The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis.

Mutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS.

Generation of two iPSC lines from adult central core disease patients with dominant missense variants in the RYR1 gene.

RYR1 variants are a common cause of congenital myopathies, including multi-minicore disease (MmD) and central core disease (CCD). Here, we generated iPSC lines from two CCD patients with dominant RYR1 missense variants that affect the transmembrane (pore) and SPRY3 protein domains (p.His4813Tyr and p.Asn1346Lys, respectively). Both lines had typical iPSC morphology, expressed canonical pluripotency markers, exhibited trilineage differentiation potential, and had normal karyotypes. Together with existing RYR1 iPSC lines, these represent important tools to study and develop treatments for RYR1-related myopathies.

Generation of two iPSC lines from patients with inherited central core disease and concurrent malignant hyperthermia caused by dominant missense variants in the RYR1 gene.

RYR1 variants are the most common genetic cause of congenital myopathies, and typically cause central core disease (CCD) and/or malignant hyperthermia (MH). Here, we generated iPSC lines from two patients with CCD and MH caused by dominant RYR1 variants within the central region of the protein (p.Val2168Met and p.Arg2508Cys). Both lines displayed typical iPSC morphology, uniform expression of pluripotency markers, trilineage differentiation potential, and had normal karyotypes. These are the first RYR1 iPSC lines from patients with both CCD and MH. As these are common CCD/MH variants, these lines should be useful to study these conditions and test therapeutics.

Generation of two induced pluripotent stem cell lines from a 33-year-old central core disease patient with a heterozygous dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene.

Central core disease (CCD) is a congenital disorder that results in hypotonia, delayed motor development, and areas of reduced oxidative activity in the muscle fibre. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 33-year-old male with CCD, caused by a previously unreported dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene. Both lines demonstrated typical morphology, pluripotency, trilineage differentiation, and had a normal karyotype. As the first published iPSC model of CCD caused by an RYR1 variant these lines are a potential resource for further investigation of RYR1-related myopathies in a human context.

Generation of an induced pluripotent stem cell line from a 3-month-old nemaline myopathy patient with a heterozygous dominant c.515C > A (p.Ala172Glu) variant in the ACTA1 gene.

Variants in the ACTA1 gene are a common cause of nemaline myopathy (NM); a muscle disease that typically presents at birth or early childhood with hypotonia and muscle weakness. Here, we generated an induced pluripotent stem cell line (iPSC) from lymphoblastoid cells of a 3-month-old female patient with intermediate NM caused by a dominant ACTA1 variant (c.515C > A (p.Ala172Glu)). iPSCs showed typical morphology, expressed pluripotency markers, demonstrated trilineage differentiation potential, and had a normal karyotype. This line complements our previously published ACTA1 iPSC lines derived from patients with typical and severe NM.

Generation of two isogenic induced pluripotent stem cell lines from a 1-month-old nemaline myopathy patient harbouring a homozygous recessive c.121C > T (p.Arg39Ter) variant in the ACTA1 gene.

Nemaline myopathy (NM) is a congenital skeletal muscle disorder that typically results in muscle weakness and the presence of rod-like structures (nemaline bodies) in the sarcoplasma and/or in the nuclei of myofibres. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 1-month-old male with severe NM caused by a homozygous recessive mutation in the ACTA1 gene (c.121C > T, p.Arg39Ter). The iPSC lines demonstrated typical morphology, expressed pluripotency markers, exhibited trilineage differentiation potential and displayed a normal karyotype. These isogenic lines represent a potential resource to investigate and model recessive ACTA1 disease in a human context.

Generation of two isogenic induced pluripotent stem cell lines from a 4-month-old severe nemaline myopathy patient with a heterozygous dominant c.553C > A (p.Arg183Ser) variant in the ACTA1 gene.

