Abstract
The regulators of complement activation (RCA) gene cluster in 1q31-1q32 includes most of the genes encoding complement regulatory proteins. Genetic variability in the RCA gene cluster frequently involve copy number variations (CNVs), a type of chromosome structural variation causing alterations in the number of copies of specific regions of DNA. CNVs in the RCA gene cluster often relate with gene rearrangements that result in the generation of novel genes, carrying internal duplications or deletions, and hybrid genes, resulting from the fusion or exchange of genetic material between two different genes. These gene rearrangements are strongly associated with a number of rare and common diseases characterized by complement dysregulation. Identification of CNVs in the RCA gene cluster is critical in the molecular diagnostic of these diseases. It can be done by bioinformatics analysis of DNA sequence data generated by massive parallel sequencing techniques (NGS, next generation sequencing) but often requires special techniques like multiplex ligation-dependent probe amplification (MLPA). This is because the currently used massive parallel DNA sequencing approaches do not easily identify all the structural variations in the RCA gene cluster. We will describe here how to use the MLPA assays and two computational tools to analyze NGS data, NextGENe and ONCOCNV, to detect CNVs and gene rearrangements in the RCA gene cluster.
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References
Rodríguez De Córdoba S, Díaz-Guillén MA, Heine-Suñer D (1999) An integrated map of the human regulator of complement activation (RCA) gene cluster on 1q32. Mol Immunol 36:803–808. https://doi.org/10.1016/S0161-5890(99)00100-5
Lambert J-C, Heath S, Even G et al (2009) Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer’s disease. Nat Genet 41:1094–1099. https://doi.org/10.1038/ng.439
Venables JP, Strain L, Routledge D et al (2006) Atypical haemolytic uraemic syndrome associated with a hybrid complement gene. PLoS Med 3:e431. https://doi.org/10.1371/journal.pmed.0030431
Valoti E, Alberti M, Tortajada A et al (2014) A novel atypical hemolytic uremic syndrome-associated hybrid CFHR1/CFH gene encoding a fusion protein that antagonizes factor H-dependent complement regulation. J Am Soc Nephrol 26:209–219. https://doi.org/10.1681/ASN.2013121339
Francis NJ, McNicholas B, Awan A et al (2012) A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome. Blood 119:591–601. https://doi.org/10.1182/blood-2011-03-339903
Goicoechea de Jorge E, Tortajada A, Pinto García S et al (2018) Factor H competitor generated by gene conversion events associates with atypical hemolytic uremic syndrome. J Am Soc Nephrol 29:1–10. https://doi.org/10.1681/ASN.2017050518
Gharavi AG, Kiryluk K, Choi M et al (2012) Genome-wide association study identifies susceptibility loci for IgA nephropathy. Nat Genet 43:321–327. https://doi.org/10.1038/ng.787.Genome-wide
Hughes AE, Orr N, Esfandiary H et al (2006) A common CFH haplotype, with deletion of CFHR1 and CFHR3, is associated with lower risk of age-related macular degeneration. Nat Genet 38:1173–1177. https://doi.org/10.1038/ng1890
Tortajada A, Yébenes H, Abarrategui-Garrido C et al (2013) C3 glomerulopathy–associated CFHR1 mutation alters FHR oligomerization and complement regulation. J Clin Invest 123:2434–2446. https://doi.org/10.1172/JCI68280DS1
Krych-Goldberg M, Atkinson JP (2001) Structure-function relationships of complement receptor type 1. Immunol Rev 180:112–122
Liu D, Niu ZX (2009) The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35). Immunopharmacol Immunotoxicol 31:524–535. https://doi.org/10.3109/08923970902845768
Klickstein LB, Wong WW, Smith JA et al (1987) HUMAN C3b/C4b RECEPTOR (CR1) demonstration of long homologous repeating domains that are composed of the short consensus repeats characteristic of C3/C4 binding proteins. J Exp Med 165:1095–1112. https://doi.org/10.1084/jem.165.4.1095
Rodriguez de Cordoba S, Rubinstein P (1986) Quantitative variations of the C3b/C4b receptor (CR1) in human erythrocytes are controlled by genes within the regulator of complement activation (RCA) gene cluster. J Exp Med 164:1274–1283. https://doi.org/10.1084/jem.164.4.1274
Brouwers N, Van Cauwenberghe C, Engelborghs S et al (2012) Alzheimer risk associated with a copy number variation in the complement receptor 1 increasing C3b/C4b binding sites. Mol Psychiatry 17:223–233. https://doi.org/10.1038/mp.2011.24
Kisserli A, Tabary T, Cohen JHM et al (2017) High-resolution melting PCR for complement receptor 1 length polymorphism genotyping: an innovative tool for Alzheimer’s disease gene susceptibility assessment. J Vis Exp 125:1–11. https://doi.org/10.3791/56012
Rodriguez de Cordoba S, Hidalgo MS, Pinto S, Tortajada A (2014) Genetics of atypical hemolytic uremic syndrome (aHUS). Semin Thromb Hemost 40:422–430. https://doi.org/10.1055/s-0034-1375296
Józsi M, Tortajada A, Uzonyi B et al (2015) Factor H-related proteins determine complement-activating surfaces. Trends Immunol 36:374–384. https://doi.org/10.1016/j.it.2015.04.008
Pérez-Caballero D, González-Rubio C, Gallardo ME et al (2001) Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome. Am J Hum Genet 68:478–484. https://doi.org/10.1086/318201
Tortajada A, Gutiérrez E, Goicoechea de Jorge E et al (2017) Elevated factor H–related protein 1 and factor H pathogenic variants decrease complement regulation in IgA nephropathy. Kidney Int 92:953–963. https://doi.org/10.1016/j.kint.2017.03.041
Zhao J, Wu H, Khosravi M et al (2011) Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility. PLoS Genet 7:1–9. https://doi.org/10.1371/journal.pgen.1002079
Abarrategui-Garrido C, Martínez-Barricarte R, López-Trascasa M et al (2009) Characterization of complement factor H-related (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome. Blood 114:4261–4271. https://doi.org/10.1182/blood-2009-05-223834
Jodele S, Licht C, Goebel J et al (2013) Abnormalities in the alternative pathway of complement in children with hematopoietic stem cell transplant-associated thrombotic microangiopathy. Blood 122:2003–2007. https://doi.org/10.1182/blood-2013-05-501445
Moore I, Strain L, Pappworth I et al (2010) Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome. Blood 115:379–387. https://doi.org/10.1182/blood-2009-05-221549
Zipfel PF, Edey M, Heinen S et al (2007) Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome. PLoS Genet 3:0387–0392. https://doi.org/10.1371/journal.pgen.0030041
Eyler SJ, Meyer NC, Zhang Y et al (2013) A novel hybrid CFHR1/CFH gene causes atypical hemolytic uremic syndrome. Pediatr Nephrol 28(11):2221–2225. https://doi.org/10.1007/s00467-013-2560-2
Chen Q, Wiesener M, Eberhardt HU et al (2014) Complement factor H-related hybrid protein deregulates complement in dense deposit disease. J Clin Invest 124:145–155. https://doi.org/10.1172/JCI71866
Malik TH, Lavin PJ, Goicoechea de Jorge E et al (2012) A hybrid CFHR3-1 gene causes familial C3 glomerulopathy. J Am Soc Nephrol 23:1155–1160. https://doi.org/10.1681/ASN.2012020166
Xiao X, Ghossein C, Tortajada A et al (2016) Familial C3 glomerulonephritis caused by a novel CFHR5-CFHR2 fusion gene. Mol Immunol 77:89–96. https://doi.org/10.1016/j.molimm.2016.07.007
Gale DP, Goicoechea de Jorge E, Cook HT et al (2010) Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis. Lancet 376:794–801. https://doi.org/10.1016/S0140-6736(10)60670-8
Medjeral-Thomas N, Malik TH, Patel MP et al (2014) A novel CFHR5 fusion protein causes C3 glomerulopathy in a family without Cypriot ancestry. Kidney Int 85:933–937. https://doi.org/10.1038/ki.2013.348
Togarsimalemath SK, Sethi SK, Duggal R et al (2017) A novel CFHR1-CFHR5 hybrid leads to a familial dominant C3 glomerulopathy. Kidney Int 92:876–887. https://doi.org/10.1016/j.kint.2017.04.025
Boeva V, Popova T, Lienard M et al (2014) Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data. Bioinformatics 30:3443–3450. https://doi.org/10.1093/bioinformatics/btu436
Wang H, Nettleton D, Ying K (2014) Copy number variation detection using next generation sequencing read counts. BMC Bioinformatics 15:109. https://doi.org/10.1186/1471-2105-15-109
Malekpour SA, Pezeshk H, Sadeghi M (2018) MSeq-CNV: accurate detection of copy number variation from sequencing of multiple samples. Sci Rep 8:1–12. https://doi.org/10.1038/s41598-018-22323-8
Samorodnitsky E, Datta J, Jewell BM et al (2015) Comparison of custom capture for targeted next-generation DNA sequencing. J Mol Diagn 17:64–75. https://doi.org/10.1016/j.jmoldx.2014.09.009
Samorodnitsky E, Jewell BM, Hagopian R et al (2015) Evaluation of hybridization capture versus amplicon-based methods for whole-exome sequencing. Hum Mutat 36:903–914. https://doi.org/10.1002/humu.22825
Acknowledgments
SRdeC is supported by the Spanish “Ministerio de Economía y Competitividad-FEDER” (SAF2015-66287R, PID2019-104912RB-I00, RTC-2016-4635-1 and the Autonomous Region of Madrid (S2017/BMD-3673). Secugen has received a soft loan from the Spanish “Ministerio de Economía y Competitividad” Retos Program RTC-2016-4635-1 cofinanced by FEDER funds.
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García-Fernández, J., Vilches-Arroyo, S., Olavarrieta, L., Pérez-Pérez, J., Rodríguez de Córdoba, S. (2021). Detection of Genetic Rearrangements in the Regulators of Complement Activation RCA Cluster by High-Throughput Sequencing and MLPA. In: Roumenina, L.T. (eds) The Complement System. Methods in Molecular Biology, vol 2227. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1016-9_16
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DOI: https://doi.org/10.1007/978-1-0716-1016-9_16
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