rs781362878
Variant summary
Our verdict is Uncertain significance. The variant received 2 ACMG points: 2P and 0B. PS3_Moderate
This summary comes from the ClinGen Evidence Repository: The NM_000527.5(LDLR):c.1586+5G>A variant is classified as Uncertain significance - insufficient evidence for Familial Hypercholesterolemia by applying ACMG/AMP evidence code PS3_Moderate as defined by the ClinGen Familial Hypercholesterolemia Expert Panel LDLR-specific variant curation guidelines (specification version 1.2) on 23 February 2024. The supporting evidence is as follows:PS3_Moderate:-Level 2, PMID 10668928 (Jensen et al., 1999), heterologous cells (COS-7), FACS and CLSM assays, mutant protein retained at ER; <2% LDLR at surface, 2-5% LDL uptake. Overall LDLR activity is below 70% of wild-type activity.-Level 3, PMID 19208450 (Holla et al., 2009), heterozygous patients' Epstein Barr virus transformed lymphocytes, RNA assays showed skipping of exon 10 (p.Thr454_Gly529del). -Level 3, PMID 10668928 (Jensen et al., 1999), heterozygous patients' Epstein Barr virus transformed lymphocytes, quantitative RT-PCR assays. Mutated transcripts were 47% of total transcripts; from mutated allele: 50% correctly spliced RNA, skipping of exon 10 (p.Thr454_Gly529del) in 25% of RNAs and retention of the first 66 nucleotides from intron 10 (p.Gly529_Phe530insCysValSerThrThrLeuArgAlaAlaGluGlyMetGluGlyAlaGlyArgSerPheArgAsnCys) in 25% of RNAs. LINK:https://erepo.genome.network/evrepo/ui/classification/CA035154/MONDO:0007750/013
Frequency
Consequence
NM_000527.5 splice_region, intron
Scores
Clinical Significance
Conservation
Publications
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ACMG classification
Our verdict: Uncertain_significance. The variant received 2 ACMG points.
Variant Effect in Transcripts
ACMG analysis was done for transcript: NM_000527.5. You can select a different transcript below to see updated ACMG assignments.
RefSeq Transcripts
| Selected | Gene | Transcript | Tags | HGVSc | HGVSp | Effect | Exon Rank | Protein | UniProt |
|---|---|---|---|---|---|---|---|---|---|
| LDLR | NM_000527.5 | MANE Select | c.1586+5G>A | splice_region intron | N/A | NP_000518.1 | |||
| LDLR | NM_001195798.2 | c.1586+5G>A | splice_region intron | N/A | NP_001182727.1 | ||||
| LDLR | NM_001195799.2 | c.1463+5G>A | splice_region intron | N/A | NP_001182728.1 |
Ensembl Transcripts
| Selected | Gene | Transcript | Tags | HGVSc | HGVSp | Effect | Exon Rank | Protein | UniProt |
|---|---|---|---|---|---|---|---|---|---|
| LDLR | ENST00000558518.6 | TSL:1 MANE Select | c.1586+5G>A | splice_region intron | N/A | ENSP00000454071.1 | |||
| LDLR | ENST00000252444.10 | TSL:1 | c.1844+5G>A | splice_region intron | N/A | ENSP00000252444.6 | |||
| LDLR | ENST00000558013.5 | TSL:1 | c.1586+5G>A | splice_region intron | N/A | ENSP00000453346.1 |
Frequencies
GnomAD3 genomes
GnomAD2 exomes AF: 0.0000319 AC: 8AN: 250846 AF XY: 0.0000369 show subpopulations
GnomAD4 exome AF: 0.0000234 AC: 34AN: 1452200Hom.: 1 Cov.: 31 AF XY: 0.0000277 AC XY: 20AN XY: 722982 show subpopulations
Age Distribution
GnomAD4 genome
ClinVar
Submissions by phenotype
Hypercholesterolemia, familial, 1 Pathogenic:8Uncertain:5
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.
subjects mutated among 2600 FH index cases screened = 2
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.
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.
