Expanding the MTM1 mutational spectrum: novel variants including the first multi-exonic duplication and development of a locus-specific database

Abstract

Myotubular myopathy (MIM#310400), the X-linked form of Centronuclear myopathy (CNM) is mainly characterized by neonatal hypotonia and inability to maintain unassisted respiration. The MTM1 gene, responsible for this disease, encodes myotubularin - a lipidic phosphatase involved in vesicle trafficking regulation and maturation. Recently, it was shown that myotubularin interacts with desmin, being a major regulator of intermediate filaments. We report the development of a locus-specific database for MTM1 using the Leiden Open Variation database software (http://www.lovd.nl/MTM1), with data collated for 474 mutations identified in 472 patients (by June 2012). Among the entries are a total of 25 new mutations, including a large deletion encompassing introns 2-15. During database implementation it was noticed that no large duplications had been reported. We tested a group of eight uncharacterized CNM patients for this specific type of mutation, by multiple ligation-dependent probe amplification (MLPA) analysis. A large duplication spanning exons 1-5 was identified in a boy with a mild phenotype, with results pointing toward possible somatic mosaicism. Further characterization revealed that this duplication causes an in-frame deletion at the mRNA level (r.343_444del). Results obtained with a next generation sequencing approach suggested that the duplication extends into the neighboring MAMLD1 gene and subsequent cDNA analysis detected the presence of a MTM1/MAMLD1 fusion transcript. A complex rearrangement involving the duplication of exon 10 has since been reported, with detection also enabled by MLPA analysis. It is thus conceivable that large duplications in MTM1 may account for a number of CNM cases that have remained genetically unresolved.

Figure 1

Mutations registered in MTM1-LOVD as distributed along the MTM1 gene. (a) Total number of small mutations is shown for each exon and intron of MTM1. (b) Large deletions and duplications identified in MTM1. Solid bars represent regions involved (blue – deletions; red – duplications); gray bars represent undelineated breakpoints. In cases with more than one independent report the total number (n) of patients is indicated.

Figure 2

Histological features of patient 1. Histological analysis of the left deltoid muscle biopsy performed using staining with hematoxylin and eosin (a, b and e), histochemical reactions for NADH-TR (c), and PAS (d). Abnormal fiber size variability is seen on all stainings. The key feature of the pathology is the central nuclei (more evident on H&E image a and e). The dark central staining areas accompanying the central nuclei are another striking feature (c and d). The ‘necklace fibers', recently described as a histopathological marker of MTM1, can be best appreciated on PAS staining (d) and faintly on H&E (e).

Figure 3

Large genomic duplication detected in patient 1. (a) MLPA analysis in patient 1 (P1) shows duplication of exons 1–5 of the MTM1 gene. Red arrows indicate the duplicated probes. (b) Southern blot technique using a cDNA probe spanning exons 2–7 (SB probe); red arrow-heads represent EcoRI restriction sites; red arrow highlight the absence of a band in the patient, corresponding to exons 5 and 6. (c) RT-PCR analysis of mRNA obtained from muscle, revealing a mutated isoform lacking exon 6; (*) – smaller faint band corresponding to an alternative splicing product (Δ exon 5 and 6, ENSEMBL transcript ID: ENST00000424519). (d) Low coverage NGS analysis of a 500 kb interval within the X chromosome. Histograms correspond to the total number of reads aligned per 10 kb interval for the patient (top), an unrelated normal control sample (middle) and ratio between the patient and control (bottom) where the line corresponds to ratio value of 1. (e) Partial sequence of the fusion transcript, showing MTM1 exon 6 adjacent to MAMLD1 exon 4. (f) Schematic representation of duplicated region (dashed rectangle); resulting transcripts are indicated by black arrows – observed (filled) and predicted (unfilled). P1, patient; C/C1/C2, controls; M, patient's mother; MW, molecular weight marker.