The Low-Density Lipoprotein Receptor
The
LDLR gene was the first gene found where mutations cause FH. It spans 45 kb (kilobases) on the short arm of chromosome 19 and comprises 18 exons that are transcribed and translated into five distinct domains which form the cell surface LDL-receptor [
5]. Any defect in the
LDLR gene can cause loss of function of LDL-receptors resulting in reduced LDL-C uptake from blood and cause FH. In mutation carriers, blood cholesterol level is usually raised two fold above the normal level. In the homozygous form, where two identical mutations have been inherited, one from each parent (usually from a consanguineous marriage), or compound heterozygous FH, where two different mutations on both alleles have been inherited, the cholesterol levels are four or five times greater than those of the heterozygous cases [
6].
FH-causing mutations in the
LDLR gene are found along the entire length of the gene. There are more than 2900 different variants identified in the
LDLR gene with majority of them being exonic substitutions and small (<100 bp) or large rearrangements (>100 bp) [
7••]. More than 90% of the reported variants are likely to be disease causing [
8]. Most FH cohort studies showed that among the variants found, a large proportion cluster in exon 4 [
9]. This could be due to the large size of exon 4 or to the highly detrimental effect of variants in this exon which encodes the ligand-binding domain, on the gene function compared to variants in other exons. Patients with a mutation in exon 4 might present with more severe FH in the clinics. In contrast, the mutation frequency in exons 15 and 16 is extremely low. The spectrum of FH mutations varies between countries; from Greece, where a relatively small numbers of mutations account for the majority of FH cases, to the Netherlands where the mutation spectrum was found to be extensive [
10]. The cause of FH in the UK is highly heterogeneous with over 200 different mutations reported [
11,
12] The information regarding molecular diagnosis of FH in some parts of the world such as Latin America and South Asia are scant. In Brazil and Mexico, the countries with the largest cohorts in Latin America, only few
LDLR mutations have been found that have been encountered in the European population previously [
13].
Predicting whether novel variants in
LDLR are pathogenic or not is not always straightforward, especially for synonymous and missense variants. In 2013, the Association for Clinical Genetic Science (ACGS) published guidelines for the classification of variants, with categories ranging from 1 and 2 (clearly not or unlikely to be pathogenic), to 3 (variants of unknown significance), to 4 and 5 (likely to be or clearly pathogenic). The recently updated
LDLR variant database with variants classified according to these guidelines may be accessed via:
http://databases.lovd.nl/shared/genes/LDLR [
7••]. All 128 nonsense substitutions, 336 small frame-shifting rearrangements and 116/117 large rearrangements were considered to be pathogenic (classes 4 and 5). Of the 795 missense variants analysed, 115were in classes 1 and 2, 605 in class 4 and 75 in class 3. One hundred eleven of the 180 intronic variants, 4 of 34 synonymous variants and 14 of 37 reported promoter variants were predicted to be likely or clearly pathogenic (classes 4 and 5). It is clearly of great importance to be able to assess whether variants identified in clinical settings or as incidental findings in genomics projects are pathogenic or not. Although 93% (1588) of
LDLR variants in the current upgrade of the database have been assigned to an ACGS pathogenicity category, 7% (115) remain as variants of unknown significance. It is hoped that as more information becomes available from in vitro functional studies, the development of additional in silico tools and from the various genomics studies, it will be possible to determine the pathogenicity of these variants, and indeed the classification of some variants may also change as our knowledge increases. The ‘gold standard’ test for pathogenicity of a variant is to carry out co-segregation studies, where the co-inheritance of the variant with elevated LDL-C levels is seen in many relatives in a family, while the relatives without the inherited variant have normal levels of LDL-C. The interpretation of family data may be complicated by the overlay of environmental factors that influence lipid levels and by the presence in the family of other genetic variants that raise or lower LDL-C.
