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Melusin gene (ITGB1BP2) nucleotide variations study in hypertensive and cardiopathic patients.

Palumbo V, Segat L, Padovan L, Amoroso A, Trimarco B, Izzo R, Lembo G, Regitz-Zagrosek V, Knoll R, Brancaccio M, Tarone G, Crovella S - BMC Med. Genet. (2009)

Bottom Line: The role of Melusin in heart function has been established both by loss and gain of function experiments in murine models.Only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11.Preliminary functional results and bioinformatic analysis seem to exclude a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism.Our analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Genetica Biologia e Biochimica, Università di Torino, via Santena 19, Torino 10126, Italy.

ABSTRACT

Background: Melusin is a muscle specific signaling protein, required for compensatory hypertrophy response in pressure-overloaded heart. The role of Melusin in heart function has been established both by loss and gain of function experiments in murine models. With the aim of verifying the hypothesis of a potential role of the Melusin encoding gene, ITGB1BP2, in the modification of the clinical phenotype of human cardiomyopathies, we screened the ITGB1BP2 gene looking for genetic variations possibly associated to the pathological phenotype in three selected groups of patients affected by hypertension and dilated or hypertrophic cardiomyopathy

Methods: We analyzed ITGB1BP2 by direct sequencing of the 11 coding exons and intron flanking sequences in 928 subjects, including 656 hypertensive or cardiopathic patients and 272 healthy individuals.

Results: Only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11.

Conclusions: The three variations of the ITGB1BP2 gene have been detected in families of patients affected either by hypertension or primary hypertrophic cardiomyopathy; however, a clear genotype/phenotype correlation was not evident. Preliminary functional results and bioinformatic analysis seem to exclude a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism.Our analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.

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A: Analysis of the splicing pattern by RT-PCR of the wild-type and IVS6+12_18dupTTTTGAG constructs after transfection in the HeLa cell line: both constructs reproduce the same splicing pattern (amplicon length 382 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: IVS6+12_18dupTTTTGAG sample. B: Analysis of the splicing pattern by RT-PCR of the wild-type and 843C>T SNP constructs after transfection in the HeLa cell line, showing that both constructs reproduce the same splicing pattern (amplicon length 432 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: 843C>T sample.
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Figure 3: A: Analysis of the splicing pattern by RT-PCR of the wild-type and IVS6+12_18dupTTTTGAG constructs after transfection in the HeLa cell line: both constructs reproduce the same splicing pattern (amplicon length 382 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: IVS6+12_18dupTTTTGAG sample. B: Analysis of the splicing pattern by RT-PCR of the wild-type and 843C>T SNP constructs after transfection in the HeLa cell line, showing that both constructs reproduce the same splicing pattern (amplicon length 432 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: 843C>T sample.

Mentions: We then performed an in-silico analysis for both the 843C>T and IVS6+12_18dupTTTTGAG, with the aim of testing the possible effects of the nucleotide variation on exon splicing using the following softwares NNsplice http://www.fruitfly.org/seq_tools/splice.html, Cryp-Skip http://www.dbass.org.uk/cryp-skip/ and NetGene2 http://www.cbs.dtu.dk/services/NetGene2/: the results obtained indicated that the mutated sequences didn't introduce or delete any donor or acceptor splice site. In order to test the effect of the mutations in-vitro, we also performed a minigene splicing assay for both 843C>T and IVS6+12_18dupTTTTGAG, showing that the transcripts obtained from the cells transfected with the wild-type sequence and the mutated one, were the same both in terms of length (Figure 3) and of sequence of transcripts.


Melusin gene (ITGB1BP2) nucleotide variations study in hypertensive and cardiopathic patients.

Palumbo V, Segat L, Padovan L, Amoroso A, Trimarco B, Izzo R, Lembo G, Regitz-Zagrosek V, Knoll R, Brancaccio M, Tarone G, Crovella S - BMC Med. Genet. (2009)

A: Analysis of the splicing pattern by RT-PCR of the wild-type and IVS6+12_18dupTTTTGAG constructs after transfection in the HeLa cell line: both constructs reproduce the same splicing pattern (amplicon length 382 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: IVS6+12_18dupTTTTGAG sample. B: Analysis of the splicing pattern by RT-PCR of the wild-type and 843C>T SNP constructs after transfection in the HeLa cell line, showing that both constructs reproduce the same splicing pattern (amplicon length 432 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: 843C>T sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803168&req=5

Figure 3: A: Analysis of the splicing pattern by RT-PCR of the wild-type and IVS6+12_18dupTTTTGAG constructs after transfection in the HeLa cell line: both constructs reproduce the same splicing pattern (amplicon length 382 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: IVS6+12_18dupTTTTGAG sample. B: Analysis of the splicing pattern by RT-PCR of the wild-type and 843C>T SNP constructs after transfection in the HeLa cell line, showing that both constructs reproduce the same splicing pattern (amplicon length 432 bp). Mk: 100 bp molecular ladder; WT: wild-type sample; MT: 843C>T sample.
Mentions: We then performed an in-silico analysis for both the 843C>T and IVS6+12_18dupTTTTGAG, with the aim of testing the possible effects of the nucleotide variation on exon splicing using the following softwares NNsplice http://www.fruitfly.org/seq_tools/splice.html, Cryp-Skip http://www.dbass.org.uk/cryp-skip/ and NetGene2 http://www.cbs.dtu.dk/services/NetGene2/: the results obtained indicated that the mutated sequences didn't introduce or delete any donor or acceptor splice site. In order to test the effect of the mutations in-vitro, we also performed a minigene splicing assay for both 843C>T and IVS6+12_18dupTTTTGAG, showing that the transcripts obtained from the cells transfected with the wild-type sequence and the mutated one, were the same both in terms of length (Figure 3) and of sequence of transcripts.

Bottom Line: The role of Melusin in heart function has been established both by loss and gain of function experiments in murine models.Only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11.Preliminary functional results and bioinformatic analysis seem to exclude a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism.Our analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Genetica Biologia e Biochimica, Università di Torino, via Santena 19, Torino 10126, Italy.

ABSTRACT

Background: Melusin is a muscle specific signaling protein, required for compensatory hypertrophy response in pressure-overloaded heart. The role of Melusin in heart function has been established both by loss and gain of function experiments in murine models. With the aim of verifying the hypothesis of a potential role of the Melusin encoding gene, ITGB1BP2, in the modification of the clinical phenotype of human cardiomyopathies, we screened the ITGB1BP2 gene looking for genetic variations possibly associated to the pathological phenotype in three selected groups of patients affected by hypertension and dilated or hypertrophic cardiomyopathy

Methods: We analyzed ITGB1BP2 by direct sequencing of the 11 coding exons and intron flanking sequences in 928 subjects, including 656 hypertensive or cardiopathic patients and 272 healthy individuals.

Results: Only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11.

Conclusions: The three variations of the ITGB1BP2 gene have been detected in families of patients affected either by hypertension or primary hypertrophic cardiomyopathy; however, a clear genotype/phenotype correlation was not evident. Preliminary functional results and bioinformatic analysis seem to exclude a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism.Our analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.

Show MeSH
Related in: MedlinePlus