NM_000527.5:c.313+1G>A
Variant summary
Our verdict is Pathogenic. Variant got 17 ACMG points: 17P and 0B. PM2PS3_ModeratePP4PP1_StrongPVS1_StrongPS4
This summary comes from the ClinGen Evidence Repository: The NM_000527.4(LDLR):c.313+1G>A variant is classified as Pathogenic for Familial Hypercholesterolemia by applying evidence codes (PVS1_Strong, PS4, PP1_Strong, PM2, PS3_Moderate and PP4) as defined by the ClinGen Familial Hypercholesterolemia Expert Panel LDLR-specific variant curation guidelines (https://doi.org/10.1101/2021.03.17.21252755). The supporting evidence is as follows: PVS1_strong - Variant is in canonical GT +1 splice site, predicted to lead to exon 3 skipping (in frame).PS4 - Variant meets PM2. Identified in at least 14 unrelated index cases from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA) with DLCS equal or above 6. PP1_strong - variant segregates with FH phenotype in 6 informative meiosis in 5 families from different laboratories. PM2 - PopMax MAF = 0.00006156 (0.006%) in European non-Finnish exomes (gnomAD v2.1.1).PS3_moderateLevel 2 assays: PMID 19361455: Hmz patients' Epstein-Barr tranformed lymphocytes, RNA and FACS assays - results - Alternative splicing: skipping of exon 3 or inclusion of intron 3; amount of cell-surface LDLR 33% of normal (Hmz); 12% LDL-LDLR uptake in Hmz ---- Aberrant transcripts confirmed by sequencing and above 25% of total transcript, and uptake is below 70% of wild-type, so PS3_moderate is Met.PP4 - Variant meets PM2. Identified in 3 FH cases from Cardiovascular Research Group,Instituto Nacional de Saude Doutor Ricardo Jorge who fulfill Simon-Broome criteria and in 13 FH cases from University of British Columbia (UBC, Canada) and 14 from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA) with clinical Dutch Lipid Clinic Network Criteria score ≥ 6. LINK:https://erepo.genome.network/evrepo/ui/classification/CA023688/MONDO:0007750/013
Frequency
Consequence
NM_000527.5 splice_donor, intron
Scores
Clinical Significance
Conservation
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ACMG classification
Verdict is Pathogenic. Variant got 17 ACMG points.
Transcripts
RefSeq
| Gene | Transcript | HGVSc | HGVSp | Effect | Exon rank | MANE | Protein | UniProt |
|---|---|---|---|---|---|---|---|---|
| LDLR | NM_000527.5 | c.313+1G>A | splice_donor_variant, intron_variant | Intron 3 of 17 | ENST00000558518.6 | NP_000518.1 |
Ensembl
Frequencies
GnomAD3 genomes 0.0000131 AC: 2AN: 152184Hom.: 0 Cov.: 31
GnomAD3 exomes AF: 0.0000278 AC: 7AN: 251424Hom.: 0 AF XY: 0.0000441 AC XY: 6AN XY: 135902
GnomAD4 exome AF: 0.0000274 AC: 40AN: 1460880Hom.: 0 Cov.: 31 AF XY: 0.0000261 AC XY: 19AN XY: 726744
GnomAD4 genome 0.0000131 AC: 2AN: 152184Hom.: 0 Cov.: 31 AF XY: 0.0000135 AC XY: 1AN XY: 74334
ClinVar
Submissions by phenotype
Hypercholesterolemia, familial, 1 Pathogenic:24Benign:1
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The splice site variant c.313+1G>A in LDLR gene has been reported previously in heterozygous state in individuals affected with Familial hypercholesterolemia (Trinder M, et al., 2019, Zakharova FM, et al., 2005). Experimental evidence shows that this variant results in two mutant transcripts, one has skipping of exon 3 and the other has inclusion of intron 3. The transcript with skipping of exon 3 was demonstrated to have considerably reduced cell-surface LDLR and total amounts of internalized LDL, indicating that the variant LDLR is markedly dysfunctional (Cameron J, et al., 2009). The variant has 0.003% allele frequency in gnomAD Exomes and is novel (not in any individuals) in 1000 Genomes. It has been submitted to ClinVar as Likely Benign, Likley Pathogenic, Pathogenic (multiple submissions).This variant is predicted to cause loss of normal protein function through protein truncation. Loss of function variants have been previously reported to be disease causing. For these reasons, this variant has been classified as Pathogenic. -
ACMG classification criteria: PVS1 strong, PS3 supporting, PS4 strong, PM2 moderate, PP1 strong, PP4 supporting -
