{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T03:11:14Z","timestamp":1760238674305,"version":"build-2065373602"},"reference-count":26,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2020,8,31]],"date-time":"2020-08-31T00:00:00Z","timestamp":1598832000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100010198","name":"Ministerio de Econom\u00eda, Industria y Competitividad, Gobierno de Espa\u00f1a","doi-asserted-by":"publisher","award":["SAF2016-80125-R"],"award-info":[{"award-number":["SAF2016-80125-R"]}],"id":[{"id":"10.13039\/501100010198","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100006371","name":"Asociaci\u00f3n Universitaria Iberoamericana de Postgrado","doi-asserted-by":"publisher","award":["Beca estudios de doctorado"],"award-info":[{"award-number":["Beca estudios de doctorado"]}],"id":[{"id":"10.13039\/501100006371","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Background: Complement C4 gene copy number variation plays an important role as a determinant of genetic susceptibility to common diseases, such as systemic lupus erythematosus, schizophrenia, rheumatoid arthritis, and infectious diseases. This study aimed to develop an assay for the quantification of copy number variations in the C4 locus. Methods: the assay was based on a gene ratio analysis copy enumeration (GRACE) PCR combined with high resolution melting (HRM) PCR. The test was optimized using samples of a known genotype and validated with 72 DNA samples from healthy blood donors. Results: to validate the assay, standard curves were generated by plotting the C4\/RP1 ratio values against copy number variation (CNV) for each gene, using genomic DNA with known C4 CNV. The range of copy numbers in control individuals was comparable to distributions observed in previous studies of European descent. Conclusions: the method herein described significantly simplifies C4 CNV diagnosis to validate the assay.<\/jats:p>","DOI":"10.3390\/ijms21176309","type":"journal-article","created":{"date-parts":[[2020,8,31]],"date-time":"2020-08-31T11:53:49Z","timestamp":1598874829000},"page":"6309","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Complement C4 Gene Copy Number Variation Genotyping by High Resolution Melting PCR"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8034-190X","authenticated-orcid":false,"given":"Claudia P.","family":"Jaimes-Bernal","sequence":"first","affiliation":[{"name":"Immunogenetics Unit, Department of Experimental Biology, Universidad de Ja\u00e9n, 23071 Ja\u00e9n, Spain"},{"name":"Faculty of Health Sciences, Universidad de Boyac\u00e1, 150003 Tunja, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2487-2041","authenticated-orcid":false,"given":"Monte","family":"Trujillo","sequence":"additional","affiliation":[{"name":"Transfusion Blood Center, 23009 Jaen, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Francisco Jos\u00e9","family":"M\u00e1rquez","sequence":"additional","affiliation":[{"name":"Immunogenetics Unit, Department of Experimental Biology, Universidad de Ja\u00e9n, 23071 Ja\u00e9n, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5788-7840","authenticated-orcid":false,"given":"Antonio","family":"Caruz","sequence":"additional","affiliation":[{"name":"Immunogenetics Unit, Department of Experimental Biology, Universidad de Ja\u00e9n, 23071 Ja\u00e9n, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,8,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"262","DOI":"10.3389\/fimmu.2015.00262","article-title":"Complement System Part I\u2014Molecular Mechanisms of Activation and Regulation","volume":"6","author":"Merle","year":"2015","journal-title":"Front. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/S0079-6603(03)75007-7","article-title":"Dancing with Complement C4 and the RP-C4-CYP21-TNX (RCCX) Modules of the Major Histocompatibility Complex","volume":"75","author":"Yu","year":"2003","journal-title":"Prog. Nucleic Acid Res. Mol. Bio."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/S1567-5769(01)00019-4","article-title":"Genetic, structural and functional diversities of human complement components C4A and C4B and their mouse homologues, Slp and C4","volume":"1","author":"Blanchong","year":"2001","journal-title":"Int. Immunopharmacol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"866","DOI":"10.1002\/humu.21259","article-title":"Assessment of complement C4 gene copy number using the paralog ratio test","volume":"31","author":"Fernando","year":"2010","journal-title":"Hum. Mutat."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1097\/MIB.0000000000000623","article-title":"Genome-Wide Copy Number Variation Scan Identifies Complement Component C4 as Novel Susceptibility Gene for Crohn\u02bcs Disease","volume":"22","author":"Cleynen","year":"2016","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1038\/nature16549","article-title":"Schizophrenia risk from complex variation of complement component 4","volume":"530","author":"Sekar","year":"2016","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Flachsbart, F., Caliebe, A., Heinsen, F.-A., Hemming-Karlsen, T., Schreiber, S., Franke, A., and Nebel, A. (2014). Investigation of Complement Component C4 Copy Number Variation in Human Longevity. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0086188"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1007\/s13353-017-0407-4","article-title":"Clinical interpretation of copy number variants in the human genome","volume":"58","author":"Nowakowska","year":"2017","journal-title":"J. Appl. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"42628","DOI":"10.1038\/srep42628","article-title":"Association between C4, C4A, and C4B copy number variations and susceptibility to autoimmune diseases: A meta-analysis","volume":"7","author":"Li","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1097\/RHU.0b013e318156b9e3","article-title":"Partial C4 Deficiency in Juvenile Idiopathic Arthritis Patients","volume":"13","author":"Gilliam","year":"2007","journal-title":"J. Clin. Rheumatol."},{"key":"ref_11","first-page":"1","article-title":"Complement C4A and C4B gene copy number study in Alzheimer\u2019s disease patients","volume":"13","author":"Zorzetto","year":"2016","journal-title":"Curr. Alzheimer Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1016\/S0168-8278(02)00237-4","article-title":"Influence of C4A deficiency on nonresponse to HBsAg vaccination: A new immune response gene","volume":"37","author":"Milich","year":"2002","journal-title":"J. Hepatol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1016\/S0168-8278(02)00205-2","article-title":"C4A deficiency and nonresponse to hepatitis B vaccination","volume":"37","author":"Starke","year":"2002","journal-title":"J. Hepatol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1016\/j.humimm.2012.02.015","article-title":"C4A deficiency in children and adolescents with recurrent respiratory infections","volume":"73","author":"Kainulainen","year":"2012","journal-title":"Hum. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3012","DOI":"10.4049\/jimmunol.179.5.3012","article-title":"Sensitive and specific real-time polymerase chain reaction assays to accurately determine copy number variations (CNVs) of human complement C4A, C4B, C4-long, C4-short, and RCCX modules: Elucidation of C4 CNVs in 50 consanguineous subjects with defined HLA genotypes","volume":"179","author":"Wu","year":"2007","journal-title":"J. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Paakkanen, R., Vauhkonen, H., Eronen, K.T., J\u00e4rvinen, A., Sepp\u00e4nen, M., and Lokki, M.-L. (2012). Copy Number Analysis of Complement C4A, C4B and C4A Silencing Mutation by Real-Time Quantitative Polymerase Chain Reaction. PLoS ONE, 7.","DOI":"10.1371\/annotation\/d19ba035-dbf0-4e58-932a-52efaa8137f3"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1186\/s12974-016-0548-0","article-title":"Multiallelic copy number variation in the complement component 4A (C4A) gene is associated with late-stage age-related macular degeneration (AMD)","volume":"13","author":"Grassmann","year":"2016","journal-title":"J. Neuroinflamm."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1217","DOI":"10.1007\/s00439-014-1457-x","article-title":"Human gene copy number variation and infectious disease","volume":"133","author":"Hollox","year":"2014","journal-title":"Hum. Genet."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1159\/000184725","article-title":"Methods to detect and analyze copy number variations at the genome-wide and locus-specific levels","volume":"123","author":"Lee","year":"2008","journal-title":"Cytogenet. Genome Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1373\/clinchem.2019.304246","article-title":"A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations","volume":"65","author":"Cassinari","year":"2019","journal-title":"Clin. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12881-015-0258-y","article-title":"Development and validation of a high throughput, closed tube method for the determination of haemoglobin alpha gene (HBA1 and HBA2) numbers by gene ratio assay copy enumeration-PCR (GRACE-PCR)","volume":"16","author":"Turner","year":"2015","journal-title":"BMC Med. Genet."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"684","DOI":"10.1373\/clinchem.2015.239368","article-title":"High-Resolution Assessment of Copy Number Variation","volume":"61","author":"Dobrovic","year":"2015","journal-title":"Clin. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2734","DOI":"10.4049\/jimmunol.171.5.2734","article-title":"Diversity in intrinsic strengths of the human complement system: Serum C4 protein concentrations correlate with C4 gene size and polygenic variations, hemolytic activities, and body mass index","volume":"171","author":"Yang","year":"2003","journal-title":"J. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Turner, A., Sasse, J., and V\u00e1radi, A. (2016). Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR). BMC Med. Genet., 17.","DOI":"10.1186\/s12881-016-0334-y"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Szilagyi, A., Blask\u00f3, B., Szilassy, D., F\u00fcst, G., Sasvari-Szekely, M., and Ronai, Z. (2006). Real-time PCR quantification of human complement C4A and C4B genes. BMC Genet., 7.","DOI":"10.1186\/1471-2156-7-1"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Real, L.M., Neukam, K., Herrero, R., Guardiola, J.M., Reiberger, T., Rivero-Ju\u00e1rez, A., Salazar, J., Mandorfer, M., Merino, D., and Sorianob, V. (2014). IFNL4 ss469415590 Variant Shows Similar Performance to rs12979860 as Predictor of Response to Treatment against Hepatitis C Virus Genotype 1 or 4 in Caucasians. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0095515"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/17\/6309\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:05:05Z","timestamp":1760177105000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/17\/6309"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,8,31]]},"references-count":26,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2020,9]]}},"alternative-id":["ijms21176309"],"URL":"https:\/\/doi.org\/10.3390\/ijms21176309","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2020,8,31]]}}}