Available online at www.sciencedirect.com
ScienceDirect Neuromuscular Disorders 24 (2014) 331–334 www.elsevier.com/locate/nmd
Case report
Novel large deletion in the ACTA1 gene in a child with autosomal recessive nemaline myopathy Bethany Friedman a, Kara Simpson b, Carolina Tesi-Rocha c, Delu Zhou d, Cheryl A. Palmer d, Sharon F. Suchy a,⇑ b
a GeneDx, Gaithersburg, MD, USA Genetics and Metabolism, Children’s National Medical Center, Washington, DC, USA c Neurology, Children’s National Medical Center, Washington, DC, USA d Department of Pathology, University of Utah, Salt Lake City, UT, USA
Received 17 July 2013; received in revised form 14 November 2013; accepted 16 December 2013
Abstract Nemaline myopathy (NM) is a genetically and clinically heterogeneous disorder resulting from a disruption of the thin filament proteins of the striated muscle sarcomere. The disorder is typically characterized by muscle weakness including the face, neck, respiratory, and limb muscles and is clinically classified based on the age of onset and severity. Mutations in the ACTA1 gene contribute to a significant proportion of NM cases. The majority of ACTA1 gene mutations are missense mutations causing autosomal dominant NM by producing an abnormal protein. However, approximately 10% of ACTA1 gene mutations are associated with autosomal recessive NM; these mutations are associated with loss of protein function. We report the first case of a large deletion in the ACTA1 gene contributing to autosomal recessive NM. This case illustrates the importance of understanding disease mechanisms at the molecular level to accurately infer the inheritance pattern and potentially aid with clinical management. Ó 2014 Published by Elsevier B.V. Keywords: Nemaline myopathy; ACTA1; Deletion; Dominant negative; Loss of function
1. Introduction Nemaline myopathy (NM) is a genetically and clinically heterogeneous myopathy and, with an incidence of approximately 1/50,000 live births, is one of the most common congenital myopathic conditions. Histologically, this disorder is characterized by the accumulation of thin filaments and aggregates of the muscle Z-disks, known as nemaline bodies, on muscle biopsy. Most of the genes that cause NM encode sarcomeric thin filament proteins. Mutations in the skeletal muscle NEB gene are the most common cause of NM, however this disorder can be caused by mutations in seven other genes: ACTA1, ⇑ Corresponding author. Address: 207 Perry Parkway, Gaithersburg, MD 20877, USA. Tel.: +1 301 519 2100; fax: +1 301 519 2892. E-mail address: [email protected] (S.F. Suchy).
0960-8966/$ - see front matter Ó 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.nmd.2013.12.006
TPM3, TNNT1, TPM2, CFL2, KBTBD13, and KLHL40 [1–3]. Mutations in the ACTA1 gene, that encodes skeletal muscle a-actin, account for about 20% of cases of NM and approximately 50% of cases of severe NM [1,4–6]. Individuals with congenital NM typically exhibit marked proximal muscle weakness, hypotonia, decreased or absent deep tendon reflexes, and myopathic facies; respiratory and feeding difficulties along with motor delays are often present due to significant muscle weakness. NM is the most common congenital myopathy caused by mutations in the ACTA1 gene. Mutations in the ACTA1 gene are associated with several other congenital myopathies, based on histologic features, with overlapping clinical phenotypes. Histologic findings on muscle biopsy due to mutations in the ACTA1 gene can include one or more of the following: nemaline bodies, intranuclear rods, actin filament aggregates, caps,
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core-like areas, dystrophic features, fiber type disproportion, zebra bodies, and/or non-specific changes [2]. The presence of nemaline bodies is diagnostic for NM. Correlation between muscle histology and clinical presentation is poor [7]. However, some correlation between the location of a mutation in the ACTA1 gene and muscle histology has been reported [5,8,9]. The ACTA1 gene encodes skeletal muscle a-actin, which is the main component of thin filaments in skeletal muscle. Mutations in the ACTA1 gene can result in defects in actin folding, actin polymerization, and muscle contraction [2,7,8]. Disease-associated mutations in ACTA1 are located throughout the gene and ultimately affect skeletal muscle contraction, typically leading to weakness of the skeletal muscles [8,10]. To date, ACTA1 gene mutations reported in the literature causing autosomal recessive NM include nonsense, splice-site, and frameshift mutations as well as missense