Novel functional APOB mutations outside LDL-binding region causing familial hypercholesterolaemia

Abstract

Familial hypercholesterolaemia (FH) is characterized by increased circulating low-density lipoprotein (LDL) cholesterol leading to premature atherosclerosis and coronary heart disease. Although FH is usually caused by mutations in LDLR, mutations in APOB and PCSK9 also cause FH but only a few mutations have been reported, APOB p.R3527Q being the most common. However, 30-80% of clinical FH patients do not present an identifiable mutation in any of the described genes. To identify the genetic cause of the hypercholesterolaemia in 65 patients without mutations in LDLR, PCSK9 or in fragments of exon 26 and 29 of APOB currently analysed, we performed whole sequencing of APOB by pyrosequencing. A total of 10 putative mutations in APOB were identified. Flow cytometry with fluorescently labelled LDL from patients and relatives showed that p.Arg1164Thr (exon 22) and p.Gln4494del (exon 29) presented a 40% decrease in internalization in lymphocytes and HepG2 cells, very similar to APOB3527. The proliferation assays with U937 cells showed reduced growth for both cases. The variant p.Tyr1247Cys was found to be neutral and other three alterations were considered polymorphisms. Our results emphasize the need to study the whole APOB in routine protocols to improve patient identification and cardiovascular risk assessment.

Mutational analysis of a cohort with clinical diagnosis of familial hypercholesterolemia: considerations for genetic diagnosis improvement

“…Phase II includes the study of large rearrangements by multiplex ligation-dependent probe amplification (MLPA) technique. 12 Phase III includes the study of five exons (1,2,4,7,9) and flanking regions of PCSK9, where the putative PCSK9 mutations have been described, and, in severely affected patients, the whole study of this gene. Phase IV comprises the study of promoter, all exons, and flanking regions of APOB in selected patients.…”

“…Phase IV comprises the study of promoter, all exons, and flanking regions of APOB in selected patients. 7 Phase V comprises the functional in vitro studies. Phases I and II are always executed for all patients, and phases III and IV are performed only if no putative mutation is detected in the previous phases.…”

“…27 In APOB, four have been shown to be present in more than 1% of the normolipidemic subjects panel and, for this reason, are considered to be a polymorphism. 7 In PCSK9, one variant has been described as non-pathogenic. 28 The remaining 7/55 (Supplementary Table S2e …”

“…1 More than 1,600 mutations associated with FH have been described globally in LDLR. 4 Mutations in APOB [5][6][7] or PCSK9 genes 8 are also associated with FH, but they are less frequent in FH patients (APOB <8% and PCSK9 <3%), 9 although the number of functional APOB mutations has been increasing in the past 2 years. 6,7 Because there is a genetic diagnosis and appropriate treatment to reduce the elevated cardiovascular risk of these patients, the World Health Organization has, since 1998, recommended universal screening for FH.…”

“…4 Mutations in APOB [5][6][7] or PCSK9 genes 8 are also associated with FH, but they are less frequent in FH patients (APOB <8% and PCSK9 <3%), 9 although the number of functional APOB mutations has been increasing in the past 2 years. 6,7 Because there is a genetic diagnosis and appropriate treatment to reduce the elevated cardiovascular risk of these patients, the World Health Organization has, since 1998, recommended universal screening for FH. 10 Based on this recommendation, the Portuguese FH Study was established in 1999 at the National Institute of Health, having implemented the molecular study of this pathology to promote early identification and characterization of FH patients and therefore to decrease their cardiovascular risk, through the implementation of early and correct counseling/treatment.…”