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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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Splicing analysis and translational effect of the c. 1746-20 C>G variant. Panel A. Schematic representation of the 5 splicing patterns generated by this variant, resulting from the use of the cryptic acceptor splice sites A1, A2, A3, A4, carrying the retention of 19, 86, 122 and 305 bp of intron 13 (dashed boxes), respectively, and WTA13, with the retention of the entire intron 13 (dashed box). The arrows indicate the localization of the primers used. Panel B. Splicing-specific PCR (designed to potentially amplify all the 5 aberrant transcripts) showing that these transcripts are variably expressed and not always detectable in the heterozygous mutant patients (Pt. 1, II-1, and Pt.3). The WTA transcript is expressed also in normal control (C). Panel C. Family pedigree (case n. 6211) and western blot showing that this intronic mutation (filled symbol) produced a deleterious effect at protein level, as demonstrated by the complete loss of calpain-3 protein in the muscle from one affected patient (II-1) who was a compound heterozygote for a second missense mutation that has a deleterious effect as well (p.L204V, dashed symbol).
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fig04: Splicing analysis and translational effect of the c. 1746-20 C>G variant. Panel A. Schematic representation of the 5 splicing patterns generated by this variant, resulting from the use of the cryptic acceptor splice sites A1, A2, A3, A4, carrying the retention of 19, 86, 122 and 305 bp of intron 13 (dashed boxes), respectively, and WTA13, with the retention of the entire intron 13 (dashed box). The arrows indicate the localization of the primers used. Panel B. Splicing-specific PCR (designed to potentially amplify all the 5 aberrant transcripts) showing that these transcripts are variably expressed and not always detectable in the heterozygous mutant patients (Pt. 1, II-1, and Pt.3). The WTA transcript is expressed also in normal control (C). Panel C. Family pedigree (case n. 6211) and western blot showing that this intronic mutation (filled symbol) produced a deleterious effect at protein level, as demonstrated by the complete loss of calpain-3 protein in the muscle from one affected patient (II-1) who was a compound heterozygote for a second missense mutation that has a deleterious effect as well (p.L204V, dashed symbol).

Mentions: Conversely, in our study, the deleterious effect of this mutation has definitely been demonstrated at both translational (when this mutation was associated with another mutant allele, it always resulted in severely reduced protein quantity; Figure 4) and transcriptional levels. Indeed, this mutation had been predicted by the HSF algorithm to create a new acceptor splice site (A1), which was detected in muscle mRNA by splicing-specific PCR. Furthermore, numerous cryptic splicing sites in the region surrounding this mutation obtained a high score by SSPPs analysis and were expected to cause the insertion of different portions of intron 13. Three such transcripts (A2, A3, A4) were detected in our patients by cDNA analysis. However, even though none of the algorithms used predicted the loss of the canonical acceptor splice site (and only a slight decrease in its score was obtained for two of them), we identified an aberrant transcript (WTA) carrying the insertion of the entire intron 13. All these transcripts were variably expressed and detected in the patients, but while 4 aberrant transcripts (A1, A2, A3, A4) were detected only in mutant patients, suggesting their pathogenetic effect, the variant WTA was also expressed in 10 normal controls, indicating its non-pathogenicity. We attribute the occurrence of this latter transcript to the result of an alternative splicing event that takes place in normal tissues, as previously reported [Kawabata et al., 2003; De Tullio et al., 2003].


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. 1746-20 C>G variant. Panel A. Schematic representation of the 5 splicing patterns generated by this variant, resulting from the use of the cryptic acceptor splice sites A1, A2, A3, A4, carrying the retention of 19, 86, 122 and 305 bp of intron 13 (dashed boxes), respectively, and WTA13, with the retention of the entire intron 13 (dashed box). The arrows indicate the localization of the primers used. Panel B. Splicing-specific PCR (designed to potentially amplify all the 5 aberrant transcripts) showing that these transcripts are variably expressed and not always detectable in the heterozygous mutant patients (Pt. 1, II-1, and Pt.3). The WTA transcript is expressed also in normal control (C). Panel C. Family pedigree (case n. 6211) and western blot showing that this intronic mutation (filled symbol) produced a deleterious effect at protein level, as demonstrated by the complete loss of calpain-3 protein in the muscle from one affected patient (II-1) who was a compound heterozygote for a second missense mutation that has a deleterious effect as well (p.L204V, dashed symbol).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Splicing analysis and translational effect of the c. 1746-20 C>G variant. Panel A. Schematic representation of the 5 splicing patterns generated by this variant, resulting from the use of the cryptic acceptor splice sites A1, A2, A3, A4, carrying the retention of 19, 86, 122 and 305 bp of intron 13 (dashed boxes), respectively, and WTA13, with the retention of the entire intron 13 (dashed box). The arrows indicate the localization of the primers used. Panel B. Splicing-specific PCR (designed to potentially amplify all the 5 aberrant transcripts) showing that these transcripts are variably expressed and not always detectable in the heterozygous mutant patients (Pt. 1, II-1, and Pt.3). The WTA transcript is expressed also in normal control (C). Panel C. Family pedigree (case n. 6211) and western blot showing that this intronic mutation (filled symbol) produced a deleterious effect at protein level, as demonstrated by the complete loss of calpain-3 protein in the muscle from one affected patient (II-1) who was a compound heterozygote for a second missense mutation that has a deleterious effect as well (p.L204V, dashed symbol).
Mentions: Conversely, in our study, the deleterious effect of this mutation has definitely been demonstrated at both translational (when this mutation was associated with another mutant allele, it always resulted in severely reduced protein quantity; Figure 4) and transcriptional levels. Indeed, this mutation had been predicted by the HSF algorithm to create a new acceptor splice site (A1), which was detected in muscle mRNA by splicing-specific PCR. Furthermore, numerous cryptic splicing sites in the region surrounding this mutation obtained a high score by SSPPs analysis and were expected to cause the insertion of different portions of intron 13. Three such transcripts (A2, A3, A4) were detected in our patients by cDNA analysis. However, even though none of the algorithms used predicted the loss of the canonical acceptor splice site (and only a slight decrease in its score was obtained for two of them), we identified an aberrant transcript (WTA) carrying the insertion of the entire intron 13. All these transcripts were variably expressed and detected in the patients, but while 4 aberrant transcripts (A1, A2, A3, A4) were detected only in mutant patients, suggesting their pathogenetic effect, the variant WTA was also expressed in 10 normal controls, indicating its non-pathogenicity. We attribute the occurrence of this latter transcript to the result of an alternative splicing event that takes place in normal tissues, as previously reported [Kawabata et al., 2003; De Tullio et al., 2003].

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