rs781362878

chr19-11113767-G-Achr19-11113767-G-C

Variant summary

Our verdict is Likely pathogenic. Variant got 7 ACMG points: 7P and 0B. PM2 PP3_Strong PP5

The NM_000527.5(LDLR):c.1586+5G>A variant causes a splice donor 5th base, intron change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.0000234 in 1,452,200 control chromosomes in the GnomAD database, including 1 homozygotes. In-silico tool predicts a benign outcome for this variant. 2/2 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.000023 (1 hom.)
• Consequence: LDLR NM_000527.5 splice_donor_5th_base, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:14 U:4
• Conservation: PhyloP100: 9.40

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 Likely_pathogenic. Variant got 7 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 19-11113767-G-A is Pathogenic according to our data. Variant chr19-11113767-G-A is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 251909.We mark this variant Likely_pathogenic, oryginal submissions are: {Pathogenic=3, Uncertain_significance=4, Likely_pathogenic=9}. Variant chr19-11113767-G-A is described in Lovd as [Pathogenic]. Variant chr19-11113767-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.1586+5G>A		splice_donor_5th_base_variant, intron_variant		ENST00000558518.6

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
LDLR	ENST00000558518.6	c.1586+5G>A		splice_donor_5th_base_variant, intron_variant		1	NM_000527.5	P3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD3 exomes AF: 0.0000319 AC: 8 AN: 250846 Hom.: 0 AF XY: 0.0000369 AC XY: 5 AN XY: 135624

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.000196

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000176

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.0000234 AC: 34 AN: 1452200 Hom.: 1 Cov.: 31 AF XY: 0.0000277 AC XY: 20 AN XY: 722982

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: 0.000302

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00000634

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

Bravo AF: 0.00000378

EpiCase AF: 0.00

EpiControl AF: 0.0000593

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:14 Uncertain:4

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Hypercholesterolemia, familial, 1 Pathogenic:8 Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	Centre de Génétique Moléculaire et Chromosomique, Unité de génétique de l'Obésité et des Dyslipidémies, APHP, GH Hôpitaux Universitaires Pitié-Salpêtrière / Charles-Foix	Dec 16, 2016	subjects mutated among 2600 FH index cases screened = 2 -
Likely pathogenic, criteria provided, single submitter	clinical testing	Illumina Laboratory Services, Illumina	Apr 29, 2022	The LDLR c.1586+5G>A variant, also known as c.1592+5G>A, occurs in a splice region and results in the substitution of a guanine at nucleotide position c.1586+5 with an adenine. Across a selection of the available literature, the c.1586+5G>A variant has been identified in at least seven individuals with clinically diagnosed familial hypercholesterolemia and was shown to segregate with the disorder (Ekstrom et al. 1995; Jensen et al. 1999; Fouchier et al. 2005; Mollaki et al. 2016; Bertolini et al. 2020; Meshkov et al. 2020; Lamiquiz-Moneo et al. 2021; Leren et al. 2021; Sturm et al. 2021). Another variant at the same nucleotide position, has been reported as c.1592+5G>C in a heterozygous state in three individuals with familial hypercholesterolemia (Yang et al. 2007; Defesche et al. 2017). This variant is reported in the Genome Aggregation Database in six alleles at a frequency of 0.000196 in the South Asian population (version 2.1.1). Analysis of mRNA from patient cells with sequence analysis found that the c.1586+5G>A variant results in alternate RNA splicing mechanisms that cause the activation of a cryptic splice site within intron 10 or skipping of exon 10 and causes defects in receptor transport in vitro (Jensen et al. 1999). Based on the available evidence, the c.1586+5G>A variant is classified as likely pathogenic for familial hypercholesterolemia. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, King Faisal Specialist Hospital and Research Center	Apr 04, 2024	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	U4M - Lille University & CHRU Lille, Université de Lille - CHRU de Lille	Mar 30, 2017	- -
Likely pathogenic, criteria provided, single submitter	literature only	LDLR-LOVD, British Heart Foundation	Mar 25, 2016	- -
Pathogenic, no assertion criteria provided	research	Laboratorium voor Moleculaire Diagnostiek Experimentele Vasculaire Geneeskunde, Academisch Medisch Centrum	-	- -
Uncertain significance, criteria provided, single submitter	research	Brunham Lab, Centre for Heart and Lung Innovation, University of British Columbia	Jun 05, 2019	- -
Uncertain significance, criteria provided, single submitter	research	Fundacion Hipercolesterolemia Familiar	Mar 01, 2016	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Aug 28, 2023	This variant causes a G to A nucleotide substitution at the +5 position of intron 10 of the LDLR gene. Splice site prediction tools predict that this variant may have a significant impact on RNA splicing. Molecular studies of the impact of this variant on RNA splicing demonstrated two aberrant mRNAs due to either in-frame skipping of exon 10 or the activation of a cryptic splice site in intron 10 inserting 66 intronic base pairs (PMID: 10668928). Additionally, this variant was shown to cause aberrant LDL receptor trafficking in transfected COS7 cells (PMID: 10668928). This variant has been reported in individuals affected with familial hypercholesterolemia (PMID: 7635461, 10668928, 16250003, 17539906, 19446849, 25463123, 32660911). This variant has also been observed in compound heterozygous state in individuals affected with homozygous familial hypercholesterolemia (PMID: 31947532, 32977124), indicating that this variant contributes to disease. It has been shown that this variant segregates with disease in multiple affected individuals in one family (PMID: 10668928). This variant has been identified in 8/250846 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Loss of LDLR function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Likely Pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	MGZ Medical Genetics Center	Feb 17, 2022	- -
Pathogenic, criteria provided, single submitter	clinical testing	Molecular Genetics, Royal Melbourne Hospital	Mar 30, 2023	This sequence change in LDLR is an intronic variant located in intron 10 (also described as c.1592+5G>A). The highest population minor allele frequency in gnomAD v2.1 is 0.02% (6/30,588 alleles) in the South Asian population, which is lower than the credible allele frequency for this LDLR. This variant has been reported in at least 10 probands/families meeting a clinical diagnosis of familial hypercholesterolaemia and segregates with disease in multiple families (PMID: 7635461, 10668928, 19208450, 19446849, 25463123, 31345425, 33418990; ClinVar: SCV000503370.1, SCV000583852.1). The results from multiple in silico splicing predictors (SpliceAI, MaxEntScan, NNSplice) indicate that this variant may impact splicing by disrupting the donor splice site of intron 10 of LDLR. This prediction is confirmed by RT-PCR of EBV-transformed lymphocytes. The assay demonstrated that the variant impacts splicing by causing in-frame exon 10 skipping and 66 bp insertion of intron 10 through cryptic donor site activation (PMID: 10668928, 19208450). Based on the classification scheme RMH Modified ACMG Guidelines v1.5.1, this variant is classified as PATHOGENIC. Following criteria are met: PS4, PP1_Strong, PS3_Supporting, PM2_Supporting, PP3. -

not provided Pathogenic:2 Uncertain:1

Likely pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Mar 27, 2023	Not observed at significant frequency in large population cohorts (gnomAD); While mRNA studies confirm the presence of one or more alternatively-spliced transcripts, functional studies at the protein level do not agree on the effect of this variant on receptor function (Jensen et al., 1999; Holla et al., 2009); Also known as c.1592+5 G>A; This variant is associated with the following publications: (PMID: 27578104, 27821657, 25463123, 19208450, 16250003, 17539906, 19446849, 31447099, 32793292, 17964958, 30710474, 7635461, 32977124, 32660911, 33740630, 34037665, 33418990, 34456049, 35913489, 31947532, 10668928) -
Pathogenic, no assertion criteria provided	provider interpretation	Stanford Center for Inherited Cardiovascular Disease, Stanford University	Dec 11, 2017	c.1586+5G>A in intron 10 of the LDLR gene (NM_00527; chr19-11224443-G-A) SCICD Classification: pathogenic variant based on strong case data, strong segregation data and strong functional data. 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): at least 25 cases, plus other family members. · ClinVar: § LDLR-LOVD, British Heart Foundation - likely pathogenic - 2 out of 2600 patients with FH have this variant § Centre de Génétique Moléculaire et Chromosomique, Unité de génétique de l'Obésité et des Dyslipidémies,APHP, GH Hôpitaux Universitaires Pitié-Salpêtrière / Charles-Foix - VUS § U4M - Lille University & CHRU Lille,Université Lille 2 - CHRU de Lille - likely pathogenic § Fundacion Hipercolesterolemia Familiar - VUS § Never seen at Invitae § Color Genomics has this variant in their "John Doe" report: they indicate that many algorithms predict a significant impact on the intron 10 donor · Laboratorium voor Moleculaire Diagnostiek Experimentele Vasculaire Geneeskunde,Academisch Medisch Centrum - pathogenic · Cases in the literature: · Yang et al 2007: 87 Taiwanese individuals with FH from 30 unrelated families: seen in 2 families. Authors report that this variant may result in the skipping of exon 10. This variant segregated with disease in family members; however, the number of family members with which it segregates is unknown. · Ekström et al 1994: this variant (noted as c.1596+5G>A in this paper) was detected in 1 out of 7 children with FH. · Fouchier et al 2005: 1 out of 325 patients in the Dutch cohort · Taylor et al 2007: Found this variant in one out of 400 patients with FH. This particular patient had possible FH. · Guardamagna et al 2009: This variant was seen in one patient of the following cohort: 264 children from 201 families, 148 affected parents and 100 unaffected siblings. · Mollaki et al 2014: Variant detected in 1 out of 561 patients from 262 families · Jensen et al 1999: This variant is known as c.1596+5G>A in this paper. This variant was present in a 36yo Danish man presenting with tendon xanthomas, severe CAD, CABG at 24yo and very high LDL cholesterol. This man had another variant as well - p.T383P. This variant segregated with disease in his family members (pedigree A, FH-DK 8). Another family had only this variant and it segregated with disease. From the first family, this variant segregated with 8 affected individuals. From the second family, this variant segregated in 4 individuals. Segregation data: See above, Jensen et al 1999 Functional data: Jenner et 1999: sequence analysis of cDNA indicates that either i) a cryptic splice site is activated or ii) exon 10 is skipped: i) the donor splice site at the +5 position is activated to create a cryptic splice site which results in an in-frame insertion of 22 amino acids; ii) in-frame deletion of 75 amino acids in the EGF precursor homology domain of the LDL receptor protein. The authors predicted about 50% of these variants were spliced correctly, and 50% spliced incorrectly. To measure the amount of LDL receptor protein on the surface of the cells, fluorescence was used. Fluorescence of both of these mutant cell lines was markedly reduced in both of these cell lines compared to wildtype. Splice data (splice variants only): Buratti et al 2007: "...point mutations leading to cryptic 5' splice site activation were most common in the first intron nucleotide, followed by the fifth nucleotide. Substititons at position +5 were exclusively G>A transitions... The frequency of point mutations at position +5 was significantly higher than ob -
Uncertain significance, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Apr 25, 2017	Variant summary: The LDLR c.1586+5G>A variant involves the alteration of a conserved intronic nucleotide that is 5 nucleotides away from the exon-intron junction. 4/5 splice prediction tools predict a significant impact on normal splicing and ESE finder predicts that the variant may cause an SRp55 ESE site to be introduced at the locus. One functional study has shown that the variant causes two aberrant splicing transcripts in several heterozygous patient cell lines and that the variant segregates with disease within two families, although several family members are discordant. This study also showed that LDL receptor was not properly trafficked to the cell surface in transfected COS7 cells (Jensen_CG_1999). However, a second functional study also detected aberrant splicing, but assessed LDL receptor protein function in heterozygous patient-derived lymphoblast cell lines and found that ~75% or greater of LDLR was at the cell surface and LDL was internalized similarly to controls, suggesting the variant transcripts do not have a significant biological impact (Holla_MGM_2009). Additionally, several studies have cited the variant in hypercholesterolemia patients, though cosegregation data is lacking in most cases. Based on the literature data, the variant may represent a mild mutation and may have a potentiated effect in compound heterozygosity with a truly pathogenic mutation, but have a less severe impact in heterozygous patients. This variant was found in the large control database ExAC at a frequency of 0.000034 (4/117662 control chromosomes), which does not exceed the estimated maximal expected allele frequency of a pathogenic LDLR variant (0.0010005). Multiple clinical diagnostic laboratories/reputable databases have classified this variant with conflicting interpretations, including likely pathogenic and uncertain significance. Taken together, this variant is classified as VUS until more conclusive family segregation data and/or control data can be assessed. -

