Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F

Abstract

Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.

Figure 1. LGMD1F family pedigree.

Squares represent male; circles represent female; white figures symbolize normal individuals; black figures indicate individuals with clinical muscular dystrophy. The original LGMD1F family has been extended from subject II,2 and now includes 64 LGMD patients of both sexes and five non-penetrant carriers (IV-4, V-26, V-29, V-33, and VI-68). The whole-exome sequencing was performed in four patients indicated by arrows (V-28, VI-36, VI-53, VII-5).

Figure 2. Sequence analyses of the TNPO3 mutations.

a) Heterozygous delA mutation in Exon 22 of the TNPO3 gene in Proband VII-5. Aligned electropherograms show mutated (top) and wild-type (bottom) sequences; b) Heterozygous. c.G2453A) in exon 21 of the TNPO3 gene; c) Pedigree of the isolated case.

Figure 3. Western blot analysis of skeletal muscle tissue with antibodies to TNPO3.

Equal amounts of muscle proteins from a LGMD1F patient and a control were run in each lane (10 µg) on a 9% SDS-polyacrylamide gel and then blotted onto nitrocellulose membrane. In this experiment, we used a monoclonal antibody that recognizes a recombinant fragment (Human) near the N terminus of TNPO3 at a 1∶100 dilution. A double band is visible in the patient only.

Figure 4. Indirect immunofluorescence analysis of the wt-hTNPO3 compared with delA p.X924C -hTNPO3.

Following transient transfections, HeLa cells were incubated for 48 h with normal DMEM and detected by anti-HA immunofluorescence. Nuclei are stained with DAPI (blue). The endogenous protein is recognized using a rabbit monoclonal anti-TNPO3 antibody (green), while the transfected TNPO3 proteins were HA-tagged (red). a) An accumulation around the nucleus is usually observed using the mutant delA p.X924C -hTNPO3. b) The typical intranuclear staining pattern can be observed in cells transfected with wt-hTNPO3 (in red) or c) in non transfected HeLa cells.