A KLHL40 3' UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism.

Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). Common features include fetal akinesia, fractures, contractures, dysphagia, respiratory failure and neonatal death. Here, we describe a 26-year-old man with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles' contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout childhood and became wheelchair bound from age 13 after prolonged immobilization. Muscle magnetic resonance imaging at age 13 indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs and vastus intermedius. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in KLHL40 mRNA and protein, respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in KLHL40; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G > T) in the 3' untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G > T variant created a cryptic donor splice site. RNA-seq and in vitro analyses indicated that the c.*152G > T variant induces multiple de novo splicing events that likely provoke nonsense mediated decay of KLHL40 mRNA explaining the loss of mRNA expression and protein abundance in the patient. Analysis of 3' UTR variants in ClinVar suggests variants that introduce aberrant 3' UTR splicing may be underrecognized in Mendelian disease. We encourage consideration of this mechanism during variant curation.

Ablation of the carboxy-terminal end of MAMDC2 causes a distinct muscular dystrophy.

The extracellular matrix (ECM) has an important role in the development and maintenance of skeletal muscle, and several muscle diseases are associated with the dysfunction of ECM elements. MAMDC2 is a putative ECM protein and its role in cell proliferation has been investigated in certain cancer types. However, its participation in skeletal muscle physiology has not been previously studied. We describe 17 individuals with an autosomal dominant muscular dystrophy belonging to two unrelated families in which different heterozygous truncating variants in the last exon of MAMDC2 co-segregate correctly with the disease. The radiological aspect of muscle involvement resembles that of COL6 myopathies with fat replacement at the peripheral rim of vastii muscles. In this cohort, a subfascial and peri-tendinous pattern is observed in upper and lower limb muscles. Here we show that MAMDC2 is expressed in adult skeletal muscle and differentiating muscle cells, where it appears to localize to the sarcoplasm and myonuclei. In addition, we show it is secreted by myoblasts and differentiating myotubes into to the extracellular compartment. The last exon encodes a disordered region with a polar residue compositional bias loss of which likely induces a toxic effect of the mutant protein. The precise mechanisms by which the altered MAMDC2 proteins cause disease remains to be determined. MAMDC2 is a skeletal muscle disease-associated protein. Its role in muscle development and ECM-muscle communication remains to be fully elucidated. Screening of the last exon of MAMDC2 should be considered in patients presenting with autosomal dominant muscular dystrophy, particularly in those with a subfascial radiological pattern of muscle involvement.

Novel ANO5 intronic Roma variant alters splicing causing muscular dystrophy.

The pathogenic role of intronic variants is generally difficult to assess, except for those near known splice sites for which aberrant splicing is suspected, although deeper intronic variants can also alter splicing. We have identified a novel (NM_213599.2:c.1180+6T>C) ANO5 variant that causes the exclusion of exon 12. The mutation, identified in a Roma individual, has an estimated carrier rate of 1.68% among the Iberian Roma population, this being the first ANO5 pathogenic variant communicated in this ethnic group. In this study, we have also characterized the ANO5 splice forms expressed in human muscle with the detection of an alternative transcript, in which exons 8 and 9 are spliced out.

Proteolytic Processing of Neurexins by Presenilins Sustains Synaptic Vesicle Release.

Proteolytic processing of synaptic adhesion components can accommodate the function of synapses to activity-dependent changes. The adhesion system formed by neurexins (Nrxns) and neuroligins (Nlgns) bidirectionally orchestrate the function of presynaptic and postsynaptic terminals. Previous studies have shown that presenilins (PS), components of the gamma-secretase complex frequently mutated in familial Alzheimer's disease, clear from glutamatergic terminals the accumulation of Nrxn C-terminal fragments (Nrxn-CTF) generated by ectodomain shedding. Here, we characterized the synaptic consequences of the proteolytic processing of Nrxns in cultured hippocampal neurons from mice and rats of both sexes. We show that activation of presynaptic Nrxns with postsynaptic Nlgn1 or inhibition of ectodomain shedding in axonal Nrxn1-ß increases presynaptic release at individual terminals, likely reflecting an increase in the number of functional release sites. Importantly, inactivation of PS inhibits presynaptic release downstream of Nrxn activation, leaving synaptic vesicle recruitment unaltered. Glutamate-receptor signaling initiates the activity-dependent generation of Nrxn-CTF, which accumulate at presynaptic terminals lacking PS function. The sole expression of Nrxn-CTF decreases presynaptic release and calcium flux, recapitulating the deficits due to loss of PS function. Our data indicate that inhibition of Nrxn processing by PS is deleterious to glutamatergic function.SIGNIFICANCE STATEMENT To gain insight into the role of presenilins (PS) in excitatory synaptic function, we address the relevance of the proteolytic processing of presynaptic neurexins (Nrxns) in glutamatergic differentiation. Using synaptic fluorescence probes in cultured hippocampal neurons, we report that trans-synaptic activation of Nrxns produces a robust increase in presynaptic calcium levels and neurotransmitter release at individual glutamatergic terminals by a mechanism that depends on normal PS activity. Abnormal accumulation of Nrxn C-terminal fragments resulting from impaired PS activity inhibits presynaptic calcium signal and neurotransmitter release, assigning synaptic defects to Nrxns as a specific PS substrate. These data may provide links into how loss of PS activity inhibits glutamatergic synaptic function in Alzheimer's disease patients.

