Background: Familial ligand-defective apolipoprotein B-100 (FDB) is characterized by elevated plasma concentrations of LDL-cholesterol and apolipoprotein (apo) B, normal triglyceride and HDL-cholesterol levels, the presence of tendon xanthomas, and premature coronary artery disease. FDB cannot be clinically distinguished from heterozygous LDL-receptor-defective familial hypercholesterolaemia (FH) without genetic testing.
Methods: Amplicons in exon 26 and exon 29 of the APOB gene were screened for established genetic variants including mutations and polymorphisms using high-resolution melting analysis. Six novel variants associated with FDB in hypercholesterolaemic Dutch patients (S3476L, S3488G, Y3533C, T3540M, I4350T, G4368D) were also studied.
Results: All positive controls, a total of 10 mutations in exon 26 and four mutations in exon 29, were readily detectable by melting curve analysis. In addition, a patient previously not known to be heterozygous for the H3543Y mutation was identified in a screen of hypercholesterolaemic subjects. The method was validated by comparison of high-resolution melting analysis with DNA sequence data in a 'blinded' manner in 35 consecutive patients attending a lipid disorders clinic. These patients were classified as 'definite FH' by the Dutch Lipid Clinic Network criteria. Five patients were found to be heterozygous for the R3500Q and one for H3543Y.
Conclusions: We have established a novel, robust method of FDB mutation detection using high-resolution melting analysis in conjunction with DNA sequencing. Compared with existing methods it is not only more cost-effective, but is also capable of detecting new sequence changes and will have importance in cascade screening of affected subjects.