Muscular Phenotype Research Articles

Using MRI to measure in vivo free radical production and perfusion dynamics in a mouse model of elevated oxidative stress and neurogenic atrophy

Mitochondrial dysfunction, reactive oxygen species (ROS) and oxidative damage have been implicated to play a causative role in age-related skeletal muscle atrophy and weakness (i.e. sarcopenia). Mice lacking the superoxide scavenger CuZnSOD (Sod1−/−) exhibit high levels of oxygen-derived radicals and oxidative damage, associated with neuronal and muscular phenotypes consistent with sarcopenia. We used magnetic resonance imaging (MRI) technology combined with immunospin-trapping (IST) to measure in vivo free radical levels in skeletal muscle from wildtype, Sod1−/− and SynTgSod1−/− mice, a mouse model generated using targeted expression of the human Sod1 transgene specifically in neuronal tissues to determine the impact of motor neuron degeneration in muscle atrophy. By combining the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) and molecular MRI (mMRI), we monitored the level of free radicals in mouse hindlimb muscle. The level of membrane-bound macromolecular radicals in the quadriceps muscle was elevated by ~3-fold in Sod1−/− mice, but normalized to wildtype levels in SynTgSod1−/− rescue mice. Skeletal muscle mass was reduced by ~25–30% in Sod1−/− mice, but fully reversed in muscle from SynTgSod1−/− mice. Using perfusion MRI we also measured the dynamics of blood flow within mouse hindlimb. Relative muscle blood flow in Sod1−/− is decreased to ~50% of wildtype and remained low in the SynTgSod1−/− mice. Our findings are significant in that we have shown for the first time that in vivo free radical production in skeletal muscle is directly correlated to muscle atrophy in an experimental model of oxidative stress. Neuron-specific expression of CuZnSOD reverses the in vivo free radical production in skeletal muscle in the Sod1−/− mouse model and prevents muscle atrophy. These results further support the feasibility of using in vivo assessments of redox status in the progression of a pathological process such as sarcopenia. This approach can also be valuable for evaluating responses to pharmacologic interventions.

Beneficial role of adipose-derived mesenchymal stem cells from microfragmented fat in a murine model of duchenne muscular dystrophy.

No etiologic therapy is available for Duchenne muscular dystrophy (DMD), but mesenchymal stem cells were shown to be effective in preclinical models of DMD. The objective of this study is to investigate the effect of microfragmented fat extracted on a murine model of DMD. Fat tissue was extracted from healthy human participants and injected IM into DMD mice. Histological analysis, cytokines, and force measurement were performed up to 4 weeks after injection. Duchenne muscular dystrophy mice injected with microfragmented fat exhibited an improved muscle phenotype (decreased necrosis and fibrosis), a decrease of inflammatory cytokines, and increased strength. Administration of microfragmented fat in key muscles may improve muscular phenotype in patients with DMD. Muscle Nerve, 2019.

NNOS/GSNOR interaction contributes to skeletal muscle differentiation and homeostasis

Neuronal nitric oxide synthase (nNOS) plays a crucial role in the maintenance of correct skeletal muscle function due, at least in part, to S-nitrosylation of specific protein targets. Similarly, we recently provided evidence for a muscular phenotype in mice lacking the denitrosylase S-nitrosoglutathione reductase (GSNOR). Here, we demonstrate that nNOS and GSNOR are concomitantly expressed during differentiation of C2C12. They colocalizes at the sarcolemma and co-immunoprecipitate in cells and in myofibers. We also provide evidence that GSNOR expression decreases in mouse models of muscular dystrophies and of muscle atrophy and wasting, i.e., aging and amyotrophic lateral sclerosis, suggesting a more general regulatory role of GSNOR in skeletal muscle homeostasis.

Parent-of-Origin Effects in 15q11.2 BP1-BP2 Microdeletion (Burnside-Butler) Syndrome.

To identify whether parent-of-origin effects (POE) of the 15q11.2 BP1-BP2 microdeletion are associated with differences in clinical features in individuals inheriting the deletion, we collected 71 individuals reported with phenotypic data and known inheritance from a clinical cohort, a research cohort, the DECIPHER database, and the primary literature. Chi-squared and Mann-Whitney U tests were used to test for differences in specific and grouped clinical symptoms based on parental inheritance and proband gender. Analyses controlled for sibling sets and individuals with additional variants of uncertain significance (VOUS). Among all probands, maternal deletions were associated with macrocephaly (p = 0.016) and autism spectrum disorder (ASD; p = 0.02), while paternal deletions were associated with congenital heart disease (CHD; p = 0.004). Excluding sibling sets, maternal deletions were associated with epilepsy as well as macrocephaly (p < 0.05), while paternal deletions were associated with CHD and abnormal muscular phenotypes (p < 0.05). Excluding sibling sets and probands with an additional VOUS, maternal deletions were associated with epilepsy (p = 0.019) and paternal deletions associated with muscular phenotypes (p = 0.008). Significant gender-based differences were also observed. Our results supported POEs of this deletion and included macrocephaly, epilepsy and ASD in maternal deletions with CHD and abnormal muscular phenotypes seen in paternal deletions.

Mitochondrial dysfunction and kidney disease

Context: Mitochondria play a vital role in producing the energy needed for different cellular activities. The role of mitochondria in different diseases and the aging process is gradually being clarified. Different studies have suggested that mitochondrial dysfunction due to mutations in genes that maintain the integrity of mitochondrial DNA (mtDNA), mitophagy, and apoptosis can lead to many neurological and muscular phenotypes as well as diseases in other organ systems including liver, gastrointestinal tract, heart, and kidneys. We examined the current knowledge of mitochondrial dysfunction and its role in renal pathophysiology. Additionally, we examined how chronic kidney diseases can lead to mitochondrial dysfunction through oxidative stress accumulation, which can subsequently lead to other pathological complications. Evidence Acquisitions: Directory of Open Access Journals (DOAJ), Google Scholar, PubMed (NLM), LISTA (EBSCO), and Web of Science have been searched. Results: The renal pathological manifestation of mitochondrial dysfunction includes tubular defects, focal segmental glomerular sclerosis (FSGS), glomerular dysfunction, interstitial nephritis, and cystic kidney disease or renal tumors. These conditions can be caused by mutations in the nuclear genes that are involved in mtDNA replication and transcription or due to mtDNA mutations in the genes involved in the respiratory chain. Conclusions: Clearly, mtDNA plays an important role in renal pathology, and mitochondria may serve as a potential therapeutic target to treat different renal pathologies.

The nuclear variant of bone morphogenetic protein 2 (nBMP2) is expressed in macrophages and alters calcium response

We previously identified a nuclear variant of bone morphogenetic protein 2 (BMP2), named nBMP2, that is translated from an alternative start codon. Decreased nuclear localization of nBMP2 in the nBmp2NLStm mouse model leads to muscular, neurological, and immune phenotypes—all of which are consistent with aberrant intracellular calcium (Ca2+) response. Ca2+ response in these mice, however, has yet to be measured directly. Because a prior study suggested impairment of macrophage function in nBmp2NLStm mutant mice, bone marrow derived (BMD) macrophages and splenic macrophages were isolated from wild type and nBmp2NLStm mutant mice. Immunocytochemistry revealed that nuclei of both BMD and splenic macrophages from wild type mice contain nBMP2, while the protein is decreased in nuclei of nBmp2NLStm mutant macrophages. Live-cell Ca2+ imaging and engulfment assays revealed that Ca2+ response and phagocytosis in response to bacterial supernatant are similar in BMD macrophages isolated from naïve (uninfected) nBmp2NLStm mutant mice and wild type mice, but are deficient in splenic macrophages isolated from mutant mice after secondary systemic infection with Staphylococcus aureus, suggesting progressive impairment as macrophages respond to infection. This direct evidence of impaired Ca2+ handling in nBMP2 mutant macrophages supports the hypothesis that nBMP2 plays a role in Ca2+ response.

Sex influences DNA methylation and gene expression in human skeletal muscle myoblasts and myotubes

