Validation of LDLr Activity as a Tool to Improve Genetic Diagnosis of Familial Hypercholesterolemia: A Retrospective on Functional Characterization of LDLr Variants

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by high blood-cholesterol levels mostly caused by mutations in the low-density lipoprotein receptor (LDLr). With a prevalence as high as 1/200 in some populations, genetic screening for pathogenic LDLr mutations is a cost-effective approach in families classified as ‘definite’ or ‘probable’ FH and can help to early diagnosis. However, with over 2000 LDLr variants identified, distinguishing pathogenic mutations from benign mutations is a long-standing challenge in the field. In 1998, the World Health Organization (WHO) highlighted the importance of improving the diagnosis and prognosis of FH patients thus, identifying LDLr pathogenic variants is a longstanding challenge to provide an accurate genetic diagnosis and personalized treatments. In recent years, accessible methodologies have been developed to assess LDLr activity in vitro, providing experimental reproducibility between laboratories all over the world that ensures rigorous analysis of all functional studies. In this review we present a broad spectrum of functionally characterized missense LDLr variants identified in patients with FH, which is mandatory for a definite diagnosis of FH.

Widening the spectrum of genetic testing in familial hypercholesterolaemia: Will it translate into better patient and population outcomes?

“…Traditionally, the uptake and degradation of 125 I‐labelled LDL in cultured skin fibroblasts was measured . A variety of methods is now used, including flow cytometry of transfected Chinese hamster ovary cells using fluorescent‐labelled LDL to determine receptor activity, with immunofluorescence detected by confocal laser scanning microscopy to assess LDL receptor expression and localisation within the cell; and mRNA studies …”

“…This is inferred from the type of sequence variant, population frequency, online prediction tools, and family studies. Truncating variants including nonsense, out‐of‐frame indels, splicing variants and deletions may not require functional confirmation, unless quantification of the degree of loss‐of‐function is required; the effects of nonsynonymous missense and other non‐truncating variants, which constitute the majority, are harder to determine from sequencing alone …”

“…The shortcomings of the available tools for determining pathogenicity are increasingly being recognised . Online prediction tools are imperfect.…”

“…Barriers to widespread functional testing include cost, complexity, and accessibility. However, there is evidence of a renewed interest in functional studies, which is likely to address some of the uncertainty gap created by increased genetic testing in patients with suspected FH …”

“…12 A variety of methods is now used, including flow cytometry of transfected Chinese hamster ovary cells using fluorescent-labelled LDL to determine receptor activity, with immunofluorescence detected by confocal laser scanning microscopy to assess LDL receptor expression and localisation within the cell; and mRNA studies. [13][14][15][16] The reality of the contemporary investigation of FH has been an explosion in the detection of LDLR variants, many of which are labelled as causative, but functional assays of LDL receptor activity, representing the gold-standard confirmation of pathogenicity, are rarely performed. Generally, the pathogenicity of a new variant is assessed by a number of means, supported by guidelines from the American College of Medical Genetics and Genomics (ACMG).…”