B l o o d G r o u p R e v i e w An update on the Chido/Rodgers group system

R. Mougey

This update of the Chido/Rodgers blood group system (Mougey R. not necessarily correlate with a particular Chido or Rodgers A review of the Chido/Rodgers blood group. Immunohematology phenotype. The CNV seen with and C4B genes has been 2010;26:30–8) summarizes the current understanding of the an important aspect of the determination that a lower or higher genetics and serology of this blood group (of which little has changed since the publication of the first review) in a table C4 gene expression can have a strong association with certain format as well as summarizes the gene frequencies and disease diseases (discussed in a later section). Table 1 provides a list of association with low copy number of C4A or C4B genes. The C4 and Chido/Rodgers blood group details. International Society of (ISBT) has designated the ISBT number 017 to this system and the abbreviation CH/RG for the antigen or antibody notation. There are currently nine Serology of Anti-Ch and Anti-Rg antigens in the CH/RG system. A brief discussion on the serologic challenges of detecting the antibodies and of newer information No new reports on the serology of anti-Ch or anti-Rg were on the disease associations is provided. This review concludes with some speculation on how our understanding of C4 genes found in an exhaustive survey of the literature. Although we may be illuminated by current investigation into complexities of suspect these antibodies are still being detected, it is tempting autoimmunity and the role of C4 and its progression to a disease to speculate that the methods of automation using solid-phase state. Immunohematology 2019;35:135–138. or gel technology do not detect them very well, making them more difficult to recognize and thus affecting the likelihood of Key Words: Chido/Rodgers, C4, complement finding additional Ch/Rg phenotypes.8 Most blood bankers consider it an advantage that current A review of the Chido/Rodgers blood group system anti-IgG reagents do not contain much reactivity against IgG4 published in 2010 summarized the discovery of this blood subclass antibodies; anti-Ch and anti-Rg are often of this group,1 with the eventual recognition that Chido/Rodgers subtype. Consequently, the combination of test method used, antigens were not intrinsic (RBC) antigens but reagents, and the low number of immunized individuals result were epitopes carried on the C4 component of complement. in fewer antibodies being detected and, if detected, they may In normal homeostasis, fragments of the C4 protein (C4b) are not be recognized for what they are because of their variable deposited on the RBC membrane and on other tissues as well. weak reactivity. Finally, if the antibodies are recognized, the C4b is then broken down to C4d. Thus, with anti-Ch or anti- resources needed to investigate and publish such studies may Rg, agglutination with indirect antiglobulin testing can be not be possible. observed with most individuals’ RBCs because the antibody When investigating a suspected anti-Ch or anti-Rg, a is detecting the deposited portion of the C4d molecule that well-characterized pool of group AB antibody-negative plasma carries either the Chido or Rodgers antigens. This discovery should be used as part of the inhibition method, since partial also explained why antibody-negative plasma could be used to inhibition could occur with single-sourced plasma unless neutralize Chido or Rodgers antibodies. the donor’s Ch or Rg antigens are known. Past experience The International Society of Blood Transfusion has has proven that tube methods are preferred to solid-phase assigned 017 to the Chido/Rodgers blood group system, and methods to obtain the best reactivity. If the antibody can be nine antigens have been described based on the serology of the neutralized, then C4-coated RBCs can be prepared to confirm antibodies.1 Additionally, based on molecular analysis, there that the weak antibody can react at immediate spin using C4- are at least 24 alleles for C4A and 27 for C4B.2 These allelic coated RBCs. The challenge of using other anti-Ch or anti-Rg forms include single nucleotide polymorphisms, insertions, sources to confirm that the individual forming the antibody is deletions, stop codons, and copy number variants (CNVs). Ch negative or Rg negative relies on the assumption that no Thus, a rich complexity of possible genotypes occurs that does other antibodies are present in the antibody source used for

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Table 1. C4 and Chido/Rodgers blood group facts

C4 genes Gene locus Each C4 gene is found in a complex called RCCX on , in a short or long form (long form has retroviral insert HERV, 76 percent of white individuals) between HLA class 1 and class 3 genes in a tandem array as C4A and/or C4B RP-C4-CYP21-TNX. The presence of the long or short form is noted by an L and/or S. In individuals with equal numbers of C4 genes, those with only the short form (no insert) had C4 protein plasma levels higher than individuals with the long form.3 Gene  expression  protein The RCCX monomodular gene normally comprises four functional genes, but if duplicate (bi- modular, tri-modular) RCCX segments are present, generally only the gene-encoding complement component C4 produces a protein with the extra RP, CYP21, and TNX usually silent.4

