Use of targeted exome sequencing as a diagnostic tool for Familial Hypercholesterolaemia.
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.
Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London WC1E 6JF, UK.
Marta
M
Futema
Vincent
V
Plagnol
Ros A
RA
Whittall
H Andrew W
HA
Neil
Steve Eric
SE
Humphries
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D001055:Apolipoproteins B / Q000235:genetics; D019295:Computational Biology / Q000379:methods; D056915:DNA Copy Number Variations; D059472:Exome; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000175:diagnosis* / Q000235:genetics*; D009154:Mutation; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D001055:Apolipoproteins B; C469745:LDLRAP1 protein, human; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases