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BACKGROUNDA key objective of the UK National Institute for Health and Care Excellence (NICE) pathway for diagnosis of familial hypercholesterolemia (FH) is the identification of affected relatives of index cases through cascade screening. At present, there is no systematic appraisal of available methodological options to identify the appropriate diagnostic testing protocol that would allow cost-effective cascade genetic screening. The majority of FH-causing mutations identified in the LDL receptor (LDLR) or apolipoprotein B (APOB) genes are single-nucleotide changes. This pattern of mutations suggests that PCR methods using melting curve-based genotyping might offer a convenient methodological approach for screening relatives.METHODSWe developed and validated one-tube PCR methods for the mutations APOB c.10580G>A (p.Arg3527Gln), LDLR c.1474G>A (p.Asp492Asn), and c.2054C>T (p.Pro685Leu) and 3 novel LDLR mutations identified in the Coventry and Warwickshire population: LDLR c.1567G>C (p.Val523Leu), c.487dupC (p.Gln163Profs17), and c.647G>C (p.Cys216Ser).RESULTSThese methods successfully amplified target sequence from genomic DNA extracted from either peripheral blood or saliva. They also demonstrated acceptable analytical performance characteristics (specificity of amplification, repeatability, and reproducibility) over a wide range of DNA concentrations and purity. This approach was used for cascade testing of relatives of index FH cases with confirmed mutations and identified family members with high plasma LDL cholesterol as heterozygous for disruptive alleles.CONCLUSIONSOur study generates proof-of-concept evidence of methods suitable for detecting single nucleotide substitutions and insertions that can deliver reliable, easy, low-cost, and rapid family screening of FH patients and can be adopted by nonspecialist molecular diagnostic laboratories.

the journal of applied laboratory medicine

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