TY - JOUR
T1 - Intronic variant screening with targeted next-generation sequencing reveals first pseudoexon in LDLR in familial hypercholesterolemia
AU - Reeskamp, Laurens F.
AU - Balvers, Manon
AU - Peter, Jorge
AU - van de Kerkhof, Laura
AU - Klaaijsen, Lisette N.
AU - Motazacker, Mahdi M.
AU - Grefhorst, Aldo
AU - van Riel, Natal A. W.
AU - Hovingh, G. Kees
AU - Defesche, Joep C.
AU - Zuurbier, Linda
N1 - Funding Information: This study was funded by a ZonMW grant (VIDI No. 016.156.445 ) obtained by G.K. Hovingh. The funder (ZonMW) was not involved in the design, data collection, analysis, interpretation or any other aspect of this study. Funding Information: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: LFR is co-founder of Lipid Tools. GKH has served as consultant and speaker for biotechnology and pharmaceutical companies that develop molecules that influence lipoprotein metabolism, including Regeneron, Aege-rion Pfizer, Merck, KOWA, Sanofi, and Amgen; has served as principal investigator for clinical trials conducted with a.o. Amgen, Sanofi, Eli Lilly, Novartis, Kowa, Genzyme, Cerenis, Pfizer, Dezima, and AstraZeneca; has received research grants from ZonMW (Vidi grant [016.156.445]), Klinkerpad fonds, the European Union, Amgen, Sanofi, AstraZeneca, Aege-rion, and Synageva; has received honoraria and investigator fees (to the Department of Vascular Medicine) for sponsor-driven studies and lectures for companies with approved lipid-lowering therapy in the Netherlands; and is partly employed by Novo Nordisk AS, Copenhagen, Denmark (0.7FTE) and the Amsterdam UMC, Amsterdam, the Netherlands (0.3FTE). Funding Information: We would like to acknowledge J.F. Los for her work as a genetic field worker involved in the expansion of the included family and we would like to thank all participants in this study for their participation. This work was partly funded by a grant from ZonMW (Vidi grant [016.156.445]). Publisher Copyright: © 2021 The Author(s)
PY - 2021/3/1
Y1 - 2021/3/1
N2 - Background and aims: Familial hypercholesterolemia (FH) is caused by pathogenic variants in LDLR, APOB, or PCSK9 genes (designated FH+). However, a significant number of clinical FH patients do not carry these variants (designated FH-). Here, we investigated whether variants in intronic regions of LDLR attribute to FH by affecting pre-mRNA splicing. Methods: LDLR introns are partly covered in routine sequencing of clinical FH patients using next-generation sequencing. Deep intronic variants, >20 bp from intron-exon boundary, were considered of interest once (a) present in FH- patients (n = 909) with LDL-C >7 mmol/L (severe FH-) or after in silico analysis in patients with LDL-C >5 mmol/L (moderate FH-) and b) absent in FH + patients (control group). cDNA analysis and co-segregation analysis were performed to assess pathogenicity of the identified variants. Results: Three unique variants were present in the severe FH- group. One of these was the previously described likely pathogenic variant c.2140+103G>T. Three additional variants were selected based on in silico analyses in the moderate FH- group. One of these variants, c.2141-218G>A, was found to result in a pseudo-exon inclusion, producing a premature stop codon. This variant co-segregated with the hypercholesterolemic phenotype. Conclusions: Through a screening approach, we identified a deep intronic variant causal for FH. This finding indicates that filtering intronic variants in FH- patients for the absence in FH + patients might enrich for true FH-causing variants and suggests that intronic regions of LDLR need to be considered for sequencing in FH- patients.
AB - Background and aims: Familial hypercholesterolemia (FH) is caused by pathogenic variants in LDLR, APOB, or PCSK9 genes (designated FH+). However, a significant number of clinical FH patients do not carry these variants (designated FH-). Here, we investigated whether variants in intronic regions of LDLR attribute to FH by affecting pre-mRNA splicing. Methods: LDLR introns are partly covered in routine sequencing of clinical FH patients using next-generation sequencing. Deep intronic variants, >20 bp from intron-exon boundary, were considered of interest once (a) present in FH- patients (n = 909) with LDL-C >7 mmol/L (severe FH-) or after in silico analysis in patients with LDL-C >5 mmol/L (moderate FH-) and b) absent in FH + patients (control group). cDNA analysis and co-segregation analysis were performed to assess pathogenicity of the identified variants. Results: Three unique variants were present in the severe FH- group. One of these was the previously described likely pathogenic variant c.2140+103G>T. Three additional variants were selected based on in silico analyses in the moderate FH- group. One of these variants, c.2141-218G>A, was found to result in a pseudo-exon inclusion, producing a premature stop codon. This variant co-segregated with the hypercholesterolemic phenotype. Conclusions: Through a screening approach, we identified a deep intronic variant causal for FH. This finding indicates that filtering intronic variants in FH- patients for the absence in FH + patients might enrich for true FH-causing variants and suggests that intronic regions of LDLR need to be considered for sequencing in FH- patients.
KW - Familial hypercholesterolemia
KW - Intron
KW - LDL cholesterol
KW - LDL receptor
KW - Next-generation sequencing
KW - RNA splicing
UR - http://www.scopus.com/inward/record.url?scp=85101036348&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.atherosclerosis.2021.02.003
DO - https://doi.org/10.1016/j.atherosclerosis.2021.02.003
M3 - Article
C2 - 33601267
SN - 0021-9150
VL - 321
SP - 14
EP - 20
JO - Atherosclerosis
JF - Atherosclerosis
ER -