G. Ravenscroft

Nemaline myopathy with stiffness and hypertonia associated with an ACTA1 mutation

Nemaline myopathy, known to be caused by mutations in 7 genes, including skeletal muscle α-actin ( ACTA1 ),1 is characterized by muscle weakness, hypotonia, and nemaline rods in muscle biopsy. Here we report a patient with nemaline rods but the opposite phenotype of hypercontractility. ### Case report. The John Radcliffe Hospital ethics review board approved the study. The first child of nonconsanguineous European parents presented at 6 weeks of age with an apneic episode thought due to bilateral strangulated inguinal hernias and an umbilical hernia. However, after herniotomy, rigidity and recurrent apneas requiring mechanical ventilation continued. The patient was born by elective Caesarean section at 39 weeks, with no perinatal complications, although the mother, with hindsight, thought there might have been stiffness in the weeks after birth as she had difficulty clothing him. Polyhydramnios had been noted. On examination, he had very stiff and hypertrophic abdominal and proximal limb muscles and elbow and knee contractures. Deep tendon reflexes showed brisk contraction and slow relaxation and percussion myotonia could be elicited. However, EMG in multiple muscles showed no evidence of myotonia. He also had normal nerve conduction studies, MRI, EEG, and cardiac assessment, including echocardiography. Neurometabolic investigations were also normal including serum and CSF lactate, CSF neurotransmitters, urinary catecholamines, carnitine profile, white cell enzymes, and screening for mucopolysaccharidosis. Serum creatine kinase was slightly raised (370 IU, range 24–195). A sleep study showed hypoventilation and carbon dioxide retention. Hyperekplexia, paroxysmal episodic pain disorder, neuromyotonia, and episodic ataxia were considered, though none exactly matched the phenotype because of the underlying stiffness, but relevant testing ( ARX , FRAXA , GLRA1 , KCN1A , POLG , SCN9A , anti GAD, and VGKC antibody studies) was negative. Pharmacologic treatment including phenytoin, carbamazepine, flecainide, baclofen, dantrolene, and acetazolamide was ineffective. He continued having recurrent episodes of stiffness, where he stopped …

G.P.273

Mutations in the gene ( KLHL40 ), encoding Kelch-like family member 40, were recently identified in patients presenting with severe autosomal recessive nemaline myopathy (NEM8). Using exome sequencing or targeted re-sequencing of 276 known neuromuscular disease genes through a TargetSeq (Life Technologies) capture panel, we have identified two additional cases with KLHL40 mutations. Both were from consanguineous kindreds. Case 1 was diagnosed with severe SMA and congenital fractures. We identified a homozygous nonsense mutation in KLHL40 (c.46C>T, p. Gln16*). Muscle biopsy identified variation in fibre size and miliary nemaline bodies by electron microscopy seen as very fine scattered granules upon Gomori trichrome staining. Case 2 presented with severe nemaline myopathy, the biopsy showing numerous tiny nemaline bodies. We identified a homozygous missense mutation in KLHL40 (c.931C>A, p. Arg311Ser). Neither of the variants was present in 1000genomes or EVS databases. These data further suggest that muscle biopsy may be useful to inform genetic testing and that KLHL40 should be considered in patients presenting with severe miliary nemaline myopathy. Nemaline myopathy should also be considered in the differential diagnosis of severe SMA, especially in patients presenting with congenital fractures and contractures. The normal biological function of KLHL40 is largely unknown and therefore the basis of the severe muscle disease resulting from mutations of KLHL40 is also unknown. In investigations of control and diseased mouse and patient muscle, we show that KLHL40 is more abundant in the mouse soleus and diaphragm (muscles that comprise oxidative type I and IIA myofibres) than in the EDL, gastrocnemius and quadriceps muscles (type II glycolytic predominant). In addition we show that KLHL40 is increased in dystrophic mouse ( mdx ) and regenerating patient muscle (including DMD), suggesting that it may serve as a novel marker of skeletal muscle regeneration.

