Synthesis of human plasminogen by the liver.

Genetic types of plasminogen were determined from a donor and a recipient before and after hepatic homotransplantation. Examination of the plasminogen types demonstrated that the liver is the principal site of synthesis of human plasminogen.

The chromosomal order of genes controlling the major histocompatibility complex, properdin factor B, and deficiency of the second component of complement.

The relationship of the genes coding for HLA to those coding for properdin Factor B allotypes and for deficiency of the second component of complement (C2) was studied in families of patients with connective tissue disorders. Patients were selected because they were heterozygous or homozygous for C2 deficiency. 12 families with 15 matings informative for C2 deficiency were found. Of 57 informative meioses, two crossovers were noted between the C2 deficiency gene and the HLA-B gene, with a recombinant fraction of 0.035. A lod score of 13 was calculated for linkage between C2 deficiency and HLA-B at a maximum likelihood value of the recombinant fraction of 0.04. 18 families with 21 informative matings for both properdin Factor B allotype and HLA-B were found. Of 72 informative meioses, three recombinants were found, giving a recombinant fraction of 0.042. A lod score of 16 between HLA-B and Factor B allotypes was calculated at a maximum likelihood value of the recombinant fraction of 0.04. A crossover was shown to have occurred between genes for Factor B and HLA-D, in which HLA-D segregared with HLA-A and B. These studies suggest that the genes for Factor B and C2 deficiency are located outside those for HLA, that the order of genese is HLA-A, -B, -D, Factor B allotype, C2 deficiency, that the genes coding for C2 deficiency and Factor B allotypes are approximately 3--5 centimorgans from the HLA-A and HLA-B loci, and that the apparent lack of recombinants between the Factor B gene and C2 deficiency gene suggests that these two genes lie in close proximity to one another.

Inherited deficiency of the second component of complement. Rheumatic disease associations.

The prevalence of homozygous and heterozygous deficiency of the second component of complement (C2) was determined in patients with rheumatic disease including 137 with systemic lupus erythematosus (SLE), 274 with juvenile rheumatoid arthritis, and 134 with rheumatoid arthritis. 1 C2 homozygous deficient and 19 possible heterozygous deficient individuals were identified by using both immunochemical and functional assays to determine C2 levels. Of the 20, 8 had SLE (5.9%), 10 had juvenile rheumatoid arthritis (3.7%), and 2 had rheumatoid arthritis (1.4%), the homozygous deficient individual having SLE. The prevalence of C2 deficiency in the SLE and juvenile rheumatoid arthritis patients was significantly increased (P = 0.0009 and P = 0.02, respectively) when compared with controls, 6 (1.2%) of 509 blood donors having C2 levels consistent with heterozygous deficiency. 15 of the 20 C2 deficient patients were HLA typed and found to have antigens A10(Aw25), B18, or both. The patients with C2 deficiency and SLE had earlier age of onset of disease and less antinuclear antibody when compared with the C2 normal SLE patients. 11 families of the propositi were studied and found to have one or more C2 heterozygous deficient individuals. The family members had an equal distribution of rheumatic disease and antinuclear antibody in the C2 deficient and C2 normal groups. C2 deficient individuals were found to have significantly lower levels of properdin Factor B (242 mug/ml+/-54) when compared with the non-C2 deficient family members (282 mug/ml+/-73). These data support the concept that inherited deficiency of C2 is significantly associated with both SLE and juvenile rheumatoid arthritis.

Extended major histocompatibility complex haplotypes in type I diabetes mellitus.

We have studied major histocompatibility complex markers in Caucasian patients with type I diabetes mellitus and their families. The frequencies of extended haplotypes that were composed of specific HLA-B, HLA-DR, BF, C2, C4A, and C4B allelic combinations, which occurred more commonly than expected, were compared on random diabetic and normal chromosomes in the study families. We demonstrated that all of the previously recognized increases in HLA-B8, B18, B15, DR3, and perhaps DR4 could be ascribed to the increase among diabetic haplotypes of a few extended haplotypes: [HLA B8, DR3, SC01, GLO2]; [HLA-B18, DR3, F1C30]; [HLA-B15, DR4, SC33]; and [HLA-BW38, DR4, SC21]. In fact, HLA-DR3 on nonextended haplotypes was "protective", with a relative risk considerably less than 1.0. There was a paucity or absence among diabetic patients of several extended haplotypes of normal chromosomes, notably [HLA-B7, DR2, SC31] and [HLA-BW44, DR4, SC30]. The extended haplotype [HLA-BW38, DR4, SC21] is found only in Ashkenazi Jewish patients, which suggests that extended haplotypes mark specific mutations that arise in defined ethnic groups. The data show that no known MHC allele, including HLA-DR3 and possibly HLA-DR4, is per se a marker for or itself a susceptibility gene for type I diabetes. Rather, extended haplotypes, with relatively fixed alleles, are either carriers or noncarriers of susceptibility genes for this disease. Thus, the increased frequency (association) or the decreased frequency (protection) of individual MHC alleles is largely explainable by these extended haplotypes.

Genetic polymorphism in C8 beta-chains. Evidence for two unlinked genetic loci for the eighth component of human complement (C8).

