A Homozygous Deep Intronic Mutation Alters the Splicing of Nebulin Gene in a Patient With Nemaline Myopathy

Figure 1. Structure of the nebulin protein and binding partners. Nebulin gene consists of multiple repeat modules (M) that contain actin-binding domain. The S21 super repeat encodes the mutually alternatively spliced exons 143 and 144 and contains the binding site for KLHL40. The C-terminal is anchored in the Z-disk region of the sarcomere and contains a conserved serine-rich homology domain (SH3). This terminal region binds many muscle proteins. Binding partners are written in blue. Adapted from Yuen and Ottenheijm (39), http://creativecommons.org/licenses/by/4.0/.

Figure 2. Pedigree of the patient's family. The proband (black square), his sister, father, and mother were recruited in the project. Due to the consanguinity, autosomal recessive type of inheritance was suspected. Pedigree symbols. Circle, female; square, male; black filled, affected; unfilled, non-affected.

Figure 3. Muscle biopsy of the patient. (A) Gömöri trichrome staining of a quadricep muscle biopsy section shows mild anisomorphism fibers. More than 90% of the fibers have red stain inclusions typical of nemaline rods. Scale bar = 50 μm. (B) Electron microscopy image shows cytoplasmic rod-shaped nemaline bodies. No intranuclear rods were observed. Magnification, ×3,200; scale bar = 2 μm.

Figure 4. In silico analysis of the effects of the mutation. Sequence of the exon 144 followed by the first part of the intronic sequence that includes the mutation. This schematic representation of the affected region of the nebulin gene shows the new splice site predicted to be created by this intronic variant. According to Human Splicing Finder, the new site has a strength of 90.24* in comparison with 85.75* for the natural site. *These numbers are relative consensus values established by the tool ranging from 1 to 100.

Figure 5. Analysis of cloned fragments ranging from exons 142–145 from the patient's biopsy or skeletal muscle control RNA. (A) Fragments with or without exons 143 and/or 144 found in the control and patient's biopsy. (B) Electrophoresis analysis of the PCR fragment of the exons 144–145 shows the presence of an additional band in the patient which corresponds to the intron containing fragment found in the biopsy. The smaller fragment found in both control and patient corresponds to the normal exons 144–145 region. (C) Comparison of part of the sequence of the normal and intron containing fragments with exon 144. The mutation disrupts the reading frame by introducing a premature termination codon that targets the mRNA for degradation or production of a truncated protein. C, control; P, patient.

Figure 6. Decreased levels of nebulin expression in the patient's skeletal muscle. Quantitative real-time RT-PCR comparative analysis on the covering region from exons 142–145. Relative expression represented as fold ratio of nebulin fragments in the patient biopsy compared with control. Fold changes were normalized using the three reference genes beta-2-microglobulin (B2M), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are represented as mean of the quadruplicate ± SD. ***P = 0.0005.