G.P.197

Muscle mass and strength are variable traits in humans. Many French Canadians (FC) became international celebrities because of their exceptional strength. Though muscle hypertrophy has been associated in many mammals with myostatin mutations, to date only a single pediatric case has been reported in humans. Dominant pathological mutations in the CLCN1, SCN4A, CAV3, ATP2A1, LPIN1, LMNA and RYR1 gene have been known to lead to muscle hypertrophy in humans, in particular selective muscle hypertrophy in certain myopathies. These conditions are associated with either weakness or increased strength. We recruited a cohort of more than 115 cases belonging to more than 65 families of a heterogeneous dominant condition that we refer to as: “Strongman syndrome” (SM). In all families, the most affected case suffers from incapacitating myalgias with variable degree of muscle cramps, and they also have a personal and familial history of above average strength. On examination these patients have large well defined muscles, above average strength, non-electric prolonged muscle contractions and progressive weakness on repeated contractions. Exome sequencing of a large FC family identified a single segregating variant predicted to be damaging in the DCST2 gene shared also by two other FC families. The DCST2 gene is expressed in skeletal muscle and is predicted to code for a six transmembrane domains channel-like protein. To better characterize the herculean strength we assesses different biometric variables in a subset of cases with and without DCST2 mutations and investigated if myofiber hypertrophy is present. Results of quantifiable methods to evaluate the clinical and pathological phenotype of strongman cases will be presented, as well as functional data about DCST2. The uncovering of the function of DCST2 and other strongman genes will contribute to a better understanding of pathways important for muscle hypertrophy and strength in health and disease.

A.P.10

Filamin proteins play an essential role in the binding of actin to the cytoskeleton of cells. Filamin C ( FLNC ) is the skeletal muscle specific isoform in this gene family. Mutations in the actin binding domain (ABD) of FLNC cause distal myopathy, whilst mutations in other FLNC domains cause myofibrillar myopathy. Until now there has not been an appropriate mouse model to observe the effects of these mutations in the ABD FLNC region and studies have been limited to in vitro analyses. We have characterised the first mouse model with an ABD FLNC mutation, specifically a knock-in mutation (p.E247K), to determine if it is a suitable mouse model for distal myopathy. KI (Flnc) E247K mice have lower bodyweight than wild-type mice, and they run less on average than wild-type mice as determined by voluntary running wheel activity. Analysis of skeletal muscle pathology by histological and immunostaining techniques at varying timepoints has revealed that the soleus in particular shows pathological features in older muscle, especially at the oldest time point examined of 1year of age. This is interesting, given that the soleus was one of the most affected muscles in distal myopathy patients with an ABD FLNC mutation. Accumulations of desmin are present in KI (Flnc) E247K mice at the later timepoints suggesting that age-related pathological changes are occurring in these mice. However levels of desmin protein expression by western blotting are not altered in the soleus of KI (Flnc) E247K mice. Further investigation of these mice, including analysis of force generation, will hopefully allow better understanding of the mechanism by which FLNC ABD mutations lead to muscle disease and may provide a valuable model for evaluating potential therapies.

P.9.14 Next generation sequencing provides diagnosis for multiple foetal akinesia disorders

Whole exome sequencing (WES) permits cost effective causative mutation identification in highly heterogeneous diseases. Foetal akinesia/hypokinesia (the absence of or decreased movement in utero) is one disease group where WES is revolutionising patient diagnosis. The aim of this study was to identify the genetic cause of disease in a cohort of patients presenting with foetal akinesia and/or arthrogryposis. The first family in which causative variants were identified consisted of two siblings diagnosed with non-lethal multiple pterygium syndrome. WES of the proband revealed two novel mutations in a recently identified (Jan 2013) disease gene (ECEL1) causing distal arthrogryposis (DA) type 5D; a missense mutation (c.1531G>A; p.Gly511Ser) and a splice site mutation (c.1797-1G>A). Further investigations confirmed that the mutations co-segregated with disease. The clinical features of the siblings are in keeping with the described clinical picture for DA5D. The second family presented with Freeman-Sheldon syndrome (DA2A). WES of this proband revealed a known mutation in MYH3 (c.2015G>A; p.Arg672His). Sanger sequencing suggests the mutation likely arose de novo as neither of the parents carried the mutation. A third family presented with two consecutive pregnancies showing limited foetal movement, polyhydramnios, oedema and camptodactlyly. Muscle biopsy showed occasional cores and minicores. WES of the proband revealed two novel RYR1 variants: c.2455C>T; p.Arg819∗ and c.5989G>A; p.Glu1997Lys, as well as a novel TPM3 variant (c.502C>G; p.Leu168Val). The pathogenic significance of the apparent double trouble is yet to be clarified. This study exemplifies the capabilities of WES in the diagnoses of cases of foetal akinesia and arthrogryposis. It also highlights apparent “double trouble” which is likely to be recognized more as the use of WES becomes more widespread. In many other probands we did not identify causative mutations indicating perhaps the limitations of WES.