mutations shown to result in functionally null a-actin. Here we report a proband with autosomal recessive NM due to two loss-of-function ACTA1 mutations, including an exon-level deletion. This is the first description of a large deletion of the ACTA1 gene. 2. Case report The proband was born at 39 weeks gestation at 6 lb 9 oz to a 29 year old G3P2 mother. Decreased fetal movements were noted during the pregnancy. The birth was complicated by a bilateral brachial plexus injury and a broken clavicle. The newborn was admitted to the neonatal intensive care unit at day one of life due to respiratory and feeding difficulties for which she was started on biphasic positive airway pressure (BiPAP) and nasogastric feeds; a gastrostomy tube was later placed. Significant hypotonia was noted. Physical examination of the proband at 6 and 27 months of age revealed myopathic facies, pectus excavatum, neuromuscular kyphoscoliosis, hyporeflexia, and marked proximal muscle hypotonia. At five years of age, the patient continues to receive physical and speech therapies for developmental delays, dysarthria, and dysphagia. She is routinely on nocturnal BiPAP. The family history was noncontributory; the proband’s parents and older brother were well with no clinical features suggestive of an underlying myopathy. The mother and father of the proband were of Scottish and Danish ancestry, respectively; there was no known consanguinity in the family. A skeletal muscle biopsy revealed numerous rounded, atrophic myofibers on the hematoxylin and eosin sections. A marked type 1 fiber hypotrophy was present, although a nearly equal distribution of type 1 and type 2 fibers were seen. Gomori trichrome stain revealed occasional darkly staining granules distributed throughout the specimen (Fig. 1A). Ultrastructural examination identified the presence of electron-dense
Fig. 1. Gomori trichrome section of the skeletal muscle reveals varying fiber size with numerous atrophic fibers as well as dark granules in scattered fibers (arrows) (Gomori trichrome 1000) (A). Electron microscopy demonstrates electron dense rod-shaped nemaline bodies (arrowheads) (28,010) (B).
nemaline bodies in the myofibers, diagnostic of NM (Fig. 1B). To identify the underlying molecular cause of NM in the proband, genetic analysis was necessary. Although mutations in the NEB gene are the most common cause of NM, at the time of testing, full sequencing was not clinically available for the very large NEB gene. Therefore, sequence analysis of the ACTA1 gene was pursued on DNA extracted from peripheral blood. Bi-directional sequence analysis of the coding region of the ACTA1 gene (exons 2–7) by Sanger sequencing initially identified an apparently homozygous, novel deletion of a single nucleotide in exon 6 (c.911delG) (Fig. 2). This mutation causes a frameshift (p.Gly304AlafsX24) which is predicted to result in nonsense-mediated mRNA decay or protein truncation. The presence of this loss-of-function mutation in a homozygous state would be consistent with autosomal recessive NM. However, given the parents are non-consanguineous, it would be unlikely for both parents to be carriers of the same
B. Friedman et al. / Neuromuscular Disorders 24 (2014) 331–334
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3. Discussion
Fig. 2. Forward (Fwd) and reverse (Rev) sequence traces of the proband surrounding and including the c.911delG mutation in the ACTA1 gene. The proband’s sequence reveals an apparent homozygous novel deletion of a single nucleotide, c.911delG. The nucleotide sequence surrounding the mutation is shown below.
mutation. Targeted sequence analysis was recommended for the parents to confirm that they were both heterozygous carriers of the mutation, and to rule out a failure to amplify both alleles and an exon-level deletion. Sequence analysis revealed that only the mother of the proband was a carrier of the mutation. Due to the absence of the c.911delG mutation in the father, an exon-level deletion of the ACTA1 gene was considered as a possible mechanism for disease in the proband. This possibility was investigated by quantitative copy number analysis of the ACTA1 gene in the proband and father by quantitative PCR (qPCR); labeled probes were located in intron 1 (35 bp before exon 2), exon 4, exon 6, and exon 7 of the ACTA1 gene. Analysis revealed only a single ACTA1 allele present at all the positions tested. This demonstrated the presence of a large deletion of the ACTA1 gene extending, at a minimum, from intron 1 to exon 7. This deletion was also identified in the proband’s father (Fig. 3).