Apolipoprotein B
Apolipoprotein B (apoB) is the major apolipoprotein on lipoprotein molecules, especially LDL-C, and functions as a ligand to the LDL-receptor. The gene is located on chromosome 2p and spans more than 43 kb. The gene comprises 29 exons and is transcribed and translated into a protein of 4563 amino acids [
14]. While truncation mutations in the
APOB gene cause hypobetalipoproteinemia, mutations causing hypercholesterolaemia are due to missense mutations that result in ligand-defective apoB protein. The LDL-C particles made from this allele are therefore not able to bind to the LDL-receptor and thus accumulate in the blood [
15]. A single mutation of the
APOB gene (p.Arg3527Gln) accounts for approximately 6–10% of all FH cases in European population, and it is located in exon 26 of
APOB gene [
16]. Other
APOB mutations in other regions of the gene such as p.Arg50Trp, p.Arg1164Thr and p.Gln4494del were also recently found to cause FH [
17,
18•]. For other variants, for example for p.Arg3531Cys, which was detected in a patient with a clinical diagnosis of FH, while initial reports showed that LDL-C from the patient had reduced binding to the LDL-receptor, later co-segregation studies found that there was no clear co-segregation [
19]. This variant is now considered to be a ‘susceptibility’ variant that raises the likelihood of hypercholesterolaemia in a carrier but does not itself cause frank FH.
Proprotein Convertase Subtilisin/Kexin Type 9
The
PCSK9 (proprotein convertase subtilisin/kexin type 9) gene encodes an enzyme that is involved in regulating the degradation of the LDL-receptor protein in the lysosome of the cell, preventing it from being recycled to the cell surface. The gene is found on chromosome 1p and comprises 12 exons, covering 39 kb [
20]. The PCSK9 molecule is synthesised as an inactive proprotein and undergoes cleavage in the endoplasmic reticulum to produce an enzyme with the prodomain noncovalently bound to the catalytic site, preventing further enzyme action. PCSK9 is secreted mostly from the liver and its binding to the LDL-receptor directs the receptor to the lysosome for degradation [
21].
Mutations in the
PCSK9 gene that cause FH are gain-of-function mutations that increase LDL-receptor degradation and consequently reduce the number of receptors on the cell surface. Although more than 20 such variants have been reported world-wide, the only common
PCSK9 variant in the UK is p.Asp374Tyr, which occurs in about 2% of the mutation-positive FH patients. This variant is associated with a raised cholesterol level and a high risk of developing premature coronary heart disease, compared with a mutation in the
LDLR gene [
22]. On the other hand, loss-of-function mutations that inactivate the PCSK9 protein lead to less degradation of the LDL-receptor [
23]. The most common of these variants, p.Arg46Leu, enhances the clearance of LDL-C from the plasma and lowers cholesterol level in the plasma. In European populations, approximately 3% of individuals are carriers of this variant, and because of their lifelong lower LDL-C levels, they have ∼28% lower CHD risk [
24].
Other Monogenic Causes of FH
A very rare autosomal recessive hypercholesterolaemia is caused by mutations in the low-density lipoprotein receptor adaptor protein 1 (
LDLRAP1) gene which encodes a cytosolic protein that interacts with the cytoplasmic tail of the LDL-receptor. Mutations in this gene that usually cause premature truncations of the protein lead to LDL-receptor malfunction and hypercholesterolaemia. This gene is located on the short arm of chromosome 1 [
25]. The LDL-C level in these cases is typically intermediate between homozygote and heterozygote autosomal dominant FH patients [
2].
Several studies have reported that a specific mutation (p.Leu167del) in
APOE gene causes autosomal dominant FH [
26]. This mutation has been previously reported to be associated with sea-blue histiocytosis and familial combined hyperlipidaemia (FCH) but overlap between the FCH and FH phenotype has been shown before as hypertriglyceridemia can be seen due to many common genetic and environmental factors [
27,
28].
Several studies have been conducted to identify new genes causing FH, using family studies and next-generation sequencing (NGS) and this has identified three genes
STAP1 (signal transducing adaptor protein family 1),
LIPA (lysosomal acid lipase) and
PNPLA5 (patatin-like phospholipase-domain-containing family) where mutations may be causing significantly elevated LDL-C and possibly the clinical phenotype of FH [
29•,
30,
31]. So far, for
STAP1 and
PNPLA5, these genes and variants in them have yet to be independently confirmed as FH-causing.