Gene (in order of gene frequency in 111 people of European ancestry)5 C4A and C4B bi-modular, high gene frequency The most common C4 gene complexes are bi-modular variants of one C4A and one C4B gene (C 4AL-BL 0.41 or C 4AL-B S 0.31), combined frequency 0.72 Bi-modular C4A only, low gene frequency Duplicated C4AL-AL gene, C4B deficient (gene frequency 0.11) Mono-modular C4B, low gene frequency A single C4BS gene, C4A deficient (gene frequency 0.07)

Gene detected in too low numbers to calculate frequency (in the same 111 people)5 Mono-modular C4A C4AL, C4B deficient Mono-modular C4B C4BL, C4A deficient Tri-modular C4A, C4B Two types of C4A and one copy of C4B with either S or L forms (C 4AL-AS-BL , C 4AL-AL-B S , or C 4AL-AL-BL) Tri-modular C4B, C4A C 4AL-BL-BL Tri-modular of C4A, no C4B C4AL-AL-AL Tri-modular C4A, C4B C 4AL-BL-B S C4AL-BS-BS Quadri-modular C4A, C4B C4AL-AS-BS-BS Bi-modular C4B C4BL-BS, C4B deficient

C4A protein C4B protein C4 deficiency for C4A or C4B Homozygous deficiency seen in 1–6 percent Homozygous deficiency seen in 1–10 percent of of populations6 populations6 Gel electrophoretic mobility A for acidic B for basic 1774 amino acid protein with changes at residue Pro-Cys-Pro-Val-Leu-Asp Leu-Ser-Pro-Val-Ile-His 1101–1106 Function7 Primary role in immune complex clearance Primary role activation by carbohydrate antigens (reacts with amino groups of antigen or immune complex) Association with common C4A or C4B isotypes Lower hemolytic activity Greater hemolytic activity Antigens, serology2 CH/RG:11, CH/RG:12 CH/RG:1, CH/RG:2, CH/RG:3, CH/RG:4, CH/RG:5, CH/RG:6, CH/RG:7 CH/RG:11,12 (Rg:1,2 old notation) phenotype CH/RG:1,2,3 (Ch1,2,3) phenotype 88.2 percent 95 percent Amino acid changes at position 1188–1191 VDLL (Rg antigens) ADLR (Ch antigens) Plasma neutralization Partial neutralization can be confusing if individual plasma is used for neutralization; a pool of antibody-negative known plasmas or a well-characterized plasma is recommended. Most reactive method Little information has been published regarding reactivity with newer methods such as gel, solid-phase, or automated gel, solid-phase. One report indicated no reactivity with antibody identification panels using multiple methods despite strong reactivity with PEG tube methods.8

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Low copy number, disease association Celiac disease, lymphoma, SLE, sarcoidosis, Intolerance to sulfonamides, increased risk post- increased autoimmunity,6 SLE,9 juvenile- infection symptoms6 onset SLE,10 improved survival of transplanted Juvenile-onset SLE but not adult-onset,10 kidneys,11 early-onset preeclampsia,12 autism,13 rheumatoid arthritis,14 possible lower overall recurrent respiratory infections,18 type 1 survival of low copy or deficient C4B individuals diabetes19 (in some populations)15,16,20 High copy number, disease association A lower risk for age-related macular degeneration21 Increased risk of schizophrenia3 Increased risk for vitiligo with Graves disease17 Null alleles, disease association Autism, autoimmunity, lymphoma, sarcoidosis, Susceptibility to bacterial and enveloped virus adverse drug reactions, celiac disease4 infection, adverse drug reactions (particularly with antibiotics)4 PEG = polyethylene glycol; SLE = systemic erythematosus. the typing. In cases where the sample typed as positive with of the role of the microbiome in health and disease provide anti-Ch and/or anti-Rg, one could also suspect a variant Ch intriguing directions to be explored in discovering the link and/or Rg antigen. between the role the genes of the immune system play along with the effect the environment and disruptions in our Disease Association microbiome have on the progression to disease.23 These are exciting times for immunology and its association with the The information in the current scientific literature relating human blood group Chido/Rodgers system. to the Chido/Rodgers blood group antigens is focused on their association with various diseases, relating to both the role of References C4 in disease and health and the effect of CNV (usually low 1. Mougey R. A review of the Chido/Rodgers blood group. copy number) in diseases known to have an association with Immunohematology 2010;26:30–8. C4. Although this is the most intriguing aspect of C4 genetics, 2. Daniels G. Chido/Rodgers blood group system in human blood it must be noted that association is not the same as causation, groups. 3rd ed. Oxford: Wiley-Blackwell, 2013. and the complexities of the interplay between the various genes 3. Castley A, Martinez O. Molecular analysis of complement component C4 gene copy number. Methods Mol Biol that play a role in immunity is far from definitive. It seems 2012;882:159–71. likely that there is an environmental effect as well. 4. Bánlaki Z, Doleschall M, Rajczy K, Fust G, Szilágyi A. Fine- The strongest increased risk with certain C4 gene variants tuned characterization of RCCX copy number variants and their relationship with extended MHC haplotypes. Genes is for systemic lupus erythematosus (SLE). Lintner et al.9 report Immun 2012;13:530–5. that in a study of 28 individuals with a complete deficiency of 5. Sekar A, Bialas AR, de Rivera H, et al. risk both C4A and C4B, 22 of the 28 individuals had SLE or lupus- from complex variation of complement component 4. Nature like disease, and an additional 4 individuals had renal disease 2016:530:177–83. including glomerulonephritis. The authors also noted that 32.9 6. Liesmaa I, Paakkanen R, Jarvinen A, Valtonen V, Lokki M-L. Clinical features of patients with homozygous complement percent of SLE patients had a low copy number for C4A. Thus, C4A or C4B deficiency. PLoS One 2018;13:e0199205. the association with CNV and autoimmunity makes it likely 7. Dodds AW, Ren XD, Willis AC, Law SK. The reaction that the functional role C4 plays with innate and acquired mechanism of the internal thioester in the human complement component C4. Nature 1996;379:177–9. immunity in various autoimmune diseases is important and 8. Kahwash E, Talwalkar S, Leonard J, Lockwood W. Atypical may provide a key understanding of autoimmunity. presentation of Anti-Rga. Ann Clin Lab Sci 2005;35:100–4. Finally, one of the newer aspects being explored is the role 9. Lintner K, Wu Y, Yang Y, et al. Early components of the of the microbiome in immunity and . As Belkaid complement classical activation pathway in human systemic and Hand22 note in their review, most of our immune system autoimmune diseases. Front Immunol 2016;7:36. 10. Pereira K, Faria A, Liphaus B, et al. Low C4, C4A, and C4B gene functions focus on controlling our relationship with our copy numbers are stronger risk factors for juvenile-onset than microbiome, and the corollary that microbiomes, in protecting for adult-onset systemic lupus erythematosus. Rheumatology their ecological niche, aim at enforcing barrier immunity and 2016;55:869–73. therefore their own containment. These emerging perceptions

