Reply : A novel MCM3AP mutation in a Lebanese family with recessive Charcot-Marie-Tooth neuropathy

Non peer reviewe

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.Peer reviewe

ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.Peer reviewe

Distinct effects on mRNA export factor GANP underlie neurological disease phenotypes and alter gene expression depending on intron content

Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.Peer reviewe

MCM3AP in recessive Charcot-Marie-Tooth neuropathy and mild intellectual disability

Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability.Peer reviewe

Identification and role of MCM3AP disease gene in neurological disease spectrum and its neuronal modelling

Rare diseases are a heterogenous group of disorders, which together affect a large number of people. These diseases typically have a genetic cause, which affects various cellular processes and pathways. One of the key processes in the cell is RNA metabolism, which is crucial for gene expression. Defects in RNA processing steps contribute to a variety of human diseases ranging from motor neuron diseases to cancer. Most rare diseases currently have no cure. In this dissertation, biallelic variants in Minichromosome maintenance complex 3 associated protein (MCM3AP) gene were found to underlie a neurological syndrome: early-onset peripheral neuropathy with/without intellectual disability. Through international collaboration, patients from multiple families and the disease variants in MCM3AP were identified. Germinal center associated nuclear protein (GANP), encoded by MCM3AP, is a large scaffold protein in the Transcription and Export 2 (TREX-2) complex, which is responsible for the transport of mRNAs from nucleus to the cytoplasm. The effects of different disease-causing variants on GANP abundance on the nuclear envelope were determined, which led to establishment of a genotype/phenotype correlation regarding the severity of the motor phenotype. This research showed that GANP regulates gene expression in a cell type specific manner. In patient fibroblasts, GANP was found to dysregulate genes depending on intron content. In addition, stem cell-based technology and genome editing was utilized to allow modeling of GANP defects in human cultured motor neurons. We found that GANP is a major regulator of gene expression in developing motor neurons, with GANP defects altering the expression of genes related to synaptic functions and resulting in compensatory induction of protein synthesis and mitochondrial pathways. Understanding of the causes and mechanisms of rare diseases is needed for improving diagnostics, therapeutic development and personalized treatments. In this dissertation, the foundation for understanding the mechanisms of MCM3AP-linked neurological syndrome has been established through identification of the causative gene and description of the disease phenotype. The different cellular models including patient-specific motor neurons used in this study have revealed molecular pathways that may be targets for treatment in preclinical trials.Harvinaissairaudet ovat laaja tautien kirjo, joita sairastaa suuri määrä ihmisiä. Kyseiset sairaudet ovat tyypillisesti perinnöllisiä, ja vaikuttavat soluissa useisiin tärkeisiin prosesseihin. Yksi tärkeistä tapahtumista soluissa on ribonukleiinihapon (RNA) kiertokulku, joka on tärkeä geenien säätelylle ja ilmaisulle. Ongelmat RNA:n prosessoinnin eri vaiheissa johtavat erilaisiin sairauksiin liikehermosairauksista syöpään. Useimpiin harvinaissairauksiin ei ole tarjolla hoitokeinoja. Tässä väitöskirjassa MCM3AP (Minichromosome maintenance complex 3 associated protein) geeni todettiin taudinaiheuttajaksi lapsuusiällä alkavalle neurologiselle oireyhtymälle. Taudinkuvaan kuului potilailla pääasiallisina oireina ääreishermoston neuropatia ja lukuisissa potilaissa myös kehitysvamma. Lisäksi taudinkuvaan kuuluu muita neurologisia ilmentymiä kuten epilepsia ja ataksia sekä laaja-alaisia muita oireita kuten liikalihavuus, oppimisvaikeudet ja muut kehitykselliset häiriöt. Kansainvälisen yhteistyön avulla tunnistimme potilaita useista perheistä, jonka avulla tunnistimme tautia aiheuttavat geenivirheet MCM3AP geenissä. GANP (Germinal center associated nuclear protein), jota koodaa MCM3AP geeni, on merkittävä proteiini TREX-2 (Transcription and Export 2) kompleksissa. Kyseinen proteiinikompleksi mahdollistaa lähetti RNA:n kuljetuksen tumasta sytoplasmaan. Geenivirheiden vaikutukset GANP proteiinin määrään tumakalvolla auttoivat määrittämään perimän ja ilmiasun välisen korrelaation potilaiden liikkumisvaikeuksien suhteen. Tämä tutkimus osoitti, että GANP säätelee geenien ilmaisua solutyypistä riippuen. Potilaiden fibroblasteissa GANP sääteli geenejä intronien määrästä riippuen. Lisäksi tässä tutkimuksessa hyödynnettiin kantasoluteknologiaa ja geenieditointia, joka mahdollisti GANP proteiinin puutteen vaikutuksien analysoimisen ihmisen liikehermosoluissa. Tässä tutkimuksessa havaitsimme, että GANP on merkittävä geenien ilmaisun säätelijä liikehermosolujen kehityksen aikana. Geenivirheet muuttivat geenien ilmaisun säätelyä erityisesti hermoliitoksiin liittyvissä geeneissä ja lisäsivät kompensaatiota proteiinisynteesiin ja mitokondrioihin liittyvissä geeneissä. Harvinaissairauksien aiheuttajien ja solutason mekanismien ymmärrystä tarvitaan diagnostiikan, hoitokeinojen ja henkilökohtaisten hoitojen kehittämiseksi. Tässä väitöskirjassa perusteet MCM3AP geenivirheiden aiheuttaman neurologisen tautikirjon ymmärtämiseksi on luotu geenivirheiden löytymisestä taudinkuvan määrittelyyn. Tässä työssä hyödynnetyt erilaiset solumallit, kuten potilaiden kantasoluista erilaistetut hermosolut, ovat lisänneet ymmärrystä molekyylitason muutoksista, joita voidaan tulevaisuudessa hyödyntää lääkekehityksessä

MCM3AP in recessive Charcot-Marie-Tooth neuropathy and mild intellectual disability

Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability

MCM3AP in recessive Charcot-Marie-Tooth neuropathy and mild intellectual disability

Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability