INF2Mutations in Charcot–Marie–Tooth Disease with Glomerulopathy

BACKGROUND Charcot–Marie–Tooth neuropathy has been reported to be associated with renal diseases, mostly focal segmental glomerulosclerosis (FSGS). However, the common mechanisms underlying the neuropathy and FSGS remain unknown. Mutations in INF2 were recently identified in patients with autosomal dominant FSGS. INF2 encodes a formin protein that interacts with the Rho-GTPase CDC42 and myelin and lymphocyte protein (MAL) that are implicated in essential steps of myelination and myelin maintenance. We therefore hypothesized that INF2 may be responsible for cases of Charcot–Marie–Tooth neuropathy associated with FSGS. METHODS We performed direct genotyping of INF2 in 16 index patients with Charcot–Marie– Tooth neuropathy and FSGS who did not have a mutation in PMP22 or MPZ, encoding peripheral myelin protein 22 and myelin protein zero, respectively. Histologic and functional studies were also conducted. RESULTS We identified nine new heterozygous mutations in 12 of the 16 index patients (75%), all located in exons 2 and 3, encoding the diaphanous-inhibitory domain of INF2. Patients presented with an intermediate form of Charcot–Marie–Tooth neuropathy as well as a glomerulopathy with FSGS on kidney biopsy. Immunohistochemical analysis revealed strong INF2 expression in Schwann-cell cytoplasm and podocytes. Moreover, we demonstrated that INF2 colocalizes and interacts with MAL in Schwann cells. The INF2 mutants perturbed the INF2–MAL–CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42 and mislocalization of INF2, MAL, and CDC42. CONCLUSIONS INF2 mutations appear to cause many cases of FSGS-associated Charcot–Marie–Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwanncell function.

Neurology, 2013

De novo INF2 mutations expand the genetic spectrum of hereditary neuropathy with glomerulopathy ABSTRACT Objective: Identification of mutations in the inverted formin-2 (INF2) gene in patients with Charcot-Marie-Tooth (CMT) disease combined with focal segmental glomerulosclerosis (FSGS) in order to expand the genetic and phenotypic spectrum. Methods: We sequenced INF2 in 5 patients with CMT disease and FSGS. Mutations were subsequently screened in family members of the index patient and 264 control individuals.

Journal of the American Society of Nephrology, 2011

The recent identification of mutations in the INF2 gene, which encodes a member of the formin family of actin-regulating proteins, in cases of familial FSGS supports the importance of an intact actin cytoskeleton in podocyte function. To determine better the prevalence of INF2 mutations in autosomal dominant FSGS, we screened 54 families (78 patients) and detected mutations in 17% of them. All mutations were missense variants localized to the N-terminal diaphanous inhibitory domain of the protein, a region that interacts with the C-terminal diaphanous autoregulatory domain, thereby competing for actin monomer binding and inhibiting depolymerization. Six of the seven distinct altered residues localized to an INF2 region that corresponded to a subdomain of the mDia1 diaphanous inhibitory domain reported to co-immunoprecipitate with IQ motif-containing GTPase-activating protein 1 (IQGAP1). In addition, we evaluated 84 sporadic cases but detected a mutation in only one patient. In conclusion, mutations in INF2 are a major cause of autosomal dominant FSGS. Because IQGAP1 interacts with crucial podocyte proteins such as nephrin and PLC1, the identification of mutations that may alter the putative INF2-IQGAP1 interaction provides additional insight into the pathophysiologic mechanisms linking formin proteins to podocyte dysfunction and FSGS.

The New England Journal of Medicine, 2011

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Brain, 2007

The neurofilament light chain (NF-L) is a major constituent of intermediate filaments and plays a pivotal function in the assembly and maintenance of axonal cytoskeleton. Mutations in the NF-L gene (NEFL) cause autosomal dominant neuropathies that are classified either as axonal Charcot-Marie-Tooth (CMT) type 2E (CMT2E) or demyelinating CMT type 1F (CMT1F). The pathophysiological bases of the disorder(s) are elusive. We performed a mutational analysis of NEFL in a series of 177 index cases with CMT and without mutations in the genes for peripheral myelin protein zero (MPZ), peripheral myelin protein 22 (PMP22) and connexin 32 (GJB1); the motor nerve conduction velocity (MNCV) at the median nerve was below 38 m/s in 76 cases and above 38 m/s in 101. We identified five new pedigrees with four mutations in the head and rod domains of NF-L, including a novel Leu268Pro substitution and a novel del322Cys_326Asn deletion. Several examined affected members exhibited marked variability in the severity of disease and age at onset. Nerve conduction alterations were consistent with an axonal neuropathy often associated with demyelinating features, such as prolonged distal latencies (DL). Pathological examination of sural nerve biopsies in the probands detected in four cases a chronic axonal neuropathy dominated by focal accumulations of NF with axonal swellings (giant axons) and significant secondary demyelination; in the fifth case no NFs accumulations were evident but many myelinated fibres consisted exclusively of microtubules with few or absent NF. The pathological phenotype correlated with the pattern of nerve conduction alterations and indicated that NEFL mutations cause a profound alteration of the cytoskeleton possibly related to defective targeting of NF.

Journal of the American Society of Nephrology, 2020

Background Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly. Why the disease-causing mutations are restricted to the N terminus and how they cause human disease has been unclear. Methods We examined INF2 isoforms present in podocytes and evaluated INF2 cleavage as an explanation for immunoblot findings. We evaluated the expression of INF2 N- and C-terminal fragments in human kidney disease conditions. We also investigated the localization and functions of the DID-containing N-terminal fragment in podocytes and assessed whether the FSGS-associated R218Q mutation impairs INF2 cleavage or the funct...

