Genetic diagnosis of familial hypercholesterolaemia by targeted next‐generation sequencing

Abstract

Maglio C., Mancina R. M., Motta B. M., Stef M., Pirazzi C., Palacios L., Askaryar N., Borén J., Wiklund O., Romeo S. (University of Gothenburg, Gothenburg, Sweden; University Magna Graecia of Catanzaro, Italy; University of Milan, Italy; Progenika Biopharma SA, Derio, Spain). Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing.ObjectivesThe aim of this study was to combine clinical criteria and next-generation sequencing (pyrosequencing) to establish a diagnosis of familial hypercholesterolaemia (FH).Design, setting and subjectsA total of 77 subjects with a Dutch Lipid Clinic Network score of ≥3 (possible, probable or definite FH clinical diagnosis) were recruited from the Lipid Clinic at Sahlgrenska Hospital, Gothenburg, Sweden. Next-generation sequencing was performed in all subjects using SEQPRO LIPO RS, a kit that detects mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLR adapter protein 1 (LDLRAP1) genes; copy-number variations in the LDLR gene were also examined.ResultsA total of 26 mutations were detected in 50 subjects (65% success rate). Amongst these, 23 mutations were in the LDLR gene, two in the APOB gene and one in the PCSK9 gene. Four mutations with unknown pathogenicity were detected in LDLR. Of these, three mutations (Gly505Asp, Ile585Thr and Gln660Arg) have been previously reported in subjects with FH, but their pathogenicity has not been proved. The fourth, a mutation in LDLR affecting a splicing site (exon 6–intron 6) has not previously been reported; it was found to segregate with high cholesterol levels in the family of the proband.ConclusionsUsing a combination of clinical criteria and targeted next-generation sequencing, we have achieved FH diagnosis with a high success rate. Furthermore, we identified a new splicing-site mutation in the LDLR gene.

Homozygous Familial Hypercholesterolemia in Spain

“…23,24 These genetic-based studies have been conducted almost exclusively in 4 research centers, with >16 000 genetic studies performed in patients with clinical suspicion of FH.…”

“…4,25 From 2012 to 2015, molecular diagnosis was performed using next-generation sequencing of the LDLR promoter, exons, and intron-exon boundaries; the APOB LDLR-binding domain; the PCSK9 promoter, exons, and intron-exon boundaries; and the LDLRAP1 promoter, exons, and intron-exon boundaries. 24 From all analyzed studies, we selected the patients carrying at least 2 mutations within the 3 genes responsible for FH or in LDLRAP1, as previously described.…”

Cascade Screening for Familial Hypercholesterolemia: PCR Methods with Melting-Curve Genotyping for the Targeted Molecular Detection of Apolipoprotein B and LDL Receptor Gene Mutations to Identify Affected Relatives

“…The wide spectrum of FHassociated mutations identified in the LDLR gene, which harbors the majority of disease-causing mutations, requires molecular methods suitable for comprehensive scanning of the nucleotide sequence of the candidate genes (19,20). Next-generation sequencing as a comprehensive genetic analysis method has demonstrated high levels of specificity and sensitivity (21)(22)(23). However, a significant drawback is the recognition that sequencing 4 genes is insufficient, and approximately 15% of people with FH do not have a mutation in LDLR, APOB, or PCSK9 genes (though estimates range from 12% to 48%) (24).…”

Clinical Management of Familial Hypercholesterolemia: New Insights from International Guidelines and Recent Studies

“…19 Costs may fall with the introduction of next generation sequencing techniques. 20 However, mutations cannot be found in the LDLR, apoB or PCSK-9 genes in all patients with an autosomal dominant pattern of inheritance of severe hypercholesterolemia. Even in patients with homozygous FH (HoFH) 25% can lack a definitive double allele pattern.…”