Background: Nemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB), which is thought to account for roughly 50% of cases. Results: We describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost. Conclusions: Our findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease. Keywords: congenital myopathy, nemaline myopathy, nemaline rod (body), thin filament, nebulin Background KBTBD13 . With the exception of KBTBD13,whose With an estimated incidence of 1 in 50,000 live births, function is unknown, these genes share the unifying fea- nemaline myopathy (NM) is the most common of the ture that they all encode proteins of the sarcomeric thin congenital myopathies, accounting for roughly one-half filament, suggesting that weakness and rod formation in of the cases of these conditions . Clinically, NM is NM is related to improper thin-filament structure and heterogeneous, producing symptoms ranging from pro- function . found perinatal weakness and hypotonia to mild, non- The skeletal muscle-specific NEB gene is large, with a progressive weakness with onset in adolescence or total of 183 exons spanning 249 kb of genomic sequence adulthood.AdiagnosisofNMrequiressymptomsof and a theoretical full-length transcript of 26 kb, and is skeletal muscle weakness and the presence of nemaline predicted to encode an approximately 800-kDa protein rods in muscle fibers, in the absence of findings diag- . Great diversity in nebulin size, and possibly func- nostic of other unrelated conditions . To date, muta- tion, is generated through alternative splicing of at least tions of seven genes have been implicated in NM, 41 NEB exons, leading to production of at least hun- including tropomyosin 3 (TPM3) , skeletal a-actin dreds of distinct isoforms [11,12]. A single nebulin (ACTA1) , nebulin (NEB) , tropomyosin 2 (TPM2) molecule spans the thin filament with its C terminus , troponin T (TNNT1) , cofilin 2 (CFL2) and anchored at the Z-disk and its N-terminal region direc- ted toward the pointed end of the thin filament. Recent * Correspondence: firstname.lastname@example.org studies have suggested that nebulin may play a role in Division of Genetics and Program in Genomics, The Manton Center for the regulation of contraction in addition to its role in Orphan Disease Research, Children’s Hospital Boston, Harvard Medical determining thin-filament length . Control of thin- School, 300 Longwood Avenue, CLSB 15026, Boston, MA 02115, USA Full list of author information is available at the end of the article © 2011 Lawlor et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lawlor et al. Skeletal Muscle 2011, 1:23 Page 2 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 filament length is essential for proper muscle contrac- disease in either parent, and neither parent had signs or tion, since the degree of overlap between thick and thin symptoms of muscle disease. filaments determines the amount of force that a muscle can produce [4,14-17]. Nebulin enhances force genera- Patient 16-2 tion by altering cross-bridge cycling kinetics to increase This baby boy was born after a pregnancy complicated the number of force-generating cross-bridges [18,19]. after 31 weeks by polyhydramnios, and fetal movements Studies of two nebulin-knockout mouse models, both of were weak and infrequent. Birth occurred at 37 weeks which exhibit severe, early, lethal phenotypes, have gestational age, and the boy required intubation in the shown that nebulin absence is associated with shorter delivery room. He was 47 cm in length, weighed 2,500 thin filaments [18,20,21] and altered cross-bridge g, and had a head circumference of 37.5 cm. The patient cycling . had facial weakness; contractures of the hips, knees, Mutations of the skeletal muscle-specific NEB gene ankles, elbows and wrists; and other abnormalities, are the most common cause of autosomal recessive including a broad, prominent forehead; downward-slant- NM . Although often associated with the nonpro- ing palpebral fissures; micrognathia; a bulbous nose; a gressive or slowly progressive “typical” form of conge- cleft palate; ears that were low-set and posteriorly nital NM , NEB mutations have also been reported rotated; cryptorchism; and a small phallus. His neurolo- in patients with “intermediate” and “severe” forms of gical findings were otherwise normal. He required tube NM, characterized by lack of ambulation or even death feeding. Echocardiography revealed a large patent ductus in infancy . To date, 64 different mutations in NEB arteriosus with pulmonary hypertension. Electromyogra- have been reported in NM probands [4,10,14]. Largely phy performed at one week of age was inconclusive. A because that portion of the gene was studied first, biopsy of the right rectus femoris muscle obtained at many of the known mutations reside in the 3’ end of eight days of life revealed myopathic muscle with the gene and may affect interactions between nebulin numerous nemaline bodies and/or rods, which are diag- and other proteins at the Z-disc. Some cases exhibit nostic for NM (see below). He was ventilator-dependent loss of immunoreactivity of some proximal epitopes, until 28 days of life, when ventilator care was withdrawn with retention