While a number of NEB
mutations have been described in patients with NM, the differences in molecular pathogenetic pathways leading to variable disease severity are currently unclear. Here we have described two siblings with compound heterozygote mutations in intron 13 and exon 81 of NEB
, resulting in severe congenital myopathy with profound muscle weakness, arthrogryposis and neonatal death. The splice site mutation in intron 13 is expected to cause exon 13 skipping, which would result in a protein lacking 39 amino acids in the N terminus disrupting simple repeat M8 and super repeat 1. The frameshift mutation in exon 81 is predicted to cause premature truncation of the protein at super repeat 14. These mutations were associated with a marked decrease in the quantity of nebulin detectable by Western blot analysis, which was found to be at even lower levels than those seen in patients with severe NM due to exon 55 mutations in NEB
]. Pathologically, the patient muscles showed a variable degree of myopathic changes and nemaline body (rod) burden. Contractility testing of skinned myofibers from the diaphragm revealed marked deficits in contractile performance.
Mutations in NEB
are the most common cause of NM [4
]. While the clinical findings in NEB
-associated NM are variable, the most frequent presentation of these patients is the so-called "typical" form of NM. Patients with typical NM have congenital onset of weakness followed by delayed attainment of gross motor milestones and a slowly progressive or nonprogressive course. Although the severe form of NM, which is associated with a lack of spontaneous movements or respiration at birth, sometimes with multiple congenital contractures or fractures [25
], is more frequently caused by mutations in ACTA1
], severe NM has been reported in 13 families as a consequence of NEB
]. In the first report of severe NM caused by NEB
mutation, three of five families had mutations located in exon 184 [24
], suggesting that this might represent a hotspot for mutations causing severe disease. However, none of the subsequently reported families had mutations of this exon [14
], although, notably, one of these families had a splice site mutation in intron 81 predicted to lead to a defect similar to the exon 81 frameshift found in our family 16.
The particularly severe phenotype of both affected children in our study is reflected by the presence of multiple contractures at birth, or arthrogryposis multiplex congenita (AMC). Rather than a specific pathological diagnosis, AMC is a description of a clinical phenotype that occurs in 1 in 3,000 live births and is a characteristic of more than 300 different disorders [26
]. In cases of AMC that are associated with neuromuscular disease, contractures are present at birth as a result of fetal muscle weakness causing insufficient movements. AMC has been reported in some cases of severe NM due to mutations in NEB
], but this is an uncommon and particularly severe presentation of this disease. Our data suggest that low levels of nebulin may be associated with such a severe NM presentation and AMC and also that quantitation of the nebulin content in skeletal muscle might be useful in the workup of patients with AMC.
A recent report described variably severe NM in patients with mutations in exon 55 of NEB
, which encodes an N-terminal portion of nebulin [10
]. Mechanical and structural analyses of muscle from these patients led to the discovery that the exon 55 deletions produced decreased contractile force because of shorter and nonuniform thin-filament lengths, despite a lack of significant changes in the molecular weight of nebulin [17
]. Contractile studies of Neb
-knockout mice [27
], the patients with exon 55 deletions [17
] and our more severely affected patients described herein all showed large reductions in maximal tension and ktr
and increases in tension cost. Whether the apparently greater diminution of ktr
in muscle from patient 16-4 is really associated with these boys' unusually severe clinical presentations will require the identification and analysis of additional similar cases.
Our Western blot studies using antibodies against the N- and C-terminal portions of nebulin revealed significant reduction of nebulin content with greater immunoreactivity detected using antibodies against the C-terminal portion of nebulin rather than the N-terminal portion. Studies of patients with deletion of exon 55 have also detected reduced quantities of nebulin of appropriate molecular size and weaker immunoreactivity to antibodies directed against the N terminus [17
]. These findings confirm earlier results in patients with other nebulin mutations and suggest that mutations affecting the N-terminal regions of nebulin can impair recognition of nebulin using antibodies against the N terminus while leaving C-terminal immunoreactivity intact [4
]. Notably, the overall quantity of nebulin in muscle from patient 16-2 (estimated to be 10% of normal) was markedly lower than the approximately 28% levels seen in four patients with exon 55 deletion, suggesting that the greater severity of symptoms in our patients might be related to the degree of nebulin deficiency. In contrast, a recent report described clinical and mechanical findings in an NM patient with > 70% normal levels of nebulin due to compound heterozygosity for two splicing mutations predicted to induce skipping of NEB
exons 3 and 22 [30
]. In that study, the patient was a 46-year-old man with the "typical" form of NM, with only mild (Medical Research Council (MRC) grade 4) weakness. Remarkably, and in contrast to our findings in patients with a greater degree of nebulin deficiency, muscle from this patient exhibited a normal force-sarcomere length relationship and normal calcium sensitivity of force production. A more complete correlation between nebulin expression and clinical severity is necessary, but, taken together, these data suggest that levels of nebulin expression detected by Western blot analysis may be indicative of the underlying molecular mechanisms of weakness and thus may be useful in predicting the prognosis of patients with NM due to mutations in NEB