized by highly variable onset and severity, proximal as well as distal and asymmetric muscle weakness, electromyography demonstrating denervation in proximal and distal muscles, and frequent progression to severe amyotrophy. FIG4 mutations should be considered in Charcot-Marie-Tooth patients with these characteristics, especially if found in combination with sporadic or recessive inheritance, childhood onset and a phase of rapid progression.

Mutations affecting the conversion of PI3P to the signaling lipid PI(3,5)P(2) result in spongiform degeneration of mouse brain and are associated with the human disorders Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis (ALS). We now report accumulation of the proteins LC3-II, p62 and LAMP-2 in neurons and astrocytes of mice with mutations in two components of the PI(3,5)P(2) regulatory complex, Fig4 and Vac14. Cytoplasmic inclusion bodies containing p62 and ubiquinated proteins are present in regions of the mutant brain that undergo degeneration. Co-localization of p62 and LAMP-2 in affected cells indicates that formation or recycling of the autolysosome is impaired. These results establish a role for PI(3,5)P(2) in autophagy in the mammalian central nervous system (CNS) and demonstrate that mutations affecting PI(3,5)P(2) can contribute to inclusion body disease.

Mutations of the lipid phosphatase FIG4 that regulates PI(3,5)P2 are responsible for the recessive peripheral-nerve disorder CMT4J. We now describe nonsynonymous variants of FIG4 in 2% (9/473) of patients with amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). Heterozygosity for a deleterious allele of FIG4 appears to be a risk factor for ALS and PLS, extending the list of known ALS genes and increasing the clinical spectrum of FIG4-related diseases., Howard Hughes Medical Institute (Investigator), National Institutes of Health (U.S.) (grant GM24872), National Institutes of Health (U.S.) (grant NS050557), National Institutes of Health (U.S.) (grant NIH NS050557), National Institutes of Health (U.S.) (grant NIH NS050641), National Institutes of Health (U.S.) (grant NIH T32 GM007544), Pierre L. de Bourgknecht ALS Research Foundation, Project ALS Foundation, Angel Fund

Contains fulltext : 70663.pdf (Publisher’s version ) (Closed access) Recently, two common sequence variants on 9p21, tagged by rs10757278-G and rs10811661-T, were reported to be associated with coronary artery disease (CAD) and type 2 diabetes (T2D), respectively. We proceeded to further investigate the contributions of these variants to arterial diseases and T2D. Here we report that rs10757278-G is associated with, in addition to CAD, abdominal aortic aneurysm (AAA; odds ratio (OR) = 1.31, P = 1.2 x 10(-12)) and intracranial aneurysm (OR = 1.29, P = 2.5 x 10(-6)), but not with T2D. This variant is the first to be described that affects the risk of AAA and intracranial aneurysm in many populations. The association of rs10811661-T to T2D replicates in our samples, but the variant does not associate with any of the five arterial diseases examined. These findings extend our insight into the role of the sequence variant tagged by rs10757278-G and show that it is not confined to atherosclerotic diseases.

Intracranial aneurysm (IA) is a complex genetic disease for which, to date, 10 loci have been identified by linkage. Identification of the risk-conferring genes in the loci has proven difficult, since the regions often contain several hundreds of genes. An approach to prioritize positional candidate genes for further studies is to use gene expression data from diseased and nondiseased tissue. Genes that are not expressed, either in diseased or nondiseased tissue, are ranked as unlikely to contribute to the disease. We demonstrate an approach for integrating expression and genetic mapping data to identify likely pathways involved in the pathogenesis of a disease. We used expression profiles for IAs and nonaneurysmal intracranial arteries (IVs) together with the 10 reported linkage intervals for IA. Expressed genes were analyzed for membership in Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways. The 10 IA loci harbor 1,858 candidate genes, of which 1,561 (84%) were represented on the microarrays. We identified 810 positional candidate genes for IA that were expressed in IVs or IAs. Pathway information was available for 294 of these genes and involved 32 KEGG biological function pathways represented on at least 2 loci. A likelihood-based score was calculated to rank pathways for involvement in the pathogenesis of IA. Adherens junction, MAPK, and Notch signaling pathways ranked high. Integration of gene expression profiles with genetic mapping data for IA provides an approach to identify candidate genes that are more likely to function in the pathology of IA.

