rs879254746

chr19-11110685-G-Achr19-11110685-G-Cchr19-11110685-G-T

Variant summary

Our verdict is Pathogenic. Variant got 18 ACMG points: 18P and 0B. PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000527.5(LDLR):c.974G>A(p.Cys325Tyr) variant causes a missense change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.00000137 in 1,461,568 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Likely pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. C325F) has been classified as Likely pathogenic.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000014 (0 hom.)
• Consequence: LDLR NM_000527.5 missense
• Clinical Significance: Likely pathogenic reviewed by expert panel P:10
• Conservation: PhyloP100: 9.68

Genes affected

LDLR (HGNC:6547): (low density lipoprotein receptor) The low density lipoprotein receptor (LDLR) gene family consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. The encoded protein is normally bound at the cell membrane, where it binds low density lipoprotein/cholesterol and is taken into the cell. Lysosomes release the cholesterol, which is made available for repression of microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting step in cholesterol synthesis. At the same time, a reciprocal stimulation of cholesterol ester synthesis takes place. Mutations in this gene cause the autosomal dominant disorder, familial hypercholesterolemia. Alternate splicing results in multiple transcript variants.[provided by RefSeq, May 2022]

ACMG classification

Verdict is Pathogenic. Variant got 18 ACMG points.

• PM1: In a hotspot region, there are 8 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 benign, 7 uncertain in NM_000527.5
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr19-11110685-G-T is described in ClinVar as [Likely_pathogenic]. Clinvar id is 579747.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.997
• PP5: Variant 19-11110685-G-A is Pathogenic according to our data. Variant chr19-11110685-G-A is described in ClinVar as [Likely_pathogenic]. Clinvar id is 251580.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr19-11110685-G-A is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
LDLR	NM_000527.5	c.974G>A	p.Cys325Tyr	missense_variant	7/18	ENST00000558518.6

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
LDLR	ENST00000558518.6	c.974G>A	p.Cys325Tyr	missense_variant	7/18	1	NM_000527.5	P3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 0.00000137 AC: 2 AN: 1461568 Hom.: 0 Cov.: 31 AF XY: 0.00000275 AC XY: 2 AN XY: 727084

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.0000252

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00

Gnomad4 OTH exome AF: 0.0000166

GnomAD4 genome Cov.: 32

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:10

Revision: reviewed by expert panel

Submissions by phenotype

Hypercholesterolemia, familial, 1 Pathogenic:6

Pathogenic, criteria provided, single submitter	research	Brunham Lab, Centre for Heart and Lung Innovation, University of British Columbia	Jun 05, 2019	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Laboratory of molecular diagnosis of dyslipidemias, Università egli studi di Napoli Federico II	May 24, 2021	Reduced activity, in stimulated T-lymphocytes and EBV-transformed B-lymphocytes. -
Likely pathogenic, criteria provided, single submitter	literature only	LDLR-LOVD, British Heart Foundation	Mar 25, 2016	- -
Pathogenic, criteria provided, single submitter	clinical testing	Fulgent Genetics, Fulgent Genetics	Apr 20, 2022	- -
Likely pathogenic, criteria provided, single submitter	research	Fundacion Hipercolesterolemia Familiar	Mar 01, 2016	- -
Likely pathogenic, reviewed by expert panel	curation	ClinGen Familial Hypercholesterolemia Variant Curation Expert Panel	Feb 09, 2022	The NM_000527.5 (LDLR): c. 974G>A (p.Cys325Tyr) variant is classified as Likely Pathogenic for Familial Hypercholesterolemia by applying evidence codes (PM2, PM1, PS4_Moderate, PP3, PP4, PS3_Supporting,) 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 Met: This variant is absent in gnomAD (gnomAD v2.1.1). PP3 Met: REVEL = 0.977, which is above the threshold of 0.75. PM1 Met: The variant meets PM2, and alters Cys325, which is located in EGF-like 1 domain and is one of 60 highly conserved cysteine residues. PS3_Supporting Met: FACS assay using heterozygous patients’ lymphocytes (level 3 functional assay) showed 50-60% LDLR activity compared to wild type, from one research lab (CEINGE-Biotecnologie Avanzate, Napoli, Italy, PMID21865347). PP4 Met: This variant meets PM2 and is identified >1 index cases who met clinical criteria for FH after alternative causes for high cholesterol were excluded. PS4_Moderate Met: Variant meets PM2 and is identified in 8 unrelated index cases. Five of the index cases fulfil DLCN criteria for FH from 5 different laboratories (1 case from Mayo Clinic Atherosclerosis and Lipid genomics Laboratory, LabID500068; 1 case from University of Genova-University of Modena and Reggio Emilia, Italy, PMID32977124; 1 case from Lipid Clinic, Fundación Jiménez Díaz, Spain, PMID19318025; 1 case from Centre for Heart Lung Innovation, University of British Columbia, Canada, PMID31345425; and 1case from Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, China, PMID30526649). One index case fulfil FH diagnosis criteria established by the Societa Italiana per lo Studio della Arteriosclerosi, CEINGE-Biotecnologie Avanzate, Napoli, Italy, PMID21865347. Two index cases fulfil Japanese FH guidelines, from Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan, PMID31491741. There are three other variants in the same codon: LDLR: NM_000527: c.973T>C (p.Cys325Arg), LDLR: NM_000527: c.974G>C (p.Cys325Ser), LDLR: NM_000527: c.974G>T (p.Cys325Phe), which are classified as Uncertain significance by these guidelines. Therefore PM5 is not met. -

