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Targeted sequence capture and GS-FLX Titanium sequencing of 23 hypertrophic and dilated cardiomyopathy genes: implementation into diagnostics.

Mook OR, Haagmans MA, Soucy JF, van de Meerakker JB, Baas F, Jakobs ME, Hofman N, Christiaans I, Lekanne Deprez RH, Mannens MM - J. Med. Genet. (2013)

Bottom Line: Multiple genes are involved but no clear genotype-phenotype correlations have been found so far.Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing.Therefore, NGS is ready for implementation in a diagnostic setting.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands. o.r.mook@amc.uva.nl

ABSTRACT

Background: Genetic evaluation of cardiomyopathies poses a challenge. Multiple genes are involved but no clear genotype-phenotype correlations have been found so far. In the past, genetic evaluation for hypertrophic (HCM) and dilated (DCM) cardiomyopathies was performed by sequential screening of a very limited number of genes. Recent developments in sequencing have increased the throughput, enabling simultaneous screening of multiple genes for multiple patients in a single sequencing run.

Objective: Development and implementation of a next generation sequencing (NGS) based genetic test as replacement for Sanger sequencing.

Methods and results: In order to increase the number of genes that can be screened in a shorter time period, we enriched all exons of 23 of the most relevant HCM and DCM related genes using on-array multiplexed sequence capture followed by massively parallel pyrosequencing on the GS-FLX Titanium. After optimisation of array based sequence capture it was feasible to reliably detect a large panel of known and unknown variants in HCM and DCM patients, whereby the unknown variants could be confirmed by Sanger sequencing.

Conclusions: The rate of detection of (pathogenic) variants in both HCM and DCM patients was increased due to a larger number of genes studied. Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing. Therefore, NGS is ready for implementation in a diagnostic setting.

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Related in: MedlinePlus

Mapping of a coding 26 bp duplication in a hypertrophic cardiomyopathy patient. In contrast with short read platforms, a heterozygous insertion of 26 bp is easily mapped and reported.
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JMEDGENET2012101231F3: Mapping of a coding 26 bp duplication in a hypertrophic cardiomyopathy patient. In contrast with short read platforms, a heterozygous insertion of 26 bp is easily mapped and reported.

Mentions: Our first pilot study with five HCM patients with known pathogenic mutations showed that array based target enrichment and NGS could easily detect different types of known mutations (substitutions, insertions and deletions) and numerous non-pathogenic variants already detected with Sanger sequencing. All 57 variants detected with Sanger sequencing were also found with GS-FLX Titanium sequencing, including a coding 26 bp insertion in the MYBPC3 gene (figure 3). Because we detected 100% of the variants present in five HCM patients we proceeded with testing of additional patients. Nevertheless, in this pilot study we have observed that exon coverage varies significantly within one sample (determined by array design) resulting in lower confidentiality of particular variants (eg, MYBPC3: NM_000256.3: c.1093-24C>T). A balanced representation of all targeted exons would reduce the average coverage needed to detect variants with high confidentiality, consequently lowering the false negative rate. Therefore, we have designed arrays with a more balanced coverage as has previously been proposed by others.4748 The rebalanced capture array has been used to analyse nine HCM patients and 19 DCM patients. The rebalanced design showed that 99.80% of the targeted coding bases were covered at least once and 99.64% at least 16×. We and others8 have calculated that at 15–16× coverage a 99% sensitivity is obtained. This means that for an experiment with a mean coverage of 100±35× the statistical chance that a variant is missed in a patient is 0.004% (for calculation see supplementary figure S2).


Targeted sequence capture and GS-FLX Titanium sequencing of 23 hypertrophic and dilated cardiomyopathy genes: implementation into diagnostics.

Mook OR, Haagmans MA, Soucy JF, van de Meerakker JB, Baas F, Jakobs ME, Hofman N, Christiaans I, Lekanne Deprez RH, Mannens MM - J. Med. Genet. (2013)

Mapping of a coding 26 bp duplication in a hypertrophic cardiomyopathy patient. In contrast with short read platforms, a heterozygous insertion of 26 bp is easily mapped and reported.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756457&req=5

JMEDGENET2012101231F3: Mapping of a coding 26 bp duplication in a hypertrophic cardiomyopathy patient. In contrast with short read platforms, a heterozygous insertion of 26 bp is easily mapped and reported.
Mentions: Our first pilot study with five HCM patients with known pathogenic mutations showed that array based target enrichment and NGS could easily detect different types of known mutations (substitutions, insertions and deletions) and numerous non-pathogenic variants already detected with Sanger sequencing. All 57 variants detected with Sanger sequencing were also found with GS-FLX Titanium sequencing, including a coding 26 bp insertion in the MYBPC3 gene (figure 3). Because we detected 100% of the variants present in five HCM patients we proceeded with testing of additional patients. Nevertheless, in this pilot study we have observed that exon coverage varies significantly within one sample (determined by array design) resulting in lower confidentiality of particular variants (eg, MYBPC3: NM_000256.3: c.1093-24C>T). A balanced representation of all targeted exons would reduce the average coverage needed to detect variants with high confidentiality, consequently lowering the false negative rate. Therefore, we have designed arrays with a more balanced coverage as has previously been proposed by others.4748 The rebalanced capture array has been used to analyse nine HCM patients and 19 DCM patients. The rebalanced design showed that 99.80% of the targeted coding bases were covered at least once and 99.64% at least 16×. We and others8 have calculated that at 15–16× coverage a 99% sensitivity is obtained. This means that for an experiment with a mean coverage of 100±35× the statistical chance that a variant is missed in a patient is 0.004% (for calculation see supplementary figure S2).

Bottom Line: Multiple genes are involved but no clear genotype-phenotype correlations have been found so far.Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing.Therefore, NGS is ready for implementation in a diagnostic setting.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands. o.r.mook@amc.uva.nl

ABSTRACT

Background: Genetic evaluation of cardiomyopathies poses a challenge. Multiple genes are involved but no clear genotype-phenotype correlations have been found so far. In the past, genetic evaluation for hypertrophic (HCM) and dilated (DCM) cardiomyopathies was performed by sequential screening of a very limited number of genes. Recent developments in sequencing have increased the throughput, enabling simultaneous screening of multiple genes for multiple patients in a single sequencing run.

Objective: Development and implementation of a next generation sequencing (NGS) based genetic test as replacement for Sanger sequencing.

Methods and results: In order to increase the number of genes that can be screened in a shorter time period, we enriched all exons of 23 of the most relevant HCM and DCM related genes using on-array multiplexed sequence capture followed by massively parallel pyrosequencing on the GS-FLX Titanium. After optimisation of array based sequence capture it was feasible to reliably detect a large panel of known and unknown variants in HCM and DCM patients, whereby the unknown variants could be confirmed by Sanger sequencing.

Conclusions: The rate of detection of (pathogenic) variants in both HCM and DCM patients was increased due to a larger number of genes studied. Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing. Therefore, NGS is ready for implementation in a diagnostic setting.

Show MeSH
Related in: MedlinePlus