ArticleRelationship Between Protein Complotypes and DNA Variant Haplotypes: Complotype-RFLP Constellations (CRC)
Introduction
There is extensive genetic polymorphism in the genes encoding the complement proteins of the major histocompatibility complex in humans. It is most extensive in the genes encoding the two isotypes of C4, C4A and C4B [1], but also involves factor B of the alternative pathway 2, 3, 4and, to a lesser extent, C2 5, 6, 7. These genes form fixed haplotypes (complotypes) defined by their BF, C2, C4A, and C4B protein alleles [8]that are population-characteristic. Among mixed European caucasians, there are about a dozen protein complotypes that occur at frequencies of 1% or more.
By the application of molecular biologic techniques, it was shown that the 4 complement genes (C2, BF, C4A, C4B, in that order from telomere to centromere) as well as 2 genes for the adrenal steroid cytochrome P450 21-hydroxylase enzyme (CYP21A, a pseudogene, and CYP21B, the expressed gene) are found within 100 to 120 kb of genomic DNA 9, 10, 11, 12, 13. A number of restriction fragment length polymorphisms (RFLP’s) have been found in the DNA of the complement and CYP21 genes. In addition, deletions of C4A, C4B and CYP21A, gene conversion-like C4B to C4A changes and duplications of C4B and CYP21B are common.
The present study was designed to determine the relationship over the whole complotype region between the polymorphisms in the major histocompatibility complex-encoded complement proteins and RFLP’s, largely in non-coding regions, detected in the corresponding genomic DNA. In this way, we hoped to define complotype-RFLP constellations or CRC’s. An analysis of these, in turn, should suggest evolutionary relationships among the genes for individual protein-defined C2, C4 and BF alleles and complotypes in caucasians.
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Subjects
Lymphoblastoid lines were established by Epstein–Barr virus transformation of peripheral blood B lymphocytes from members of 52 families in which at least 1 member had type 1 diabetes mellitus. All subjects were studied for C2, C4 and BF protein types and complotypes were assigned from studies in immediate relatives. In addition, lymphoblastoid lines were established from peripheral blood B lymphocytes of MHC homozygous individuals either by us or by the 10th International Histocompatibility
Results
The complotype-RFLP constellations (CRC’s) are defined by their C2/Sst I (2.75, 2.70, 2.65, and 2.40 kb), BF/Taq I (6.6 and 4.5 kb), C4A/Bgl II (15 kb, designated I, and 4.5 kb, designated II), 5′ C4 (7.0, 6.4, 6.0 and 5.4 kb) and 3′ C4/Xba I/BamH I (11 and 4+7 kb) RFLP variants, by the presence or absence of C4A, C4B, CYP21A and CYP21B genes and by the presence or absence of duplications (Fig. 1 and Table 2). Of the more than 1000 theoretically possible combinations of these variants, only 19
Discussion
It is remarkable that only 19 of the more than 1000 theoretically possible CRC’s comprise the major part of caucasian complotypes. Since most of the RFLP’s that characterize the CRC’s represent macrostructural features such as the presence or absence of an intron derived from an endogenous retrovirus as in the long and short C4B genes 32, 35, 36, 37, the presence or absence of C4 or CYP21 genes 10, 31, and the size of an intron containing retroviral sequences in the C2 gene 22, 38, this speaks
Acknowledgements
We thank Barbara Moore, Susan Mrose and Carroll Goldsmith for expert technical assistance and Dr. Devendra Dubey for thoughtful comments. This work was supported by National Institutes of Health grants AI 14157, HL 29583, HL 48675, HD 17461, and DK 26844, and by the Swedish Medical Research Council and by the Alfred Österlund Fund. Louise Viehmann provided outstanding secretarial help. Drs. Michael Carroll, A. Steven Whitehead, and R. Duncan Campbell kindly provided DNA probes for these studies.
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