Autonomous microfluidic multi-channel chip for real-time PCR with integrated liquid handling

We report on a novel, polymer-based, multi-channel device for polymerase chain reaction that combines, for the first time, rapid sample processing in less than 5min with high throughput at low costs. This is achieved by sample shuttling, during which submicroliter sample plugs (∼100nl) are oscillated rapidly over three constant-temperature zones by pneumatic actuation with integrated system. The accuracy and the speed of the liquid handling have been significantly increased, while the design of the device can be kept very simple and allows for mass production using conventional low-cost polymer fabrication processes. Massive parallelization can lead to a throughput up to 100 samples in 10min including the preparation time. The amplification can be optically monitored by means of online fluorescence detection. Successful real-time PCR and the determination of the threshold cycle, C t, using the developed device were demonstrated with plasmid DNA in a fluorescent real-time forma

Loss of phosphatase activity in myotubularin-related protein 2 is associated with Charcot-Marie-Tooth disease type 4B1

Mutations in the gene encoding myotubularin-related protein 2 (MTMR2) are responsible for autosomal recessive Charcot-Marie-Tooth disease type 4B1 (CMT4B1), a severe hereditary motor and sensory neuropathy characterized by focally folded myelin sheaths and demyelination. MTMR2 belongs to the myotubularin family, which is characterized by the presence of a phosphatase domain. Myotubularin (MTM), the archetype member of this family, is mutated in X-linked myotubular myopathy. Although MTMR2 and MTM are closely related, they are likely to have different functions. Recent studies revealed that MTM dephosphorylates specifically phosphatidylinositol 3-phosphate. Here we analyze the biochemical properties of the mouse Mtmr2 protein, which shares 97% amino acid identity with human MTMR2. We show that phosphatidylinositol-3-phosphate is also a substrate for Mtmr2, but, unlike myotubularin, Mtmr2 dephosphorylates phosphatidylinositol 3,5-bisphosphate with high efficiency and peak activity at neutral pH. We demonstrate that the known disease-associated MTMR2 mutations lead to dramatically reduced phosphatase activity, suggesting that the MTMR2 phosphatase activity is crucial for the proper function of peripheral nerves in CMT4B1. Expression analysis of Mtmr2 suggests particularly high levels in neurons. Thus, the demyelinating neuropathy CMT4B1 might be triggered by the malfunction of neural membrane recycling, membrane trafficking, and/or endocytic or exocytotic processes, combined with altered axon-Schwann cell interactions. Furthermore, the different biochemical properties of MTM and MTMR2 offer a potential explanation for the different human diseases caused by mutations in their respective gene

An animal model for Charcot-Marie-Tooth disease type 4B1

Charcot-Marie-Tooth disease (CMT) comprises a family of clinically and genetically very heterogeneous hereditary peripheral neuropathies and is one of the most common inherited neurological disorders. We have generated a mouse model for CMT type 4B1 using embryonic stem cell technology. To this end, we introduced a stop codon into the Mtmr2 locus within exon 9, at the position encoding amino acid 276 of the MTMR2 protein (E276X). Concomitantly, we have deleted the chromosomal region immediately downstream of the stop codon up to within exon 13. The resulting allele closely mimics the mutation found in a Saudi Arabian CMT4B1 patient. Animals homozygous for the mutation showed various degrees of complex myelin infoldings and outfoldings exclusively in peripheral nerves, in agreement with CMT4B1 genetics and pathology. Mainly, paranodal regions of the myelin sheath were affected, with a high degree of quantitative and qualitative variability between individuals. This pathology was progressive with age, and axonal damage was occasionally observed. Distal nerve regions were more affected than proximal parts, in line with the distribution in CMT. However, we found no significant electrophysiological changes, even in aged (16-month-old) mice, suggesting that myelin infoldings and outfoldings per se are not invariably associated with detectable electrophysiological abnormalities. Our animal model provides a basis for future detailed molecular and cellular studies on the underlying disease mechanisms in CMT4B1. Such an analysis will reveal how the disease develops, in particular, the enigmatic myelin infoldings and outfoldings as well as axonal damage, and provide mechanistic insights that may aid in the development of potential therapeutic approache

