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 man in his 20s 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 during adolescence after prolonged immobilisation. Muscle MRI during adolescence 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 SNPs that introduce aberrant 3’ UTR splicing may be underrecognised in Mendelian disease. We encourage consideration of this mechanism during variant curation.This study was funded by an Australian NHMRC Investigator Grant (APP2007769), Fellowship (APP1117510) and Ideas Grant (APP2002640). The study was supported by an Australian Government Research Training Program (RTP) Scholarship, as well as resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia.N

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.LD is supported by an Australian Government Research Training Program (RTP) Scholarship. GR (Investigator Grant, APP2007769) and NGL (Fellowship APP1117510) are supported by the Australian National Health and Medical Research Council (NHMRC). This work is funded by NHMRC Ideas Grant (APP2002640). This work was supported by resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia.Peer reviewe

Heterozygous CAPN3 missense variants causing autosomal-dominant calpainopathy in seven unrelated families

[Aims] Recessive variants in CAPN3 gene are the cause of the commonest form of autosomal recessive limb girdle muscle dystrophy. However, two distinct in-frame deletions in CAPN3 (NM_000070.3:c.643_663del21 and c.598_621del15) and more recently, Gly445Arg and Arg572Pro substitutions have been linked to autosomal dominant (AD) forms of calpainopathy. We report 21 affected individuals from seven unrelated families presenting with an autosomal dominant form of muscular dystrophy associated with five different heterozygous missense variants in CAPN.[Methods] We have used massively parallel gene sequencing (MPS) to determine the genetic basis of a dominant form of limb girdle muscular dystrophy in affected individuals from seven unrelated families.[Results] The c.700G> A, [p.(Gly234Arg)], c.1327T> C [p.(Ser443Pro], c.1333G> A [p.(Gly445Arg)], c.1661A> C [p.(Tyr554Ser)] and c.1706T> C [p.(Phe569Ser)] CAPN3 variants were identified. Affected individuals presented in young adulthood with progressive proximal and axial weakness, waddling walking and scapular winging or with isolated hyperCKaemia. Muscle imaging showed fatty replacement of paraspinal muscles, variable degrees of involvement of the gluteal muscles, and the posterior compartment of the thigh and minor changes at the mid-leg level. Muscle biopsies revealed mild myopathic changes. Western blot analysis revealed a clear reduction in calpain 3 in skeletal muscle relative to controls. Protein modelling of these variants on the predicted structure of calpain 3 revealed that all variants are located in proximity to the calmodulin-binding site and are predicted to interfere with proteolytic activation.[Conclusions] We expand the genotypic spectrum of CAPN3-associated muscular dystrophy due to autosomal dominant missense variants.This study was funded in part by Instituto de Salud Carlos III through the project PI14/00738 to M. O. (co-funded by European Regional Development Fund. ERDF, a way to build Europe). We thank CERCA Programme / Generalitat de Catalunya for institutional support NGL (APP1117510) and GR (APP1122952) are supported by the Australian National Health and Medical Research Council (NHMRC). This work is also funded by an NHMRC Project Grant (APP1080587).Peer reviewe

Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts

9 páginas.Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mtDNA. Here, we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from 2 patients with MELAS who harbored the A3243G mutation. Both mitochondrial respiratory chain enzyme activities and coenzyme Q(10) (CoQ) levels were significantly decreased in MELAS fibroblasts. A similar decrease in mitochondrial membrane potential was found in intact MELAS fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and the activation of mitochondrial permeability transition (MPT), which triggered the degradation of impaired mitochondria. Furthermore, we found defective autophagosome elimination in MELAS fibroblasts. Electron and fluorescence microscopy studies confirmed a massive degradation of mitochondria and accumulation of autophagosomes, suggesting mitophagy activation and deficient autophagic flux. Transmitochondrial cybrids harboring the A3243G mutation also showed CoQ deficiency and increased autophagy activity. All these abnormalities were partially restored by CoQ supplementation. Autophagy in MELAS fibroblasts was also abolished by treatment with antioxidants or cyclosporine, suggesting that both reactive oxygen species and MPT participate in this process. Furthermore, prevention of autophagy in MELAS fibroblasts resulted in apoptotic cell death, suggesting a protective role of autophagy in MELAS fibroblasts.This work was supported by Ministerio de Sanidad, Spain, grants FIS PI080500 and FIS EC08/00076, and by Asociación de Enfermos de Patología Mitocondrial (AEPMI) and Fundación Española de Enfermedades Lisosomales (FEEL). M. de la Mata is the recipient of a fellowship from Colegio Oficial de Farmacéuticos de Sevilla. This group was founded by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII).Peer reviewe