of distal epitopes, suggesting that com- and he was taken home. He died a few hours thereafter. plex patterns of alternative or abnormal splicing allow production of internally deleted, but stable and par- Patient 16-4 tially functional, proteins [4,17]. These studies focused This baby boy was born two years later to the same par- on the presence of immunohistochemical staining in ents at 31 weeks gestational age by spontaneous vaginal patient muscle biopsies, however, and did not assess delivery, with Apgar scores of 1, 1 and 2 at one, five and the quantity of nebulin present. A relatively common ten minutes, respectively. The pregnancy was compli- in-frame deletion of NEB exon 55, identified in Ashke- cated by oligohydramnios and preterm precipitous onset nazi Jewish NM patients with variable forms of NM, of labor. In utero monitoring demonstrated poor fetal has been studied extensively at the genetic and physio- movement and contractures of the upper and lower logical levels and has been shown to result in moder- extremities. At birth, contractures were present at the ately reduced levels of nebulin [10,15,17]. In this elbows, wrists, fingers, hips, knees and feet, with the report, we describe clinical, histological, genetic, pro- first, second and fifth digits overlapping the third and tein expression and muscle fiber contractility studies in fourth digits of the hands. The infant had significant a sibling pair with multiple congenital contractures and respiratory distress at delivery, with no respiratory effort neonatal death due to a particularly severe form of and poor color despite administration of 100% oxygen. NM resulting from two compound heterozygous muta- Prior to delivery, the parents had requested supportive tions in NEB. measures only, and active treatment was discontinued because of the patient’s clinical findings and significant Results respiratory distress during his first day of life. The A North American family (family “16”) with two affected patient died shortly thereafter. siblings with severe NM was referred for research stu- dies to determine the genetic basis for their condition. Pathological studies Patient 16-2 Parental history A right rectus femoris muscle biopsy taken at eight At the time of the birth of patient 16-2, the mother was days of life revealed skeletal muscle with small, round 25-year-old G7, P2-2-2-4 (seven pregnancies, two term fibers and excessive variation in fiber size (Figure 1A). births, two preterm births, two abortions and four living Numerous cells of all sizes and fiber types contained children). There was no family history of neuromuscular granular red material in a diffuse cytoplasmic and Lawlor et al. Skeletal Muscle 2011, 1:23 Page 3 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 Figure 1 Histological findings in two brothers with severe NM. Gomori trichrome staining (A) through (D) of frozen muscle tissue reveals punctate red inclusions (nemaline rods) within the cytoplasm of skeletal muscle fibers. These structures are readily identifiable in a biopsy specimen from the rectus femoris muscle of patient 16-2 (A), an autopsy specimen of the psoas muscle from patient 16-2 (B) and autopsy specimens from the diaphragm (C) and abdominal wall (D) muscles of patient 16-4. Toluidine blue staining of Epon-embedded quadriceps muscle from patient 16-2 (E) reveals a diffuse distribution of nemaline rods and marked variation in myofiber size. Note the variation in nemaline rod burden, fiber size variation and fibrosis between individual muscles within the same patient. Ultrastructural examination of this tissue (F) confirms the identity of the dense cytoplasmic inclusions as nemaline rods. Scale bars = 200 μm for (A) through (E) and 40 μm for (F). NM, nemaline myopathy. Lawlor et al. Skeletal Muscle 2011, 1:23 Page 4 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 subsarcolemmal distribution, consistent with the pre- Histologically, minimally expanded alveoli contained sence of nemaline rods. No nuclear rods were seen. scattered squamous cells and possible early hyaline mem- There was a mild focal increase in perimysial fibrosis. branes. The right and left pulmonary veins were signifi- The nicotinamide adenine dinucleotide (NADH) and cantly smaller than expected (1 or 2 mm), but the succinic dehydrogenase (SDH) stains revealed type 1 relationship and size of the vasculature were otherwise fiber predominance with appropriately sized type 2 normal. Sections taken from the thymus, trachea, esopha- fibers. No inflammation, excessive central nucleation, gus, adrenal gland, spleen, kidney, pancreas, bone, bone corelike structures or myonecrosis was seen. marrow and brain were unremarkable when visualized by The causes of death reported on the basis of autopsy light microscopy. The heart was structurally normal for findings were severe congenital NM and patchy acute gestational age, with a patent ductus arteriosus and fora- bronchopneumonia of the left lung. The lungs appeared men ovale. mildly edematous but without discrete lesions on the Evaluation of skeletal muscle from the diaphragm cut surface. The heart and all other organs were report- (Figure 1C) revealed small, round myofibers containing edly unremarkable. The histological findings in cardiac large, peripherally placed nuclei consistent with neonatal muscle were normal at both light and ultrastructural muscle. Gomori trichrome staining revealed numerous levels. nemaline rods in most myofibers, with a diffuse distri- Frozen muscle from the psoas, quadriceps, diaphragm bution of rods within the myofibers. NADH and ATPase and cardiac muscles collected at the time of autopsy staining allowed only poor differentiation between oxi- were available for histological review. Gomori trichrome dative and glycolytic fibers, but nemaline rods were pre- staining of the psoas muscle (Figure 1B) revealed skele- sent in both fiber types. Skeletal muscle from the tal muscle with small, round fibers and excessive varia- abdominal wall (Figure 1D) showed increased variation tion in fiber size. The findings in this specimen were in fiber size, with numerous small, round fibers and a similar to those seen in the patient’s earlier biopsy, greater degree of fiber size variation than was seen in except that the degree of perimysial and endomysial the diaphragm. The myofibers had large, peripherally fibrosis was more marked here and a greater proportion placed nuclei with coarse granular basophilic staining in of the fibers (approximately 80% to 90%) contained a many fibers. Gomori trichrome staining revealed nema- diffuse distribution of nemaline rods. No nuclear rods line rods within many fibers, which were arranged in a were seen. The quadriceps muscle had moderate to predominantly diffuse distribution within myocytes. The severe variation in fiber size, mild perimysial fibrosis rods were readily found in both large and small fiber and a diffuse and subsarcolemmal distribution of nema- populations. NADH and ATPase staining revealed that line rods in some fibers (Figure 1E). Nemaline rods the small fibers were of both oxidative and glycolytic were found in fewer fibers of the quadriceps muscle fiber types, and nemaline rods were located within both compared to the psoas muscle. Ultrastructural examina- fiber types. tion of the quadriceps muscle further confirmed the presence of nemaline rods in muscle fibers (Figure 1F). Mutation analysis Focal Z-band streaming was also present. The mito- Genetic studies of the ACTA1, TPM2, TPM3 and CFL2 chondria were appropriate with respect to their size, genes, as well as the recurrent NEB exon 55 deletion, shape, complexity and distribution. The diaphragm dis- were all negative in one or the other of the two affected played marked variation in fiber size with scattered patients. Genomic PCR and denaturing high-perfor- round small fibers and a mild, focal increase in perimy- mance liquid chromatography (dHPLC) analysis of 159 sial fibrosis. Most muscle fibers in the diaphragm were NEB gene (GenBank:NG_009382.1) exons revealed two much larger than the fibers seen in any of the other spe- distinct mutations, one each in the patients’ father and cimens from this patient, including the prior muscle mother. Both affected boys were compound heterozy- biopsy. Scattered fibers contained subsarcolemmal gotes for these two autosomal recessive mutations. The aggregates of granular red material, suggestive of nema- mutation inherited from the father was a splice site line rods. It should be noted, however, that these aggre- mutation in the 5’ splice site of intron 13 (GT > TT) gates were much smaller and less numerous than those (g.32596G > T, c.1152 + 1G > T) (Figure 2A), and the seen in the other skeletal muscle specimens. No inflam- mutation inherited from the mother was a deletion AG mation, corelike structures or myonecrosis was seen in leading to a frameshift in exon 81 (g.129384_129385del, any of the specimens. c.11318_11319del, p.Lys3774Argfs*10) (Figure 2B). Patient 16-4 Neither of these mutations was identified in 236 control At autopsy, pulmonary findings included bilobed right chromosomes analyzed by sequencing. Exons 13 and 81 lung, markedly hypoplastic lungs bilaterally, and imma- are both constitutively expressed, so patients with this ture, minimally expanded lung parenchyma. Lawlor et al. Skeletal Muscle 2011, 1:23 Page 5 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 Figure 2 Severe NM in two brothers caused by compound heterozygous mutations for an exon 13 splice site and an exon 81 frameshift. DNA sequence analysis of genomic PCR products illustrates the two mutations found in these patients. (A) A splice site mutation in the 5’ splice site of intron 13 (GT > TT)(g.32596G > T) was found in both reported patients and their father, but was not seen in their mother. (B) A deletion of two nucleotides was found in exon 81 (g.129384_129385del) of both patients and their mother, but was not present in their father. The results of PCR assays run in forward and reverse are shown to confirm the presence of a frameshift mutation. The sequencing results for patient 16-4 were identical to those shown for patient 16-2. Arrows indicate mutation sites. Lawlor et al. Skeletal Muscle 2011, 1:23 Page 6 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 combination of mutations would not be expected to was seen in control samples (Figure 3A). However, produce normal nebulin. when normalized to myosin heavy chain (MHC), label- ing of nebulin’s C terminus was approximately 90% less Nebulin expression intense in the NM patient compared with control (Fig- Western blot analysis performed using protein extracted ure 3D). Also, the antibody against nebulin’s C terminus from quadriceps muscle from patient 16-2 revealed sig- detected a doublet (Figure 3C), which might be a reflec- nificantly lower nebulin contentincomparisontocon- tion of the different mutations on each of the alleles. In trol patients and patients with exon 55 mutations who contrast, four patients with NM due to homozygous had been evaluated in a prior report . Protein from deletion of exon 55 of NEB showed labeling of nebulin’s patient 16-4 could not be evaluated. The antibody C terminus approximately 72% reduced in comparison against nebulin’s N terminus showed no detectable to control . Prior work using these samples [10,17] labeling, whereas the antibody against nebulin’s C termi- described a less severe clinical course and less severe nus detected a nebulin isoform at the appropriate mole- abnormalities seen on contractile studies using myofi- cular weight (approximately 773 kDa), similar to what bers from these patients. Figure 3 Compound heterozygosity for the exon 13 and exon 81 mutations is associated with dramatic reduction of nebulin protein levels in NM muscle. (A) Nebulin expression in comparison to myosin heavy chain (MHC) expression is shown using extracted protein from a control patient (Con) or from patient 16-2 (16-2). (B) Nebulin was detected using antibodies directed against either the N-terminal (N-term) or the C-terminal (C-term) regions of nebulin. Only the C-terminal antibody reacted with nebulin in the NM sample and reveals a barely detectable doublet. (C) Comparison of nebulin expression in control patients (Con), patient 16-2 (16-2) and a patient with nemaline myopathy caused by a mutation in exon 55 (Ex 55). (D) Quantification of nebulin expression (normalized to MHC expression and expressed as a percentage of the values seen in controls) in four patients with nemaline myopathy caused by mutations in exon 55 in comparison to the expression measured in patient 16-2. Values shown are means ± standard error of the mean (SEM). Lawlor et al. Skeletal Muscle 2011, 1:23 Page 7 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 Muscle mechanics due to exon 55 mutations in NEB . Pathologically, Recent studies using tissue from nebulin-knockout mice the patient muscles showed a variable degree of myo- [18-22] and humans with NM due to deletions of NEB pathic changes and nemaline body (rod) burden. Con- exon 55 [17,22] have demonstrated an impaired capacity tractility testing of skinned myofibers from the to generate force because of shorter thin filaments and diaphragm revealed marked deficits in contractile altered cross-bridge cycling. Here we used skinned fiber performance. preparations to determine the contractile properties of Mutations in NEB are the most common cause of NM myofibers from patient 16-4, whose clinical presentation . While the clinical findings in NEB-associated NM and quantity of nebulin represent an intermediate phe- are variable, the most frequent presentation of these notype between the knockout mice and patients with patients is the so-called “typical” form of NM. Patients the exon 55 deletion. Fibers isolated from a diaphragm with typical NM have congenital onset of weakness fol- 2+ specimen had severely reduced maximal Ca -activated lowed by delayed attainment of gross motor milestones active tension (4 ± 0.5 mN/mm compared with 88 ± 5 and a slowly progressive or nonprogressive course. mN/mm in control) (Figure 4A), which is similar to the Although the severe form of NM, which is associated recent report of contractile function in fibers of NM with a lack of spontaneous movements or respiration at patients with NEB exon 55 deletions . Because of birth, sometimes with multiple congenital contractures theuse of autopsytissueinthese studies, we cannot or fractures , is more frequently caused by muta- rule out that postmortem protein degradation contribu- tions in ACTA1 , severe NM has been reported in ted to the decrease in contractile performance, but the 13 families as a consequence of NEB mutation [14,24]. decrease was consistent among fibers and similar in In the first report of severe NM caused by NEB muta- scale to findings in our previous studies of surgical spe- tion, three of five families had mutations located in cimens. The rate of force redevelopment (k ) was dra- exon 184 , suggesting that this might represent a tr -1 matically decreased from 3.2 ± 0.2 s in control hotspot for mutations causing severe disease. However, -1 myofibers to 0.4 ± 0.04 s ,evenmoresothaninthe none of the subsequently reported families had muta- previously described patients  (Figure 4C). There tions of this exon , although, notably, one of these was also a significant increase in tension cost (adenosine families had a splice site mutation in intron 81 predicted triphosphate (ATP) consumption rate normalized to to lead to a defect similar to the exon 81 frameshift force) in fibers isolated from our patients (9.7 ± 2.3 found in our family 16. pmol/mN/mm/second) in comparison to controls (6.1 ± The particularly severe phenotype of both affected 0.6 pmol/mN/mm/second) (Figure 4B). These findings children in our study is reflected by the presence of are consistent with severely impaired force generation multiple contractures at birth, or arthrogryposis multi- that can be partly explained by altered cross-bridge plex congenita (AMC). Rather than a specific pathologi- cycling kinetics (slower cross-bridge attachment and fas- cal diagnosis, AMC is a description of a clinical ter detachment), resulting in a lower fraction of bound phenotype that occurs in 1 in 3,000 live births and is a force-generating cross-bridges [17,22]. characteristic of more than 300 different disorders . In cases of AMC that are associated with neuromuscular Discussion disease, contractures are present at birth as a result of While a number of NEB mutations have been described fetal muscle weakness causing insufficient movements. in patients with NM, the differences in molecular patho- AMC has been reported in some cases of severe NM genetic pathways leading to variable disease severity are due to mutations in NEB , but this is an uncommon currently unclear. Here we have described two siblings and particularly severe presentation of this disease. Our with compound heterozygote mutations in intron 13 data suggest that low levels of nebulin may be associated and exon 81 of NEB, resulting in severe congenital myo- with such a severe NM presentation and AMC and also pathy with profound muscle weakness, arthrogryposis that quantitation of the nebulin content in skeletal mus- and neonatal death. The splice site mutation in intron cle might be useful in the workup of patients with 13 is expected to cause exon 13 skipping, which would AMC. result in a protein lacking 39 amino acids in the N ter- A recent report described variably severe NM in minus disrupting simple repeat M8 and super repeat 1. patients with mutations in exon 55 of NEB,which Theframeshiftmutationinexon81ispredicted to encodes an N-terminal portion of nebulin . Mechan- cause premature truncation of the protein at super ical and structural analyses of muscle from these repeat 14. These mutations were associated with a patients led to the discovery that the exon 55 deletions marked decrease in the quantity of nebulin detectable produced decreased contractile force because of shorter by Western blot analysis, which was found to be at even and nonuniform thin-filament lengths, despite a lack of lower levels than those seen in patients with severe NM significant changes in the molecular weight of nebulin Lawlor et al. Skeletal Muscle 2011, 1:23 Page 8 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 Figure 4 Weakness in severe NM with greatly reduced nebulin levels is associated with reduced force generation with increased tension cost and slower force redevelopment. Skinned myofibers from control patients (Ctrl) and patient 16-4 (16-4) reveal (A) a large reduction in maximal tension measured at -log[Ca2+] (pCa) 4.5, (B) an increase in tension cost and (C) a large reduction in the rate constant of force development (k ). Control myofibers were taken from three normal quadriceps biopsies, and analysis was limited to type 2 fibers, since the tr patient 16-4 biopsy expressed mainly type 2 myosin heavy chains. Values are the means ± SEM of measurements from five myofibers. *P < 0.05. Lawlor et al. Skeletal Muscle 2011, 1:23 Page 9 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 . Contractile studies of Neb-knockout mice , the report represent another scenario in which NEB muta- patients with exon 55 deletions [17,22] and our more tions can cause severe NM that is associated with an severely affected patients described herein all showed even shorter life expectancy than that of the most large reductions in maximal tension and k and severely affected patients with exon 55 mutations. Sin- tr increases in tension cost. Whether the apparently gle-fiber contractile studies from patient 16-2 in this greater diminution of k in muscle from patient 16-4 is study revealed marked decreases in contractile force tr really associated with these boys’ unusually severe clini- compared with control fibers, and these decreases cal presentations will require the identification and ana- exceeded the contractile force deficits reported in stu- lysis of additional similar cases. dies using myofibers from NM patients with mutations Our Western blot studies using antibodies against the in exon 55 of NEB . Overall, the cases described in N- and C-terminal portions of nebulin revealed signifi- this study represent the severe end of the clinical spec- cant reduction of nebulin content with greater immu- trum of NM, exceeding the clinical and physiological noreactivity detected using antibodies against the C- deficits observed in patients with deletion of exon 55 of terminal portion of nebulin rather than the N-terminal NEB. Additionally, our results imply that low levels of portion. Studies of patients with deletion of exon 55 nebulin protein detected by Western blot analysis may have also detected reduced quantities of nebulin of correlate with a poor prognosis for patients with NM appropriate molecular size and weaker immunoreactivity due to NEB mutations, which would be diagnostically to antibodies directed against the N terminus . useful if this finding holds true in studies of larger num- These findings confirm earlier results in patients with bers of patients. other nebulin mutations and suggest that mutations affecting the N-terminal regions of nebulin can impair Methods recognition of nebulin using antibodies against the N Pathological evaluation terminus while leaving C-terminal immunoreactivity Muscle biopsy and autopsy tissues were obtained and intact [4,28,29]. Notably, the overall quantity of nebulin prepared using standard histological protocols . The in muscle from patient 16-2 (estimated to be 10% of rectus femoris, psoas, quadriceps, diaphragm and cardiac normal) was markedly lower than the approximately muscles were obtained from patient 16-2, and the dia- 28% levels seen in four patients with exon 55 deletion, phragm and abdominal wall muscles were obtained suggesting that the greater severity of symptoms in our from patient 16-4. Briefly, fresh muscle was frozen in patients might be related to the degree of nebulin defi- isopentane and stored at -80°C until sectioning. Eight- ciency. In contrast, a recent report described clinical micrometer cryostat sections were stained with hema- and mechanical findings in an NM patient with > 70% toxylin and eosin, Gomori trichrome, ATPase (at pH 4.3 normal levels of nebulin due to compound heterozygos- and pH 9.2) or NADH. Photomicrographs were ity for two splicing mutations predicted to induce skip- obtained by using a Nikon Eclipse 50i microscope (Mel- ping of NEB exons 3 and 22 . In that study, the ville, NY, USA) equipped with a SPOT Insight 4 Meg patient was a 46-year-old man with the “typical” form of FW Color Mosaic camera and SPOT 220.127.116.11 software NM, with only mild (Medical Research Council (MRC) from Diagnostic Instruments (Sterling Heights, MI, grade 4) weakness. Remarkably, and in contrast to our USA). For electron microscopy, samples were fixed and findings in patients with a greater degree of nebulin processed according to standard histological techniques, deficiency, muscle from this patient exhibited a normal and ultrastructural examination was performed on lead- force-sarcomere length relationship and normal calcium stained, 95-nm sections at the time of autopsy and sensitivity of force production. A more complete corre- repeated during the preparation of this article. lation between nebulin expression and clinical severity is necessary, but, taken together, these data suggest that Mutation analysis levels of nebulin expression detected by Western blot Mutation analysis of the NEB gene (GenBank: analysis may be indicative of the underlying molecular NG_009382-1) was performed by dHPLC and sequen- mechanisms of weakness and thus may be useful in pre- cing as previously described . The dHPLC analyses dicting the prognosis of patients with NM due to muta- were carried out using the automated Transgenomic tions in NEB. WAVE Nucleic Acid Fragment Analysis System (Trans- genomic, Omaha, NE, USA) with associated Navigator Conclusions software. Primer data are available upon request (from Our studies provide further evidence that NEB muta- VLL). All 159 of the 183 nebulin exons that could be tions can cause marked impairment of contractile per- analyzed by dHPLC were amplified using 148 primer formance that causes severe myopathic disease. The pairs. PCR reactions were performed in 96-well plates compound heterozygous mutations described in this suitable for dHPLC equipment. Each 35-μL reaction Lawlor et al. Skeletal Muscle 2011, 1:23 Page 10 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 mixture contained 60 to 90 ng of genomic DNA (3 μL), polyclonal antibody, x35-x36a 1843x, provided by Dr 10× PCR buffer supplied with the AmpliTaq Gold PCR Carol C Gregorio) and its C terminus (rabbit polyclonal Master Mix (Applied Biosystems, Carlsbad, CA, USA) antibody 6963, provided by Dr Siegfried Labeit) [21,32] containing 15 mmol MgCl2, 5 nmol each of deoxyribo- or against MHC. To control for loading differences, nucleotide triphosphate, 20 pmol forward primer, 20 nebulin labeling was normalized to MHC as determined pmol reverse primer and 0.8 U AmpliTaq Gold poly- from the Ponceau S-stained membrane. Secondary anti- merase enzyme (Applied Biosystems, Carlsbad, CA, bodies conjugated with fluorescent dyes with infrared USA). Reactions were carried out in a PTC-225 DNA excitation spectra were used for detection. One-color IR Engine Tetrad Thermocycler (MJ Research, Waltham, western blots were scanned (Odyssey Infrared Imaging MA, USA) starting with denaturation for 10 minutes at System, LI-COR Biosciences, Lincoln, NE, USA) and the 95°C, followed by annealing at 55°C to 60°C depending images analyzed with One-D scan EX. on the amplicon and extension at 72°C. The lengths of the denaturation, annealing and extension steps varied Muscle mechanics depending on the amplicon. A final extension was per- Small strips dissected from muscle biopsies were formed at 72°C for 10 minutes. Amplification of the skinned overnight at about 4°C in relaxing solution (20 PCR products was confirmed by agarose gel electro- mmol N,N-Bis-(2-hydroxyethyl)-2-aminoethane sulfonic phoresis before dHPLC analysis. acid (BES), 10 mmol ethylene glycol tetraacetic acid Before dHPLC analysis, PCR samples were denatured (EGTA), 6.56 mmol MgCl2, 5.88 mmol Na -ATP, 1 for 3 minutes at 95°C and then slowly reannealed by mmol dithiothreitol, 46.35 mmol K -propionate, 15 lowering the temperature from 95°C to 37°C over a per- mmol creatine phosphate, pH 7.0, at 20°C) containing iod of 1 hour. Two to five microliters of the PCR ampli- 1% (vol/vol) Triton X-100. Control samples for muscle con on the 96-well plate were injected into a heated mechanics studies were isolated from the quadriceps reverse-phase DNASep Column (Transgenomic, Omaha, muscles of three living individuals between 30 and 40 NE, USA). The column temperature of the dHPLC was years of age, and all results were comparable to pre- set for partially denaturing conditions. The melting pro- viously published results for control and experimental files of the amplicons were calculated using the Naviga- specimens representing a variety of ages, muscle groups tor software, but the exact temperature was determined and postmortem or postbiopsy intervals [17,22]. To empirically. Conditions used for dHPLC analysis of each ensure that measurements were taken from representa- amplicon are available on request (from VLL). tive and comparable fiber types, fibers and fiber bundles Following dHPLC analysis, samples showing abnormal were typed and analysis was restricted to type 2 fibers peaks were sequenced. The PCR products were purified (the predominant type found in the patient specimens). using Exonuclease I and shrimp alkaline phosphatase The skinning procedure renders the membranous struc- (USB Corp., Cleveland, OH, USA), and the purified pro- tures in the muscle fibers permeable, which enables acti- 2+ ducts were sequenced using BigDye version 3.1 sequen- vation of the myofilaments with exogenous Ca . cing chemistry and an ABI 3730 DNA Analyzer Preparations were washed thoroughly with relaxing solu- (Applied Biosystems, Carlsbad, CA, USA). Sequences tion and stored in 50% glycerol relaxing solution at -20° were analyzed using Sequencher 4.1 software (Gene C for up to approximately 8 weeks. Small muscle bun- Codes Corp, Ann, Arbor, MI, USA). dles (diameter approximately 0.07 mm) were dissected from the skinned strips and mounted between a displa- Western blot analysis cement generator and a force transducer element (AE For determination of nebulin content, muscle samples (a 801; SensoNor, Horten, Norway) using aluminum T biopsy of the quadriceps muscle from patient 16-2 and clips. Sarcomere length (SL) was set using a He-Ne laser an autopsy specimen from the abdominal wall of patient diffraction system. Mechanical experiments performed 16-4) were first homogenized in buffers containing pro- on contracting muscle were carried out at a SL of about tease inhibitors (phenylmethylsulfonyl fluoride, 0.5 2.5 μm for control muscle and at just over slack length mmol; leupeptin, 0.04 mmol; E64, 0.01 mmol) to pre- for NM muscle, a length selected on the basis of our vent protein degradation during the homogenization prior studies. By constructing force-SL relationships, we process. The homogenized muscle samples were run on previously showed that at a SL of 2.5 μm, human mus- 2.6% to 7% SDS-PAGE gels, and transferred onto polyvi- cle fibers from controls produced maximal force, nylidene fluoride membrane using a semidry transfer whereas nebulin-deficient muscle fibers from NM unit (Bio-Rad Laboratories, Hercules, CA, USA). Blots patients produced maximal force just over slack length were stained with Ponceau S to visualize total trans- because of their shorter thin filaments . Thus, by ferred protein. The blots were then probed with primary performing our mechanical studies on NM muscle set antibodies against nebulin’sNterminus (rabbit just over slack length, we aimed to minimize force Lawlor et al. Skeletal Muscle 2011, 1:23 Page 11 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 differences due to shorter thin-filament lengths. Fiber the skinned muscle fibers from the preactivation width and diameter were measured at three points along solution containing a low concentration of EGTA (pCa the fiber, and the cross-sectional area was determined by 9.0) to a pCa 4.5 activating solution. Once the steady assuming an elliptical cross-section. Three different state was reached, a slack equivalent to 10% of the bathing solutions were used during the experimental muscle length was rapidly induced at one end of the protocols: a relaxing solution, a preactivating solution muscle using the motor. This was followed immediately with low EGTA concentration and an activating solu- by an unloaded shortening lasting 30 milliseconds. The tion. The composition of these solutions was described remaining bound cross-bridges were mechanically previously . detached by rapidly (1 millisecond) restretching the muscle fiber to its original length, after which tension Simultaneous force-ATPase measurement redeveloped. The rate constant of monoexponential k tr We used the system described by Stienen et al.tomea- wasdeterminedbyfitting theriseintension to the - tr· sure simultaneous force-ATPase activity . To mea- following equation: F = Fss(1 - e k t), where F is force sure ATPase activity, a nearby UV light was projected at time t and k is the rate constant of tension tr through the quartz window of the bath (30 μLvolume redevelopment. and temperature controlled at 20°C) and detected at 340 nm. The maximum activation buffer (at -log[Ca2+] Abbreviations (pCa) 4.5) contained 10 mmol phosphoenol pyruvate -1 -1 dHPLC: denaturing high-performance liquid chromatography; MHC: myosin with 4 mg mL pyruvate kinase (500 U mg ), 0.24 mg heavy chain; NM: nemaline myopathy; PCR: polymerase chain reaction. -1 -1 mL lactate dehydrogenase (870 U mg )and 20 μmol Acknowledgements diadenosine 5’-pentaphosphate. For efficient mixing, the This work was supported by National Institutes of Health (NIH) grants solution in the bath was continuously stirred by means K08 AR059750 and L40 AR057721 (to MWL); a VENI grant from the of motor-driven vibration of a membrane positioned at Dutch Organization for Scientific Research (to CAO); grants from the Academy of Finland, the Association Francaisecontreles Myopathies, thebaseofthe bath.ATPaseactivityofthe skinned the Sigrid Jusélius Foundation, the Finska Läkaresällskapet and the fiber bundles was measured as follows. ATP regenera- Medicinska understödsföreningen Liv och Hälsa (to CWP and VLL); NIH tion from adenosine diphosphate (ADP) was coupled to grant R01 AR053897 (to HLG); and NIH grant R01 AR044345 and P50 NS040828 as well as grants from the Joshua Frase Foundation and Lee the breakdown of phosphoenol pyruvate to pyruvate and and Penny Anderson Family Foundation (to AHB). We also thank ATP was catalyzed by pyruvate kinase, which is linked Elizabeth DeChene for assistance with clinical data collection and to the synthesis of lactate catalyzed by lactate dehydro- analysis and Dr Stephen Smith and Beth Conrad of the Hennepin County Medical Center, Minneapolis, MN, USA, for patient referral and genase. The breakdown of NADH, which is proportional clinical and pathological data. to the amount of ATP consumed, was measured online by UV absorbance at 340 nm. The ratio of light inten- Author details Division of Genetics and Program in Genomics, The Manton Center for sity at 340 nm (sensitive to NADH concentration) and Orphan Disease Research, Children’s Hospital Boston, Harvard Medical the light intensity at 410 nm (reference signal) was School, 300 Longwood Avenue, CLSB 15026, Boston, MA 02115, USA. obtained by means of an analog divider. After each Department of Physiology, University of Arizona, 1501 N. Campbell, Rm. 4104, Tucson, AZ, 85724, USA. Laboratory for Physiology, Institute for recording, the UV absorbance signal of NADH was cali- Cardiovascular Research, VU University Medical Center, Van der brated by multiple rapid injections of 0.25 nmol of ADP Boechorststraat 7, Amsterdam 1081 BT, The Netherlands. The Folkhälsan (0.025 μLof10mmolADP)intothe bathingsolution Institute of Genetics and Department of Medical Genetics, Haartman Institute, P.O. Box 63 (Haartmaninkatu 8), FI-00014, University of Helsinki, with a stepper motor-controlled injector. The slope of Helsinki, Finland. Division of Genetics, Department of Biosciences, P.O. Box the ATP concentration versus time trace during steady- 56 (Viikinkaari 9), FI-00014, University of Helsinki, Helsinki, Finland. state tension development of a calcium-induced contrac- Authors’ contributions tion (Figure 4B) was determined from a linear fit, and MWL interpreted the clinical information, performed the pathological the value was divided by the fiber volume (in cubic analysis, and prepared the manuscript. CAO and HG performed the millimeters) to determine the fiber’s ATPase rate. contractile studies and composed the section of the results and discussion pertaining to contractile performance. VLL, KP, and CWP carried out the ATPase rates were corrected for the basal ATPase mea- molecular genetics analyses, assisted in analyzing the clinical and genetic sured in relaxing solution. The ATPase rate was divided data and composed sections of the introduction and discussion related to by tension (force ÷ cross sectional area (CSA) to deter- these topics. KC performed genetic analysis on the patients and created the figure related to these data. AHB conceived of the study, participated in its mine the tension cost. design and data interpretation, and helped to draft the manuscript. All authors read and approved the final manuscript. k measurements tr Competing interests To measure k , we used the large slack/release approach tr The authors declare that they have no competing interests.  to disengage force-generating cross-bridges from the thin filaments, which were isometrically activated. Received: 15 April 2011 Accepted: 20 June 2011 Published: 20 June 2011 Fast activation of the fiber was achieved by transferring Lawlor et al. Skeletal Muscle 2011, 1:23 Page 12 of 12 http://www.skeletalmusclejournal.com/content/1/1/23 References 18. 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Published: Jun 20, 2011