This is the first case report of a large deletion of the ACTA1 gene contributing to autosomal recessive NM. Mutations in the ACTA1 gene have been shown to cause disease by two different mechanisms. Most ACTA1 mutations are de novo missense mutations hypothesized to cause autosomal dominant NM by a gain-of-function mechanism (dominant negative), producing a protein with abnormal function [5,6,11]. However, approximately 10% of ACTA1 gene mutations cause autosomal recessive NM [9] due to the loss of skeletal muscle a-actin function [7,9,12,13]. The presence of the deletion along with the identified frameshift mutation in the proband is consistent with the loss-of-function mechanism associated with autosomal recessive NM. Carrier parents of an affected child with recessive NM have not been reported to exhibit clinical features or symptoms of NM, demonstrating that one null, loss-of-function, ACTA1 allele is not sufficient to cause disease [12,13]. Many of the reported cases of autosomal recessive NM occur in families with known consanguinity [13]. In the absence of known consanguinity, the presence of an apparently homozygous mutation in an affected individual must be further evaluated by parental studies to exclude the possibility of a large gene deletion or the failure to amplify one allele. As Sanger sequencing is performed on amplified segments of each allele, a large deletion of one allele would not amplify, resulting in apparent homozygosity, whereas complete failure to amplify a segment, or all, of a gene may suggest a homozygous deletion. Exon-level deletion testing should be considered in individuals with only one loss-of-function mutation detected by sequence analysis of the ACTA1 gene. In
Fig. 3. Relative quantity histograms of qPCR data for probes located in intron 1 (A) and exon 7 (B) of the ACTA1 gene. The controls are compared to a calibrator reference run with each assay (assigned 1.0 on the quantity scale) and fall within pre-determined acceptable limits (0.8–1.1) on the Relative Quantity scale. The decreased relative quantity (copy number) in the proband and father’s samples compared to control samples indicates the presence of only one intact ACTA1 allele.
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addition, it may be prudent to pursue deletion studies if one missense mutation is identified in an affected individual and the functional consequences of the mutation are not known. Furthermore, as several other genes have been identified as causative of NM, additional genetic evaluation may be warranted if studies fail to confirm the expected inheritance pattern. Identifying the correct underlying molecular cause of NM is important for genetic counseling in assessing recurrence risk. Additionally, proposed therapeutics, such as the up-regulation of cardiac a-actin (ACTC), for affected individuals may require knowledge of the underlying molecular mechanism to determine if therapy is appropriate [2,4,12,14,15]. In summary, this first report of a large deletion in the ACTA1 gene illustrates the importance of understanding disease mechanism in nemaline myopathy at the molecular level and highlights the need to pursue parental testing in cases of apparent homozygosity in a proband. Acknowledgments With thanks to the proband’s family for their cooperation and to Stephanie Warren and Melissa Tomcavage for assistance with Fig. 3. References [1] North K, Ryan MM. Nemaline myopathy. 2002 June 19 [updated 2012 March 15]. In: Pagon RA, Adam MP, Bird TD, et al., editors. GeneReviewse [Internet]. Seattle, WA: University of Washington; 1993–2013. Available from: http://www.ncbi.nlm.nih.gov/books/ NBK1288. [2] Nowak KJ, Ravenscroft G, Laing NG. Skeletal muscle a-actin diseases (actinopathies): pathology and mechanisms. Acta Neuropathol 2013;125:19–32.