The lipid phosphatase FIG4 is a subunit of the protein complex that regulates biosynthesis of the signaling lipid PI(3,5)P2. Mutations of FIG4 result in juvenile lethality and spongiform neurodegeneration in the mouse, and are responsible for the human disorders Charcot-Marie-Tooth disease, Yunis-Varon syndrome and polymicrogyria with seizures. We previously demonstrated that conditional expression of a wild-type FIG4 transgene in neurons is sufficient to rescue most of the abnormalities of Fig4 null mice, including juvenile lethality and extensive neurodegeneration. To evaluate the contribution of the phosphatase activity to the in vivo function of Fig4, we introduced the mutation p.Cys486Ser into the Sac phosphatase active-site motif CX5RT. Transfection of the Fig4(Cys486Ser) cDNA into cultured Fig4(-/-) fibroblasts was effective in preventing vacuolization. The neuronal expression of an NSE-Fig4(Cys486Ser) transgene in vivo prevented the neonatal neurodegeneration and juvenile lethality seen in Fig4 null mice. These observations demonstrate that the catalytically inactive FIG4 protein provides significant function, possibly by stabilization of the PI(3,5)P2 biosynthetic complex and/or localization of the complex to endolysosomal vesicles. Despite this partial rescue, later in life the NSE-Fig4(Cys486Ser) transgenic mice display significant abnormalities that include hydrocephalus, defective myelination and reduced lifespan. The late onset phenotype of the NSE-Fig4(Cys486Ser) transgenic mice demonstrates that the phosphatase activity of FIG4 has an essential role in vivo.

Familial aggregation of abdominal aortic aneurysms (AAAs) is now widely recognized, however, susceptibility genes have not yet been identified. Our approach to find susceptibility genes has been to collect families with AAAs and to perform DNA linkage analyses to identify regions on the human chromosomes that are linked to AAAs and could harbor susceptibility genes for AAAs. We identified 233 families with at least two individuals diagnosed with AAAs. These families had 653 AAA patients and an average of 2.8 cases per family. When evaluating mode of inheritance, 167 (72%) families were consistent with autosomal recessive inheritance, whereas 58 (25%) families were consistent with autosomal dominant inheritance and in 8 families the familial aggregation could be explained by autosomal dominant inheritance with incomplete penetrance. Blood samples from 235 affected relative pairs (ARPs) and their unaffected relatives were collected for DNA isolation and the DNA used for genotyping with highly variable microsatellite markers. We included covariates in the statistical analyses to allow for genetic heterogeneity. The results for chromosomes 19g13 and 4q31 were significant with sex, number of affected first-degree relatives, and their interaction as covariates. These chromosomal regions contain many plausible candidate genes, and the future research will include more detailed analyses of these positional candidate genes.

Charcot-Marie-Tooth disease (CMT) is genetically heterogeneous and classification based on motor nerve conduction velocity and inheritance is used to direct genetic testing. With the less common genetic forms of CMT, identifying the causative genetic mutation by Sanger sequencing of individual genes can be time-consuming and costly. Next-generation sequencing technologies show promise for clinical testing in diseases where a similar phenotype is caused by different genes. We report the unusual occurrence of CMT4J, caused by mutations in FIG4, in a apparently dominant pedigree. The affected proband and her mother exhibit different disease severities associated with different combinations of compound heterozygous FIG4 mutations, identified by whole exome sequencing. The proband was also shown to carry a de novo nonsense mutation in the dystrophin gene, which may contribute to her more severe phenotype. This study is a cautionary reminder that in families with two generations affected, explanations other than dominant inheritance are possible, such as recessive inheritance due to three mutations segregating in the family. It also emphasises the advantages of next-generation sequencing approaches that screen multiple CMT genes at once for patients in whom the common genes have been excluded.

The endo-lysosomal system and autophagy are essential components of macromolecular turnover in eukaryotic cells. The low-abundance signaling lipid PI(3,5)P2 is a key regulator of this pathway. Analysis of mouse models with defects in PI(3,5)P2 biosynthesis have revealed the unique dependence of the mammalian nervous system on this signaling pathway. This insight led to the discovery of the molecular basis for several human neurological disorders, including Charcot-Marie-Tooth Disease and Yunis-Varon Syndrome. Spontaneous mutants, conditional knockouts, transgenic lines and gene-trap alleles of Fig4, Vac14 and Pikfyve (Fab1) in the mouse have provided novel information regarding the role of PI(3,5)P2 in vivo. This review summarizes what has been learned from mouse models and highlights the utility of manipulating complex signaling pathways in vivo.

