BACKGROUND: Large deletions and duplications within the low-density lipoprotein receptor (LDLR) gene make up approximately 10% of LDLR pathogenic variants found in Czech patients with familial hypercholesterolemia. The goal of this study was to test the hypothesis that all probands with each rearrangement share identical breakpoints inherited from a common ancestor and to determine the role of Alu repetitive elements in the generation of these rearrangements. METHODS: The breakpoint sequence was determined by PCR amplification and Sanger sequencing. To confirm the breakpoint position, an NGS analysis was performed. Haplotype analysis of common LDLR variants was performed using PCR and Sanger sequencing. RESULTS: The breakpoints of 8 rearrangements within the LDLR gene were analysed, including the four most common LDLR rearrangements in the Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1-4 probands). Probands with a specific rearrangement shared identical breakpoint positions and haplotypes associated with the rearrangement, suggesting a shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 6 out of 8 breakpoints, there was high homology (≥ 70%) between the two Alu repeats in which the break occurred. CONCLUSIONS: The most common rearrangements of the LDLR gene in the Czech population likely arose from one mutational event. Alu elements likely played a role in the generation of the majority of rearrangements inside the LDLR gene.

Centre for Cardiovascular Surgery and Transplantation Pekařská 53 656 91 Brno Czech Republic

Centre of Molecular Biology and Genetics University Hospital Brno Jihlavská 20 Brno 625 00 Czech Republic

Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 Brno 625 00 Czech Republic

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Beheshti SO, Madsen CM, Varbo A, Nordestgaard BG. Worldwide Prevalence of Familial Hypercholesterolemia. J Am Coll Cardiol. 2020;75(20):2553–2566. doi: 10.1016/j.jacc.2020.03.057. PubMed DOI

Hu P, Dharmayat KI, Stevens CAT, Sharabiani MTA, Jones RS, Watts GF, Genest J, Ray KK, Vallejo-Vaz AJ. Prevalence of Familial Hypercholesterolemia Among the General Population and Patients With Atherosclerotic Cardiovascular Disease: A Systematic Review and Meta-Analysis. Circulation. 2020;141(22):1742–1759. doi: 10.1161/CIRCULATIONAHA.119.044795. PubMed DOI

Yu Y, Chen L, Zhang H, Fu Z, Liu Q, Zhao H, Liu Y, Chen Y. Association Between Familial Hypercholesterolemia and Risk of Cardiovascular Events and Death in Different Cohorts A Meta-Analysis of 1.1 Million Subjects Front Cardiovasc Med. 20229860196 doi 10.3389/fcvm.2022.860196 PubMed PMC

Slack J. Risks of ischaemic heart-disease in familial hyperlipoproteinaemic states. Lancet. 1969;2(7635):1380–1382. doi: 10.1016/s0140-6736(69)90930-1. PubMed DOI

Stone NJ, Levy RI, Fredrickson DS, Verter J. Coronary artery disease in 116 kindred with familial type II hyperlipoproteinemia. Circulation. 1974;49(3):476–488. doi: 10.1161/01.cir.49.3.476. PubMed DOI

Cuchel M, Raal FJ, Hegele RA, Al-Rasadi K, Arca M, Averna M, et al. 2023 Update on European Atherosclerosis Society Consensus Statement on Homozygous Familial Hypercholesterolaemia: new treatments and clinical guidance. Eur Heart J. 2023;44(25):2277–2291. doi: 10.1093/eurheartj/ehad197. PubMed DOI PMC

McGowan MP, Hosseini Dehkordi SH, Moriarty PM, Duell PB. Diagnosis and Treatment of Heterozygous Familial Hypercholesterolemia. J Am Heart Assoc. 2019;8(24):e013225. doi: 10.1161/JAHA.119.013225. PubMed DOI PMC

Tichý L, Fajkusová L, Zapletalová P, Schwarzová L, Vrablík M, Freiberger T. Molecular genetic background of an autosomal dominant hypercholesterolemia in the Czech Republic. Physiol Res. 2017;66(Suppl 1):S47–S54. doi: 10.33549/physiolres.933587. PubMed DOI

Bertolini S, Pisciotta L, Rabacchi C, Cefalù AB, Noto D, Fasano T, Signori A, Fresa R, Averna M, Calandra S. Spectrum of mutations and phenotypic expression in patients with autosomal dominant hypercholesterolemia identified in Italy. Atherosclerosis. 2013;227(2):342–348. doi: 10.1016/j.atherosclerosis.2013.01.007. PubMed DOI

van der Graaf A, Avis HJ, Kusters DM, Vissers MN, Hutten BA, Defesche JC, Huijgen R, Fouchier SW, Wijburg FA, Kastelein JJ, Wiegman A. Molecular basis of autosomal dominant hypercholesterolemia: assessment in a large cohort of hypercholesterolemic children. Circulation. 2011;123(11):1167–1173. doi: 10.1161/CIRCULATIONAHA.110.979450. PubMed DOI

Hobbs HH, Russell DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet. 1990;24:133–170. doi: 10.1146/annurev.ge.24.120190.001025. PubMed DOI