BackgroundSex differences are known to impact muscle phenotypes, metabolism, and disease risk. Skeletal muscle stem cells (satellite cells) are important for muscle repair and to maintain functional skeletal muscle. Here we studied, for the first time, effects of sex on DNA methylation and gene expression in primary human myoblasts (activated satellite cells) before and after differentiation into myotubes.MethodWe used an array-based approach to analyse genome-wide DNA methylation and gene expression in myoblasts and myotubes from 13 women and 13 men. The results were followed up with a reporter gene assay.ResultsGenome-wide DNA methylation and gene expression differences between the sexes were detected in both myoblasts and myotubes, on the autosomes as well as the X-chromosome, despite lack of exposure to sex hormones and other factors that differ between sexes. Pathway analysis revealed higher expression of oxidative phosphorylation and other metabolic pathways in myoblasts from women compared to men. Oxidative phosphorylation was also enriched among genes with higher expression in myotubes from women. Forty genes in myoblasts and 9 in myotubes had differences in both DNA methylation and gene expression between the sexes, including LAMP2 and SIRT1 in myoblasts and KDM6A in myotubes. Furthermore, increased DNA methylation of LAMP2 promoter had negative effects on reporter gene expression. Five genes (CREB5, RPS4X, SYAP1, XIST, and ZRSR2) showed differential DNA methylation and gene expression between the sexes in both myoblasts and myotubes. Interestingly, differences in DNA methylation and expression between women and men were also found during differentiation (myoblasts versus myotubes), e.g., in genes involved in energy metabolism. Interestingly, more DNA methylation changes occur in women compared to men on autosomes.ConclusionAll together, we show that epigenetic and transcriptional differences exist in human myoblasts and myotubes as well as during differentiation between women and men. We believe that these intrinsic differences might contribute to sex dependent differences in muscular phenotypes.

Refining the spinobulbar muscular atrophy phenotype by quantitative MRI and clinical assessments.

To investigate the phenotypic features, with emphasis on muscle, in 40 patients with spinobulbar muscular atrophy (SBMA) using quantitative MRI, stationary dynamometry, questionnaires, and functional tests. Patients with genetically confirmed SBMA were included. MRI was used to describe muscle involvement and quantify muscle fat fractions of arm, back, and leg muscles. Muscle strength was assessed with a stationary dynamometer. All patients were evaluated with the SBMA functional rating scale and the 6-minute walk test among others. MRI and muscle strength results were compared with healthy controls. Forty patients with SBMA were included. The muscle fat content was significantly higher in patients with SBMA than in controls: paraspinal fat fraction was 45% vs 33% in controls, thigh fat fraction 36% vs 14%, calf fat fraction 37% vs 15%, upper arm fat fraction 20% vs 8%, and forearm fat fraction was 20% vs 9%. Muscle strength in patients was reduced to approximately half of that in controls in all muscles. Muscle fat content correlated with muscle strength, SBMA functional rating scale score, and 6-minute walk test distance. Our results show that there is a diffuse muscle involvement pattern in SBMA. Leg muscles are more vulnerable than arm muscles, especially the posterior flexor muscles. The muscle fat content correlates with muscle function and disease severity.

A Review on the Genetic Basis of Growth and Prolificacy Traits in Sheep

The performance of an animal for a particular trait is the result of its genetic merit and the effects of the environment where it exists. To set up genetic improvement in sheep, the genetic component attributed to the trait of interest need to be defined. The aim of this review was to describe major candidate genes influencing growth traits and prolificacy in sheep. Although growth and prolificacy are quantitative traits and are expected to be influenced by many genes with individual genes contributing small effects, there are major genes that have been identified with significant influence on growth and prolificacy. The CLPG, GDF8 and CAST genes are some of the major genes that have strong influence on sheep growth and carcass quality. The CLPG mutation can cause pronounced effect in the muscle found in the hindquarter and is responsible for the muscular hypertrophy phenotype in sheep. The GDF8 gene also play important role in increasing muscle depth due to mutation in the regulatory region and coding sequences. The CAST gene is an endogenous and specific inhibitor of calpain enzyme and thereby regulates the rate and extent of muscle tenderization following slaughtering. For prolificacy, BMP15, GDF9 and BMPR1B have been shown to exert significant influence on ovulation rate and litter size in various sheep breeds in the world. Both of the three genes are member of the TGF-beta family protein that encode protein product responsible for growth, differentiation and proliferation of ovarian follicles. The mechanism of action for such major genes are associated with the existence of mutation in the coding sequence resulting amino acid change as well as in the regulatory region that vary the expression level and inheritance of the genes. Up to now, better attempts have been made to describe the genetic basis of growth and prolificacy in sheep. However, more works are needed to characterize other genes influencing these traits. More importantly, making use of the identified genes in sheep breeding program through marker assisted and genomic selection should receive due attentions.

The HDAC3 enzymatic activity regulates skeletal muscle fuel metabolism.