Muscle MRI characteristic pattern for late-onset TK2 deficiency diagnosis.

BACKGROUND AND OBJECTIVE: TK2 deficiency (TK2d) is a rare mitochondrial disorder that manifests predominantly as a progressive myopathy with a broad spectrum of severity and age of onset. The rate of progression is variable, and the prognosis is poor due to early and severe respiratory involvement. Early and accurate diagnosis is particularly important since a specific treatment is under development. This study aims to evaluate the diagnostic value of lower limb muscle MRI in adult patients with TK2d. METHODS: We studied a cohort of 45 genetically confirmed patients with mitochondrial myopathy (16 with mutations in TK2, 9 with mutations in other nuclear genes involved in mitochondrial DNA [mtDNA] synthesis or maintenance, 10 with single mtDNA deletions, and 10 with point mtDNA mutations) to analyze the imaging pattern of fat replacement in lower limb muscles. We compared the identified pattern in patients with TK2d with the MRI pattern of other non-mitochondrial genetic myopathies that share similar clinical characteristics. RESULTS: We found a consistent lower limb muscle MRI pattern in patients with TK2d characterized by involvement of the gluteus maximus, gastrocnemius medialis, and sartorius muscles. The identified pattern in TK2 patients differs from the known radiological involvement of other resembling muscle dystrophies that share clinical features. CONCLUSIONS: By analyzing the largest cohort of muscle MRI from patients with mitochondrial myopathies studied to date, we identified a characteristic and specific radiological pattern of muscle involvement in patients with TK2d that could be useful to speed up its diagnosis.

NOVEL intronic CAPN3 Roma mutation alters splicing causing RNA mediated decay.

CAPN3 mutations cause a limb girdle muscular dystrophy. Functional characterization of novel mutations facilitates diagnosis of future cases. We have identified a novel (c.1992 + 2T>G) CAPN3 mutation that disrupts the donor splice site of intron 17 splicing out exon 17, with mRNA levels severely reduced or undetectable. The mutation induces a strong change in the 3D structure of the mRNA which supports no-go mRNA decay as the probable mechanism for RNA degradation. The mutation was identified in two unrelated Roma individuals showing a common ancestral origin and founder effect. This is the first Roma CAPN3 mutation to be reported.

Generation of an induced pluripotent stem cell line (CSCRMi001-A) from a patient with a new type of limb-girdle muscular dystrophy (LGMD) due to a missense mutation in POGLUT1 (Rumi).

Recently, a new type of limb-girdle muscular dystrophy (LGMD type 2Z) has been identified due to a missense mutation in POGLUT1 (protein O-glucosyltransferase-Rumi), an enzyme capable of adding glucose to a distinct serine residue of epidermal growth factor-like repeats containing a C-X-S-X-(P/A)-C consensus sequence such as Notch receptors. Affected patients demonstrate reduced Notch signaling, decreased muscle stem cell pool and hypoglycosylation of α-dystroglycan, leading to LGMD phenotype. Here we report the generation and characterization of an iPSC line (CSCRMi001-A) from a LGMD-2Z patient with missense mutation in POGLUT1 which can be used for in vitro disease modeling.

A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss.

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb-girdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O-glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O-glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle-specific α-dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch-dependent loss of satellite cells.

Presenilin/γ-secretase regulates neurexin processing at synapses.

Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing ß-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect ß-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS-/- cells rescues the processing of ß-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD.