19-11100349-A-T

Position:

• chr19-11100349-A-T

Variant summary

Our verdict is Likely pathogenic. Variant got 9 ACMG points: 9P and 0B. PS4 PM2 PP4 PS3_Moderate

This summary comes from the ClinGen Evidence Repository: NM_000527.5(LDLR):c.190+4A>T variant is classified as Likely pathogenic for Familial Hypercholesterolemia by applying evidence codes (PM2, PP4, PS4 and PS3_Moderate) 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 = 0.00020 (0.02%) in East Asian exomes+genomes (gnomAD v2.1.1).PP4 - Variant meets PM2. Variant identified in 11 unrelated index cases (1 case fulfilling Simon-Broome criteria published in PMID:21418584; 3 cases fulfilling Simon-Broome criteria published in PMID:20236128; 5 cases with Dutch lipid clinic network >=6 from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA); 1 case with Dutch lipid clinic network >=6 from Robarts Research Institute; 1 case with MedPed definite from Mayo Clinic Atherosclerosis and Lipid Genomics Laboratory.PS3_Moderate - Level 2 assays: PMID 19208450:Heterozygous patient cells, FACS/RT-PCR/Nothern blotting assays - result - 47% low-density lipoprotein particle receptor activity (LDLR activity reported as a mean value of cell-surface LDLR and LDL internalization measurements); aberrant transcript is not confirmed by sequencing (NMD expected) and is 40% of total transcripts.PS4 - Variant meets PM2. Variant identified in 11 unrelated index cases (1 case fulfilling Simon-Broome criteria published in PMID:21418584; 3 cases fulfilling Simon-Broome criteria published in PMID:20236128; 5 cases with Dutch lipid clinic network >=6 from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA); 1 case with Dutch lipid clinic network >=6 from Robarts Research Institute; 1 case with MedPed definite from Mayo Clinic Atherosclerosis and Lipid Genomics Laboratory. LINK:https://erepo.genome.network/evrepo/ui/classification/CA042227/MONDO:0007750/013

• Frequency:
  • Genomes: 𝑓 0.0000066 (0 hom., cov: 33)
  • Exomes 𝑓: 0.0000048 (0 hom.)
• Consequence: LDLR NM_000527.5 splice_region, intron
• Scores: Splicing: ADA: 0.9996
• Clinical Significance: Likely pathogenic reviewed by expert panel P:17 U:3
• Conservation: PhyloP100: 4.69

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]

LDLR (HGNC:):

ACMG classification

Verdict is Likely_pathogenic. Variant got 9 ACMG points.

• PS3: For more information check the summary or visit ClinGen Evidence Repository.
• PS4: For more information check the summary or visit ClinGen Evidence Repository.
• PM2: For more information check the summary or visit ClinGen Evidence Repository.
• PP4: For more information check the summary or visit ClinGen Evidence Repository.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
LDLR	NM_000527.5	c.190+4A>T		splice_region_variant, intron_variant		ENST00000558518.6	NP_000518.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
LDLR	ENST00000558518.6	c.190+4A>T		splice_region_variant, intron_variant		1	NM_000527.5	ENSP00000454071.1

Frequencies

GnomAD3 genomes AF: 0.00000657 AC: 1 AN: 152108 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.00

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.000193

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.00

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000160 AC: 4 AN: 250582 Hom.: 0 AF XY: 0.00000738 AC XY: 1 AN XY: 135492

Gnomad AFR exome AF: 0.0000617

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.000163

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000479 AC: 7 AN: 1461062 Hom.: 0 Cov.: 31 AF XY: 0.00000413 AC XY: 3 AN XY: 726816

Gnomad4 AFR exome AF: 0.0000299

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00

Gnomad4 OTH exome AF: 0.0000994

GnomAD4 genome AF: 0.00000657 AC: 1 AN: 152108 Hom.: 0 Cov.: 33 AF XY: 0.0000135 AC XY: 1 AN XY: 74286

Gnomad4 AFR AF: 0.00

Gnomad4 AMR AF: 0.00

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.000193

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.00

Gnomad4 OTH AF: 0.00

Bravo AF: 0.0000302

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:17 Uncertain:3

Revision: reviewed by expert panel

Submissions by phenotype

Hypercholesterolemia, familial, 1 Pathogenic:8 Uncertain:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Oct 28, 2022	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Dec 14, 2023	This variant has been reported in multiple individuals with familial hypercholesterolemia (PMID: 21418584, 20236128, 34573395). It is present in 5 of 281964 total alleles in the Genome Aggregation Database (http://gnomad.broadinstitute.org/). This variant is predicted to be deleterious by in silico analysis. Well-established functional studies suggest that this variant has a deleterious effect on the protein sufficient to be disease-causing (PMID: 19208450). Different variants at the same splice site motif have been reported in association with disease and are independently classified as likely pathogenic or pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Robarts Research Institute, Western University	-	- -
Uncertain significance, criteria provided, single submitter	research	Brunham Lab, Centre for Heart and Lung Innovation, University of British Columbia	Jun 05, 2019	- -
Pathogenic, criteria provided, single submitter	clinical testing	Victorian Clinical Genetics Services, Murdoch Childrens Research Institute	Jul 17, 2023	Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with familial hypercholesterolemia 1 (FH) (MIM#143890). (I) 0108 - This gene is associated with both recessive and dominant disease. Variants have been reported in heterozygous, compound heterozygous and homozygous states with the recessive disease being more severe (OMIM, PMID: 10978268). (I) 0112 - The condition associated with this gene has incomplete penetrance (GeneReviews). (I) 0209 - Splice site variant proven to affect splicing of the transcript with uncertain effect on protein sequence. Functional analysis of this variant using transfected lymphocytes has demonstrated that 40% of transcripts are defective and were unable to be isolated suggesting degradation (PMID: 19208450). (SP) 0251 - This variant is heterozygous. (I) 0302 - Variant is present in gnomAD (v2) <0.001 for a dominant condition (5 heterozygotes, 0 homozygotes). (SP) 0311 - An alternative nucleotide change at the same splice site, is present in gnomAD (v2) (1 heterozygote, 0 homozygotes). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported multiple times as likely pathogenic and pathogenic in a clinical setting, and has been reported in many patients with familial hypercholesterolemia (ClinVar, PMID: 19208450, PMID: 31345425). (SP) 1102 - Strong phenotype match for this individual. (SP) 1205 - This variant has been shown to be maternally inherited. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign -
Uncertain significance, criteria provided, single submitter	literature only	LDLR-LOVD, British Heart Foundation	Mar 25, 2016	- -
Likely pathogenic, reviewed by expert panel	curation	ClinGen Familial Hypercholesterolemia Variant Curation Expert Panel	Feb 09, 2022	NM_000527.5(LDLR):c.190+4A>T variant is classified as Likely pathogenic for Familial Hypercholesterolemia by applying evidence codes (PM2, PP4, PS4 and PS3_Moderate) 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 = 0.00020 (0.02%) in East Asian exomes+genomes (gnomAD v2.1.1). PP4 - Variant meets PM2. Variant identified in 11 unrelated index cases (1 case fulfilling Simon-Broome criteria published in PMID: 21418584; 3 cases fulfilling Simon-Broome criteria published in PMID: 20236128; 5 cases with Dutch lipid clinic network >=6 from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA); 1 case with Dutch lipid clinic network >=6 from Robarts Research Institute; 1 case with MedPed definite from Mayo Clinic Atherosclerosis and Lipid Genomics Laboratory. PS3_Moderate - Level 2 assays: PMID 19208450: Heterozygous patient cells, FACS/RT-PCR/Nothern blotting assays - result - 47% low-density lipoprotein particle receptor activity (LDLR activity reported as a mean value of cell-surface LDLR and LDL internalization measurements); aberrant transcript is not confirmed by sequencing (NMD expected) and is 40% of total transcripts. PS4 - Variant meets PM2. Variant identified in 11 unrelated index cases (1 case fulfilling Simon-Broome criteria published in PMID: 21418584; 3 cases fulfilling Simon-Broome criteria published in PMID: 20236128; 5 cases with Dutch lipid clinic network >=6 from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA); 1 case with Dutch lipid clinic network >=6 from Robarts Research Institute; 1 case with MedPed definite from Mayo Clinic Atherosclerosis and Lipid Genomics Laboratory. -
Pathogenic, no assertion criteria provided	clinical testing	Cardiovascular Genetics Laboratory, PathWest Laboratory Medicine WA - Fiona Stanley Hospital	Mar 18, 2010	- -
Pathogenic, no assertion criteria provided	research	Laboratorium voor Moleculaire Diagnostiek Experimentele Vasculaire Geneeskunde, Academisch Medisch Centrum	-	- -
Likely pathogenic, criteria provided, single submitter	curation;literature only	Cardiovascular Research Group, Instituto Nacional de Saude Doutor Ricardo Jorge	Mar 01, 2016	- -

Familial hypercholesterolemia Pathogenic:5

Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Sep 22, 2022	This variant causes an A>T nucleotide substitution at the +4 position of intron 2 of the LDLR gene. Splice site prediction tools predict that this variant may have a significant impact on RNA splicing. A functional RNA study has demonstrated that this variant causes aberrant splicing and degradation of the mutant transcript (PMID: 19208450). Lymphocytes from a heterozygous carrier showed that the levels of cell-surface LDLR protein and internalized LDL were decreased by ~40% and ~55%, respectively, compared to normal cells (PMID: 19208450). This variant has been reported in at least fifteen individuals diagnosed with familial hypercholesterolemia (PMID: 16205024, 16250003, 16792510, 21418584, 22883975, 23680767, 24075752, 25682442, 27765764, 31345425). This variant has been identified in 5/281964 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Loss of LDLR function is a known mechanism of disease. Based on available evidence, this variant is classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Feb 06, 2019	Variant summary: The variant, LDLR c.190+4A>T alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes a 5 splicing donor site. Two predict the variant weakens a 5' donor site. A functional study, Holla_2009, detected aberrant splicing and found the variant to cause a decrease in LDLR at the cell surface (~40% of normal) and internalization (~55% of normal). The variant allele was found at a frequency of 1.8e-05 in 276542 control chromosomes (gnomAD). c.190+4A>T has been reported in the literature in multiple individuals affected with Familial Hypercholesterolemia (Al-Khateeb_2011, Fouchier_2005, Taylor_2010, Leren_2004, Punzalan_2005). These data indicate that the variant is very likely to be associated with disease. Four ClinVar submissions from clinical diagnostic laboratories (evaluation after 2014) cite the variant three times as likely pathogenic/pathogenic and once as uncertain significance. Based on the evidence outlined above, the variant was classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Rady Children's Institute for Genomic Medicine, Rady Children's Hospital San Diego	Sep 09, 2019	This variant has been previously reported as a heterozygous change in patients with familial hypercholesterolemia (PMID: 15199436, 16205024, 16250003, 21418584, 27765764). Functional studies conducted in EBV-transformed lymphocytes demonstrated that this variant leads to reduced LDLR transcript levels, reduced LDLR protein present at the cell surface, and reduced levels of internalized LDL (PMID: 19208450). This variant is present in the heterozygous state in the gnomAD population database at a frequency of 0.002% (5/276382) and thus is presumed to be rare. Multiple splice prediction tools suggest this variant is likely to interfere with normal splicing. Based on the available evidence, the c.190+4A>T variant is classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Jan 19, 2024	This sequence change falls in intron 2 of the LDLR gene. It does not directly change the encoded amino acid sequence of the LDLR protein. It affects a nucleotide within the consensus splice site. This variant is present in population databases (rs769446356, gnomAD 0.02%). This variant has been observed in individuals with familial hypercholesterolemia (PMID: 15199436, 16205024, 16250003, 21418584, 27765764). ClinVar contains an entry for this variant (Variation ID: 225097). Studies have shown that this variant alters LDLR gene expression (PMID: 19208450). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Human Genome Sequencing Center Clinical Lab, Baylor College of Medicine	Apr 30, 2019	The c.190+4A>T variant in the LDLR gene in intron 2 is predicted to disrupts splicing resulting in an abnormal mRNA transcript. This variant has been reported in multiple unrelated individuals with familial hypercholesterolemia (PMID: 15199436, 16205024, 21418584, 27765764). Functional studies have shown reduced internalization and cell surface LDLR protein when this variant is present (PMID: 19208450). The c.190+4A>T variant in the LDLR gene is classified as likely pathogenic. -

not provided Pathogenic:2 Uncertain:1

Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jun 20, 2022	Not observed at significant frequency in large population cohorts (gnomAD); Published functional studies suggest a damaging effect leading to reduction in LDLR protein (Holla et al., 2009); In silico analysis supports a deleterious effect on splicing; This variant is associated with the following publications: (PMID: 25525159, 26795593, 15199436, 16205024, 21418584, 16250003, 22883975, 25682442, 23680767, 27765764, 24075752, 19208450, 34037665, 32719484, 33740630, 34573395) -
Pathogenic, no assertion criteria provided	provider interpretation	Stanford Center for Inherited Cardiovascular Disease, Stanford University	Jul 05, 2016	Intronic variant c.190+4A>T in the LDLR gene (NM_000527.4) Given moderate to strong case data and functional studies showing deleterious impact on the protein, we consider this variant likely pathogenic and we feel it is suitable for assessing risk in healthy relatives ("predictive genetic testing"). We have seen this variant in a person with FH. Testing was done by Ambry. The variant has been seen in at least 6 unrelated cases of FH (not including this patient's family). There is moderate case data, though no segregation has been demonstrated. The c.190+4A>T variant is listed by Ambry in ClinVar, but without any case data. It was reported in the literature by Khateeb et al., 2011 (1 person with FH), by Punzalan et al., 2005 (3 people with FH), and Vandrovcova et al (1 person with FH). It was also reported in Holla et al., 2009 in a Dutch individual reported to have confirmed FH and in Wald et al., 2015 in a 50 year-old with a total cholesterol level of 294 mg/dL and history of myocardial infarction. No other case data was provided. None of the available studies have performed segregation analysis. Holla et al., 2009 performed some functional studies on the c.190+4A>T variant. Semi-quantitative PCR showed a reduced amount of mRNA transcript from the variant allele compared to control cell lines (less than a 42% cut-off). They did not see exon skipping or activation of a cryptic splice site by RT-PCR or activation of a cryptic splice site by Northern Blot for this variant. They speculated that the mutant transcript containing this variant was degraded. Per the Ambry report, this nucleotide position is well conserved in available vertebrate species. The c.190+4A>T variant was reported online in 2 of 60,098 individuals in the Exome Aggregation Consortium dataset (http://exac.broadinstitute.org/), which currently includes variant calls on ~64,000 individuals of European, African, Latino and Asian descent (as of 4/19/2016). Specifically, the variant was observed in 2 of 4297 East Asian individuals. 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. An allelic c.190+4A>G variant was also listed in ExAC 1 of 60,098 individuals, specifically in 1 of 4297 East Asian individuals. Note that other variants with strong evidence for pathogenicity have been seen at similar frequencies in datasets like this so this does not necessarily rule out pathogenicity (Pan et al 2012). This is especially true for a relatively common disorder like FH. -
Uncertain significance, criteria provided, single submitter	clinical testing	AiLife Diagnostics, AiLife Diagnostics	May 18, 2020	- -

Homozygous familial hypercholesterolemia Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine

Feb 08, 2018

The c.190+4A>T variant in LDLR has been reported in at least 7 individuals with hypercholesterolemia (Fouchier 2005, Punzalan 2005, Al-Khateeb 2011, Vandrovcova 2013, Wang 2016). It has also been identified in 4/18862 East Asian chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/; dbSNP rs769446356). This variant is located in the 5' splice region, and computational tools do suggest an impact to splicing. In addition, in vitro functional studies provide some evidence that the c.190+4A>T variant may impact protein function (Holla 2009). However, these computational and functional studies may not accurately represent biological function. This variant has been reported in ClinVar (Variation ID: 225097). In summary, although additional studies are required to fully establish its clinical significance, the c.190+4A>T variant is likely pathogenic. ACMG/AMP Criteria applied: PS3_Moderate; PS4_Moderate; PM2; PP3. -

Cardiovascular phenotype Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jan 30, 2024

The c.190+4A>T intronic variant results from an A to T substitution 4 nucleotides downstream of coding exon 2 in the LDLR gene. This alteration was previously detected in Malaysian and Filipino individuals reported to have familial hypercholesterolemia (FH) (Punzalan FE et al. J Atheroscler Thromb. 2005;12(5):276-83; Khateeb A et al. BMC Med Genet. 2011;12:40), and has been detected in other individuals with FH from additional cohorts (Leren TP et al. Semin Vasc Med. 2004;4(1):75-85; Hooper AJ et al. Atherosclerosis. 2012;224(2):430-4; Vandrovcova J et al. Genet Med. 2013;15(12):948-57). One in vitro study indicated this alteration results in reduced LDL receptor activity and possible degradation of mutant transcript (Holla ØL et al. Mol Genet Metab. 2009;96(4):245-52). This nucleotide position is well conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.53
CADD	Benign	18
DANN	Benign	0.78
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.0

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.83
SpliceAI score (max)		0.84		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.84		Position offset: -4

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

Publications

LitVar

Other links and lift over

dbSNP: rs769446356; hg19: chr19-11211025;