Biljana Ilkovski

Nemaline Myopathy Caused by Mutations in the Muscle α-Skeletal-Actin Gene

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder characterized by muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle. Disease-causing mutations have been reported in five genes, each encoding a protein component of the sarcomeric thin filament. Recently, we identified mutations in the muscle α-skeletal-actin gene (ACTA1) in a subset of patients with NM. In the present study, we evaluated a new series of 35 patients with NM. We identified five novel missense mutations in ACTA1, which suggested that mutations in muscle α-skeletal actin account for the disease in ∼15% of patients with NM. The mutations appeared de novo and represent new dominant mutations. One proband subsequently had two affected children, a result consistent with autosomal dominant transmission. The seven patients exhibited marked clinical variability, ranging from severe congenital-onset weakness, with death from respiratory failure during the 1st year of life, to a mild childhood-onset myopathy, with survival into adulthood. There was marked variation in both age at onset and clinical severity in the three affected members of one family. Common pathological features included abnormal fiber type differentiation, glycogen accumulation, myofibrillar disruption, and “whorling” of actin thin filaments. The percentage of fibers with rods did not correlate with clinical severity; however, the severe, lethal phenotype was associated with both severe, generalized disorganization of sarcomeric structure and abnormal localization of sarcomeric actin. The marked variability, in clinical phenotype, among patients with different mutations in ACTA1 suggests that both the site of the mutation and the nature of the amino acid change have differential effects on thin-filament formation and protein-protein interactions. The intrafamilial variability suggests that α-actin genotype is not the sole determinant of phenotype.

Clinical course correlates poorly with muscle pathology in nemaline myopathy

Objective: To report pathologic findings in 124 Australian and North American cases of primary nemaline myopathy. Methods: Results of 164 muscle biopsies from 124 Australian and North American patients with primary nemaline myopathy were reviewed, including biopsies from 19 patients with nemaline myopathy due to α-actin (ACTA1) mutations and three with mutations in α-tropomyosinSLOW (TPM3). For each biopsy rod number per fiber, percentage of fibers with rods, fiber-type distribution of rods, and presence or absence of intranuclear rods were documented. Results: Rods were present in all skeletal muscles and diagnosis was possible at all ages. Most biopsies contained nemaline bodies in more than 50% of fibers, although rods were seen only on electron microscopy in 10 patients. Rod numbers and localization correlated poorly with clinical severity. Frequent findings included internal nuclei and increased fiber size variation, type 1 fiber predominance and atrophy, and altered expression of fiber type specific proteins. Marked sarcomeric disruption, increased glycogen deposition, and intranuclear rods were associated with more severe clinical phenotypes. Serial biopsies showed progressive fiber size variation and increasing numbers of rods with time. Pathologic findings varied widely in families with multiple affected members. Conclusions: Very numerous nemaline bodies, glycogen accumulation, and marked sarcomeric disruption were common in nemaline myopathy associated with mutations in skeletal α-actin. Nemaline myopathy due to mutations in α-tropomyosinSLOW was characterized by preferential rod formation in, and atrophy of, type 1 fibers. Light microscopic features of nemaline myopathy correlate poorly with disease course. Electron microscopy may correlate better with disease severity and genotype.

Dominant Mutations in KBTBD13, a Member of the BTB/Kelch Family, Cause Nemaline Myopathy with Cores

We identified a member of the BTB/Kelch protein family that is mutated in nemaline myopathy type 6 (NEM6), an autosomal-dominant neuromuscular disorder characterized by the presence of nemaline rods and core lesions in the skeletal myofibers. Analysis of affected families allowed narrowing of the candidate region on chromosome 15q22.31, and mutation screening led to the identification of a previously uncharacterized gene, KBTBD13, coding for a hypothetical protein and containing missense mutations that perfectly cosegregate with nemaline myopathy in the studied families. KBTBD13 contains a BTB/POZ domain and five Kelch repeats and is expressed primarily in skeletal and cardiac muscle. The identified disease-associated mutations, C.742C>A (p.Arg248Ser), c.1170G>C (p.Lys390Asn), and c.1222C>T (p.Arg408Cys), located in conserved domains of Kelch repeats, are predicted to disrupt the molecules beta-propeller blades. Previously identified BTB/POZ/Kelch-domain-containing proteins have been implicated in a broad variety of biological processes, including cytoskeleton modulation, regulation of gene transcription, ubiquitination, and myofibril assembly. The functional role of KBTBD13 in skeletal muscle and the pathogenesis of NEM6 are subjects for further studies.

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Expanding the clinical, pathological and MRI phenotype of DNM2-related centronuclear myopathy

Mutations in dynamin-2 (DNM2) cause autosomal dominant centronuclear myopathy (CNM). We report a series of 12 patients from eight families with CNM in whom we have identified a number of novel features that expand the reported clinicopathological phenotype. We identified two novel and five recurrent missense mutations in DNM2. Early clues to the diagnosis include relative weakness of neck flexors, external ophthalmoplegia and ptosis, although these are not present in all patients. Pes cavus was present in two patients, and in another two members of one family there was mild slowing of nerve conduction velocities. Whole-body MRI examination in two children and one adult revealed a similar pattern of involvement of selective muscles in head (lateral pterygoids), neck (extensors), trunk (paraspinal) and upper limbs (deep muscles of forearm). Findings in lower limbs and pelvic region were similar to that previously reported in adults with DNM2 mutations. Two patients presented with dystrophic changes as the predominant pathological feature on muscle biopsies; one of whom had a moderately raised creatine kinase, and both patients were initially diagnosed as congenital muscular dystrophy. DNM2 mutation analysis should be considered in patients with a suggestive clinical phenotype despite atypical histopathology, and MRI findings can be used to guide genetic testing. Subtle neuropathic features in some patients suggest an overlap with the DNM2 neuropathy phenotype. Missense mutations in the C-terminal region of the PH domain appear to be associated with a more severe clinical phenotype evident from infancy.

