ClinVar Genomic variation as it relates to human health

NM_000527.5(LDLR):c.682G>A (p.Glu228Lys) variant is classified as pathogenic for Familial Hypercholesterolemia by applying evidence code PS3, PS4, PM1, PM2, PP1_Strong, PP3 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 = 0.0001105 (0.01%) in East Asian exomes (gnomAD v2.1.1). PP3 - REVEL = 0.972. It is above 0.75. PM1 - Variant meets PM2 and is missense located in exon 4 . PS3 - Three studies contribute to PS3 attribution. One (PMID: 10978268) report a level 3 assay performed on heterozygous patient's fibroblasts with radiolabeled LDL consistent with damaging effect of the variant (50% LDLR activity). The second reports a level 2 assay perfomed on homozygous patient's fibroblasts with radiolabeled LDL consistent with damaging effect of the variant (< 2% LDLR activity).The third is permformed on Heterologous cells (COS-7). FACS, CLSM and WB results in 24% LDLR expression and 21% LDL clearance. LDLR is retained in the ER. PS4 - Variant meets PM2 and is identified in 13 index cases who fulfil SB criteria for FH (n=1 CGMC, UFGOD, APHP.Sorbonne Université, Hôpital de la Pitié-Salpêtrière; n=1 Cardiovascular Research Group,Instituto Nacional de Saude Doutor Ricardo Jorge; n=1 Molecular Genetics Laboratory (Centre for Cardiovascular Surgery and Transplantation)) or DLCN criteria for FH (n=1 Robarts Research Institute; n=9 CGMC, UFGOD, APHP.Sorbonne Université, Hôpital de la Pitié-Salpêtrière). PP1_Strong - Variant segregate with FH in 10 informatives meiosis (6 relatives positive LDL-C > 75th percentile and 4 relatives negative LDLC < 50th percentile) from 2 families from Laboratory of Genetics and Molecular Cardiology, University of São Paulo and in 1 relative positive for variant (LDL-C > 75th percentile) from Cardiovascular Research Group,Instituto Nacional de Saude Doutor Ricardo Jorge. PP4 - Variant meets PM2 and is identified in 13 index cases who fulfil SB criteria for FH (n=1 CGMC, UFGOD, APHP.Sorbonne Université, Hôpital de la Pitié-Salpêtrière; n=1 Cardiovascular Research Group,Instituto Nacional de Saude Doutor Ricardo Jorge; n=1 Molecular Genetics Laboratory (Centre for Cardiovascular Surgery and Transplantation)) or DLCN criteria for FH (n=1 Robarts Research Institute; n=9 CGMC, UFGOD, APHP.Sorbonne Université, Hôpital de la Pitié-Salpêtrière).

0/190 non-FH alleles; 0/100 Chinese normolipidemic individuals; 0/100 healthy control individuals

subjects mutated among 2600 FH index cases screened = 9 , family members = 3 with co-segregation / FH-Canada-3, < 2% LDLR Activity / Software predictions: Damaging

Disrupt SDE motif. SDE bind structural Ca2+.

Variant summary: LDLR c.682G>A (p.Glu228Lys) results in a conservative amino acid change located in the Low-density lipoprotein (LDL) receptor class A repeat of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 1.6e-05 in 244390 control chromosomes. c.682G>A has been reported in the literature in numerous individuals affected with Familial Hypercholesterolemia. These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function, showing the variant to result in impaired lipoprotein binding (Hobbs_1992). In addition, other variants at this codon have been reported as pathogenic (p.Glu228Gln, p.Glu228X). 12 clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This variant (also known as p.Glu207Lys in the mature protein and as FH Canadian-3, FH Modena, and FH Mexico-3) is a missense variant located in the fifth LDLR type A repeat of the ligand binding domain of the LDLR protein that forms a calcium binding pocket. Computational prediction tools and conservation analyses suggest that this variant may have deleterious impact on the protein function. Computational splicing tools suggest that this variant may not impact the RNA splicing. In experimental studies, the mutant protein has shown normal LDL binding affinity but reduced affinity for _x0001_beta-VLDL (PMID: 18677035) and delayed processing to the mature form (PMID: 2318961). This has been identified in numerous individuals diagnosed with familial hypercholesterolemia (PMID: 2318961, 15359125, 21475731, 21722902, 22390909, 23375686). This variant has been identified in 4/247898 chromosomes by the general population by the Genome Aggregation Database (gnomAD). Based on available evidence, this variant is classified as Likely Pathogenic.

Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Published functional studies suggest a damaging effect with reduced mature protein, reduced cell surface localization, and reduced ligand binding (Chang et al., 2003); Also known as Glu207Lys, FH French Canadian-3, FH Mexico; This variant is associated with the following publications: (PMID: 2318961, 28965616, 22390909, 21475731, 21722902, 9484998, 31447099, 30592178, 33111339, 31372158, 34011801, 31491741, 30755392, 34037665, 33458634, 33418990, 35339733, 35480308, 32041611, 32977124, 32331935, 30710474, 33519890, 1301956, 2088165, 15359125, 18677035, 10978268, 12837857, 23375686, 12827279)

PP1_strong, PP3, PP4, PM2_supporting, PS3, PS4

This sequence change replaces glutamic acid, which is acidic and polar, with lysine, which is basic and polar, at codon 228 of the LDLR protein (p.Glu228Lys). This variant is present in population databases (rs121908029, gnomAD 0.01%). This missense change has been observed in individual(s) with familial hypercholesterolemia (PMID: 2318961, 21475731, 21722902, 22390909, 23375686). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. This variant is also known as p.Glu207Lys. ClinVar contains an entry for this variant (Variation ID: 3691). 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: 18677035). This variant disrupts the p.Glu228 amino acid residue in LDLR. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 1301956, 8882879, 16250003, 17094996). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The p.Glu228Lys variant in LDLR is a common pathogenic variant that has been reported in a large number of individuals across several studies (Kusters 2011, Hujgen 2012, Bertolini 2013, Leitersdorf 1990). It is one of the 12 most common LDLR variants in the Dutch (Kusters 2011). This variant has been identified in 2/17176 East Asian chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs121908029). In vitro functional studies support that the p.Glu228Lys impacts protein function (Leitersdorf 1990). In addition, several other variants at the same position (p.Glu228Gln, p.Glu228Ala, p.Glu228Gly) have been reported with evidence supporting a disease causing role (ClinVar ID: 251393, 251394, 375796), suggesting that a change at this position is not tolerated. In summary, this variant meets criteria to be classified as pathogenic for familial hypercholesterolemia in an autosomal dominant manner based upon functional studies, presence in multiple affected individuals, low frequency in controls and pathogenicity of other variants at the same amino acid position.

LDLR: PP1:Strong, PM1, PM2, PM5, PS4:Moderate, PS3:Supporting

The p.E228K pathogenic mutation (also known as c.682G>A), located in coding exon 4 of the LDLR gene, results from a G to A substitution at nucleotide position 682. The glutamic acid at codon 228 is replaced by lysine, an amino acid with similar properties. This alteration, historically described as p.E207K, FH French Canadian, FH Mexico-3, and FH-Modena, has been detected in numerous individuals with familial hypercholesterolemia (FH) across multiple ethnicities (Leitersdorf E et al. J. Clin. Invest., 1990 Apr;85:1014-23; Bertolini S et al. Arterioscler. Thromb. Vasc. Biol., 2000 Sep;20:E41-52; Kim HN et al. Chonnam Med J, 2018 Jan;54:31-35). Functional studies have demonstrated decreased amounts of mature mRNA, reduced protein transport from the endoplasmic reticulum, and LDLR activity that is 40-45% of wild-type levels (Leitersdorf E et al. J. Clin. Invest., 1990 Apr;85:1014-23; Bertolini S et al. Arterioscler. Thromb. Vasc. Biol., 2000 Sep;20:E41-52; Chang JH et al. J. Lipid Res., 2003 Oct;44:1850-8). Furthermore, an alternate nucleotide change at this position, c.682G>C p.E228Q (also known as p.E207Q, FH Tulsa-2, FH Iraq) has been well-described as a mutation in numerous individuals with FH, suggesting a likely hotspot location (Hobbs HH et al. Hum. Mutat., 1992;1:445-66). In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

