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

Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F, Rispoli R, Savarese M, Garofalo A, Piluso G, Morandi L, Ricci G, Siciliano G, Angelini C, Nigro V - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: TIGEM (Telethon Institute of Genetics and Medicine), Napoli, Italy.

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.

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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).
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pone-0063536-g001: 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).

Mentions: The original LGMD1F family has been extended (Figure 1) to include additional family members in seven generations starting from subject II, 2. The updated pedigree includes 64 LGMD patients of both sexes and five non-penetrant carriers (93% penetrance). To perform an informative exome sequencing analysis, we selected four affected family members (VII-5, VI-53, V-28, and VI-36) with a manifest LGMD phenotype separated by the largest number of meioses. Interestingly, two family members (VI-53 and V-28) were absent from the original family used for the linkage analyses. DNA samples of three individuals (V-28, VI-53, VII-5) were fragmented, enriched using the SureSelect whole exome kit and sequenced by SOliD. DNA, muscle RNA and proteins were extracted for the studies. We found ∼20,000 exonic variations for each sample, 5,722 of which were common to all three (Table 1 and Table S1) of which 2,471 were non synonymous. Considering the dominant mode of inheritance of LGMD1F, we focused on the heterozygous calls and discarded all variants present with a frequency higher than 1% in the NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS) or 1000genomes [32] large scale projects. The resulting filtered list of 273 variants was composed of 253 missense, 14 stopgain, 2 frameshift deletions, 2 nonframeshift insertions/deletions and 2 stoploss variations. Only two variants were mapped into the disease interval between D7S1822 and D7S2519 (positions: 126,287,140-129,964,025) [11]: a nonsynonymous SNV in the gene IRF5 and a frame-shift deletion that modify the termination codon in the exon 22 (stoploss) in the TNPO3 on chromosome 7q32.1 at position 128,597,310 (GRCh37/hg19). To verify whether we could have missed by NGS other shared variants, we resequenced by the dideoxy-chain termination method all the coding exons and flanking introns of the full 7q32 region with lower/absent coverage (Table S3). No other shared unknown variant was found. In addition, the DNA sample of VI-36 was sent to a commercial provider for exome sequencing using the Illumina platform HiSeq2000. Among 153 variations that were shared by all, the only one in the disease interval was that in the TNPO3 gene (Table 1). Interestingly, this was the only variation of the whole exome that resulted absent in dbSNP137. We also refined the interval: the SNP rs45445295 at the SMO gene at position 128,845,555 was present in some affected members (V-8, VI-60, V-14, VI-11, V-25, V-12), but it was absent in other affected members (VI-57, VI-27, VI-56) and in all non-affected individuals. Therefore, the linked region associated with disease locus was ∼1.1 Mb smaller (126,287,140-128,845,555) than that reported by Palenzuela [11].


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

Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F, Rispoli R, Savarese M, Garofalo A, Piluso G, Morandi L, Ricci G, Siciliano G, Angelini C, Nigro V - PLoS ONE (2013)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646821&req=5

pone-0063536-g001: 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).
Mentions: The original LGMD1F family has been extended (Figure 1) to include additional family members in seven generations starting from subject II, 2. The updated pedigree includes 64 LGMD patients of both sexes and five non-penetrant carriers (93% penetrance). To perform an informative exome sequencing analysis, we selected four affected family members (VII-5, VI-53, V-28, and VI-36) with a manifest LGMD phenotype separated by the largest number of meioses. Interestingly, two family members (VI-53 and V-28) were absent from the original family used for the linkage analyses. DNA samples of three individuals (V-28, VI-53, VII-5) were fragmented, enriched using the SureSelect whole exome kit and sequenced by SOliD. DNA, muscle RNA and proteins were extracted for the studies. We found ∼20,000 exonic variations for each sample, 5,722 of which were common to all three (Table 1 and Table S1) of which 2,471 were non synonymous. Considering the dominant mode of inheritance of LGMD1F, we focused on the heterozygous calls and discarded all variants present with a frequency higher than 1% in the NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS) or 1000genomes [32] large scale projects. The resulting filtered list of 273 variants was composed of 253 missense, 14 stopgain, 2 frameshift deletions, 2 nonframeshift insertions/deletions and 2 stoploss variations. Only two variants were mapped into the disease interval between D7S1822 and D7S2519 (positions: 126,287,140-129,964,025) [11]: a nonsynonymous SNV in the gene IRF5 and a frame-shift deletion that modify the termination codon in the exon 22 (stoploss) in the TNPO3 on chromosome 7q32.1 at position 128,597,310 (GRCh37/hg19). To verify whether we could have missed by NGS other shared variants, we resequenced by the dideoxy-chain termination method all the coding exons and flanking introns of the full 7q32 region with lower/absent coverage (Table S3). No other shared unknown variant was found. In addition, the DNA sample of VI-36 was sent to a commercial provider for exome sequencing using the Illumina platform HiSeq2000. Among 153 variations that were shared by all, the only one in the disease interval was that in the TNPO3 gene (Table 1). Interestingly, this was the only variation of the whole exome that resulted absent in dbSNP137. We also refined the interval: the SNP rs45445295 at the SMO gene at position 128,845,555 was present in some affected members (V-8, VI-60, V-14, VI-11, V-25, V-12), but it was absent in other affected members (VI-57, VI-27, VI-56) and in all non-affected individuals. Therefore, the linked region associated with disease locus was ∼1.1 Mb smaller (126,287,140-128,845,555) than that reported by Palenzuela [11].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: TIGEM (Telethon Institute of Genetics and Medicine), Napoli, Italy.

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.

Show MeSH
Related in: MedlinePlus