The c.313+1G>A variant in LDLR gene destroys the canonical splice donor site in intron 3 and is predicted to result in abnormal splicing of the LDLR message. Functional studies have shown that the c.313+1G>A variant disrupts mRNA splicing and produces a protein with abnormal function (PMID 19361455). This variant has been reported in multiple unrelated individuals with hypercholesterolemia (PMID 7718019, 22390909, 22883975, 20045108, 20145306, 15523646) and individuals with early-onset myocardial infarction (PMID 25487149). This variant is classified as pathogenic. -
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The NM_000527.4(LDLR):c.313+1G>A variant is classified as Pathogenic for Familial Hypercholesterolemia by applying evidence codes (PVS1_Strong, PS4, PP1_Strong, PM2, PS3_Moderate and PP4) as defined by the ClinGen Familial Hypercholesterolemia Expert Panel LDLR-specific variant curation guidelines (https://doi.org/10.1101/2021.03.17.21252755). The supporting evidence is as follows: PVS1_strong - Variant is in canonical GT +1 splice site, predicted to lead to exon 3 skipping (in frame). PS4 - Variant meets PM2. Identified in at least 14 unrelated index cases from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA) with DLCS equal or above 6. PP1_strong - variant segregates with FH phenotype in 6 informative meiosis in 5 families from different laboratories. PM2 - PopMax MAF = 0.00006156 (0.006%) in European non-Finnish exomes (gnomAD v2.1.1). PS3_moderate Level 2 assays: PMID 19361455: Hmz patients' Epstein-Barr tranformed lymphocytes, RNA and FACS assays - results - Alternative splicing: skipping of exon 3 or inclusion of intron 3; amount of cell-surface LDLR 33% of normal (Hmz); 12% LDL-LDLR uptake in Hmz ---- Aberrant transcripts confirmed by sequencing and above 25% of total transcript, and uptake is below 70% of wild-type, so PS3_moderate is Met. PP4 - Variant meets PM2. Identified in 3 FH cases from Cardiovascular Research Group,Instituto Nacional de Saude Doutor Ricardo Jorge who fulfill Simon-Broome criteria and in 13 FH cases from University of British Columbia (UBC, Canada) and 14 from Cardiovascular Genetics Laboratory (PathWest Laboratory Medicine WA) with clinical Dutch Lipid Clinic Network Criteria score ≥ 6. -
PVS1_strong, PP1_strong, PS4, PM2, PP4, PS3_moderate -
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0/208 non-FH alleles; 0/100 healthy control individuals -
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The c.313+1G>A variant in the LDLR gene is located at the canonical splice site of intron 3 and is predicted to result in abnormal splicing, leading to an absent or disrupted protein product. This variant has been identified in 14 unrelated index cases and it segregates with familial hypercholesterolemia phenotype in 6 informative meiosis in 5 families from different laboratories according to ClinGen Familial Hypercholesterolemia Variant Curation Expert Panel. Additionally, in a study focusing on the Norwegian population, this variant has been identified as one of the most prevalent variants identified in individuals with autosomal dominant hypercholesterolemia (PMID: 33740630). Experimental study of this variant has confirmed the skipping of exon 3 and reduced expression of LDLR (33% of wild-type) on the cell surface (PMID: 19361455). Experiment with heterozygous patient lymphocytes proved the defective LDL uptake (<20%) in comparison to the wild-type cells (PMID: 19148831). The variant is reported in ClinVar (ID: 3736) and evaluated as pathogenic by the ClinGen Expert Panel. The variant is rare in the general population according to gnomAD (7/251424). Therefore, the c.313+1G>A variant of LDLR has been classified as pathogenic. -
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subjects mutated among 2600 FH index cases screened = 4/FH-Europe -