BNIP3 Is Involved in Muscle Fiber Atrophy in Late-Onset Pompe Disease Patients

Late-onset Pompe disease (LOPD) is a rare genetic disorder produced by mutations in the GAA gene and is characterized by progressive muscle weakness. LOPD muscle biopsies show accumulation of glycogen along with the autophagic vacuoles associated with atrophic muscle fibers. The expression of molecules related to muscle fiber atrophy in muscle biopsies of LOPD patients was studied using immunofluorescence and real-time PCR. BCL2 and adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a well-known atrogene, was identified as a potential mediator of muscle fiber atrophy in LOPD muscle biopsies. Vacuolated fibers in LOPD patient muscle biopsies were smaller than nonvacuolated fibers and expressed BNIP3. The current data suggested that BNIP3 expression is regulated by inhibition of the AKT-mammalian target of rapamycin pathway, leading to phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) at Ser317 by AMP-activated protein kinase. Myoblasts and myotubes obtained from LOPD patients and age-matched controls were studied to confirm these results using different molecular techniques. Myotubes derived from LOPD patients were likewise smaller and expressed BNIP3. Conclusively, transfection of BNIP3 into control myotubes led to myotube atrophy. These findings suggest a cascade that starts with the inhibition of the AKT-mammalian target of rapamycin pathway and activation of BNIP3 expression, leading to progressive muscle fiber atrophy. These results open the door to potential new treatments targeting BNIP3 to reduce its deleterious effects on muscle fiber atrophy in Pompe disease.Peer reviewe

Método para obtener información útil para el diagnóstico de enfermedades neuromusculares

Procedimiento para obtener, a partir de la biopsia de un paciente, nuevos parámetros que permiten diagnosticar de manera objetiva diferentes enfermedades neuromusculares y su grado de afectación al paciente, que comprende: - realizar una tinción de la biopsia para resaltar las fibras musculares tipo I, las fibras musculares tipo II y el endomisio; - obtener una imagen de la biopsia tras la tinción; - segmentar la imagen para identificar los contornos de las fibras musculares; - formar una red donde las fibras musculares constituyen nodos y los contactos entre fibras musculares constituyen las uniones entre los nodos; - formar un vector característico de la biopsia cuyos elementos se eligen entre parámetros geométricos de las fibras y parámetros de la red construida; y - comparar biopsias control y afectas por medio de ACP utilizando en cada caso el vector característico seleccionadoPeer reviewedServicio Andaluz de Salud, Universidad de Sevilla, Consejo Superior de Investigaciones CientíficasB1 Patente sin examen previ

Quantifiable diagnosis of muscular dystrophies and neurogenic atrophies through network analysis