Amsellem S, Briffaut D, Carrié A, Rabès JP, Girardet JP, Fredenrich A, Moulin P, Krempf M, Reznik Y, Vialettes B, de Gennes JL, Brukert E, Benlian P. Intronic mutations outside of Alu-repeat-rich domains of the LDL receptor gene are a cause of familial hypercholesterolemia. Hum Genet. 2002;111(6):501–510. doi: 10.1007/s00439-002-0813-4. PubMed DOI

Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001;409(6846):860–921. doi: 10.1038/35057062. PubMed DOI

Houck CM, Rinehart FP, Schmid CW. A ubiquitous family of repeated DNA sequences in the human genome. J Mol Biol. 1979;132(3):289–306. doi: 10.1016/0022-2836(79)90261-4. PubMed DOI

Ullu E, Tschudi C. Alu sequences are processed 7SL RNA genes. Nature. 1984;312(5990):171–172. doi: 10.1038/312171a0. PubMed DOI

Batzer MA, Deininger PL. Alu repeats and human genomic diversity. Nat Rev Genet. 2002;3(5):370–379. doi: 10.1038/nrg798. PubMed DOI

Lehrman MA, Schneider WJ, Südhof TC, Brown MS, Goldstein JL, Russell DW. Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains. Science. 1985;227(4683):140–146. doi: 10.1126/science.3155573. PubMed DOI PMC

Gu W, Zhang F, Lupski JR. Mechanisms for human genomic rearrangements. Pathogenetics. 2008;1(1):4. doi: 10.1186/1755-8417-1-4. PubMed DOI PMC

de Smith AJ, Walters RG, Coin LJ, Steinfeld I, Yakhini Z, Sladek R, Froguel P, Blakemore AI. Small deletion variants have stable breakpoints commonly associated with alu elements. PLoS ONE. 2008;3(8):e3104. doi: 10.1371/journal.pone.0003104. PubMed DOI PMC

Boone PM, Yuan B, Campbell IM, Scull JC, Withers MA, Baggett BC, Beck CR, Shaw CJ, Stankiewicz P, Moretti P, Goodwin WE, Hein N, Fink JK, Seong MW, Seo SH, Park SS, Karbassi ID, Batish SD, Ordóñez-Ugalde A, Quintáns B, Sobrido MJ, Stemmler S, Lupski JR. The Alu-rich genomic architecture of SPAST predisposes to diverse and functionally distinct disease-associated CNV alleles. Am J Hum Genet. 2014;95(2):143–161. doi: 10.1016/j.ajhg.2014.06.014. PubMed DOI PMC

Gu S, Yuan B, Campbell IM, Beck CR, Carvalho CM, Nagamani SC, Erez A, Patel A, Bacino CA, Shaw CA, Stankiewicz P, Cheung SW, Bi W, Lupski JR. Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3. Hum Mol Genet. 2015;24(14):4061–77. doi: 10.1093/hmg/ddv146 PubMed PMC

Morales ME, White TB, Streva VA, DeFreece CB, Hedges DJ, Deininger PL. The contribution of alu elements to mutagenic DNA double-strand break repair. PLoS Genet. 2015;11(3):e1005016. doi: 10.1371/journal.pgen.1005016. PubMed DOI PMC

Goldmann R, Tichý L, Freiberger T, Zapletalová P, Letocha O, Soska V, Fajkus J, Fajkusová L. Genomic characterization of large rearrangements of the LDLR gene in Czech patients with familial hypercholesterolemia. BMC Med Genet. 2010;11:115. doi: 10.1186/1471-2350-11-115. PubMed DOI PMC

Vrablík M, Vaclová M, Tichý L, Soška V, Bláha V, Fajkusová L, Češka R, Šatný M, Freiberger T. Familial hypercholesterolemia in the Czech Republic: more than 17 years of systematic screening within the MedPed project. Physiol Res. 2017;66(Suppl 1):S1–S9. doi: 10.33549/physiolres.933600. PubMed DOI

Tichý L, Freiberger T, Zapletalová P, Soška V, Ravčuková B, Fajkusová L. The molecular basis of familial hypercholesterolemia in the Czech Republic: spectrum of LDLR mutations and genotype-phenotype correlations. Atherosclerosis. 2012;223(2):401–408. doi: 10.1016/j.atherosclerosis.2012.05.014. PubMed DOI

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. PubMed DOI

Smit AFA, Hubley R, Green P. RepeatMasker. https://www.repeatmasker.org/cgi-bin/WEBRepeatMasker [Accessed 25 October2020]

Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019;47(W1):W636–W641. doi: 10.1093/nar/gkz268. PubMed DOI PMC

Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24–26. doi: 10.1038/nbt.1754. PubMed DOI PMC

Vocke CD, Ricketts CJ, Schmidt LS, Ball MW, Middelton LA, Zbar B, Linehan WM. Comprehensive characterization of Alu-mediated breakpoints in germline VHL gene deletions and rearrangements in patients from 71 VHL families. Hum Mutat. 2021;42(5):520–529. doi: 10.1002/humu.2419431. PubMed DOI PMC

Konečná K, Tichý L, Freiberger T. Common rearrangements of the LDLR gene in the Czech population likely arise from one mutational event. Poster session presented at European Human Genetics Conference; June 11–14 2022; Vienna, Austria. Abstracts from the 55th European Society of Human Genetics (ESHG) Conference: Hybrid Posters. Eur J Hum Genet. 2023;31:412–3. 10.1038/s41431-023-01338-4.