The NM_000527.5(LDLR):c.1586+5G>A variant is classified as Uncertain significance - insufficient evidence for Familial Hypercholesterolemia by applying ACMG/AMP evidence code PS3_Moderate as defined by the ClinGen Familial Hypercholesterolemia Expert Panel LDLR-specific variant curation guidelines (specification version 1.2) on 23 February 2024. The supporting evidence is as follows: PS3_Moderate: -Level 2, PMID 10668928 (Jensen et al., 1999), heterologous cells (COS-7), FACS and CLSM assays, mutant protein retained at ER; <2% LDLR at surface, 2-5% LDL uptake. Overall LDLR activity is below 70% of wild-type activity. -Level 3, PMID 19208450 (Holla et al., 2009), heterozygous patients' Epstein Barr virus transformed lymphocytes, RNA assays showed skipping of exon 10 (p.Thr454_Gly529del). -Level 3, PMID 10668928 (Jensen et al., 1999), heterozygous patients' Epstein Barr virus transformed lymphocytes, quantitative RT-PCR assays. Mutated transcripts were 47% of total transcripts; from mutated allele: 50% correctly spliced RNA, skipping of exon 10 (p.Thr454_Gly529del) in 25% of RNAs and retention of the first 66 nucleotides from intron 10 (p.Gly529_Phe530insCysValSerThrThrLeuArgAlaAlaGluGlyMetGluGlyAlaGlyArgSerPheArgAsnCys) in 25% of RNAs.
A heterozygous 5’ splice site proximal variant in intron 10 of the LDLR gene that affects the position 5 nucleotides downstream of exon 10 was detected. The observed variant c.1586+5G>A has not been reported in the 1000 genomes, gnomAD (v3.1) and gnomAD (v2.1) databases and has a minor allele frequency of 0.0004% in the topmed database. The reference base is conserved across species. In summary, the variant meets our criteria to be classified as a variant of uncertain significance.
not provided Pathogenic:3
The LDLR c.1586+5G>A variant has been reported in the published literature in individuals affected with familial hypercholesterolemia (PMIDs: 33418990 (2021), 32977124 (2020), 31947532 (2020), 31345425 (2019)). Additionally, functional evidence suggests that this variant may impact protein function (PMIDs: 19208450 (2009), 10668928 (1999)). The frequency of this variant in the general population, 0.0002 (6/30588 chromosomes (Genome Aggregation Database, http://gnomad.broadinstitute.org)), is uninformative in the assessment of its pathogenicity. Analysis of this variant using software algorithms for the prediction of the effect of nucleotide changes on splicing yielded predictions that this variant may affect proper LDLR mRNA splicing. Based on the available information, this variant is classified as pathogenic.
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
Not observed at significant frequency in large population cohorts (gnomAD); 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, 32977124, 32660911, 33740630, 34037665, 33418990, 34456049, 35913489, 7635461, 10668928, 33955087, 37589137, 31947532, 36960729, 31345425, 28964736)
Familial hypercholesterolemia Pathogenic:3
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.
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). Invitae Evidence Modeling of clinical and family history, age, sex, and reported ancestry of multiple individuals with this LDLR variant has been performed. This variant is expected to be pathogenic with a positive predictive value of at least 99%. This is a validated machine learning model that incorporates the clinical features of 363,995 individuals referred to our laboratory for LDLR testing. 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+5G nucleotide in the LDLR gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 17964958; internal data). 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.
Variant summary: LDLR c.1586+5G>A, also reported as "1592+5G>A", 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: Four predict the variant abolishes a 5' splicing donor site. At least one publication reports experimental evidence that this variant affects mRNA splicing in patient cells, resulting in an in-frame deletion of exon 10 (contains pathogenic missense) and an in-frame insertion of 22 amino acids (example, Jensen_1999). The variant allele was found at a frequency of 2.1e-05 in 1604438 control chromosomes in the gnomAD database, including 1 homozygotes. This frequency is not significantly higher than estimated for a pathogenic variant in LDLR causing Familial Hypercholesterolemia (2.1e-05 vs 0.0013). c.1586+5G>A has been reported in the heterozygous state in the literature in multiple individuals affected with Familial Hypercholesterolemia (example, Jensen_1999). 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. The most pronounced variant effect results in <10% of wild type receptor surface expression in vitro (example, Jensen_1999). The following publications have been ascertained in the context of this evaluation (PMID: 10668928, 19208450). ClinVar contains an entry for this variant (Variation ID: 251909). Based on the evidence outlined above, the variant was classified as pathogenic.
Homozygous familial hypercholesterolemia Pathogenic:1
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
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:
Splicing
Find out detailed SpliceAI scores and Pangolin per-transcript scores at