Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for LGMD1F

<div><p>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 (<i>TNPO3</i>) gene, encoding a member of the importin-β super-family. The <i>TNPO3</i> 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.</p></div

Total and Shared Variants in Patients with LGMD1F.

<p>Total and Shared Variants in Patients with LGMD1F.</p

LGMD1F family pedigree.

<p>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).</p

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

<p>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). <b>a</b>) An accumulation around the nucleus is usually observed using the mutant delA p.X924C -hTNPO3. <b>b</b>) The typical intranuclear staining pattern can be observed in cells transfected with wt-hTNPO3 (in red) or <b>c)</b> in non transfected HeLa cells.</p

Sequence variants of USH patients identified by whole exome sequencing.

(1)<p>We annotated the resulting variation according the USHbase database (<a href="https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html" target="_blank">https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html</a>) and the 9 variants not present in the database have been classified as unreported.</p>(2)<p>Clinical diagnosis compatible with USH type 3.</p

Coverage data for long-PCR NGS sequencing.

<p>Relationship between the minimum depth coverage and the extent of basepairs of Usher exons sequenced. Solid colored lines represent sample sequenced on different platforms, whereas the dotted line is the average representation obtained from the nine sample of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043799#pone-0043799-g001" target="_blank">Figure 1</a>. x axis indicates the minimum coverage increasing from left to right up to 50× while the y axis indicates the percentage of Usher exon basepairs sequenced.</p

Workflow of the next generation sequencing strategies used.

<p>A) whole exome sequencing workflow. Samples have been pre-screened using an Apex-based Usher genotyping microarray; library preparations prior to enrichment include fragment single reads or Paired-End preparation. Three different types of enrichment methods have been used; each enrichment probe sets overlap at different extent to the RefSeq coding regions of Usher genes (horizontal bars). Sequencing protocols include single 50 bp reads on the Solid3 System, single 50 bp read on Solid4 System, Paired-end reads 50 bp+35 bp on Solid4 System. B) Long-PCR sequencing workflow. Samples have been pre-screened using Usher Apex microarray, Long-PCR approach produced 218 PCR amplicons used as input for the for <i>Fragment</i> and <i>Paired-End</i> library preparation. Sequencing was performed using both GS-FLX and GAII Systems.</p

Coverage data for whole exome sequencing.

<p>A) Relationship between the minimum depth coverage and the extent of basepairs of RefSeq exons sequenced (shown in percentage). B) Relationship between the minimum depth coverage and the extent of basepairs of Usher exons sequenced. Solid colored lines represent different samples, x axis: minimum coverage increasing from left to right up to 50×; y axis: percentage of exons sequenced.</p

Sequence variants of USH patients identified by Long-PCR sequencing.

1<p>Classification based on the USHbase database <a href="https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html" target="_blank">https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html</a>.</p