Histone deacetylase 3 (HDAC3) is a major HDAC, whose enzymatic activity is targeted by small molecule inhibitors for treating a variety of conditions. However, its enzymatic activity is largely dispensable for its function in embryonic development and hepatic lipid metabolism. HDAC3 plays a pivotal role in regulating muscle fuel metabolism and contractile function. Here, we address whether these muscular functions of HDAC3 require its enzymatic activity. By mutating the NCoR/SMRT corepressors in a knock-in mouse model named NS-DADm, we ablated the enzymatic activity of HDAC3 without affecting its protein levels. Compared to the control mice, skeletal muscles from NS-DADm mice showed lower force generation, enhanced fatigue resistance, enhanced fatty acid oxidation, reduced glucose uptake during exercise, upregulated expression of metabolic genes involved in branched-chain amino acids catabolism, and reduced muscle mass during aging, without changes in the muscle fiber-type composition or mitochondrial protein content. These muscular phenotypes are similar to those observed in the HDAC3-depleted skeletal muscles, which demonstrates that, unlike that in the liver or embryonic development, the metabolic function of HDAC3 in skeletal muscles requires its enzymatic activity. These results suggest that drugs specifically targeting HDAC3 enzyme activity could be developed and tested to modulate muscle energy metabolism and exercise performance.

Calpain Inhibition Increases SMN Protein in Spinal Cord Motoneurons and Ameliorates the Spinal Muscular Atrophy Phenotype in Mice

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is caused by the loss of survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration and loss of spinal cord motoneurons (MNs), muscular atrophy, and weakness. SMN2 is the centromeric duplication of the SMN gene, whose numbers of copies determine the intracellular levels of SMN protein and define the disease onset and severity. It has been demonstrated that elevating SMN levels can be an important strategy in treating SMA and can be achieved by several mechanisms, including promotion of protein stability. SMN protein is a direct target of the calcium-dependent protease calpain and induces its proteolytic cleavage in muscle cells. In this study, we examined the involvement of calpain in SMN regulation on MNs. In vitro experiments showed that calpain activation induces SMN cleavage in CD1 and SMA mouse spinal cord MNs. Additionally, calpain 1 knockdown or inhibition increased SMN level and prevent neurite degeneration in these cells. We examined the effects of calpain inhibition on the phenotype of two severe SMA mouse models. Treatment with the calpain inhibitor, calpeptin, significantly improved the lifespan and motor function of these mice. Our observations show that calpain regulates SMN level in MNs and calpeptin administration improves SMA phenotype demonstrating the potential utility of calpain inhibitors in SMA therapy.

Single Cell Analysis Identifies the miRNA Expression Profile of a Subpopulation of Muscle Precursor Cells Unique to Humans With Type 2 Diabetes.

MicroRNAs (miRNAs) take part in regulating central cellular processes such as differentiation and metabolism. We have previously shown that muscle progenitor cells derived from individuals with type 2 diabetes (T2DM) have a dysregulated miRNA profile. We hypothesized that the T2DM muscle progenitor population is heterogeneous in its miRNA expression and differs from the progenitor population of healthy controls. MiRNA expression profiles of CD56+ muscle progenitor cells from people with T2DM and from healthy controls were therefore investigated at a single cell level. Single-cell analysis revealed three subpopulations expressing distinct miRNA profiles: two subpopulations including both T2DM and healthy control muscle precursors presented miRNA expression profiles mostly overlapping between groups. A distinct third subpopulation consisted solely of cells from donors with T2DM and showed enriched expression of miRNAs previously shown to be associated with type 2 diabetes. Among the enriched miRNAs was miR-29, a regulator of GLUT4 mRNA expression. Interestingly, this subpopulation also revealed several miRNAs with predicted targets in the PI3K/Akt pathway, not previously described in relation to T2DM muscle dysfunction. We concluded that a subpopulation of T2DM muscle precursor cells is severely dysregulated in terms of their miRNA expression, and accumulation of this population might thus contribute to the dysfunctional muscular phenotype in type 2 diabetes.

A Mouse Model of Creatine Transporter Deficiency Reveals Impaired Motor Function and Muscle Energy Metabolism.