Familial hypercholesterolemia Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Invitae	Oct 28, 2023	This sequence change falls in intron 10 of the LDLR gene. It does not directly change the encoded amino acid sequence of the LDLR protein. RNA analysis indicates that this variant induces altered splicing and likely results in a shortened protein product. This variant is present in population databases (rs781362878, gnomAD 0.02%). This variant has been observed in individuals with hypercholesterolemia (PMID: 7635461, 10668928, 17539906, 19208450, 19446849, 25463123). This variant is also known as c.1592+5G>A. ClinVar contains an entry for this variant (Variation ID: 251909). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in skipping of 10, but is expected to preserve the integrity of the reading-frame (PMID: 10668928, 19208450). This variant disrupts the c.1586+5 nucleotide in the LDLR gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 17964958; Invitae). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Oct 16, 2023	This variant causes a G to A nucleotide substitution at the +5 position of intron 10 of the LDLR gene. Splice site prediction tools predict that this variant may have a significant impact on RNA splicing. Molecular studies of the impact of this variant on RNA splicing demonstrated two aberrant mRNAs due to either in-frame skipping of exon 10 or the activation of a cryptic splice site in intron 10 inserting 66 intronic base pairs (PMID: 10668928). Additionally, this variant was shown to cause aberrant LDL receptor trafficking in transfected COS7 cells (PMID: 10668928). This variant has been reported in over 10 individuals affected with familial hypercholesterolemia (PMID: 7635461, 10668928, 16250003, 17539906, 19446849, 25463123, 31947532, 32660911, 32977124, 33418990, 34037665, 36960729). It has been shown that this variant segregates with disease in multiple affected individuals in one family (PMID: 10668928). This variant has been identified in 8/250846 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Loss of LDLR function is a known mechanism of disease (clinicalgenome.org). A different variant occurring at the same position, c.1586+5G>C, is known to be pathogenic (ClinVar variation ID: 440652), indicating that c.G nucleotide at this position is important for normal RNA splicing. Based on the available evidence, this variant is classified as Pathogenic. -

Homozygous familial hypercholesterolemia Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine

Aug 26, 2022

The c.1586+5G>A variant in LDLR has been reported in 7 individuals with familial hypercholesterolemia (FH) and segregated with disease in at least 7 affected relatives from 2 families (Ekstrom 1995, Jensen 1999, Fouchier 2005, Taylor 2007, Guardamagna 2009, Mollaki 2014). Additionally, this variant has been identified in 6/30752 South Asian chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/) and is present in ClinVar (Variation ID: 251909). Please note that for diseases with clinical variability, reduced penetrance, or recessive inheritance, pathogenic variants may be present at a low frequency in the general population. In vitro functional studies provide some evidence that the c.1586+5G>A variant may impact protein function (Jensen 1999. This variant is located in the 5' splice region. Computational tools do suggest an impact to splicing. In summary, although additional studies are required to fully establish its clinical significance, the c.1586+5G>A variant is likely pathogenic for FH in an autosomal dominant manner based on cased observations, segregation studies, functional and computational evidence. The ACMG/AMP Criteria applied: PP1_Strong, PS4_Moderate, PP3, PS3_Supporting. -

Cardiovascular phenotype Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jan 15, 2018

The c.1586+5G>A intronic variant results from a G to A substitution 5 nucleotides after coding exon 10 in the LDLR gene. This alteration has been detected in individuals with confirmed familial hypercholesterolemia (FH) and in FH cohorts (Fouchier SW et al. Hum. Mutat., 2005 Dec;26:550-6; Taylor A et al. Clin. Genet., 2007 Jun;71:561-8; Guardamagna O et al. J. Pediatr., 2009 Aug;155:199-204.e2; Mollaki V et al. Atherosclerosis, 2014 Dec;237:798-804). Segregation with hypercholesterolemia has been observed with this alteration (reported as c.1592+5G>A); of note, in at least one family some heterozygotes had normal or near normal cholesterol levels at the time of testing (Jensen HK et al. Clin. Genet., 1999 Nov;56:378-88). RNA splicing studies have revealed exon-skipping and use of a cryptic donor site in mutant transcripts, while functional studies demonstrated some reduction in LDLR activity and conflicting membrane-trafficking results (Jensen HK et al. Clin. Genet., 1999 Nov;56:378-88; Holla ØL et al. Mol. Genet. Metab., 2009 Apr;96:245-52). This nucleotide position is highly conserved in available vertebrate species. Using the BDGP and ESEfinder splice site prediction tools, this alteration is predicted to abolish the native splice donor site; however, direct evidence is unavailable. 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	-0.58
Cadd	Benign	23
Dann	Benign	0.71
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.99
SpliceAI score (max)		0.22		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.22		Position offset: -5

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

Publications

LitVar

Other links and lift over

dbSNP: rs781362878; hg19: chr19-11224443;