ClinVar Genomic variation as it relates to human health

The NM_000527.5(LDLR):c.2098G>A (p.Asp700Asn) variant is classified as Likely pathogenic for Familial Hypercholesterolemia by applying evidence codes PM2, PS3_Moderate, PS4_supporting and PP4 as defined by the ClinGen Familial Hypercholesterolemia Expert Panel LDLR-specific variant curation guidelines (https://doi.org/10.1016/j.gim.2021.09.012). The supporting evidence is as follows: PM2 - PopMax MAF = 0.00006199 (0.006199%) in European (non-Finnish) exomes+genomes (gnomAD v2.1.1), so PM2 is Met. PS3_Moderate - Level 2 assays: PMID 34869944: Heterologous cells (CHO), FACS assays - result - 20% uptake. ---- functional study is consistent with damaging effect, so PS3_Moderate is Met. PS4_supporting - Variant meets PM2 and is identified in 2 unrelated index cases: 1 index case with DLCN>=6 from Color Health, Inc, and 1 index case with Simon Broome criteria (TC of 9.8 mmol/L and personal or family history of CHD) from UK (PMID: 23669246), so PS4_Supporting is Met. PP4 - Variant meets PM2 and is identified in 2 unrelated index cases (see PS4 for details), so PP4 is Met.

Variant identified in a genome or exome case(s) and assessed due to predicted null impact of the variant or pathogenic assertions in the literature or databases. Disclaimer: This variant has not undergone full assessment. The following are preliminary notes: This variant has been reported in 2 publications: one describing a method for detecting variants in individuals heterozygous for LDLR variants (no phenotype information) and one where it was found in a control subject. The variant is classified in ClinVar as VUS by GeneDx and British Heart Foundation. The variant is present in ExAC with a Max MAF of 0.006% (4 European alleles).

Variant summary: LDLR c.2098G>A (p.Asp700Asn) results in a conservative amino acid change located in the EGF-like domain (IPR000742) of the encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 2e-05 in 251024 control chromosomes (gnomAD). The variant, c.2098G>A, has been reported in the literature in multiple individuals affected with (familial) hypercholesterolemia (e.g. Leren_2004, Laurie_2009, Futema_2013, Defesche_2017, Trinder_2020, Dron_2020), however, it was also found in controls (Do_2015, Narang_2020). These data indicate that the variant maybe associated with disease. A recent publication reported experimental evidence evaluating an impact on protein function, and demonstrated a decreased LDLR activity (~20% of normal) in an in vitro expression system (Larrea-Sebal_2021). Seven other submitters, including a variant curation expert panel (ClinGen), have provided clinical-significance assessments for this variant in ClinVar after 2014, and classified the variant as VUS (n=5), while ClinGen classified it as likely pathogenic. Based on the evidence outlined above, the variant was classified as likely pathogenic.

The frequency of this variant in the general population, 0.000062 (8/129062 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. In the published literature, the variant has been reported in individuals with familial hypercholesterolemia (PMIDs: 28964736 (2017), 23669246 (2013), 19118540 (2009), and 15199436 (2004)). In addition, a functional study showed significantly reduced low-density lipoprotein uptake in cells with this variant (PMID: 34869944 (2021)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, this variant is classified as likely pathogenic.

This sequence change replaces aspartic acid, which is acidic and polar, with asparagine, which is neutral and polar, at codon 700 of the LDLR protein (p.Asp700Asn). This variant is present in population databases (rs375009082, gnomAD 0.007%). This missense change has been observed in individuals with clinical features of familial hypercholesterolemia (PMID: 15199436, 19118540, 23669246; Invitae). This variant is also known as p.D679N. ClinVar contains an entry for this variant (Variation ID: 200923). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt LDLR protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects LDLR function (PMID: 34869944). This variant disrupts the p.Asn700 amino acid residue in LDLR. Other variant(s) that disrupt this residue have been observed in individuals with LDLR-related conditions (PMID: 22353362, 32015373, 34456049; Invitae), which suggests that this may be a clinically significant amino acid residue. For these reasons, this variant has been classified as Pathogenic.

This missense variant (also known as p.Asp679Asn in the mature protein) replaces aspartic acid with asparagine at codon 700 in the EGF precursor homology domain of the LDLR protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). Functional studies have shown that this variant causes a significant decrease in LDLR uptake (PMID: 34869944). This variant has been reported in individuals affected with familial hypercholesterolemia (PMID: 15199436, 23669246, 28008010; Color internal data). This variant has been identified in 8/282422 chromosomes in the general population by the Genome Aggregation Database (gnomAD). This variant is classified as likely pathogenic by the ClinGen Familial Hypercholesterolemia VCEP (ClinVar variation ID: 200923). Additionally, a different variant occurring at the same codon, p.Asp700Gly, is considered to be disease-causing (Clinvar variation ID: 252220), indicating that aspartic acid at this position is important for LDLR protein function. Based on the available evidence, this variant is classified as Likely Pathogenic.

The p.D700N variant (also known as c.2098G>A), located in coding exon 14 of the LDLR gene, results from a G to A substitution at nucleotide position 2098. The aspartic acid at codon 700 is replaced by asparagine, an amino acid with highly similar properties. This alteration (also referred to as p.D679N) has been detected in familial hypercholesterolemia cohorts; however, detail was limited (Leren TP et al. Semin Vasc Med, 2004;4:75-85; Futema M et al. Atherosclerosis, 2013;229:161-8; Defesche JC et al. J Clin Lipidol. 2017;11(6):1338-1346.e7). This variant has also been reported as a secondary finding in an exome cohort, and in a population-based cohort (Retterer K et al. Genet. Med. 2016;18:696-704; Abul-Husn NS. Science. 2016;354(6319)). Studies suggest this alteration may have an impact on protein function (Larrea-Sebal A et al. JACC Basic Transl Sci, 2021 Nov;6:815-827). Alterations affecting the same amino acid (p.D700E, c.2100C>G and p.D700G, c.2099A>G) have been reported in patients with familial hypercholesterolemia; although, one was noted to co-segregate with a second LDLR alteration (Cenarro A et al. Hum. Mutat., 1998;11:413; Garc&iacute;a-Garc&iacute;a AB et al. Hum. Mutat., 2001;18:458-9). This amino acid position is highly conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.