[3] Ravenscroft G, Miyatake S, Lehtokari VL, et al. Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy. Am J Hum Genet 2013;93:6–18. [4] Ilkovski B, Cooper ST, Nowak K, et al. Nemaline myopathy caused by mutations in the muscle a-skeletal-actin gene. Am J Hum Genet 2001;68:1333–43. [5] Sparrow JC, Nowak KJ, Durling HJ, et al. Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1). Neuromuscul Disord 2003;13:519–31. [6] Ilkovski B, Nowak KJ, Domazetovska A, et al. Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms. Hum Mol Genet 2004;13:1727–43. [7] Costa CF, Rommelaere H, Waterschoot D, et al. Myopathy mutations in a-skeletal-muscle actin cause a range of molecular defects. J Cell Sci 2004;117:3367–77. [8] Feng J, Marston S. Genotype–phenotype correlations in ACTA1 mutations that cause congenital myopathies. Neuromuscul Disord 2009;19:6–16. [9] Laing NG, Dye DE, Wallgren-Pettersson C, et al. Mutations and polymorphisms of the skeletal muscle a-actin gene (ACTA1). Hum Mutat 2009;30:1267–77. [10] Ochala J. Thin filament proteins mutations associated with skeletal myopathies: defective regulation of muscle contraction. J Mol Med 2008;86:1197–204. [11] Vandamme D, Lambert E, Waterschoot D, et al. a-Skeletal muscle actin nemaline myopathy mutants cause cell death in cultured muscle cells. Biochim Biophys Acta 2009;1793:1259–71. [12] Crawford K, Flick R, Close L, et al. Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and die during the neonatal period. Mol Cell Biol 2002;22:5887–96. [13] Nowak KJ, Sewry CA, Navarro C, et al. Nemaline myopathy caused by absence of a-skeletal muscle actin. Ann Neurol 2007;61:175–84. [14] Nowak KJ, Ravenscroft G, Jackaman C, et al. Rescue of skeletal muscle a-actin-null mice by cardiac (fetal) a-actin. J Cell Biol 2009;185:903–15. [15] Ravenscroft G, Jackaman C, Bringans S, et al. Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies. Brain 2011;134:1101–15.
ScienceDirect Neuromuscular Disorders 24 (2014) 331–334 www.elsevier.com/locate/nmd
Case report
Novel large deletion in the ACTA1 gene in a child with autosomal recessive nemaline myopathy Bethany Friedman a, Kara Simpson b, Carolina Tesi-Rocha c, Delu Zhou d, Cheryl A. Palmer d, Sharon F. Suchy a,⇑ b
a GeneDx, Gaithersburg, MD, USA Genetics and Metabolism, Children’s National Medical Center, Washington, DC, USA c Neurology, Children’s National Medical Center, Washington, DC, USA d Department of Pathology, University of Utah, Salt Lake City, UT, USA
Received 17 July 2013; received in revised form 14 November 2013; accepted 16 December 2013
Abstract Nemaline myopathy (NM) is a genetically and clinically heterogeneous disorder resulting from a disruption of the thin filament proteins of the striated muscle sarcomere. The disorder is typically characterized by muscle weakness including the face, neck, respiratory, and limb muscles and is clinically classified based on the age of onset and severity. Mutations in the ACTA1 gene contribute to a significant proportion of NM cases. The majority of ACTA1 gene mutations are missense mutations causing autosomal dominant NM by producing an abnormal protein. However, approximately 10% of ACTA1 gene mutations are associated with autosomal recessive NM; these mutations are associated with loss of protein function. We report the first case of a large deletion in the ACTA1 gene contributing to autosomal recessive NM. This case illustrates the importance of understanding disease mechanisms at the molecular level to accurately infer the inheritance pattern and potentially aid with clinical management. Ó 2014 Published by Elsevier B.V. Keywords: Nemaline myopathy; ACTA1; Deletion; Dominant negative; Loss of function
1. Introduction Nemaline myopathy (NM) is a genetically and clinically heterogeneous myopathy and, with an incidence of approximately 1/50,000 live births, is one of the most common congenital myopathic conditions. Histologically, this disorder is characterized by the accumulation of thin filaments and aggregates of the muscle Z-disks, known as nemaline bodies, on muscle biopsy. Most of the genes that cause NM encode sarcomeric thin filament proteins. Mutations in the skeletal muscle NEB gene are the most common cause of NM, however this disorder can be caused by mutations in seven other genes: ACTA1, ⇑ Corresponding author. Address: 207 Perry Parkway, Gaithersburg, MD 20877, USA. Tel.: +1 301 519 2100; fax: +1 301 519 2892. E-mail address: [email protected] (S.F. Suchy).
0960-8966/$ - see front matter Ó 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.nmd.2013.12.006
TPM3, TNNT1, TPM2, CFL2, KBTBD13, and KLHL40 [1–3]. Mutations in the ACTA1 gene, that encodes skeletal muscle a-actin, account for about 20% of cases of NM and approximately 50% of cases of severe NM [1,4–6]. Individuals with congenital NM typically exhibit marked proximal muscle weakness, hypotonia, decreased or absent deep tendon reflexes, and myopathic facies; respiratory and feeding difficulties along with motor delays are often present due to significant muscle weakness. NM is the most common congenital myopathy caused by mutations in the ACTA1 gene. Mutations in the ACTA1 gene are associated with several other congenital myopathies, based on histologic features, with overlapping clinical phenotypes. Histologic findings on muscle biopsy due to mutations in the ACTA1 gene can include one or more of the following: nemaline bodies, intranuclear rods, actin filament aggregates, caps,
332
B. Friedman et al. / Neuromuscular Disorders 24 (2014) 331–334
core-like areas, dystrophic features, fiber type disproportion, zebra bodies, and/or non-specific changes [2]. The presence of nemaline bodies is diagnostic for NM. Correlation between muscle histology and clinical presentation is poor [7]. However, some correlation between the location of a mutation in the ACTA1 gene and muscle histology has been reported [5,8,9]. The ACTA1 gene encodes skeletal muscle a-actin, which is the main component of thin filaments in skeletal muscle. Mutations in the ACTA1 gene can result in defects in actin folding, actin polymerization, and muscle contraction [2,7,8]. Disease-associated mutations in ACTA1 are located throughout the gene and ultimately affect skeletal muscle contraction, typically leading to weakness of the skeletal muscles [8,10]. To date, ACTA1 gene mutations reported in the literature causing autosomal recessive NM include nonsense, splice-site, and frameshift mutations as well as missense mutations shown to result in functionally null a-actin. Here we report a proband with autosomal recessive NM due to two loss-of-function ACTA1 mutations, including an exon-level deletion. This is the first description of a large deletion of the ACTA1 gene. 2. Case report The proband was born at 39 weeks gestation at 6 lb 9 oz to a 29 year old G3P2 mother. Decreased fetal movements were noted during the pregnancy. The birth was complicated by a bilateral brachial plexus injury and a broken clavicle. The newborn was admitted to the neonatal intensive care unit at day one of life due to respiratory and feeding difficulties for which she was started on biphasic positive airway pressure (BiPAP) and nasogastric feeds; a gastrostomy tube was later placed. Significant hypotonia was noted. Physical examination of the proband at 6 and 27 months of age revealed myopathic facies, pectus excavatum, neuromuscular kyphoscoliosis, hyporeflexia, and marked proximal muscle hypotonia. At five years of age, the patient continues to receive physical and speech therapies for developmental delays, dysarthria, and dysphagia. She is routinely on nocturnal BiPAP. The family history was noncontributory; the proband’s parents and older brother were well with no clinical features suggestive of an underlying myopathy. The mother and father of the proband were of Scottish and Danish ancestry, respectively; there was no known consanguinity in the family. A skeletal muscle biopsy revealed numerous rounded, atrophic myofibers on the hematoxylin and eosin sections. A marked type 1 fiber hypotrophy was present, although a nearly equal distribution of type 1 and type 2 fibers were seen. Gomori trichrome stain revealed occasional darkly staining granules distributed throughout the specimen (Fig. 1A). Ultrastructural examination identified the presence of electron-dense
Fig. 1. Gomori trichrome section of the skeletal muscle reveals varying fiber size with numerous atrophic fibers as well as dark granules in scattered fibers (arrows) (Gomori trichrome 1000) (A). Electron microscopy demonstrates electron dense rod-shaped nemaline bodies (arrowheads) (28,010) (B).
nemaline bodies in the myofibers, diagnostic of NM (Fig. 1B). To identify the underlying molecular cause of NM in the proband, genetic analysis was necessary. Although mutations in the NEB gene are the most common cause of NM, at the time of testing, full sequencing was not clinically available for the very large NEB gene. Therefore, sequence analysis of the ACTA1 gene was pursued on DNA extracted from peripheral blood. Bi-directional sequence analysis of the coding region of the ACTA1 gene (exons 2–7) by Sanger sequencing initially identified an apparently homozygous, novel deletion of a single nucleotide in exon 6 (c.911delG) (Fig. 2). This mutation causes a frameshift (p.Gly304AlafsX24) which is predicted to result in nonsense-mediated mRNA decay or protein truncation. The presence of this loss-of-function mutation in a homozygous state would be consistent with autosomal recessive NM. However, given the parents are non-consanguineous, it would be unlikely for both parents to be carriers of the same
B. Friedman et al. / Neuromuscular Disorders 24 (2014) 331–334
333
3. Discussion
Fig. 2. Forward (Fwd) and reverse (Rev) sequence traces of the proband surrounding and including the c.911delG mutation in the ACTA1 gene. The proband’s sequence reveals an apparent homozygous novel deletion of a single nucleotide, c.911delG. The nucleotide sequence surrounding the mutation is shown below.
mutation. Targeted sequence analysis was recommended for the parents to confirm that they were both heterozygous carriers of the mutation, and to rule out a failure to amplify both alleles and an exon-level deletion. Sequence analysis revealed that only the mother of the proband was a carrier of the mutation. Due to the absence of the c.911delG mutation in the father, an exon-level deletion of the ACTA1 gene was considered as a possible mechanism for disease in the proband. This possibility was investigated by quantitative copy number analysis of the ACTA1 gene in the proband and father by quantitative PCR (qPCR); labeled probes were located in intron 1 (35 bp before exon 2), exon 4, exon 6, and exon 7 of the ACTA1 gene. Analysis revealed only a single ACTA1 allele present at all the positions tested. This demonstrated the presence of a large deletion of the ACTA1 gene extending, at a minimum, from intron 1 to exon 7. This deletion was also identified in the proband’s father (Fig. 3).
This is the first case report of a large deletion of the ACTA1 gene contributing to autosomal recessive NM. Mutations in the ACTA1 gene have been shown to cause disease by two different mechanisms. Most ACTA1 mutations are de novo missense mutations hypothesized to cause autosomal dominant NM by a gain-of-function mechanism (dominant negative), producing a protein with abnormal function [5,6,11]. However, approximately 10% of ACTA1 gene mutations cause autosomal recessive NM [9] due to the loss of skeletal muscle a-actin function [7,9,12,13]. The presence of the deletion along with the identified frameshift mutation in the proband is consistent with the loss-of-function mechanism associated with autosomal recessive NM. Carrier parents of an affected child with recessive NM have not been reported to exhibit clinical features or symptoms of NM, demonstrating that one null, loss-of-function, ACTA1 allele is not sufficient to cause disease [12,13]. Many of the reported cases of autosomal recessive NM occur in families with known consanguinity [13]. In the absence of known consanguinity, the presence of an apparently homozygous mutation in an affected individual must be further evaluated by parental studies to exclude the possibility of a large gene deletion or the failure to amplify one allele. As Sanger sequencing is performed on amplified segments of each allele, a large deletion of one allele would not amplify, resulting in apparent homozygosity, whereas complete failure to amplify a segment, or all, of a gene may suggest a homozygous deletion. Exon-level deletion testing should be considered in individuals with only one loss-of-function mutation detected by sequence analysis of the ACTA1 gene. In
Fig. 3. Relative quantity histograms of qPCR data for probes located in intron 1 (A) and exon 7 (B) of the ACTA1 gene. The controls are compared to a calibrator reference run with each assay (assigned 1.0 on the quantity scale) and fall within pre-determined acceptable limits (0.8–1.1) on the Relative Quantity scale. The decreased relative quantity (copy number) in the proband and father’s samples compared to control samples indicates the presence of only one intact ACTA1 allele.