Abdominal aortic aneurysms (AAA) are the thirteenth cause of death in the United States. The etiology of the disease is yet largely unknown, although several environmental risk factors (e.g., smoking) have been identified and the search for finding genetic risk factors has been initiated. The purpose of our study was to gain insight into the pathobiology of AAA by determining which genes are expressed in the abdominal aorta under either the diseased or normal states, thereby generating the whole-genome-wide expression profiles for these conditions.

Objective Dravet syndrome is a severe form of intractable pediatric epilepsy with a high incidence of SUDEP: Sudden Unexpected Death in epilepsy. Cardiac arrhythmias are a proposed cause for some cases of SUDEP, yet the susceptibility and potential mechanism of arrhythmogenesis in Dravet syndrome remain unknown. The majority of Dravet syndrome patients have de novo mutations in SCN1A, resulting in haploinsufficiency. We propose that, in addition to neuronal hyperexcitability, SCN1A haploinsufficiency alters cardiac electrical function and produces arrhythmias, providing a potential mechanism for SUDEP. Methods Postnatal day 15-21 heterozygous SCN1A-R1407X knock-in mice, expressing a human Dravet syndrome mutation, were used to investigate a possible cardiac phenotype. A combination of single cell electrophysiology and in vivo electrocardiogram (ECG) recordings were performed. Results We observed a 2-fold increase in both transient and persistent Na(+) current density in isolated Dravet syndrome ventricular myocytes that resulted from increased activity of a tetrodotoxin-resistant Na(+) current, likely Nav1.5. Dravet syndrome myocytes exhibited increased excitability, action potential duration prolongation, and triggered activity. Continuous radiotelemetric ECG recordings showed QT prolongation, ventricular ectopic foci, idioventricular rhythms, beat-to-beat variability, ventricular fibrillation, and focal bradycardia. Spontaneous deaths were recorded in 2 DS mice, and a third became moribund and required euthanasia. Interpretation These data from single cell and whole animal experiments suggest that altered cardiac electrical function in Dravet syndrome may contribute to the susceptibility for arrhythmogenesis and SUDEP. These mechanistic insights may lead to critical risk assessment and intervention in human patients.

SummaryPhagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

Mutations that cause defects in levels of the signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] lead to profound neurodegeneration in mice. Moreover, mutations in human FIG4 predicted to lower PI(3,5)P 2 levels underlie Charcot–Marie–Tooth type 4J neuropathy and are present in selected cases of amyotrophic lateral sclerosis. In yeast and mammals, PI(3,5)P 2 is generated by a protein complex that includes the lipid kinase Fab1/Pikfyve, the scaffolding protein Vac14, and the lipid phosphatase Fig4. Fibroblasts cultured from Vac14 −/− and Fig4 −/− mouse mutants have a 50% reduction in the levels of PI(3,5)P 2 , suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we characterize a Pikfyve gene-trap mouse ( Pikfyve β -geo/ β -geo ), a hypomorph with ∼10% of the normal level of Pikfyve protein. shRNA silencing of the residual Pikfyve transcript in fibroblasts demonstrated that Pikfyve is required to generate all of the PI(3,5)P 2 pool. Surprisingly, Pikfyve also is responsible for nearly all of the phosphatidylinositol-5-phosphate (PI5P) pool. We show that PI5P is generated directly from PI(3,5)P 2 , likely via 3′-phosphatase activity. Analysis of tissues from the Pikfyve β -geo/ β -geo mouse mutants reveals that Pikfyve is critical in neural tissues, heart, lung, kidney, thymus, and spleen. Thus, PI(3,5)P 2 and PI5P have major roles in multiple organs. Understanding the regulation of these lipids may provide insights into therapies for multiple diseases.

