RESUMEN
Mutations in APOB are the second most frequent cause of familial hypercholesterolemia (FH). APOB is highly polymorphic, and many variants are benign or of uncertain significance, so functional analysis is necessary to ascertain their pathogenicity. Our aim was to identify and characterize APOB variants in patients with hypercholesterolemia. Index patients (n = 825) with clinically suspected FH were analyzed using next-generation sequencing. In total, 40% of the patients presented a variant in LDLR, APOB, PCSK9 or LDLRAP1, with 12% of the variants in APOB. These variants showed frequencies in the general population lower than 0.5% and were classified as damaging and/or probably damaging by 3 or more predictors of pathogenicity. The variants c.10030A>G;p.(Lys3344Glu) and c.11401T>A;p.(Ser3801Thr) were characterized. The p.(Lys3344Glu) variant co-segregated with high low-density lipoprotein (LDL)-cholesterol in 2 families studied. LDL isolated from apoB p.(Lys3344Glu) heterozygous patients showed reduced ability to compete with fluorescently-labelled LDL for cellular binding and uptake compared with control LDL and was markedly deficient in supporting U937 cell proliferation. LDL that was carrying apoB p.(Ser3801Thr) was not defective in competing with control LDL for cellular binding and uptake. We conclude that the apoB p.(Lys3344Glu) variant is defective in the interaction with the LDL receptor and is causative of FH, whereas the apoB p.(Ser3801Thr) variant is benign.
Asunto(s)
Hiperlipoproteinemia Tipo II , Proproteína Convertasa 9 , Humanos , Proproteína Convertasa 9/genética , Apolipoproteínas B/genética , LDL-Colesterol/genética , Células U937 , Hiperlipoproteinemia Tipo II/genéticaRESUMEN
BACKGROUND AMD AIMS: APOB mutations are a rare cause of familial hypercholesterolaemia (FH) and, until recently, routine genetic diagnosis only included the study of two small APOB fragments. In previous years, 5 novel functional mutations have been described in APOB fragments not routinely studied, our group having functionally characterized 2 of them. The main aim of this work was to identify and characterize novel alterations in APOB to assess the genetic cause of hypercholesterolemia in patients with a clinical diagnosis of FH. METHODS: We performed next generation sequencing of 48 Portuguese clinical FH patients, who were apparently mutation negative. All variants found in APOB were annotated. For functional studies, LDL from index patients and relatives was separated and marked with FITC-LDL for flow cytometry assays in lymphocytes and U937 growth assays. RESULTS: A total of 11 potential pathogenic variants were identified. Variants p.(Pro994Leu) and p.(Thr3826Met) in exons 19 and 26 were found in 4 patients, and in vitro analysis was performed for these variants. An exon 26 alteration (p.(Thr3826Met)) showed a decrease in binding and internalization of LDL, and in U937 growth assays that was similar to the effect with p.(Arg3527Gln). An alteration in exon 19 had a neutral effect. CONCLUSIONS: The spectrum of functional alterations in APOB outside the fragments routinely screened is slowly growing. Screening of all 29 exons of APOB is advised for FH routine diagnosis, but functional characterization is necessary for pathogenicity assessment. It is expected that the number of patients with functional APOB mutations will increase in the near future.