[Background] The diagnosis of neuromuscular diseases is strongly based on the histological characterization of muscle biopsies. However, this morphological analysis is mostly a subjective process and difficult to quantify. We have tested if network science can provide a novel framework to extract useful information from muscle biopsies, developing a novel method that analyzes muscle samples in an objective, automated, fast and precise manner.[Methods] Our database consisted of 102 muscle biopsy images from 70 individuals (including controls, patients with neurogenic atrophies and patients with muscular dystrophies). We used this to develop a new method, Neuromuscular DIseases Computerized Image Analysis (NDICIA), that uses network science analysis to capture the defining signature of muscle biopsy images. NDICIA characterizes muscle tissues by representing each image as a network, with fibers serving as nodes and fiber contacts as links.[Results] After a ‘training’ phase with control and pathological biopsies, NDICIA was able to quantify the degree of pathology of each sample. We validated our method by comparing NDICIA quantification of the severity of muscular dystrophies with a pathologist’s evaluation of the degree of pathology, resulting in a strong correlation (R = 0.900, P <0.00001). Importantly, our approach can be used to quantify new images without the need for prior ‘training’. Therefore, we show that network science analysis captures the useful information contained in muscle biopsies, helping the diagnosis of muscular dystrophies and neurogenic atrophies.[Conclusions] Our novel network analysis approach will serve as a valuable tool for assessing the etiology of muscular dystrophies or neurogenic atrophies, and has the potential to quantify treatment outcomes in preclinical and clinical trials.LME and AM were supported by the Miguel Servet (Instituto Carlos III) program that also funded the work. LME is funded by the Spanish Ministry of Science (BFU2011-25734), and AP is funded by the Spanish Ministry of Science and the Andalusian Government.LME and AM were supported by the Miguel Servet (Instituto Carlos III) program that also funded the work. LME is funded by the Spanish Ministry of Science (BFU2011-25734), and AP is funded by the Spanish Ministry of Science and the Andalusian Government. AS is funded by the Consejería de Innovación, Ciencia y Empresa of the Junta de Andalucía.Peer Reviewe

Método para obtener información útil para el diagnóstico de enfermedades neuromusculares

Método para obtener información útil para el diagnóstico de enfermedades neuromusculares. Procedimiento para obtener, a partir de la biopsia de un paciente, nuevos parámetros que permiten diagnosticar de manera objetiva diferentes enfermedades neuromusculares y su grado de afectación al paciente, que comprende: realizar una tinción de la biopsia para resaltar las fibras musculares tipo I, las fibras musculares tipo II y el endomisio; obtener una imagen de la biopsia tras la tinción; segmentar la imagen para identificar los contornos de las fibras musculares; formar una red donde las fibras musculares constituyen nodos y los contactos entre fibras musculares constituyen las uniones entre los nodos; formar un vector característico de la biopsia cuyos elementos se eligen entre parámetros geométricos de las fibras y parámetros de la red construida; y comparar biopsias control y afectas por medio de ACP utilizando en cada caso el vector característico seleccionado.Peer reviewedServicio Andaluz de Salud, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

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.This project has been founded by ISCIII and FEDER “a way to achieve Europe”; Grant number PI16/00612(MC‐S) and PI16/01843 (CP). MC‐S was supported by ISCIII (JR15/00042) and Junta de Andalucia‐Consejeria de Salud (B‐0005‐2017)

Hemimegalencephaly: Prenatal diagnosis and outcome

Hemimegalencephaly (HME) is a developmental abnormality of the central nervous system (CNS) which may present as either a syndromic or isolated case. Here, we present two cases of early prenatal diagnosis of HME. Prenatal CNS ultrasound and MRI in the first case revealed ventricular asymmetry, midline shift with displacement of the occipital lobe across the midline, large dilatation mainly at the posterior horn of the left lateral ventricle, and a head circumference in the 90th percentile without involvement of the brain stem and cerebellum, as well as abdominal lymphangioma. Right hemispherectomy was performed at 3 months of age due to intractable seizures. The pathological specimen showed findings characteristic of HME, including a disorganized cytoarchitecture with lack of neuronal lamination, focal areas of polymicrogyria, and neuronal heterotopias with dysplastic cells. In the second case, 2D and 3D neurosonography demonstrated similar findings (asymmetry of cerebral hemispheres, midline shift, and dilation of the posterior horn of the left lateral cerebral ventricle). Posterior fossa structures were unremarkable. HME was diagnosed and the pregnancy was terminated. Autopsy findings confirmed the diagnosis of HME.Peer Reviewe