Objective To investigate the role in bipolar disorder of the C9ORF72 hexanucleotide repeat expansion responsible for frontotemporal lobe dementia and amyotrophic lateral sclerosis. Methods Eighty-nine subjects from a previously described panel of individuals with bipolar disorder ascertained for genetic studies were screened to detect expansion of the C9ORF72 repeat. One two-generation family with bipolar disorder and an expanded repeat was characterized in depth using molecular diagnostics, imaging, histopathology, and neurological and neuropsychological evaluation. Results One proband, with the typical clinical presentation of bipolar disorder, carried an expanded C9ORF72 allele of heterogeneous length between 14 and 20 kilobases (kb) as assessed by Southern blot. The expanded allele was inherited from a parent with atypical, late onset clinical features of bipolar disorder, who subsequently progressed to frontotemporal lobe dementia. The expansion in peripheral blood of the parent ranged from 8.5 to 20 kb. Cultured lymphoblastoid cells from this parent exhibited a homogeneous expansion of only 8.5 kb. Conclusions The disease course in the two generations described here demonstrates that expansion of the C9ORF72 may be associated with a form of bipolar disorder that presents clinically with classic phenomenology and progression to neurodegenerative disease. The frequency in our bipolar disorder cohort was only 1%, indicating that C9ORF72 is not a major contributor to bipolar disorder. DNA from cultured cells may be biased towards shorter repeats and nonrepresentative of the endogenous C9ORF72 expansion.

The plt (pale tremor) mouse carries a null mutation in theFig4(Sac3)gene that results in tremor, hypopigmentation, spongiform degeneration of the brain, and juvenile lethality. FIG4 is a ubiquitously expressed phosphatidylinositol 3,5-bisphosphate phosphatase that regulates intracellular vesicle trafficking along the endosomal–lysosomal pathway. In humans, the missense mutationFIG4I41Tcombined with aFIG4null allele causes Charcot-Marie-Tooth 4J disease, a severe form of peripheral neuropathy. Here we show thatFig4null mice exhibit a dramatic reduction of myelin in the brain and spinal cord. In the optic nerve, smaller-caliber axons lack myelin sheaths entirely, whereas many large- and intermediate-caliber axons are myelinated but show structural defects at nodes of Ranvier, leading to delayed propagation of action potentials. In theFig4null brain and optic nerve, oligodendrocyte (OL) progenitor cells are present at normal abundance and distribution, but the number of myelinating OLs is greatly compromised. The total number of axons in theFig4null optic nerve is not reduced. Developmental studies reveal incomplete myelination rather than elevated cell death in the OL linage. Strikingly, there is rescue of CNS myelination and tremor in transgenic mice with neuron-specific expression ofFig4, demonstrating a non-cell-autonomous function ofFig4in OL maturation and myelin development. In transgenic mice with global overexpression of the human pathogenicFIG4variant I41T, there is rescue of the myelination defect, suggesting that the CNS of CMT4J patients may be protected from myelin deficiency by expression of the FIG4I41Tmutant protein.

Charcot-Marie-Tooth disease is a genetically heterogeneous group of motor and sensory neuropathies associated with mutations in more than 30 genes. Charcot-Marie-Tooth disease type 4J (OMIM 611228) is a recessive, potentially severe form of the disease caused by mutations of the lipid phosphatase FIG4. We provide a more complete view of the features of this disorder by describing 11 previously unreported patients with Charcot-Marie-Tooth disease type 4J. Three patients were identified from a small cohort selected for screening because of their early onset disease and progressive proximal as well as distal weakness. Eight patients were identified by large-scale exon sequencing of an unselected group of 4000 patients with Charcot-Marie-Tooth disease. In addition, 34 new FIG4 variants were detected. Ten of the new CMT4J cases have the compound heterozygous genotype FIG4(I41T/null) described in the original four families, while one has the novel genotype FIG4(L17P/nul)(l). The population frequency of the I41T allele was found to be 0.001 by genotyping 5769 Northern European controls. Thirty four new variants of FIG4 were identified. The severity of Charcot-Marie-Tooth disease type 4J ranges from mild clinical signs to severe disability requiring the use of a wheelchair. Both mild and severe forms have been seen in patients with the same genotype. The results demonstrate that Charcot-Marie-Tooth disease type 4J is characterized by highly variable onset and severity, proximal as well as distal and asymmetric muscle weakness, electromyography demonstrating denervation in proximal and distal muscles, and frequent progression to severe amyotrophy. FIG4 mutations should be considered in Charcot-Marie-Tooth patients with these characteristics, especially if found in combination with sporadic or recessive inheritance, childhood onset and a phase of rapid progression.

CMT4J is a severe form of Charcot-Marie-Tooth neuropathy caused by mutation of the phosphoinositide phosphatase FIG4/SAC3. Affected individuals are compound heterozygotes carrying the missense allele FIG4-I41T in combination with a null allele. Analysis using the yeast two-hybrid system demonstrated that the I41T mutation impairs interaction of FIG4 with the scaffold protein VAC14. The critical role of this interaction was confirmed by the demonstration of loss of FIG4 protein in VAC14 null mice. We developed a mouse model of CMT4J by expressing a Fig4-I41T cDNA transgene on the Fig4 null background. Expression of the mutant transcript at a level 5× higher than endogenous Fig4 completely rescued lethality, whereas 2× expression gave only partial rescue, providing a model of the human disease. The level of FIG4-I41T protein in transgenic tissues is only 2% of that predicted by the transcript level, as a consequence of the protein instability caused by impaired interaction of the mutant protein with VAC14. Analysis of patient fibroblasts demonstrated a comparably low level of mutant I41T protein. The abundance of FIG4-I41T protein in cultured cells is increased by treatment with the proteasome inhibitor MG-132. The data demonstrate that FIG4-I41T is a hypomorphic allele encoding a protein that is unstable in vivo. Expression of FIG4-I41T protein at 10% of normal level is sufficient for long-term survival, suggesting that patients with CMT4J could be treated by increased production or stabilization of the mutant protein. The transgenic model will be useful for testing in vivo interventions to increase the abundance of the mutant protein., Author Summary Charcot-Marie-Tooth disease type 4J is a severe neurological disorder with childhood or adult onset and progression to loss of mobility and death. Patients inherit a mutation that changes amino acid residue 41 of the FIG4 protein from isoleucine to threonine. We report that this mutation destabilizes the FIG4 protein by blocking its interaction with a stabilizing protein partner. We developed a mouse model of CMT4J and found that a low level of expression of the mutant protein, 10% of wildtype level, is sufficient to prevent lethality. This work provides the scientific basis for development of a directed treatment for this rare, lethal disorder.

Background Abdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database. Methods Nine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies. Results Several SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples. Conclusions Association testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression.

Recent work from our laboratory has demonstrated that the phosphoinositide PtdIns(3,5)P2 has an essential role in autophagy in the mammalian nervous system. This low abundance, signaling lipid is synthesized by an enzyme complex that is localized on the vacuole membrane in yeast and in the endosome/lysosome compartment in mammalian cells. In mice with mutations in FIG4 and VAC14, two components of the PtdIns(3,5)P2 regulatory complex, autophagy intermediates accumulate in brain and spinal cord. The data indicate that PtdIns(3,5)P2 is required for completion of basal autophagy in mammalian cells.

Background—Previously, we identified 3274 distinct differentially expressed genes in abdominal aortic aneurysm (AAA) tissue compared with nonaneurysmal controls. As transcriptional control is responsible for these expression changes, we sought to find common transcriptional elements in the promoter regions of the differentially expressed genes.Methods and Results—We analyzed the up- and downregulated gene sets with Whole Genome rVISTA to determine the transcription factor (TF) binding sites overrepresented in the 5-kb promoter regions of the 3274 genes. The downregulated gene set yielded 144 TF binding sites that were overrepresented in the subset when compared with the entire genome. In contrast, the upregulated gene set yielded only 13 distinct overrepresented TF binding sites. Interestingly, as classified by TRANSFAC, 8 of the 13 TFs binding to these regions belong to the ETS family. Additionally, nuclear factor kB and its subunits p50 and p65 showed enrichment. Immunohistochemical analyses of 10 TFs from the upregulated set showed 9 to be present in AAA tissue. Based on gene ontology analysis of biological process categories of the upregulated target genes of enriched TFs, 10 TFs had enrichment in immune system process among their target genes.Conclusions—Our genome-wide analysis provides further evidence of ETS and nuclear factor kB involvement in AAA. Additionally, our results provide novel insight for future studies aiming to dissect the pathogenesis of AAA and have uncovered potential therapeutic targets for AAA prevention.

