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Efficiency of exome sequencing for the molecular diagnosis of pseudoxanthoma elasticum.

Hosen MJ, Van Nieuwerburgh F, Steyaert W, Deforce D, Martin L, Leftheriotis G, De Paepe A, Coucke PJ, Vanakker OM - J. Invest. Dermatol. (2014)

Bottom Line: Exomes with insufficient reads (⩽20 depth) for the four genes and patients with single mutations were further evaluated by Sanger sequencing (SS), but no additional mutations were found.The potential of WES compared with targeted NGS is the ease to examine target genes and the opportunity to search for novel genes when targeted analysis is negative.Together with low cost, rapid and less laborious workflow, we conclude that WES complemented with SS can provide a tiered approach to molecular diagnostics of PXE.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium [2] Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet, Bangladesh.

ABSTRACT
The molecular etiology of pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disorder, has become increasingly complex as not only mutations in ATP-binding cassette family C member 6 (ABCC6) but also ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and gamma-glutamyl carboxylase (GGCX) can cause resembling phenotypes. Identification of modifier genes, such as vascular endothelial growth factor A, has further contributed to the molecular heterogeneity of PXE. In such heterogeneous diseases, next-generation sequencing (NGS) allows to perform mutation screening of several genes in a single reaction. We explored whole-exome sequencing (WES) as an efficient diagnostic tool to identify the causal mutations in ABCC6, GGCX, ENPP1, and vitamin K epoxide reductase complex, subunit 1 (VKORC1) in 16 PXE patients. WES identified a causal ABCC6 mutation in 30 out of 32 alleles and one GGCX mutation, whereas no causal mutations in ENPP1 or VKORC1 were detected. Exomes with insufficient reads (⩽20 depth) for the four genes and patients with single mutations were further evaluated by Sanger sequencing (SS), but no additional mutations were found. The potential of WES compared with targeted NGS is the ease to examine target genes and the opportunity to search for novel genes when targeted analysis is negative. Together with low cost, rapid and less laborious workflow, we conclude that WES complemented with SS can provide a tiered approach to molecular diagnostics of PXE.

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Percentage (%) of exons covered in four genes using ⩾5 and ⩾20 depth as a filter.   
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fig3: Percentage (%) of exons covered in four genes using ⩾5 and ⩾20 depth as a filter.   

Mentions: In order to identify the disease-causing mutations in all four genes, we analyzed all the exons that were 100% covered with ⩾1 and ⩾5 reads (Figures 1 and 2) and found that 93, 84, 84, and 33% of the exons for ABCC6, GGCX, ENPP1, and VKORC1, respectively, met this criteria for the overall sequencing depth of ⩾5 reads (Figure 2). Our further strategy was to investigate all exons with a depth lower compared with 20 by SS. Previously, we demonstrated that variants with a coverage <20 might be missed in 0.1% of the cases (Leeneer et al., 2011; Figure 3 and Supplementary Tables S5—S8 online). The latter analysis was performed for patients with only a single mutation (Table 2). For patients 1 and 2, coverage was poor for all four genes, which are in correlation with the poor overall sequencing depth of their exomes.


Efficiency of exome sequencing for the molecular diagnosis of pseudoxanthoma elasticum.

Hosen MJ, Van Nieuwerburgh F, Steyaert W, Deforce D, Martin L, Leftheriotis G, De Paepe A, Coucke PJ, Vanakker OM - J. Invest. Dermatol. (2014)

Percentage (%) of exons covered in four genes using ⩾5 and ⩾20 depth as a filter.   
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4378258&req=5

fig3: Percentage (%) of exons covered in four genes using ⩾5 and ⩾20 depth as a filter.   
Mentions: In order to identify the disease-causing mutations in all four genes, we analyzed all the exons that were 100% covered with ⩾1 and ⩾5 reads (Figures 1 and 2) and found that 93, 84, 84, and 33% of the exons for ABCC6, GGCX, ENPP1, and VKORC1, respectively, met this criteria for the overall sequencing depth of ⩾5 reads (Figure 2). Our further strategy was to investigate all exons with a depth lower compared with 20 by SS. Previously, we demonstrated that variants with a coverage <20 might be missed in 0.1% of the cases (Leeneer et al., 2011; Figure 3 and Supplementary Tables S5—S8 online). The latter analysis was performed for patients with only a single mutation (Table 2). For patients 1 and 2, coverage was poor for all four genes, which are in correlation with the poor overall sequencing depth of their exomes.

Bottom Line: Exomes with insufficient reads (⩽20 depth) for the four genes and patients with single mutations were further evaluated by Sanger sequencing (SS), but no additional mutations were found.The potential of WES compared with targeted NGS is the ease to examine target genes and the opportunity to search for novel genes when targeted analysis is negative.Together with low cost, rapid and less laborious workflow, we conclude that WES complemented with SS can provide a tiered approach to molecular diagnostics of PXE.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium [2] Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet, Bangladesh.

ABSTRACT
The molecular etiology of pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disorder, has become increasingly complex as not only mutations in ATP-binding cassette family C member 6 (ABCC6) but also ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and gamma-glutamyl carboxylase (GGCX) can cause resembling phenotypes. Identification of modifier genes, such as vascular endothelial growth factor A, has further contributed to the molecular heterogeneity of PXE. In such heterogeneous diseases, next-generation sequencing (NGS) allows to perform mutation screening of several genes in a single reaction. We explored whole-exome sequencing (WES) as an efficient diagnostic tool to identify the causal mutations in ABCC6, GGCX, ENPP1, and vitamin K epoxide reductase complex, subunit 1 (VKORC1) in 16 PXE patients. WES identified a causal ABCC6 mutation in 30 out of 32 alleles and one GGCX mutation, whereas no causal mutations in ENPP1 or VKORC1 were detected. Exomes with insufficient reads (⩽20 depth) for the four genes and patients with single mutations were further evaluated by Sanger sequencing (SS), but no additional mutations were found. The potential of WES compared with targeted NGS is the ease to examine target genes and the opportunity to search for novel genes when targeted analysis is negative. Together with low cost, rapid and less laborious workflow, we conclude that WES complemented with SS can provide a tiered approach to molecular diagnostics of PXE.

Show MeSH
Related in: MedlinePlus