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Use of targeted exome sequencing as a diagnostic tool for Familial Hypercholesterolaemia.

Futema M, Plagnol V, Whittall RA, Neil HA, Simon Broome Register GroupHumphries SE, UK1 - J. Med. Genet. (2012)

Bottom Line: Exome sequencing detected 17 LDLR mutations, including three copy number variants, two APOB mutations, missed by the standard techniques, two LDLR novel variants likely to be FH-causing, and five APOB variants of uncertain effect.One heterozygous mutation was found in LDLRAP1.However, the poor coverage of gene promoters and repetitive, or GC-rich sequences, remains problematic, and validation of all identified variants is currently required.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London WC1E 6JF, UK.

ABSTRACT

Background: Familial Hypercholesterolaemia (FH) is an autosomal dominant disease, caused by mutations in LDLR, APOB or PCSK9, which results in high levels of LDL-cholesterol (LDL-C) leading to early coronary heart disease. An autosomal recessive form of FH is also known, due to homozygous mutations in LDLRAP1. This study assessed the utility of an exome capture method and deep sequencing in FH diagnosis.

Methods: Exomes of 48 definite FH patients, with no mutation detected by current methods, were captured by Agilent Human All Exon 50Mb assay and sequenced on the Illumina HiSeq 2000 platform. Variants were called by GATK and SAMtools.

Results: The mean coverage of FH genes varied considerably (PCSK9=23x, LDLRAP1=36x, LDLR=56x and APOB=93x). Exome sequencing detected 17 LDLR mutations, including three copy number variants, two APOB mutations, missed by the standard techniques, two LDLR novel variants likely to be FH-causing, and five APOB variants of uncertain effect. Two variants called in PCSK9 were not confirmed by Sanger sequencing. One heterozygous mutation was found in LDLRAP1.

Conclusions: High-throughput DNA sequencing demonstrated its efficiency in well-covered DNA regions, in particular LDLR. This highly automated technology is proving to be effective for heterogeneous diseases and may soon replace laborious conventional methods. However, the poor coverage of gene promoters and repetitive, or GC-rich sequences, remains problematic, and validation of all identified variants is currently required.

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Related in: MedlinePlus

Copy Number Variants (CNVs) in LDLR gene. A: heterozygous duplication of exons 3–8. B: heterozygous deletion of exons 11 and 12. C: heterozygous duplication of exons 13–15. All identified by ExomeDepth in the exome sequencing data. The crosses show the ratio of observed/expected number of reads for the test sample. The grey shaded region shows the estimated 99% CI for this observed ratio in the absence of CNV call. The presence of contiguous exons with read count ratio located outside of the CI is indicative of a heterozygous deletion or duplication in a sample. Exons 1 and 18 were excluded from the analysis (not shown on the graph) as they did not reach the threshold of 100 for the total number of reads. All CNVs were confirmed by MLPA experiment (see online supplementary figure S3).
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JMEDGENET2012101189F2: Copy Number Variants (CNVs) in LDLR gene. A: heterozygous duplication of exons 3–8. B: heterozygous deletion of exons 11 and 12. C: heterozygous duplication of exons 13–15. All identified by ExomeDepth in the exome sequencing data. The crosses show the ratio of observed/expected number of reads for the test sample. The grey shaded region shows the estimated 99% CI for this observed ratio in the absence of CNV call. The presence of contiguous exons with read count ratio located outside of the CI is indicative of a heterozygous deletion or duplication in a sample. Exons 1 and 18 were excluded from the analysis (not shown on the graph) as they did not reach the threshold of 100 for the total number of reads. All CNVs were confirmed by MLPA experiment (see online supplementary figure S3).

Mentions: CNV calling identified one deletion of exons 11 and 12 (c. 1587−?_1845+?del), and two duplications of exons 3–8 (191−?_1186+?dup), and exons 13–15 (c.1846−?_2311+?dup), as shown in figure 2. All CNVs were confirmed by MLPA (see online supplementary figure S3).


