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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

The exonic coverage of FH genes: APOB, LDLR, LDLRAP1 and PCSK9 in a standard box plot (the minimum, lower quartile, median, upper quartile and maximum for each gene exon). The horizontal dashed line indicates the 16x coverage, when the probability of observing a rare allele at least 3 times is 99%. The additional Y-axis describes GC content (0.4=40%, 0.5=50%, etc) for a given exon, shown as rhombuses.
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JMEDGENET2012101189F1: The exonic coverage of FH genes: APOB, LDLR, LDLRAP1 and PCSK9 in a standard box plot (the minimum, lower quartile, median, upper quartile and maximum for each gene exon). The horizontal dashed line indicates the 16x coverage, when the probability of observing a rare allele at least 3 times is 99%. The additional Y-axis describes GC content (0.4=40%, 0.5=50%, etc) for a given exon, shown as rhombuses.

Mentions: The average read depth of LDLR exons varied from 136x for exon 12, to 4x for exon 18 (figure 1). Using a 16x coverage threshold, which would give a 99% probability of observing a rare allele at least three times, all except exons 1 and 18 showed adequate coverage. Exons 3 and 4 contain the largest number of reported FH-causing mutations,24 and both these exons were well covered (mean depth 92 and 57, respectively).


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)

The exonic coverage of FH genes: APOB, LDLR, LDLRAP1 and PCSK9 in a standard box plot (the minimum, lower quartile, median, upper quartile and maximum for each gene exon). The horizontal dashed line indicates the 16x coverage, when the probability of observing a rare allele at least 3 times is 99%. The additional Y-axis describes GC content (0.4=40%, 0.5=50%, etc) for a given exon, shown as rhombuses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

JMEDGENET2012101189F1: The exonic coverage of FH genes: APOB, LDLR, LDLRAP1 and PCSK9 in a standard box plot (the minimum, lower quartile, median, upper quartile and maximum for each gene exon). The horizontal dashed line indicates the 16x coverage, when the probability of observing a rare allele at least 3 times is 99%. The additional Y-axis describes GC content (0.4=40%, 0.5=50%, etc) for a given exon, shown as rhombuses.
Mentions: The average read depth of LDLR exons varied from 136x for exon 12, to 4x for exon 18 (figure 1). Using a 16x coverage threshold, which would give a 99% probability of observing a rare allele at least three times, all except exons 1 and 18 showed adequate coverage. Exons 3 and 4 contain the largest number of reported FH-causing mutations,24 and both these exons were well covered (mean depth 92 and 57, respectively).

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