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The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.003%). Canonical splice site is predicted to alter splicing and result in a loss or disruption of normal protein function. Multiple pathogenic loss-of-function variants are reported downstream of the variant. Functional studies provide moderate evidence of the variant having a damaging effect on the gene or gene product (PMID: 19361455). The variant has been reported multiple times as an established pathogenic variant (ClinVar ID: VCV000003736). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline. -
not provided Pathogenic:7Other:1
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This variant disrupts a canonical splice-donor site and interferes with normal LDLR mRNA splicing (PMID: 19361455 (2009)). The frequency of this variant in the general population, 0.000062 (7/113710 chromosomes, http://gnomad.broadinstitute.org), is consistent with pathogenicity. In the published literature, the variant has been reported in multiple individuals with familial hypercholesterolemia (FH) (PMID: 25487149 (2015), 22390909 (2012), 20045108 (2010), 14974088 (2004), 7616128 (1995)), and has been shown to segregate with disease (PMID: 8829662 (1996)). Based on the available information, this variant is classified as pathogenic. -
PP1, PM2, PS3_moderate, PS4, PVS1_strong -
Described as a founder mutation in the Norwegian population (Leren et al., 1994); Published functional studies demonstrate that c.313+1 G>A leads to the production of two abnormal transcripts which impair LDL receptor function (Cameron et al., 1999); Reported in ClinVar but additional evidence is not available (ClinVar Variant ID#3736; ClinVar); Not observed at significant frequency in large population cohorts (gnomAD); Deletions involving coding exons of this gene are a known mechanism of disease (HGMD; other references); This variant is associated with the following publications: (PMID: 25525159, 25487149, 10735632, 22883975, 30592178, 30795984, 31048103, 22390909, 10634824, 17765246, 7616128, 20145306, 20045108, 10532689, 14974088, 7718019, 25468658, 29233637, 31447099, 8911609, 32041611, 33303402, 32719484, 33226606, 33740630, 34037665, 19361455) -
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The LDLR c.313+1G>A variant (rs112029328) is a Norwegian founder variant reported in the literature in numerous heterozygous, homozygous, and compound heterozygous individuals affected with hypercholesterolemia (Chan 2019, Di Taranto 2021, Ibarretxe 2018, Leren 2021). This variant is found in the non-Finnish European population with an allele frequency of 0.006% (7/113,710 alleles) in the Genome Aggregation Database (v2.1.1). This variant disrupts the canonical splice donor site of intron 3, which is likely to negatively impact gene function. Functional studies in patient-derived cells indicate this variant results in several aberrant transcripts and is associated with reduced levels of LDLR protein at the cell surface and overall (Cameron 2009). Based on available information, this variant is considered to be pathogenic. References: Cameron J et al. Splice-site mutation c.313+1, G>A in intron 3 of the LDL receptor gene results in transcripts with skipping of exon 3 and inclusion of intron 3. Clin Chim Acta. 2009 May;403(1-2):131-5. PMID: 19361455. Chan M et al. Genetic variations in familial hypercholesterolemia and cascade screening in East Asians. Mol Genet Genomic Med. 2019 Feb;7(2):e00520. PMID: 30592178. Di Taranto MD et al. Genetic spectrum of familial hypercholesterolemia and correlations with clinical expression: Implications for diagnosis improvement. Clin Genet. 2021 Nov;100(5):529-541. PMID: 34297352. Ibarretxe D et al. Detecting familial hypercholesterolemia earlier in life by actively searching for affected children:The DECOPIN project. Atherosclerosis. 2018 Nov;278:210-216. PMID: 30312929. Leren TP and Bogsrud MP. Molecular genetic testing for autosomal dominant hypercholesterolemia in 29,449 Norwegian index patients and 14,230 relatives during the years 1993-2020. Atherosclerosis. 2021 Apr;322:61-66. PMID: 33740630. -
