Nemaline Myopathy (NEM2) via the Nebulin (NEB) Gene

Mutations in the nebulin gene (NEB) are the most common cause of autosomal recessive nemaline myopathy (NEM). NEM is a genetically and clinically heterogeneous disorder characterized by muscle weakness, hypotonia and the presence of nemaline bodies in skeletal muscle fibers. Muscle weakness is typically observed in affected neonates or infants, although later onset cases are reported (Ryan et al. 2001). The most severely affected muscle groups are proximal limb, facial, bulbar, and respiratory muscles. Deep tendon reflexes are absent or depressed. Histologically, NEM is characterized by type 1 fiber predominance and the presence of rod-like structures called nemaline bodies upon Gomori trichrome staining of skeletal muscle (Ryan et al. 2003). Six clinical types of NEM have been delineated based on age of onset, severity and distribution of weakness, and respiratory function (Ryan et al. 2001; North and Ryan 2002). Nebulin gene mutations more often cause typical neonatal onset disease, although NEB mutations have been found in every clinical form of NEM (Lehtokari et al. 2006). Overlap among the six clinical groups is significant, and adults are sometimes diagnosed only after another family member has presented with typical signs.

To date, mutations in six genes have been shown to cause nemaline myopathy. Mutations in ACTA1 (NEM3) and NEB (NEM2) are the only relatively common causes, accounting for 15-25% and up to 50%, respectively, of all studied cases (Ryan et al. 2001; North and Ryan. 2002). All reported cases of NEB-associated NEM demonstrate autosomal recessive inheritance (Lehtokari et al. 2006; Lehtokari et al. 2014). In the largest cohort published to date, Lehtokari et al. (2014) identified 212 NEB mutations segregating with autosomal recessive NEM in 159 families. In this study, pathogenic mutations are found in most exons of the NEB gene, including the triplicate region (exons 82-105). The majority of these mutations (Lehtokari et al. 2014) were splice site variants (34%), frameshift mutations (32%), and nonsense mutations (23%). Reported pathogenic missense variants appear to be less common (~7%). The only common NEB mutation is an exon 55 deletion found at a carrier frequency of about 1% among people of Ashkenazi Jewish ancestry (Anderson et. al. 2004).

Up to 50% of all known nemaline myopathy cases are caused by mutations in NEB (North and Ryan 2002).

Gross deletions/duplications account for ~4% of all reported NEB mutations (Lehtokari et al. 2014). An exon 55 deletion is the only common deletion found at a carrier frequency of about 1% among people of Ashkenazi Jewish ancestry (Anderson et. al. 2004). A separate PCR based assay for this common deletion is available (#356).

This test provides full coverage of all coding exons of the NEB gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Exons 82-105 of the NEB gene are organized in three highly homologous blocks of 8 exons each making this region difficult to analyze by NextGen Sequencing. Due to the homology, there are mapping and alignment issues which may cause variants in this region to go undetected. We employ a novel long-range 2-step PCR with Sanger sequencing to analyze this region of the NEB gene which allows correct exon and zygosity assignment.