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Array comparative genomic hybridization identifies a heterozygous deletion of exon 3 of the RYR2 gene.

Leong IU, Sucich J, Prosser DO, Skinner JR, Crawford JR, Higgins C, Love DR - Ups. J. Med. Sci. (2015)

Bottom Line: The standard diagnostic screening involves Sanger-based sequencing of 45 of the 105 translated exons of the RYR2 gene, and copy number changes of a limited number of exons that are detected using multiplex ligation-dependent probe amplification (MLPA).The CGH array detected a 1.1 kb deletion encompassing exon 3 of the RYR2 gene.The aCGH method offers significant advantages over MLPA in genetic screening for heritable cardiac disorders.

View Article: PubMed Central - PubMed

Affiliation: Diagnostic Genetics, LabPLUS, Auckland City Hospital , PO Box 110031, Auckland 1142 , New Zealand.

ABSTRACT

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a heritable cardiac disorder characterized by life-threatening ventricular tachycardia caused by exercise or acute emotional stress. The standard diagnostic screening involves Sanger-based sequencing of 45 of the 105 translated exons of the RYR2 gene, and copy number changes of a limited number of exons that are detected using multiplex ligation-dependent probe amplification (MLPA).

Methods: In the current study, a previously validated bespoke array comparative genomic hybridization (aCGH) technique was used to detect copy number changes in the RYR2 gene in a 43-year-old woman clinically diagnosed with CPVT.

Results: The CGH array detected a 1.1 kb deletion encompassing exon 3 of the RYR2 gene. This is the first report using the aCGH technique to screen for mutations causing CPVT.

Conclusions: The aCGH method offers significant advantages over MLPA in genetic screening for heritable cardiac disorders.

No MeSH data available.


Related in: MedlinePlus

PCR amplification of the region encompassing exon 3 of the RYR2 gene. A: Amplicon sizes of the expected PCR products using DNA from an unaffected individual carrying no deletion of exon 3 of the RYR2 gene (left), and for DNA with the exon 3 deletion (right). The deletion size according to aCGH and the actual deletion size confirmed by Sanger-based sequencing are shown (above red lines). The expected PCR amplicon size of the deletion mutant according to the aCGH data and the actual product size are both shown. B: 2% agarose gel showing the results of PCR amplification of the genomic region encompassing exon 3 of the RYR2 gene for the proband (II-3) and her four children (III-1 to III-4). Chromatogram of the control and the proband showing where the breakpoint is (indicated by the red line).
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Figure 4: PCR amplification of the region encompassing exon 3 of the RYR2 gene. A: Amplicon sizes of the expected PCR products using DNA from an unaffected individual carrying no deletion of exon 3 of the RYR2 gene (left), and for DNA with the exon 3 deletion (right). The deletion size according to aCGH and the actual deletion size confirmed by Sanger-based sequencing are shown (above red lines). The expected PCR amplicon size of the deletion mutant according to the aCGH data and the actual product size are both shown. B: 2% agarose gel showing the results of PCR amplification of the genomic region encompassing exon 3 of the RYR2 gene for the proband (II-3) and her four children (III-1 to III-4). Chromatogram of the control and the proband showing where the breakpoint is (indicated by the red line).

Mentions: The aCGH analysis of the proband identified a heterozygous deletion on chromosome 1 (1q43) that was approximately 816 bp in size (hg18 co-ordinates chr1:235,560,602-235,561,416) (Figure 3). This deletion encompasses the whole of exon 3 of the RYR2 gene with flanking intronic regions (Figure 4). Sanger-based sequencing was used to confirm the aCGH results and also to determine the exact breakpoints of the deletion. Primers were designed to flank the region of interest (Figure 4A), and subsequent Sanger-based sequencing of the resulting smaller amplicon confirmed the aCGH results but with greater resolution such that the deletion proved to be 1,126 bp in size (Figure 4A). This mutation (c.169-198_c.273+823 del1126) has been previously reported by Ohno et al. (12) and Szentpali et al. (Figure 5) (13). Exon 3 of the RYR2 gene encodes for a highly conserved region of the RYR2 protein, and the mutation is an in-frame deletion of 35 amino acids (p.Asn57-Gly91).


Array comparative genomic hybridization identifies a heterozygous deletion of exon 3 of the RYR2 gene.

Leong IU, Sucich J, Prosser DO, Skinner JR, Crawford JR, Higgins C, Love DR - Ups. J. Med. Sci. (2015)

PCR amplification of the region encompassing exon 3 of the RYR2 gene. A: Amplicon sizes of the expected PCR products using DNA from an unaffected individual carrying no deletion of exon 3 of the RYR2 gene (left), and for DNA with the exon 3 deletion (right). The deletion size according to aCGH and the actual deletion size confirmed by Sanger-based sequencing are shown (above red lines). The expected PCR amplicon size of the deletion mutant according to the aCGH data and the actual product size are both shown. B: 2% agarose gel showing the results of PCR amplification of the genomic region encompassing exon 3 of the RYR2 gene for the proband (II-3) and her four children (III-1 to III-4). Chromatogram of the control and the proband showing where the breakpoint is (indicated by the red line).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: PCR amplification of the region encompassing exon 3 of the RYR2 gene. A: Amplicon sizes of the expected PCR products using DNA from an unaffected individual carrying no deletion of exon 3 of the RYR2 gene (left), and for DNA with the exon 3 deletion (right). The deletion size according to aCGH and the actual deletion size confirmed by Sanger-based sequencing are shown (above red lines). The expected PCR amplicon size of the deletion mutant according to the aCGH data and the actual product size are both shown. B: 2% agarose gel showing the results of PCR amplification of the genomic region encompassing exon 3 of the RYR2 gene for the proband (II-3) and her four children (III-1 to III-4). Chromatogram of the control and the proband showing where the breakpoint is (indicated by the red line).
Mentions: The aCGH analysis of the proband identified a heterozygous deletion on chromosome 1 (1q43) that was approximately 816 bp in size (hg18 co-ordinates chr1:235,560,602-235,561,416) (Figure 3). This deletion encompasses the whole of exon 3 of the RYR2 gene with flanking intronic regions (Figure 4). Sanger-based sequencing was used to confirm the aCGH results and also to determine the exact breakpoints of the deletion. Primers were designed to flank the region of interest (Figure 4A), and subsequent Sanger-based sequencing of the resulting smaller amplicon confirmed the aCGH results but with greater resolution such that the deletion proved to be 1,126 bp in size (Figure 4A). This mutation (c.169-198_c.273+823 del1126) has been previously reported by Ohno et al. (12) and Szentpali et al. (Figure 5) (13). Exon 3 of the RYR2 gene encodes for a highly conserved region of the RYR2 protein, and the mutation is an in-frame deletion of 35 amino acids (p.Asn57-Gly91).

Bottom Line: The standard diagnostic screening involves Sanger-based sequencing of 45 of the 105 translated exons of the RYR2 gene, and copy number changes of a limited number of exons that are detected using multiplex ligation-dependent probe amplification (MLPA).The CGH array detected a 1.1 kb deletion encompassing exon 3 of the RYR2 gene.The aCGH method offers significant advantages over MLPA in genetic screening for heritable cardiac disorders.

View Article: PubMed Central - PubMed

Affiliation: Diagnostic Genetics, LabPLUS, Auckland City Hospital , PO Box 110031, Auckland 1142 , New Zealand.

ABSTRACT

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a heritable cardiac disorder characterized by life-threatening ventricular tachycardia caused by exercise or acute emotional stress. The standard diagnostic screening involves Sanger-based sequencing of 45 of the 105 translated exons of the RYR2 gene, and copy number changes of a limited number of exons that are detected using multiplex ligation-dependent probe amplification (MLPA).

Methods: In the current study, a previously validated bespoke array comparative genomic hybridization (aCGH) technique was used to detect copy number changes in the RYR2 gene in a 43-year-old woman clinically diagnosed with CPVT.

Results: The CGH array detected a 1.1 kb deletion encompassing exon 3 of the RYR2 gene. This is the first report using the aCGH technique to screen for mutations causing CPVT.

Conclusions: The aCGH method offers significant advantages over MLPA in genetic screening for heritable cardiac disorders.

No MeSH data available.


Related in: MedlinePlus