Genetic testing: The patient had genetic testing for the familial hypercholesterolemia panel. The test included sequencing of three genes associated with familial hypercholesterolemia: LDLR, APOB and PCSK9. Results reported on October 17, 2017 showed that the following variant was identified (see report below): c.313+1G>A in the LDLR gene (NM_000527.4) Ambry classifies this variant as pathogenic. Given sufficient case data and functional in vitro studies, we consider this variant very likely disease causing and we feel it is suitable for assessing risk in healthy relatives ("predictive genetic testing"). This variant is a common cause of FH around the world. It is referred to as the FH-Elverum allele and the FH-Olbia allele. It's been identified in multiple populations around the world including Austria, Belgium, Denmark, Germany, Italy, Spain, Korea, Norway, The Netherlands, Russia, UK, Sweden, and South Africa (black). It's been identified in up to 25% of Norwegian FH patients and 5% of Western Australian patients. IT has also been reported in a case of homozygous FH. It has been written about in 14 publications and is listed as pathogenic or likely pathogenic by all seven entries in clinvar. An in vitro study found this mutation leads to several alternatively spliced transcripts, one of which skips exon 3 and can result in an abnormal LDLR protein with reduced function (Cameron et al. Clin Chim Acta. 2009. 403(1-2):13-15). This variant is located near a CpG enriched region which may be a mutational hot spot. This variant likely leads to loss of function of the LDL-receptor given its exon skipping nature. There are seven individuals with variation at c.313+1 position listed in the Genome Aggregation Consortium Dataset (gnomAD; http://gnomad.broadinstitute.org/), which currently includes variant calls on 126,184 unrelated individuals at this position who are of African, Asian, European, Latino, and Ashkenazi descent. All 7 individuals are of European descent. This corresponds to about 1 in 8,015 European individuals in the general population which confirms that it's likely a common cause of FH in the European populations. -
Familial hypercholesterolemia Pathogenic:4
This variant (also known as FH-Elverum) causes a G to A nucleotide substitution at the +1 position of intron 3 of the LDLR gene, and is predicted to abolish the intron 3 canonical splice donor site. An experimental study has shown that this variant causes skipping of exon 3 and inclusion of intron 3, and no functional LDLR protein was produced from the mutant transcript (PMID: 19361455). The variant has been reported in over 500 individuals affected with familial hypercholesterolemia (PMID: 7616128, 7718019, 10532689, 14974088, 15241806, 16159606, 17539906, 17765246, 20045108, 20145306, 22883975, 28502510, 29233637, 29353225, 30795984, 33740630, 34037665) and is considered to be a founder mutation in the Norwegian population (PMID: 7718019, 33740630). In addition, a large case-control study has shown that this variant is associated with early-onset myocardial infarction (PMID: 25487149). This variant has been identified in 7/251424 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 Pathogenic. -
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Variant summary: LDLR c.313+1G>A is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Four predict the variant abolishes a 5 splicing donor site. Experimental evidence supports these predictions indicating that this variant results in two mutant transcripts, one has skipping of exon 3 and the other has inclusion of intron 3 (Cameron_2009, Jensen_1996). The transcript with skipping of exon 3 was demonstrated to have considerably reduced cell-surface LDLR and total amounts of internalized LDL, indicating that the variant LDLR is markedly dysfunctional (Cameron_2009). The variant allele was found at a frequency of 2.8e-05 in 251424 control chromosomes (gnomAD). c.313+1G>A has been reported in the literature in multiple individuals affected with Familial Hypercholesterolemia (e.g. Deiana_2000, Jensen_1996, Mozas_2004, Leren_1994). These data indicate that the variant is very likely to be associated with disease. Fourteen ClinVar submitters (evaluation after 2014) cite the variant as pathogenic/likely pathogenic (n=13) and as likely benign (n=1). Based on the evidence outlined above, the variant was classified as pathogenic. -
