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Transcriptional and translational effects of intronic CAPN3 gene mutations.

Nascimbeni AC, Fanin M, Tasca E, Angelini C - Hum. Mutat. (2010)

Bottom Line: The absence or severe reduction of protein demonstrated their deleterious effect at translational level.We concluded that bioinformatic tools are valuable to suggest the potential effects of intronic variants; however, the experimental demonstration of the pathogenicity is not always easy to do even when using RNA analysis (low abundance, degradation mechanisms), and it might not be successful unless splicing-specific-PCR tests are used.A comprehensive approach is therefore recommended to identify and describe unclassified variants in order to offer essential data for basic and clinical geneticists.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of Padova, Italy.

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Related in: MedlinePlus

Splicing analysis and translational effect of the c. 1193+6 T>A variant. Panel A. Schematic representation of the aberrant splicing product (D1) generated by this variant, carrying the insertion of 31bp of intron 9 (dashed box) and resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequence from PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the co-amplification of the WT and the alternately spliced mRNA (D1). Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (II-1) who shows the WT product and one additional low-abundance product corresponding to the alternately spliced mRNA (D1). Family pedigree (case n. 7652) and western blot show that this intronic mutation (filled symbol) produced a reduction of calpain-3 protein of about 50% of control (C) after myosin normalization, as assessed in the muscle biopsy from both the heterozygous father (I-1) and his affected daughter (II-1), who was a compound heterozygote for a second missense mutation (p.E435K, dashed symbol).
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fig03: Splicing analysis and translational effect of the c. 1193+6 T>A variant. Panel A. Schematic representation of the aberrant splicing product (D1) generated by this variant, carrying the insertion of 31bp of intron 9 (dashed box) and resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequence from PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the co-amplification of the WT and the alternately spliced mRNA (D1). Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (II-1) who shows the WT product and one additional low-abundance product corresponding to the alternately spliced mRNA (D1). Family pedigree (case n. 7652) and western blot show that this intronic mutation (filled symbol) produced a reduction of calpain-3 protein of about 50% of control (C) after myosin normalization, as assessed in the muscle biopsy from both the heterozygous father (I-1) and his affected daughter (II-1), who was a compound heterozygote for a second missense mutation (p.E435K, dashed symbol).

Mentions: This mutation was identified in 6 unrelated LGMD2A patients in our series, all from the same administrative district of the Veneto Region (a possible founder effect followed by genetic isolation might have occurred). Among the 6 patients with this mutation, one was the object of a family study (Figure 3): we conducted both a segregation analysis of the mutant allele and a study of the effect of this mutation at calpain-3 protein level in 2 different family members (a muscle biopsy was obtained from both an affected girl and her heterozygote father, who reported hyperCKemia before the diagnosis was obtained in his daughter).


Transcriptional and translational effects of intronic CAPN3 gene mutations.

Nascimbeni AC, Fanin M, Tasca E, Angelini C - Hum. Mutat. (2010)

Splicing analysis and translational effect of the c. 1193+6 T>A variant. Panel A. Schematic representation of the aberrant splicing product (D1) generated by this variant, carrying the insertion of 31bp of intron 9 (dashed box) and resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequence from PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the co-amplification of the WT and the alternately spliced mRNA (D1). Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (II-1) who shows the WT product and one additional low-abundance product corresponding to the alternately spliced mRNA (D1). Family pedigree (case n. 7652) and western blot show that this intronic mutation (filled symbol) produced a reduction of calpain-3 protein of about 50% of control (C) after myosin normalization, as assessed in the muscle biopsy from both the heterozygous father (I-1) and his affected daughter (II-1), who was a compound heterozygote for a second missense mutation (p.E435K, dashed symbol).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2966865&req=5

fig03: Splicing analysis and translational effect of the c. 1193+6 T>A variant. Panel A. Schematic representation of the aberrant splicing product (D1) generated by this variant, carrying the insertion of 31bp of intron 9 (dashed box) and resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequence from PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the co-amplification of the WT and the alternately spliced mRNA (D1). Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (II-1) who shows the WT product and one additional low-abundance product corresponding to the alternately spliced mRNA (D1). Family pedigree (case n. 7652) and western blot show that this intronic mutation (filled symbol) produced a reduction of calpain-3 protein of about 50% of control (C) after myosin normalization, as assessed in the muscle biopsy from both the heterozygous father (I-1) and his affected daughter (II-1), who was a compound heterozygote for a second missense mutation (p.E435K, dashed symbol).
Mentions: This mutation was identified in 6 unrelated LGMD2A patients in our series, all from the same administrative district of the Veneto Region (a possible founder effect followed by genetic isolation might have occurred). Among the 6 patients with this mutation, one was the object of a family study (Figure 3): we conducted both a segregation analysis of the mutant allele and a study of the effect of this mutation at calpain-3 protein level in 2 different family members (a muscle biopsy was obtained from both an affected girl and her heterozygote father, who reported hyperCKemia before the diagnosis was obtained in his daughter).

Bottom Line: The absence or severe reduction of protein demonstrated their deleterious effect at translational level.We concluded that bioinformatic tools are valuable to suggest the potential effects of intronic variants; however, the experimental demonstration of the pathogenicity is not always easy to do even when using RNA analysis (low abundance, degradation mechanisms), and it might not be successful unless splicing-specific-PCR tests are used.A comprehensive approach is therefore recommended to identify and describe unclassified variants in order to offer essential data for basic and clinical geneticists.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of Padova, Italy.

Show MeSH
Related in: MedlinePlus