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addition, it may be prudent to pursue deletion studies if one missense mutation is identified in an affected individual and the functional consequences of the mutation are not known. Furthermore, as several other genes have been identified as causative of NM, additional genetic evaluation may be warranted if studies fail to confirm the expected inheritance pattern. Identifying the correct underlying molecular cause of NM is important for genetic counseling in assessing recurrence risk. Additionally, proposed therapeutics, such as the up-regulation of cardiac a-actin (ACTC), for affected individuals may require knowledge of the underlying molecular mechanism to determine if therapy is appropriate [2,4,12,14,15]. In summary, this first report of a large deletion in the ACTA1 gene illustrates the importance of understanding disease mechanism in nemaline myopathy at the molecular level and highlights the need to pursue parental testing in cases of apparent homozygosity in a proband. Acknowledgments With thanks to the proband’s family for their cooperation and to Stephanie Warren and Melissa Tomcavage for assistance with Fig. 3. References [1] North K, Ryan MM. Nemaline myopathy. 2002 June 19 [updated 2012 March 15]. In: Pagon RA, Adam MP, Bird TD, et al., editors. GeneReviewse [Internet]. Seattle, WA: University of Washington; 1993–2013. Available from: http://www.ncbi.nlm.nih.gov/books/ NBK1288. [2] Nowak KJ, Ravenscroft G, Laing NG. Skeletal muscle a-actin diseases (actinopathies): pathology and mechanisms. Acta Neuropathol 2013;125:19–32.
[3] Ravenscroft G, Miyatake S, Lehtokari VL, et al. Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy. Am J Hum Genet 2013;93:6–18. [4] Ilkovski B, Cooper ST, Nowak K, et al. Nemaline myopathy caused by mutations in the muscle a-skeletal-actin gene. Am J Hum Genet 2001;68:1333–43. [5] Sparrow JC, Nowak KJ, Durling HJ, et al. Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1). Neuromuscul Disord 2003;13:519–31. [6] Ilkovski B, Nowak KJ, Domazetovska A, et al. Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms. Hum Mol Genet 2004;13:1727–43. [7] Costa CF, Rommelaere H, Waterschoot D, et al. Myopathy mutations in a-skeletal-muscle actin cause a range of molecular defects. J Cell Sci 2004;117:3367–77. [8] Feng J, Marston S. Genotype–phenotype correlations in ACTA1 mutations that cause congenital myopathies. Neuromuscul Disord 2009;19:6–16. [9] Laing NG, Dye DE, Wallgren-Pettersson C, et al. Mutations and polymorphisms of the skeletal muscle a-actin gene (ACTA1). Hum Mutat 2009;30:1267–77. [10] Ochala J. Thin filament proteins mutations associated with skeletal myopathies: defective regulation of muscle contraction. J Mol Med 2008;86:1197–204. [11] Vandamme D, Lambert E, Waterschoot D, et al. a-Skeletal muscle actin nemaline myopathy mutants cause cell death in cultured muscle cells. Biochim Biophys Acta 2009;1793:1259–71. [12] Crawford K, Flick R, Close L, et al. Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and die during the neonatal period. Mol Cell Biol 2002;22:5887–96. [13] Nowak KJ, Sewry CA, Navarro C, et al. Nemaline myopathy caused by absence of a-skeletal muscle actin. Ann Neurol 2007;61:175–84. [14] Nowak KJ, Ravenscroft G, Jackaman C, et al. Rescue of skeletal muscle a-actin-null mice by cardiac (fetal) a-actin. J Cell Biol 2009;185:903–15. [15] Ravenscroft G, Jackaman C, Bringans S, et al. Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies. Brain 2011;134:1101–15.