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11. Bay JT, Schejbel L, Madsen HO, Sørensen SS, Hansen JM, 19. Rich S, O’Neill G, Dalmasso AP, Nerl C, Barbosa J. Complement Garred P. Low C4 gene copy numbers are associated with and HLA: further definition of high-risk haplotypes in insulin- superior graft survival in patients transplanted with a deceased dependent diabetes. Diabetes 1985;34:504–9. donor kidney. Kidney Int 2013;84:562–9. 20. Flachsbart F, Caliebe A, Heinsen F-A, et al. Investigation of 12. Lokki A, Heikkinen-Eloranta J, Jarva H, Saisto T. Complement complement component C4 copy number variation in human activation and regulation in preeclamptic placenta. Front longevity. PLoS One 2014;9:e86188. Immunol 2014;5:312. 21. Grassmann F, Cantsilieris S, Schulz-Kuhnt A-S, et al. Multi- 13. Mostafa G, Shehab A. The link of C4B null allele to autism allelic copy number variation in the complement component and to a family history of autoimmunity in Egyptian autistic 4A (C4A) gene is associated with late-stage age-related macular children. J Neuroimmunol 2010;223:115–9. degeneration (AMD). J Neuroinflamm 2016;13:81. 14. Rigby W, Wu Y, Zan M, et al. Increased frequency of 22. Belkaid Y, Hand T. Role of the microbiota in immunity and complement C4B deficiency in rheumatoid arthritis. Arthritis inflammation. Cell 2014;157:121–41. Rheum 2012;64:1338–44. 23. Albert-Vega C, Tawfik DM, Trouillet-Assant S, Vachot L, 15. Kramer J, Fülöp T, Rajczy K, Nguyen AT, Füst G. A marked Mallet F, Textoris J. Immune functional assays, from custom drop in the incidence of the null allele of the B gene of the to standardized tests for precision medicine. Front Immunol fourth component of complement (C4B*Q0) in elderly subjects: 2018;9:2367. C4B*Q0 as a probable negative selection factor for survival. Hum Genet 1991;86:595–8. Ruth Mougey, MT(ASCP)SBB, President, Mougey, Inc., 2106 NW 16. Szilagyi A, Fust G. Diseases associated with the low copy number of the C4B gene encoding C4, the fourth component of 88th St., Vancouver, WA 98665, [email protected]. complement. Cytogenet Genome Res 2008;123:118–30. 17. Liu Y-H, Wan L, Chang CT, et al. Association between copy number variation of complement component C4 and Graves’ disease. J Biomed Sci 2011;18:71. 18. Kainulainen L, Peltola V, Seppänen M, et al. C4A deficiency in children and adolescents with recurrent respiratory infections. Hum Immunol 2012;73:498–501.

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