p.Glu228Lys (c.682G>A) in exon 4 of the LDLR gene (NM_000527.4; chr19-11216264-G-A) This variant is also reported as p.Glu207Lys in the literature. SCICD Classification: pathogenic variant based on strong case data and low frequency in unselected populations. We do feel it is suitable for assessing risk in healthy relatives ("predictive genetic testing"). Gene-level evidence: LDLR: LDL receptors are located on the surface of the liver and play an important role in LDL recycling. Pathogenic variants in LDLR account for 80% of cases of familial hypercholesterolemia. Both missense and truncating/frameshift variants can be pathogenic. Case data (not including our patient): over 400 individuals with FH (mostly of Dutch ancestry) have this variant. · ClinVar: 12 labs § Invitae, British Heart Foundation Study, CV Research Group, Robarts Research Institute, Centre de Génétique Moléculaire et Chromosomique, Molecular Genetics Laboratory,Centre for Cardiovascular Surgery and Transplantation, U4M - Lille University & CHRU Lille,Université Lille 2 - CHRU de Lille, Laboratory of Genetics and Molecular Cardiology,University of São Paulo, Fundacion Hipercolesterolemia Familiar, Cardiovascular Genetics Laboratory,PathWest Laboratory Medicine WA - Fiona Stanley Hospital, Laboratorium voor Moleculaire Diagnostiek Experimentele Vasculaire Geneeskunde,Academisch Medisch Centrum: at least 34 people from 9 families. · Cases in the literature: · Leitersdorf et al 1990: Reported in 3 of 130 French-Canadian patients with heterozygous FH and in 2 of 11 homozygotes. · Kusters et al 2011: Determined that this variant is a Dutch founder variant. 437 out of 10,899 patients had this variant. · Vaca et al 2011: Identified this variant in 5 out of 59 Mexican patients with FH. · Huijgen et al 2012: the purpose of this study was to analyze previously-documented variants using in silico prediction tools. · Bertolini et al 2013: This variant was identified in 31 subjects from 16 families of Italian descent. Segregation data: reported (Bertolini et al 2013) but no large families with this variant have been studied for segregation data. Functional data: Zhao and Michaely (2008): The substitution of a lysine for a glutamine at this codon destroys an important binding site. The binding affinity of a nearby residue, E208, to a VLDL molecule is reduced when substituted (E208K). Conservation data: The glutamic acid at codon 228 is completely conserved across species. Nearby pathogenic variants at this codon or neighboring codons: Per the test report, "a different missense substitution at this codon (p.Glu228Gln) has been determined to be pathogenic (PMID: 1301956, 8882879, 16250003, 17094996). This variant has not been reviewed by our team. Population data: Highest MAF in East Asian population: 0.011%. The variant was reported online in 4 of 122,190 individuals in the Genome Aggregation Consortium Dataset (gnomAD; http://gnomad.broadinstitute.org/), which currently includes variant calls on >140,000 unrelated individuals of African, Asian, European, Ashkenazi, Latino descent. Specifically, the variant was observed in 2 of 8,588 individuals of East Asian descent (MAF=0.011%), 1 of 16,777 individuals of Latino descent and 1 of 55,223 individuals of European descent. The phenotype of those individuals is not publicly available. The dataset is comprised of multiple cohorts, some of which were recruited from the general population, others were enriched for common cardiovascular disease.