Abdominal aortic aneurysm (AAA) is a complex multifactorial disease with life-threatening implications. Aneurysms typically have no signs or symptoms, and rupture of AAA has a high mortality rate. Multiple environmental and genetic risk factors are involved in aneurysm formation and progression making it a complicated disease to study. Little is understood about the mechanisms in disease initiation, thus there are currently no therapeutic approaches to prevent AAA, leaving patients with surgery as their only option. Ongoing research into the genetic components of AAA using a candidate gene approach has been overall unsuccessful. A more promising approach to study complex diseases involves genome-wide techniques such as DNA linkage analysis, genetic association studies and microarray expression profiling. Furthermore, studies involving inhibition of AAA progression, rather than formation, have a potentially promising outcome. Targeting biological pathways in AAA pathogenesis may benefit patients by slowing the growth and possibly preventing the rupture of AAA. Critical pathways involved in AAA pathogenesis include immunological processes, such as T-cell and natural killer cell pathways, oxidative stress, depletion of vascular smooth muscle cells through the process of apoptosis and the destruction of the extracellular matrix by matrix metalloproteinases.

Pre-eclampsia (PE) affects 5–7% of pregnancies in the US, and is a leading cause of maternal death and perinatal morbidity and mortality worldwide. To identify genes with a role in PE, we conducted a large-scale association study evaluating 775 SNPs in 190 candidate genes selected for a potential role in obstetrical complications. SNP discovery was performed by DNA sequencing, and genotyping was carried out in a high-throughput facility using the MassARRAYTM System. Women with PE (n = 394) and their offspring (n = 324) were compared with control women (n = 602) and their offspring (n = 631) from the same hospital-based population. Haplotypes were estimated for each gene using the EM algorithm, and empirical p values were obtained for a logistic regression-based score test, adjusted for significant covariates. An interaction model between maternal and offspring genotypes was also evaluated. The most significant findings for association with PE were COL1A1 (p = 0.0011) and IL1A (p = 0.0014) for the maternal genotype, and PLAUR (p = 0.0008) for the offspring genotype. Common candidate genes for PE, including MTHFR and NOS3, were not significantly associated with PE. For the interaction model, SNPs within IGF1 (p = 0.0035) and IL4R (p = 0.0036) gave the most significant results. This study is one of the most comprehensive genetic association studies of PE to date, including an evaluation of offspring genotypes that have rarely been considered in previous studies. Although we did not identify statistically significant evidence of association for any of the candidate loci evaluated here after adjusting for multiple testing using the false discovery rate, additional compelling evidence exists, including multiple SNPs with nominally significant p values in COL1A1 and the IL1A region, and previous reports of association for IL1A, to support continued interest in these genes as candidates for PE. Identification of the genetic regulators of PE may have broader implications, since women with PE are at increased risk of death from cardiovascular diseases later in life.

Abdominal aortic aneurysm (AAA) is a complex multifactorial disease with life-threatening implications. Aneurysms typically have no signs or symptoms, and rupture of AAA has a high mortality rate. Multiple environmental and genetic risk factors are involved in aneurysm formation and progression making it a complicated disease to study. Little is understood about the mechanisms in disease initiation, thus there are currently no therapeutic approaches to prevent AAA, leaving patients with surgery as their only option. Ongoing research into the genetic components of AAA using a candidate gene approach has been overall unsuccessful. A more promising approach to study complex diseases involves genome-wide techniques such as DNA linkage analysis, genetic association studies and microarray expression profiling. Furthermore, studies involving inhibition of AAA progression, rather than formation, have a potentially promising outcome. Targeting biological pathways in AAA pathogenesis may benefit patients by slowing the growth and possibly preventing the rupture of AAA. Critical pathways involved in AAA pathogenesis include immunological processes, such as T-cell and natural killer cell pathways, oxidative stress, depletion of vascular smooth muscle cells through the process of apoptosis and the destruction of the extracellular matrix by matrix metalloproteinases.

Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P(2) levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P(2) signaling in skeletal development and maintenance.

Background The infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression. Results We analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007). Conclusions Our results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.