Creatine serves as fast energy buffer in organs of high-energy demand such as brain and skeletal muscle. L-Arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase are responsible for endogenous creatine synthesis. Subsequent uptake into target organs like skeletal muscle, heart and brain is mediated by the creatine transporter (CT1, SLC6A8). Creatine deficiency syndromes are caused by defects of endogenous creatine synthesis or transport and are mainly characterized by intellectual disability, behavioral abnormalities, poorly developed muscle mass, and in some cases also muscle weakness. CT1-deficiency is estimated to be among the most common causes of X-linked intellectual disability and therefore the brain phenotype was the main focus of recent research. Unfortunately, very limited data concerning muscle creatine levels and functions are available from patients with CT1 deficiency. Furthermore, different CT1-deficient mouse models yielded conflicting results and detailed analyses of their muscular phenotype are lacking. Here, we report the generation of a novel CT1-deficient mouse model and characterized the effects of creatine depletion in skeletal muscle. HPLC-analysis showed strongly reduced total creatine levels in skeletal muscle and heart. MR-spectroscopy revealed an almost complete absence of phosphocreatine in skeletal muscle. Increased AGAT expression in skeletal muscle was not sufficient to compensate for insufficient creatine transport. CT1-deficient mice displayed profound impairment of skeletal muscle function and morphology (i.e., reduced strength, reduced endurance, and muscle atrophy). Furthermore, severely altered energy homeostasis was evident on magnetic resonance spectroscopy. Strongly reduced phosphocreatine resulted in decreased ATP/Pi levels despite an increased inorganic phosphate to ATP flux. Concerning glucose metabolism, we show increased glucose transporter type 4 expression in muscle and improved glucose clearance in CT1-deficient mice. These metabolic changes were associated with activation of AMP-activated protein kinase – a central regulator of energy homeostasis. In summary, creatine transporter deficiency resulted in a severe muscle weakness and atrophy despite different compensatory mechanisms.

Blocking p62-dependent SMN degradation ameliorates spinal muscular atrophy disease phenotypes

Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease, caused by loss of functional survival of motor neuron (SMN) protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for patients with SMA. The ubiquitin/proteasome system is known to regulate SMN protein levels; however, whether autophagy controls SMN levels remains poorly explored. Here, we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increases SMN levels, while induction of autophagy decreases these levels. SMN degradation occurs via its interaction with the autophagy adapter p62 (also known as SQSTM1). We also show that SMA neurons display reduced autophagosome clearance, increased p62 and ubiquitinated proteins levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival, and extends lifespan in fly and mouse SMA models, revealing p62 as a potential new therapeutic target for the treatment of SMA.

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5′ splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Skeletal muscle-specific Sidt2 knockout in mice induced muscular dystrophy-like phenotype

BackgroundSidt2 is an integral lysosomal membrane protein. Previously, we generated a Sidt2 global knockout mouse and found impaired insulin secretion, along with skeletal muscle pathology. MethodsA mouse model with a muscle-specific knockout of the Sidt2 gene (Sidt2f/fCre) had been generated, to which extensive morphologic study as well as functional study was applied to investigate the direct role of Sidt2 on skeletal muscle tissue in vivo. Secondly, the autophagy-lysosomal pathway was examined by Western blot and immunostaining. Additionally, RNA expression changes in Sidt2f/fCre mice were analyzed by genechip. ResultsSidt2 deficiency in skeletal muscle results in pathognomonic hallmarks of muscular dystrophy, including muscle mass decrease, muscle weakness, fibrosis, central nucleation, fiber regeneration, mildly elevated serum creatine kinase, and dramatically elevated sarcolipin mRNA. Along with accumulation of autophagolysomes, LC3-II, adaptor protein p62, ubiquitinated aggregates, and Lamp2-positive vacuoles were increased significantly in Sidt2f/fCre skeletal muscle fibers. However, only lysosomal-related genes were upregulated, while the genes upstream of the autophagy pathway were unchanged. Simultaneously, the proteasome chymotryptic activity and the lysosomal soluble enzyme activity were unimpaired, which largely excluded the possibility of proteasome chymotryptic activity defect and the lysosomal soluble enzyme defect leading to ubiquitinated aggregates accumulation. ConclusionWe concluded that Sidt2 deficiency leads to muscular dystrophy-like phenotype in mice and Sidt2 plays a critical role in the late stage of autophagy.

Childhood cardiorespiratory fitness, muscular fitness and adult measures of glucose homeostasis

