The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class‐specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class‐specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg‐positive C4A 3a and Ch‐positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101‐1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion‐like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.

C4 is the only component of the human complement system that is coded for by two genes, C4A and C4B, showing 99% homology. The genes for the two C4 isotypes are located with the genes for the second component (C2), factor B (BF), and steroid 21-hydroxylase (21-OHA and 21-OHB) between HLA-B and -DR in the MHC on chromosome six (1-4). The C4 and 21-OH genes are tandemly arranged and have probably arisen by duplication (Fig . 1 a). Based on the direction of transcription, C4A is usually expressed at C4 locus I, whereas C4B is usually expressed at locus II (5).C4A and C4B are highly polymorphic with more than 35 alleles including null alleles (C4QO) at both loci (6). The polymorphism can be defined by electrophoretic mobility ofthe intact protein or its subunits (7-9), by serology of Rodgers (Rg) and Chido (Ch) determinants (10), and by functional studies of complement activation and binding characteristics (11,12). A sequence of four amino acids in the C4d region (Chido 4 determinant on C4B molecules) is responsible for the major structural and functional differences of the C4 isotypes (Fig. 1 c; 9, 11-13) . The antigenic determinants Rodgers and Chido are generally expressed on the C4A and C4B isotypes, respectively, but rare reversed associations have been described (14). Gene conversion has been discussed as a possible mechanism for the generation of aberrant allotypes (15,16).Null alleles of C4A or C4B (AQO or BQO) occur at frequencies of 0.1-0 .3 in the normal population (17), and are assessed by the absence of gene products. The structural analysis of the C4 genes at the DNA level has revealed that only a proportion of C4 null alleles result from gene deletions affecting an entire C4 gene and one adjacent 21-OH gene, and consequently other null alleles were due to nonexpressed genes (pseudogenes) (18,19). Alternatively, it has been suggested that null alleles represent the expression of two identical, and therefore undistinguishable, allotypes on a haplotype, and this results from gene conversion of C4A to C4B and the reverse (5, 20).

A new and distinct partial inhibition pattern was observed for Chido during routine typing; it is found in 3% of random samples, shows recessive inheritance and is linked to HLA. Polyspecificity in anti-Ch reagents, selected to detect partial inhibition, is demonstrated.

Applying a combined technology for the detection of allotypic variation of the fourth component of human complement (C4), including immunofixation with anti-C4 and C4-dependent lysis after agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C4 to separate the C4A and B alpha-chains, and the determination of Rodgers (Rg) and Chido (Ch) determinants of C4 in serum and at the blotted C4 alpha-chains, we detected rare human C4 allotypes and studied the genetic linkage. Partial inhibitors (p.i.) of anti-Rg and anti-Ch sera were found; the C4A51 allotype characterized as Rg p.i. and the C4A1 and C4B51 allotypes as Ch p.i. were genetically inherited. The C4A1 allotype has a unique Rg- Ch+ C4A alpha-chain. Duplicated C4A loci, A*3, A*2, and A*5, A*2 were both associated with a C4BQO and the HLA haplotype A3-Cw4-Bw35-DR1. These additions to the already known extensive C4 polymorphism may help to sort out their significance for the biological functions of human C4.

Summary A Japanese blood donor (H. T.) and his brother (M. S.) are the first homozygous MkMk individuals described; their red cells lack, as expected, known antigens of the MNSs blood group system and also have no demonstrable MN‐active and Ss‐active glycoproteins. Both MkMk individuals have a naturally occurring atypical antibody in their serum. The antibody in the serum of H. T. is inhibited by MNSs‐active glycoprotein preparations from normal erythrocytes.

The antigenic determinants of human C4 have been defined by human IgG antisera, Rodgers (Rg) and Chido (Ch), in hemagglutination-inhibition assays (HAI). Eight (2 Rg and 6 Ch) are of high frequency, greater than 90%, and 1, WH, is of low frequency, 15%. The phenotypic combinations are complex; generally, C4A expresses Rg, and C4B has Ch, but reverse antigenicities have been established both by HAI and by sequence data of selected C4 allotypes. A study of 325 families provides data on the antigenic expression of each C4 allotype and demonstrates strong associations. A structural model for the antigenic determinants of C4 proteins has been proposed and is completely supported by the family material. Of the 16 possible antigenic combinations for C4 proteins, only 3 are undetected. A new Ch combination has been recorded in two French families. The reported sequence variation within the C4d region can account for the antigenic determinants but leaves the location of electrophoretic variation in C4 still unclear.

Human C4 is most polymorphic at the protein level, distinction between allotypes of the C4A and C4B proteins resting on electrophoretic migration patterns and difference in hemolytic activity. The aim of the C4 reference typing has been the definition of reference variants, the assignment of rare variants, and the investigation of duplicated, deleted, or non-expressed and hybrid genes. Samples from 136 individuals, predominantly with known segregation, from 16 laboratories were investigated by standard electrophoretic techniques, for their relative hemolytic activity, reactivity with monoclonal antibodies and Rg/Ch reagents, a-, and ß-chain types, relative electrophoretic migration distance, as well as the C4/21-OH-TaqI RFLPs. The results were evaluated in three groups; they consisted in the definition of the eight most common C4 alleles, and the ten Rg/Ch standard phenotypes in group I. In group II twelve C4A and fourteen C4B duplications among 96 complotypes, as well as eighteen deleted/non-expressed C4A and twenty-two C4B alleles, and hybrid alleles were seen by correlation of lytic activity, electrophoretic mobility, and monoclonal and/or Rg/Ch reactivity. Group III consisted of the newly defined allotypes A 8, A 7, A 58, A 55, A 45, B 45, B 35, and B 22, furthermore of alleles subdividing the A 1/A 91, and the B 13/ B 12/B 11 regions. The reference typing has allowed reclassification of the majority of described C4 allotypes and resulted in a revision of the C4 nomenclature.

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