Defining α-skeletal and α-cardiac actin expression in human heart and skeletal muscle explains the absence of cardiac involvement in ACTA1 nemaline myopathy

Abstract Mutations in α-skeletal actin ( ACTA1 ) underlie several congenital muscle disorders including nemaline myopathy (NM). Almost all ACTA1 -NM patients have normal cardiac function, and, even lethally affected congenital NM patients exhibit an unremarkable gestation with decreased foetal movement just prior to birth. Although α-skeletal actin is thought to be the predominant sarcomeric actin in human heart (Boheler KR, Carrier L, de la Bastie D, et al. Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. J Clin Invest 1991;88:323–30 [1]), ACTA1 -NM patients almost never exhibit a cardiac phenotype. In this study, we define the relative expression of skeletal and cardiac actin proteins in human heart and skeletal muscle. We show that α-cardiac actin is the predominant sarcomeric isoform in human donor hearts and in early foetal skeletal muscle development. Skeletal actin is the predominant isoform from 25 to 27 weeks gestation and is the exclusive isoform expressed in muscle from infancy through to adulthood. These findings are consistent with clinical observations of NM patients and assist us to better understand the pathogenesis of inherited myopathies and cardiomyopathies with mutations in actin.

The pathogenesis of ACTA1-related congenital fiber type disproportion.

Mutations in ACTA1 have been associated with a variety of changes in muscle histology that likely result from fundamental differences in the way that ACTA1 mutations disrupt muscle function. Recently, we reported three patients with congenital fiber type disproportion (CFTD) caused by novel heterozygous missense mutations in ACTA1 (D292V, L221P, P332S) with marked type 1 fiber hypotrophy as the only pathological finding on muscle biopsy. We have investigated the basis for the histological differences between these CFTD patients and patients with ACTA1 nemaline myopathy (NM).

Mutations in Contactin-1, a Neural Adhesion and Neuromuscular Junction Protein, Cause a Familial Form of Lethal Congenital Myopathy

We have previously reported a group of patients with congenital onset weakness associated with a deficiency of members of the syntrophin-alpha-dystrobrevin subcomplex and have demonstrated that loss of syntrophin and dystrobrevin from the sarcolemma of skeletal muscle can also be associated with denervation. Here, we have further studied four individuals from a consanguineous Egyptian family with a lethal congenital myopathy inherited in an autosomal-recessive fashion and characterized by a secondary loss of beta2-syntrophin and alpha-dystrobrevin from the muscle sarcolemma, central nervous system involvement, and fetal akinesia. We performed homozygosity mapping and candidate gene analysis and identified a mutation that segregates with disease within CNTN1, the gene encoding for the neural immunoglobulin family adhesion molecule, contactin-1. Contactin-1 transcripts were markedly decreased on gene-expression arrays of muscle from affected family members compared to controls. We demonstrate that contactin-1 is expressed at the neuromuscular junction (NMJ) in mice and man in addition to the previously documented expression in the central and peripheral nervous system. In patients with secondary dystroglycanopathies, we show that contactin-1 is abnormally localized to the sarcolemma instead of exclusively at the NMJ. The cntn1 null mouse presents with ataxia, progressive muscle weakness, and postnatal lethality, similar to the affected members in this family. We propose that loss of contactin-1 from the NMJ impairs communication or adhesion between nerve and muscle resulting in the severe myopathic phenotype. This disorder is part of the continuum in the clinical spectrum of congenital myopathies and congenital myasthenic syndromes.

Autosomal dominant nemaline myopathy with intranuclear rods due to mutation of the skeletal muscle ACTA1 gene: clinical and pathological variability within a kindred.

Nemaline Myopathy with Intranuclear Rods is a rare variant of nemaline myopathy, due in almost all instances to mutation of ACTA1, the gene encoding skeletal muscle alpha-actin. We describe the novel autosomal dominant occurrence in a three-generation kindred, and review previously reported cases. Onset of myopathic symptoms in our kindred was in infancy or early childhood. Beyond infancy, limb muscle weakness was non-disabling and minimally progressive. A tall thin face and facial myopathy were prominent features in the affected adults. By light microscopy, muscle biopsies ranged from almost normal, to chronic myopathy with sarcoplasmic and intranuclear rods. A heterozygous GTG-ATG mutation (Val163Met) was found in exon 4 of ACTA1 in affected individuals. Actin is normally present within the nucleus in only trace amounts. Mutation at postion 163 may result in intranuclear rods by virtue of its close proximity to a nuclear export signal within the actin molecule.

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20–50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5′-UTR regions despite their typically low coverage in exome data.