ObjectivesTo assess whether childhood cardiorespiratory fitness (CRF) and muscular fitness phenotypes (strength, power, endurance) predict adult glucose homeostasis measures. DesignProspective longitudinal study. MethodsStudy examining participants who had physical fitness measured in childhood (aged 7–15 years) and who attended follow-up clinics approximately 20 years later and provided a fasting blood sample which was tested for glucose and insulin. Physical fitness measurements included muscular strength (right and left grip, shoulder flexion, shoulder and leg extension), power (standing long jump distance) and endurance (number of push-ups in 30s), and CRF (1.6km run duration). In adulthood, fasting glucose and insulin levels were used to derive glucose homeostasis measures of insulin resistance (HOMA2-IR) and beta cell function (HOMA2-β). ResultsA standard deviation increase in childhood CRF or muscular strength (males) was associated with fasting glucose (CRF: β=−0.06mmol/L), fasting insulin (CRF: β=−0.73mU/L; strength: β=−0.40mU/L), HOMA2-IR (CRF: β=−0.06; strength: β=−0.05) and HOMA2-β (CRF: β=−3.06%; strength: β=−2.62%) in adulthood, independent of the alternative fitness phenotype (all p<0.01). Adjustment for childhood waist circumference reduced the effect by 17–35% for CRF and 0–15% for muscular strength (males) and statistical significance remained for all associations expect between CRF, fasting glucose and HOMA2-β (p>0.06). ConclusionsCRF and muscular fitness in childhood were inversely associated with measures of fasting insulin, insulin resistance and beta cell function in adulthood. Childhood CRF and muscular fitness could both be potential independent targets for strategies to help reduce the development of adverse glucose homeostasis.

B3GALNT2 mutations associated with non-syndromic autosomal recessive intellectual disability reveal a lack of genotype\u2013phenotype associations in the muscular dystrophy-dystroglycanopathies

BackgroundThe phenotypic severity of congenital muscular dystrophy-dystroglycanopathy (MDDG) syndromes associated with aberrant glycosylation of α-dystroglycan ranges from the severe Walker-Warburg syndrome or muscle-eye-brain disease to mild, late-onset, isolated limb-girdle muscular dystrophy without neural involvement. However, muscular dystrophy is invariably found across the spectrum of MDDG patients.MethodsUsing linkage mapping and whole-exome sequencing in two families with an unexplained neurodevelopmental disorder, we have identified homozygous and compound heterozygous mutations in B3GALNT2.ResultsThe first family comprises two brothers of Dutch non-consanguineous parents presenting with mild ID and behavioral problems. Immunohistochemical analysis of muscle biopsy revealed no significant aberrations, in line with the absence of a muscular phenotype in the affected siblings. The second family includes five affected individuals from an Iranian consanguineous kindred with mild-to-moderate intellectual disability (ID) and epilepsy without any notable neuroimaging, muscle, or eye abnormalities. Complementation assays of the compound heterozygous mutations identified in the two brothers had a comparable effect on the O-glycosylation of α-dystroglycan as previously reported mutations that are associated with severe muscular phenotypes.ConclusionsIn conclusion, we show that mutations in B3GALNT2 can give rise to a novel MDDG syndrome presentation, characterized by ID associated variably with seizure, but without any apparent muscular involvement. Importantly, B3GALNT2 activity does not fully correlate with the severity of the phenotype as assessed by the complementation assay.

P.133 - Identification of variants that affect severity of the spinal muscular atrophy phenotype within and outside of the SMN2 gene

SMA is caused by loss or mutation of the SMN1 gene and retention of SMN2. The SMN2 gene copy number correlates with phenotypic severity. However, there are exceptions, with type 3 cases having 2 copies of SMN2 or type 1 cases with 3 copies as well as discordant siblings with the same copy number. With effective therapeutics for SMA early introduction of therapy is important in achieving maximum benefit. Newborn screening is progressing towards implementation but precise prediction of phenotype from genotype is critical to optimize timing of treatment. It is often not known if the SMN2 gene is intact or contains variants which can affect the amount of SMN produced. In order to identify deletion junctions as well as intact SMN2 copies and variants within the gene that affect phenotypic severity we have sequenced the entire SMN2/SMN1gene present in 60 patients. In addition, we have employed genomic sequencing in a set of discordant siblings in comparison to concordant siblings in order to identify variants in the genome associated with the milder sibling. The entire SMN2/SMN1 gene along with the CFTR and PLS3 gene was captured and sequenced with an adapted Multiplexed direct genomic selection (MDiGS) technique. The sequences were aligned and variants were determined. To determine SMN2 copy number the SMN read count was split into bins and normalized to the CFTR and PLS3 reads. We defined a deletion occurring in 8 patients, the deletion occurred between two nearly identical repeats located in intron 6 and in sequence located 3 prime of SMN2. We detected a cryptic splice site in intron 6 of a patient with one copy of SMN1 and a missense mutation (A75T) in the SMN2 gene of a patient. We detected 3 variants associated with a milder phenotype in 3 copy SMN2 individuals. Lastly, we have performed genomic sequencing of select discordant and concordant families to identify modifiers of phenotype that act in trans.

A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot–Marie–Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.