This sequence change affects a donor splice site in intron 3 of the LDLR gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in a shortened protein product. This variant is present in population databases (rs112029328, gnomAD 0.006%). Disruption of this splice site has been observed in individuals with familial hypercholesterolemia (PMID: 10532689, 15701167, 19361455). It has also been observed to segregate with disease in related individuals. 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. ClinVar contains an entry for this variant (Variation ID: 3736). Studies have shown that disruption of this splice site results in skipping of exon 3, but is expected to preserve the integrity of the reading-frame (PMID: 19361455). For these reasons, this variant has been classified as Pathogenic. -
Homozygous familial hypercholesterolemia Pathogenic:1
The c.313+1G>A variant in LDLR has been reported in >140 individuals with familial hypercholesterolemia (FH) and segregated with disease in at least 5 affected relatives from 2 families (Leren 1994, Sun 1995, Lombardi 2000, Hooper 2012). This variant has also been identified in 7/111670 European chromosomes by gnomAD (http://gnomad.broadinstitute.org). This frequency is low enough to be consistent with the frequency of FH in the general population. The c.313+1G>A variant occurs in the invariant region (+/- 1,2) of the splice consensus sequence and is predicted to cause altered splicing that would preserve the protein reading frame, leading to an abnormal protein. Furthermore, in vitro functional studies provide some evidence that this variant may impact protein function (Sun 1995, Cameron 2009). In summary, this variant meets criteria to be classified as pathogenic for FH in an autosomal dominant manner based upon presence in multiple affected individuals, segregation studies, low frequency in the general population, and impact to the protein. The ACMG/AMP Criteria applied: PS4, PM2, PP1_Moderate, PVS1_Moderate. -
LDLR-related disorder Pathogenic:1
The LDLR c.313+1G>A variant is predicted to disrupt the GT donor site and interfere with normal splicing. This variant is a well-documented pathogenic variant for familial hypercholesterolemia (see for example Chan et al. 2018. PubMed ID: 30592178; Luirink et al. 2018. PubMed ID: 30795984; Jensen et al. 1999. PubMed ID: 10532689; Lombardi et al. 2000. PubMed ID: 10735632; Chmara et al. 2010. PubMed ID: 20145306; Mozas et al. 2004. PubMed ID: 15241806; Alonso et al. 2009. PubMed ID: 19318025; Lombardi et al. 1995. PubMed ID: 7616128). This variant is reported in 0.0062% of alleles in individuals of European (Non-Finnish) descent in gnomAD. Variants that disrupt the consensus splice donor site in LDLR are expected to be pathogenic. This variant is interpreted as pathogenic. -
See cases Pathogenic:1
ACMG categories: PVS1,PM2,PP5 -
Cardiovascular phenotype Pathogenic:1
The c.313+1G>A intronic variant results from a G to A substitution one nucleotide after coding exon 3 of the LDLR gene. Alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. Based on data from gnomAD, the A allele has an overall frequency of 0.003% (7/251424) total alleles studied. The highest observed frequency was 0.006% (7/113710) of European (non-Finnish) alleles. This mutation has been identified in both heterozygous and homozygous states in patients with classic familial hypercholesterolemia (FH) (Leren, 1994; Hartgers, 2018; Ibarretxe, 2018; Chan, 2019). This nucleotide position is highly conserved in available vertebrate species. An in vitro study found this mutation leads to several alternatively spliced transcripts, one of which skips exon 3 and can result in an abnormal LDLR protein with reduced function (Cameron, 2009). In silico splice site analysis predicts that this alteration will weaken the native splice donor site. Based on the available evidence, this alteration is classified as pathogenic. -
Computational scores
Source:
Splicing
Find out detailed SpliceAI scores and Pangolin per-transcript scores at