G.P.126 “Strongman syndrome”: A new autosomal dominant herculean painful myopathy

Abstract The objective was to define clinical criteria for a novel autosomal dominant (AD) herculean painful myopathy. We reviewed the clinical assessment of 74 cases that presented with myalgia, muscle cramps, increased muscle bulk, above normal strength with a negative electrophysiological and genetic workup. We evaluated 42 members of the largest family and defined a clinical scale to establish affection status. Review of the 74 cases demonstrated the majority had a positive family history. The larger families support AD transmission with a variable phenotype. Cases presented with combinations of the following major symptoms and signs: above average strength, myalgia post-activity, deltoid fatigability after repeated contractions and percussion pseudomyotonia in more than two limbs. Many also presented with some of the following minor findings: poor quality sleep, excessive movements at night, weakness after repetitive movements, very large muscle bulk, calf hypertrophy, well defined muscles, clear superior strength by confrontation and proximal weakness affecting the iliopsoas. By doubling the weight given to the major findings we developed a 20-point affection scale. In the largest family this confirmed AD transmission. Few cases had elevated serum CPK levels. None had electrical myotonia, or decremental response on EMG. Muscle biopsies were unremarkable. Mutations in MYSN were excluded in 13 cases. No mutations were found in CAV3, CLCN1 or SCN4A. Though weakness after repeated contractions was common, none tested had electrophysiological evidence of myasthenia, nor DOK7 mutations. In some older cases a proximal weakness developed such that the condition evolved into a late-onset LGMD. We have identified a large number of Canadian and Australian cases affected by a variably painful herculean myopathy apparently transmitted in an AD fashion. Further genetic characterization of the larger families should uncover candidate loci and gene.

D.P.6 Whole exome sequencing applied to foetal akinesia

Abstract Foetal akinesia/hypokinesia is an absence or decrease of movement in utero resulting in a spectrum of anomalies characterised by intrauterine growth retardation, contractures, craniofacial anomalies, limb anomalies, pulmonary hypoplasia and polyhydramnios. Despite a large number of disease genes (>30) being associated with this heterogenous group of disorders, the majority of cases are without a genetic diagnosis. The aim of this study was to investigate the use of whole exome sequencing (WES) to identify the causative gene/s in a preliminary set of four families with foetal akinesia. In three families presenting with foetal akinesia and nemaline bodies, all the known nemaline myopathy genes were excluded by WES. The fourth family presented with lethal multiple pterygium syndrome. WES of DNA from two affected sibs revealed compound heterozygous mutations in the glycogen branching enzyme-1 gene ( GBE1 ): a known splice site mutation (c.691+2T>C) and a novel missense mutation (c.956A>G; p.His319Arg) at a highly conserved residue. Each parent carried one of the mutations. GBE1 mutations are known to cause glycogen storage disease IV (GSD IV), a subset of which present with a severe neuromuscular foetal akinesia phenotype. In light of the genetic findings, the muscle pathology was reviewed and PAS positive, diastase resistant faintly refractile material was observed, consistent with GSD IV. Our data highlight that novel nemaline myopathy gene/s remain to be identified and that WES can be used successfully for the diagnosis of foetal akinesias. This study expands the genotype–phenotype correlation of GBE1 mutations: (1) since GSD IV should now be considered in cases presenting as lethal multiple pterygium syndrome and (2) since the vast majority of known foetal akinesia GSD IV cases are due to large indels or frameshift mutations and it is generally considered that the type of the mutations corresponds with clinical severity.