Kidney International, 2013

Focal and segmental glomerulosclerosis (FSGS) is a histological pattern that has several etiologies, including genetics. The autosomal dominant form of FSGS is a heterogenic disease caused by mutations within three known genes: a-actinin 4 (ACTN4), canonical transient receptor potential 6 (TRPC6), and the inverted formin 2 (INF2) gene. More recently, INF2 mutations have also been attributed to Charcot-Marie-Tooth neuropathy associated with FSGS. Here we performed direct sequencing, histological characterization, and functional studies in a cohort of families with autosomal dominant FSGS. We detected a novel mutation in exon 6 of the INF2 gene outside of the exon 2-4 candidate region used for rapid diagnosis of autosomal dominant FSGS. This new mutation is predicted to alter a highly conserved amino-acid residue within the 17th a-helix of the diaphanous inhibitory domain of the protein. A longterm follow-up of this family indicated that all patients were diagnosed in adulthood, as opposed to early childhood, and progression to end-stage renal disease was at different times without clinical or electrodiagnostic evidence of neuropathy. Thus, this novel mutation in INF2 linked to nonsyndromic FSGS indicates the necessity for full gene sequencing if no mutation is found in the current rapid-screen region of the gene.

Orphanet Journal of Rare Diseases, 2019

Background: Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder characterized by wide clinical, genetic and pathomechanistic heterogeneity. Recently, the gene encoding peripheral myelin protein 2 (PMP2) was identified as a novel cause for CMT neuropathy with three mutations that structurally cluster together (p.Ile43Asn, p.Thr51Pro, p.Ile52Thr) reported in five families. Results: Using whole exome sequencing and cohort screening we identified two novel missense substitutions in PMP2 in Bulgarian (p.Met114Thr, c.341C > T) and German (p.Val115Ala, c.344 T > C) families. The mutations affect adjacent and highly conserved amino acid residues outside of the known mutation-rich region in the protein. Crystal structure analysis positions the affected residues within a cluster of highly conserved fatty acid coordinating residues implying their functional significance. The clinical, electrophysiological and imaging features in both families were consistent with a childhood onset polyneuropathy with variable patterns of demyelination, slow to very slow progression, and most severe involvement of the peroneal muscles. Conclusions: We expand the genetic and phenotypic spectrum of PMP2-related peripheral neuropathy. Our findings reveal a second mutational cluster in the protein.

Nature Medicine, 2014

Duplication of the gene encoding the peripheral myelin protein of 22kDA (PMP22) underlies the most common inherited neuropathy, Charcot-Marie-Tooth disease 1A (CMT1A) 1-3 , a disease without known cure 4-6 . Although demyelination represents a characteristic feature, the clinical phenotype of CMT1A is determined by the degree of axonal loss, and patients suffer from progressive muscle weakness and impaired sensation 4,7 . CMT1A disease manifests within the first two decades of life 8,9 and walking disabilities, foot deformities and electrophysiological abnormalities are already present in childhood . Here, we show in transgenic rodent models of CMT1A, that Schwann cells acquire a persistent differentiation defect during early postnatal development, caused by an imbalanced activity of the PI3K/AKT and the MEK/ERK signaling pathways. We demonstrate that enhanced PI3K/AKT signaling by axonally overexpressed Neuregulin-1 (Nrg1) type I drives diseased Schwann cells towards differentiation and preserves peripheral nerve axons. Importantly, in a preclinical experimental therapy using a CMT1A rat model, soluble NRG1 effectively overcomes impaired peripheral nerve development and restores nerve function until adulthood, when treatment is restricted to early postnatal development. Our findings suggest a model in which Schwann cell differentiation within a limited time window is crucial for the long-term maintenance of axonal support.

Scientific Reports

Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the β barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding. A multilamellar membrane structure, myelin, enwraps selected axons in the nervous system, enabling the prompt transmission of nerve impulses along the axonal membrane. Myelin contains a compact lipid-rich compartment with a unique set of membrane-associated proteins, such as myelin protein zero (MPZ), peripheral myelin protein of 22 kDa (PMP22), myelin basic protein (MBP), and myelin protein P2, and more loosely packed regions that also encompass cytoplasm. Close interplay between myelin proteins is important for the formation of cytoplasmic channels within myelin 1. In the peripheral nervous system (PNS), Schwann cells envelop axons and form insulating myelin sheaths. Defects in this fundamental structure result in chronic, severe neuropathological conditions, affecting nerve conduction velocity and neuron viability. Charcot-Marie-Tooth disease (CMT) is one of the most common inherited human neuropathies (prevalence of 1:2500), affecting both motor and sensory nerve conduction in the PNS 2. Several types of CMT exist; the most common form, autosomal dominant CMT1, influences Schwann cells and myelin. In CMT1, motor and sensory nerve conduction velocities are remarkably reduced, leading to muscle weakness and atrophy in the feet and occasionally also in upper limbs. Demyelination and onion bulb formation are present in most CMT1 patients. Most commonly, CMT1a is caused by a duplication of or a point mutation in the pmp22 gene, and 70-80% of all CMT1 cases are associated with pmp22 mutations 3. CMT1b results from mutations in the mpz gene and comprises 4% of all CMT1 cases. A number of other CMT1-associated genes have been described 2. Recently, three point mutations