Mtmr13/Sbf2-deficient mice: an animal model for CMT4B2

Charcot-Marie-Tooth (CMT) disease denotes a large group of genetically heterogeneous hereditary motor and sensory neuropathies and ranks among the most common inherited neurological disorders. Mutations in the Myotubularin-Related Protein-2 (MTMR2) or MTMR13/Set-Binding Factor-2 (SBF2) genes are associated with the autosomal recessive disease subtypes CMT4B1 or CMT4B2. Both forms of CMT share similar features including a demyelinating neuropathy associated with reduced nerve conduction velocity (NCV) and focally folded myelin. Consistent with a common disease mechanism, the homodimeric MTMR2 acts as a phosphoinositide D3-phosphatase with phosphatidylinositol (PtdIns) 3-phosphate and PtdIns 3,5-bisphosphate as substrates while MTMR13/SBF2 is catalytically inactive but can form a tetrameric complex with MTMR2, resulting in a strong increase of the enzymatic activity of complexed MTMR2. To prove that MTMR13/SBF2 is the disease-causing gene in CMT4B2 and to provide a suitable animal model, we have generated Mtmr13/Sbf2-deficient mice. These animals reproduced myelin outfoldings and infoldings in motor and sensory peripheral nerves as the pathological hallmarks of CMT4B2, concomitant with decreased motor performance. The number and complexity of myelin misfoldings increased with age, associated with axonal degeneration, and decreased compound motor action potential amplitude. Prolonged F-wave latency indicated a mild NCV impairment. Loss of Mtmr13/Sbf2 did not affect the levels of its binding partner Mtmr2 and the Mtmr2-binding Dlg1/Sap97 in peripheral nerves. Mice deficient in Mtmr13/Sbf2 together with known Mtmr2-deficient animals will be of major value to unravel the disease mechanism in CMT4B and to elucidate the critical functions of protein complexes that are involved in phosphoinositide-controlled processes in peripheral nerve

Charcot-Marie-Tooth type 4B2 demyelinating neuropathy in miniature Schnauzer dogs caused by a novel splicing SBF2 (MTMR13) genetic variant: a new spontaneous clinical model

This study reports the first genetic variant in Miniature Schnauzer dogs responsible for the occurrence of a demyelinating peripheral neuropathy with abnormally folded myelin. This discovery establishes a genotype/phenotype correlation in affected Miniature Schnauzers that can be used for the diagnosis of these dogs. It further supports the dog as a natural model of a human disease; in this instance, Charcot-Marie-Tooth disease. It opens avenues to search the biological mechanisms responsible for the disease and to test new therapies in a non-rodent large animal model. In particular, recent gene editing methods that led to the restoration of dystrophin expression in a canine model of muscular dystrophy could be applied to other canine models such as this before translation to humans

Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J

Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells(1). Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate ( PtdIns( 3,5)P-2), a phospholipid present in small quantities that regulates membrane trafficking in the endosome - lysosome axis in yeast(2). Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2 beta ( early transposon 2 beta)(3) into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC ( suppressor of actin) domain PtdIns(3,5) P-2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns( 3,5) P2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosome - lysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated patients with hereditary motor and sensory neuropathy. This novel form of autosomal recessive Charcot - Marie - Tooth disorder is designated CMT4J.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62835/1/nature05876.pd

Endosomal Phosphoinositides and Human Diseases

Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking. Seven distinct PIs can be synthesized by phosphorylation of the inositol ring of phosphatidylinositol (PtdIns), and their metabolism is accurately regulated by PI kinases and phosphatases. Two of the PIs, PtdIns3P and PtdIns(3,5)P2, are present on intracellular endosomal compartments, and several studies suggest that they have a role in membrane remodeling and trafficking. We refer to them as ‘endosomal PIs’. An increasing number of human genetic diseases including myopathy and neuropathies are associated to mutations in enzymes regulating the turnover of these endosomal PIs. The PtdIns3P and PtdIns(3,5)P2 3-phosphatase myotubularin gene is mutated in X-linked centronuclear myopathy, whereas its homologs MTMR2 and MTMR13 and the PtdIns(3,5)P2 5-phosphatase SAC3/FIG4 are implicated in Charcot–Marie–Tooth peripheral neuropathies. Mutations in the gene encoding the PtdIns3P5-kinase PIP5K3/PIKfyve have been found in patients affected with François–Neetens fleck corneal dystrophy. This review presents the roles of the endosomal PIs and their regulators and proposes defects of membrane remodeling as a common pathological mechanism for the corresponding diseases

Defective Membrane Remodeling in Neuromuscular Diseases: Insights from Animal Models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches

Autonomous microfluidic multi-channel chip for real-time PCR with integrated liquid handling

ISSN:1387-2176ISSN:1572-878