Familial hypercholesterolemia Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	May 16, 2023	Variant summary: LDLR c.974G>A (p.Cys325Tyr) results in a non-conservative amino acid change located in the EGF-like domain (IPR000742) of the encoded protein sequence. Cys325 is a highly conserved residue located in the EGF-like calcium-binding domain of the LDL receptor. HGMD lists >50 Cys missense mutations reported in the literature as associated with hypercholesterolaemia, and five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251090 control chromosomes (gnomAD). c.974G>A has been reported in the literature in individuals affected with Familial Hypercholesterolemia and coronary artery disease (Romano_2011, Alonso_2009, Rubba_2017, Cao_2018, Trinder_2019, Hori_2019, Di Taranto_2020, Wang_2020, Bertolini_2020, Doi_2021), including at least two homozygotes. One homozygous patient had this variant in cis with c.[974G>A];[(940+1_941-1)_(2311+1_2312-1)dup (Gly314_Gln770dup, described as pathogenic) (Di Taranto_2020). These data indicate that the variant may be associated with disease. Two functional studies report experimental evidence evaluating an impact on protein function and shown to have significantly decreased LDLR residual activity in EBV-transformed B-lymphocytes and stimulated T-lymphocytes (Romano_LDLR_JLR_2011, Bertolini_2020). The following publications have been ascertained in the context of this evaluation (PMID: 19318025, 29874871, 32977124, 30526649, 34496902, 31947532, 34297352, 33533259, 35929461, 31491741, 33418990, 21865347, 28353356, 33079599, 31345425, 32759540). Nine ClinVar submitters have assessed the variant since 2014: five classified the variant as likely pathogenic, and four as pathogenic. Based on the evidence outlined above, the variant was classified as likely pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Invitae	Oct 23, 2023	This sequence change replaces cysteine, which is neutral and slightly polar, with tyrosine, which is neutral and polar, at codon 325 of the LDLR protein (p.Cys325Tyr). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with clinical features of familial hypercholesterolemia (PMID: 21865347, 30526649, 32759540, 32977124, 33418990; Invitae). ClinVar contains an entry for this variant (Variation ID: 251580). 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. This variant affects a cysteine residue located within an LDLRA or epidermal-growth-factor (EGF)-like domains of the LDLR protein. Cysteine residues in these domains have been shown to be involved in the formation of disulfide bridges, which are critical for protein structure and stability (PMID: 7548065, 7603991, 7979249). In addition, missense substitutions within the LDLRA and EGF-like domains affecting cysteine residues are overrepresented among patients with hypercholesterolemia (PMID: 18325082). This variant disrupts the p.Cys325 amino acid residue in LDLR. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 26802169). 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. -

not provided Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

GeneDx

Feb 17, 2023

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 LDLR activity compared to wild type (Romano et al., 2011; Pfisterer SG et al., 2022); Also known as p.(C304Y); This variant is associated with the following publications: (PMID: 30526649, 19318025, 31491741, 21865347, 33079599, 2988123, 12459547, 29874871, 34297352, 34496902, 35474963, 32759540, 32977124, 31345425, 33418990, 31947532, 33533259) -

Cardiovascular phenotype Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jan 24, 2022

The p.C325Y pathogenic mutation (also known as c.974G>A), located in coding exon 7 of the LDLR gene, results from a G to A substitution at nucleotide position 974. The cysteine at codon 325, located in the EGF-like 1 domain, is replaced by tyrosine, an amino acid with highly dissimilar properties. Pathogenic LDLR mutations that result in the substitution or generation of cysteine residues within the cysteine-rich LDLR class A repeats and EGF-like domains are common in familial hypercholesterolemia (FH) (Villéger L. Hum Mutat. 2002;20(2):81-7). This particular alteration has been detected in multiple individuals reported to have familial hypercholesterolemia (FH), and in individuals from FH cohorts (Alonso R et al. Clin Biochem. 2009;42:899-903; Romano M et al. J Lipid Res. 2011;52:2095-100; Hori M et al. Atherosclerosis. 2019 10;289:101-108; Wang H et al. J Atheroscler Thromb. 2020 Dec;27(12):1288-1298; Meshkov A et al. Genes (Basel). 2021 01;12(1); Ambry internal data). Internal structural analysis indicates this alteration eliminates a disulfide bond critical for the structural integrity of the EGF-like 1 domain (Ambry internal data). In a study of patient-derived lymphocytes, this alteration was reported to reduce LDLR activity to approximately 50% of wildtype (Romano M et al. J Lipid Res. 2011;52:2095-100). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.99
BayesDel_addAF	Pathogenic	0.58	D
BayesDel_noAF	Pathogenic	0.60
Cadd	Pathogenic	28
Dann	Uncertain	0.99
DEOGEN2	Pathogenic	0.98	D;.;.;.;.;.
Eigen	Pathogenic	0.99
Eigen_PC	Pathogenic	0.84
FATHMM_MKL	Pathogenic	1.0	D
LIST_S2	Pathogenic	0.99	D;D;D;D;D;D
M_CAP	Pathogenic	0.94	D
MetaRNN	Pathogenic	1.0	D;D;D;D;D;D
MetaSVM	Pathogenic	0.91	D
MutationAssessor	Pathogenic	4.8	H;.;.;.;.;H
MutationTaster	Benign	1.0	D;D;D;D;D;D;D
PrimateAI	Benign	0.40	T
PROVEAN	Pathogenic	-11	D;D;D;D;D;D
Sift	Uncertain	0.0010	D;D;D;D;D;D
Sift4G	Pathogenic	0.0	D;D;D;D;D;D
Polyphen		1.0	D;.;.;.;.;.
Vest4		0.98
MutPred		0.97		Loss of disorder (P = 0.1727);Loss of disorder (P = 0.1727);.;.;.;Loss of disorder (P = 0.1727);
MVP		1.0
MPC		0.61
ClinPred		1.0	D
GERP RS		5.4
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.99
gMVP		0.99

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs879254746; hg19: chr19-11221361;