Genetic testing for familial hypercholesterolemia-past, present, and future

Abstract

In the early 1980s, the Nobel Prize winning cellular and molecular work of Mike Brown and Joe Goldstein led to the identification of the LDL receptor gene as the first gene where mutations cause the familial hypercholesterolemia (FH) phenotype. We now know that autosomal dominant monogenic FH can be caused by pathogenic variants of three additional genes (APOB/PCSK9/APOE) and that the plasma LDL-C concentration and risk of premature coronary heart disease differs according to the specific locus and associated molecular cause. It is now possible to use next-generation sequencing to sequence all exons of all four genes, processing 96 patient samples in one sequencing run, increasing the speed of test results, and reducing costs. This has resulted in the identification of not only many novel FH-causing variants but also some variants of unknown significance, which require further evidence to classify as pathogenic or benign. The identification of the FH-causing variant in an index case can be used as an unambiguous and rapid test for other family members. An FH-causing variant can be found in 20-40% of patients with the FH phenotype, and we now appreciate that in the majority of patients without a monogenic cause, a polygenic etiology for their phenotype is highly likely. Compared with those with a monogenic cause, these patients have significantly lower risk of future coronary heart disease. The use of these molecular genetic diagnostic methods in the characterization of FH is a prime example of the utility of precision or personalized medicine.

Keywords: LDL-C; LDLR; SNP score; clinical utility; coronary heart disease; index case; monogenic; next-generation sequencing; polygenic; variants of unknown significance.

Fig. 1

The first DNA diagnosis in relatives of an FH index case (data from Humphries et al. (10)). A: Southern blot using a radioactive probe for human LDLR, hybridized to a Southern blot filter of a digestion with the restriction enzyme PvuII of three samples of human DNA, showing the RFLP. The detection of an RFLP is due to a sequence change altering the restriction site and usually occurs in flanking regions or introns of the gene and is not usually itself the FH-causing variant. Identifying a potentially useful RFLP was a time-consuming and rate-limiting step in this methodology. The common allele designated V1 contains a cutting site for PvuII and is seen as a band of 14 kb. The rare allele V2 lacks the cutting site and is seen as a band of 16.5 kb. Individuals shown are homozygous for the V1 allele (genotype V1V1), homozygous for the V2 allele (genotype V2V2), and heterozygous for both alleles (genotype V1V2). B: Segregation of the PvuII RFLP in family S. The proband (deceased from an early myocardial infarction) is indicated by an arrow. It can be deduced from the genotypes of his children that this individual must have had the genotype V1V2, as do both his older sister and younger brother. From inspection of the child of the older sister, in this family, the FH causing variant is inherited with the V1 allele, since the child has inherited the V1 allele from his Fh mother and has the FH phenotype. This is confirmed in the children of the proband, where those who have inherited the V2 allele from the deceased father do not have FH, whereas the child who inherited the V1 allele has FH. In the children of the youngest brother, all have inherited the V1 allele and can all be predicted to have FH.

Fig. 2

Box and whisker plot of untreated TC before and after statin treatment and odds ratio for CHD by FH-causing gene (data from Huijgen et al. (46)). Boxes show median and interquartile range, with 95% range shown by bars. Outliers (more than 1.5 times the interquartile range from the edge of the box) are shown as dots. ∗ indicate P < 0.005 versus none, values from two-tailed unpaired Student's t-tests.

Fig. 3

Distribution of ClinVar benign, VUS, pathogenic, and unclear variants in LDLR, APOB, and PCSK9 (data from Iacocca et al. (4)).

Fig. 4

Flowchart showing the risk stratification clinical management for adding the 12-SNP polygenic risk score to the NGS strategy used in Diagnostic Laboratories.