Nemaline myopathy (NM) is a congenital myopathy typically characterized by skeletal muscle weakness and the presence of abnormal thread- or rod-like structures (nemaline bodies) in myofibres. Pathogenic variants in the skeletal muscle alpha actin gene, ACTA1, cause approximately 25% of all NM cases. We generated two induced pluripotent stem cell lines from lymphoblastoid cells of a 4-month-old female with severe NM harbouring a dominant variant in ACTA1 (c.553C > A). The isogenic lines displayed characteristic iPSC morphology, expressed pluripotency markers, differentiated into cells of all three germ layers, and possessed normal karyotypes. These lines could be useful models of human ACTA1 disease.

Generation of two isogenic induced pluripotent stem cell lines from a 10-year-old typical nemaline myopathy patient with a heterozygous dominant c.541G>A (p.Asp179Asn) pathogenic variant in the ACTA1 gene.

Nemaline myopathy (NM) is a congenital myopathy typically characterized by skeletal muscle weakness and the presence of nemaline bodies in myofibres. Approximately 25% of NM cases are caused by variants in ACTA1. We generated two induced pluripotent stem cell lines from lymphoblastoid cells of a 10-year-old female with typical NM harbouring a dominant pathogenic variant in ACTA1 (c.541C>A). The isogenic lines displayed typical iPSC morphology, expressed pluripotency markers, and could differentiate into each of the three germ layers. Although the lines have partial or complete X chromosome duplication, they may still prove useful as models of human ACTA1 disease.

Genetic screening of ANXA11 revealed novel mutations linked to amyotrophic lateral sclerosis.

ANXA11 mutations have previously been discovered in amyotrophic lateral sclerosis (ALS) motor neuron disease. To confirm the contribution of ANXA11 mutations to ALS, a large exome data set obtained from 330 French patients, including 150 familial ALS index cases and 180 sporadic ALS cases, was analyzed, leading to the identification of 3 rare ANXA11 variants in 5 patients. The novel p.L254V variant was associated with early onset sporadic ALS. The novel p.D40Y mutation and the p.G38R variant concerned patients with predominant pyramidal tract involvement and cognitive decline. Neuropathologic findings in a p.G38R carrier associated the presence of ALS typical inclusions within the spinal cord, massive degeneration of the lateral tracts, and type A frontotemporal lobar degeneration. This mutant form of annexin A11 accumulated in various brain regions and in spinal cord motor neurons, although its stability was decreased in patients' lymphoblasts. Because most ANXA11 inclusions were not colocalized with transactive response DNA-binding protein 43 or p62 deposits, ANXA11 aggregation does not seem mandatory to trigger neurodegeneration with additional participants/partner proteins that could intervene.

Novel UBQLN2 mutations linked to amyotrophic lateral sclerosis and atypical hereditary spastic paraplegia phenotype through defective HSP70-mediated proteolysis.

Mutations in UBQLN2 have been associated with rare cases of X-linked juvenile and adult forms of amyotrophic lateral sclerosis (ALS) and ALS linked to frontotemporal dementia (FTD). Here, we report 1 known (c.1489C>T, p.Pro497Ser, P497S) and 3 novel (c.1481C>T, p.Pro494Leu, P494L; c.1498C>T, p.Pro500Ser, P500S; and c.1516C>G, p.Pro506Ala, P506A) missense mutations in the PXX domain of UBQLN2 in familial motor neuron diseases including ALS and spastic paraplegia (SP). A novel missense mutation (c.1462G>A, p.Ala488Thr, A488T) adjacent to this hotspot UBQLN2 domain was identified in a sporadic case of ALS. These mutations are conserved in mammals, are absent from ExAC and gnomAD browsers, and are predicted to be deleterious by SIFT in silico analysis. Patient lymphoblasts carrying a UBQLN2 mutation showed absence of ubiquilin-2 accumulation, disrupted binding with HSP70, and impaired autophagic pathway. Our results confirm the role of PXX repeat in ALS pathogenesis, show that UBQLN2-linked disease can manifest like a SP phenotype, evidence a highly reduced disease penetrance in females carrying UBQLN2 mutations, which is important information for genetic counseling, and underline the pivotal role of ubiquilin-2 in proteolysis regulation pathways.