Use of targeted exome sequencing as a diagnostic tool for Familial Hypercholesterolaemia.

Futema M, Plagnol V, Whittall RA, Neil HA, Simon Broome Register GroupHumphries SE, UK1 - J. Med. Genet. (2012)

Copy Number Variants (CNVs) in LDLR gene. A: heterozygous duplication of exons 3–8. B: heterozygous deletion of exons 11 and 12. C: heterozygous duplication of exons 13–15. All identified by ExomeDepth in the exome sequencing data. The crosses show the ratio of observed/expected number of reads for the test sample. The grey shaded region shows the estimated 99% CI for this observed ratio in the absence of CNV call. The presence of contiguous exons with read count ratio located outside of the CI is indicative of a heterozygous deletion or duplication in a sample. Exons 1 and 18 were excluded from the analysis (not shown on the graph) as they did not reach the threshold of 100 for the total number of reads. All CNVs were confirmed by MLPA experiment (see online supplementary figure S3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3475071&req=5

JMEDGENET2012101189F2: Copy Number Variants (CNVs) in LDLR gene. A: heterozygous duplication of exons 3–8. B: heterozygous deletion of exons 11 and 12. C: heterozygous duplication of exons 13–15. All identified by ExomeDepth in the exome sequencing data. The crosses show the ratio of observed/expected number of reads for the test sample. The grey shaded region shows the estimated 99% CI for this observed ratio in the absence of CNV call. The presence of contiguous exons with read count ratio located outside of the CI is indicative of a heterozygous deletion or duplication in a sample. Exons 1 and 18 were excluded from the analysis (not shown on the graph) as they did not reach the threshold of 100 for the total number of reads. All CNVs were confirmed by MLPA experiment (see online supplementary figure S3).
Mentions: CNV calling identified one deletion of exons 11 and 12 (c. 1587−?_1845+?del), and two duplications of exons 3–8 (191−?_1186+?dup), and exons 13–15 (c.1846−?_2311+?dup), as shown in figure 2. All CNVs were confirmed by MLPA (see online supplementary figure S3).

Bottom Line: Exome sequencing detected 17 LDLR mutations, including three copy number variants, two APOB mutations, missed by the standard techniques, two LDLR novel variants likely to be FH-causing, and five APOB variants of uncertain effect.One heterozygous mutation was found in LDLRAP1.However, the poor coverage of gene promoters and repetitive, or GC-rich sequences, remains problematic, and validation of all identified variants is currently required.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London WC1E 6JF, UK.

ABSTRACT

Background: Familial Hypercholesterolaemia (FH) is an autosomal dominant disease, caused by mutations in LDLR, APOB or PCSK9, which results in high levels of LDL-cholesterol (LDL-C) leading to early coronary heart disease. An autosomal recessive form of FH is also known, due to homozygous mutations in LDLRAP1. This study assessed the utility of an exome capture method and deep sequencing in FH diagnosis.

Methods: Exomes of 48 definite FH patients, with no mutation detected by current methods, were captured by Agilent Human All Exon 50Mb assay and sequenced on the Illumina HiSeq 2000 platform. Variants were called by GATK and SAMtools.

Results: The mean coverage of FH genes varied considerably (PCSK9=23x, LDLRAP1=36x, LDLR=56x and APOB=93x). Exome sequencing detected 17 LDLR mutations, including three copy number variants, two APOB mutations, missed by the standard techniques, two LDLR novel variants likely to be FH-causing, and five APOB variants of uncertain effect. Two variants called in PCSK9 were not confirmed by Sanger sequencing. One heterozygous mutation was found in LDLRAP1.

Conclusions: High-throughput DNA sequencing demonstrated its efficiency in well-covered DNA regions, in particular LDLR. This highly automated technology is proving to be effective for heterogeneous diseases and may soon replace laborious conventional methods. However, the poor coverage of gene promoters and repetitive, or GC-rich sequences, remains problematic, and validation of all identified variants is currently required.

Show MeSH
Related in: MedlinePlus