Genetic polymorphism in the beta-subunit of the eighth component of human complement, C8, was defined by isoelectric focusing of serum in polyacrylamide gel in the presence of urea and development of specific patterns of hemolysis in an overlay gel containing antibody-sensitized erythrocytes and C8 beta-chain-deficient serum. Bands of hemolysis induced by serum from unrelated Caucasians suggested autosomal codominant inheritance of three structural alleles at a single locus, C82: C82 degrees A (acidic), C82 degrees B (basic), and C82 degrees A1 (very acidic) with frequencies of 0.952, 0.044, and 0.004, as well as the probable null allele C82 degrees Q0. The distribution of phenotypes agreed with the Hardy-Weinberg equilibrium. The previously described genetic polymorphism in human C8 defined with the use of "complete" C8 (C8 alpha-gamma-chain)-deficient serum was distinct from and independent of the inherited structural variation at C82. Therefore, the locus for C8 alpha-gamma-chains has been redesignated C81, and has the alleles C81 degrees A, C81 degrees A1, and C81 Q0. Linkage studies failed to show close linkage between the two loci for C8, C81, and C82, and between C82 and the major histocompatibility complex or C6.

Genetic control of the eighth component of complement.

Using isoelectric focusing in polyacrylamide gel and a hemolytic assay for development of patterns, extensive, structural polymorphism in human C8 has been delineated. Two alleles, C8A and C8B, have been identified in orientals, with gene frequencies of 0.655 and 0.345. In blacks, what appears to be a third common allele was found, so that frequencies were 0.692, 0.259, and 0.049 for C8A, C8B, and C8A1. In whites, C8A1 was rare with a frequency of 0.003, and frequencies for C8A and C8B were 0.649 and 0.349. Inheritance was autosomal codominant in family studies and the distribution of types in random unrelated populations fit the Hardy-Weinberg equilibrium in all groups. C8 allotypes have been determined for two previously studied families, each with a homozygous C8-deficient propositus. This study suggests that C8 deficiency is a silent or null allele of the C8 structural locus, and that half normal levels of C8 cannot be used as a single criterion for the establishment of heterozygous C8 deficiency. C8 allotypes, as well as 18 other autosomal markers, were also determined for 24 families. The C8 structural locus is not closely linked to these markers, including the human histocompatibility loci complex.

Complement-human histocompatibility antigen haplotypes in C2 deficiency.

C4 allotyping 13 homozygous C2-deficient individuals demonstrated 23 of 25 haplotypes to be of the relatively rare type C4A4 B2. This is of the same magnitude as the association of C2Q0 with HLA-DW2/DR2.

Complement genes of the major histocompatibility complex (complotypes), extended haplotypes and disease markers.

The human major histocompatibility complex (MHC)-linked genes C2,BF,C4A,C4B occur in populations and segregate in families as single genetic units or complotypes. Analysis for significant three-point linkage disequilibrium between HLA-B, DR and complotype on normal caucasian chromosomes 6p yields about a dozen haplotypes that account for most of the known HLA-B/HLA-DR linkage disequilibrium pairs previously noted in normal caucasian populations. We refer to the HLA-B/DR/complotype sets with significant linkage disequilibrium as extended haplotypes since they often show limited variation at other MHC-linked loci. From the study of MHC haplotypes in 21-hydroxylase deficiency, C2 deficiency and type 1 diabetes, it is becoming apparent that it is extended haplotypes rather than their individual alleles that are markers for these MHC-associated diseases.

BF types and the mode of inheritance of insulin-dependent diabetes mellitus (IDDM).

Insulin-dependent diabetes mellitus (IDDM) has been found to be highly associated with a rare allele of the complement protein, properdin factor B (BF). Assuming that there is a susceptibility gene for IDDM tightly linked to the genetic locus for BF and the major histocompatibility complex (MHC), the distribution of BF types in more than 1100 North American IDDM patients strongly argues for the rejection of dominant, epistatic, and overdominant modes of inheritance. Other evidence suggesting complex modes of inheritance for IDDM is reviewed and it is concluded that our observations and published data are consistent with the idea of susceptibility to IDDM being inherited as a simple autosomal recessive trait. C4 and C2 types, also linked to BF and the MHC, were investigated too. C4 Fs0 was found to be increased in association with BF F1, while C4 f0S and C2 b were each found to occur twice as frequently as in a control population and will be of value in defining haplotypes associated with susceptibility to IDDM.

Genetic polymorphism of human plasminogen.

Using isoelectric focusing (IEF) in polyacrylamide gel of neuraminidase-treated serum or plasma samples and immunofixation or caseinolytic overlay after urokinase activation of gels, a common genetic polymorphism in human plasminogen has been delineated. Two alleles PLGN*A and PLGN*B, were observed with gene frequencies in whites of .69 and .30; in Orientals of .96 and .03; and in blacks of .80 and .18. Several rare alleles were also found. The distribution of phenotypes fits the Hardy-Weinberg equilibrium. Inheritance is autosomal codominant and fits the expectations of Mendelian inheritance. There is fetal synthesis, but no transplacental passage of plasminogen in either direction.

Genetic marker for insulin-dependent diabetes mellitus.

A rare genetic type (Bf F1) of properdin factor B is found in 22.6% of patients with insulin-dependent diabetes mellitus but in only 1.9% of the general population, yielding a relative risk of 15.0. This indicates that a genetic locus for insulin-dependent diabetes mellitus is very close on chromosome 6 to Bf, and that Bf F1 is a marker for nearly 1 out of 4 insulin-dependent diabetic patients.

Possible human analogs of the murine T/t complex.

After a short description of the T-t complex of the mouse, a comparison between the MHCs of mouse and man and HLA marker-disease associations, possible candidates for human t-analogs are discussed. The hypothesis is put forward that some extended MHC haplotypes might be human analogs of murine t-mutants.

Serum complement 'supergenes' of the major histocompatibility complex in man (complotypes).

The loci for the complement proteins C2 and BF, and the two loci for C4 are closely linked to one another. In many hundreds of meioses no crossing over has been detected between these loci. In addition, the alleles of these four loci occur in specific combinations not predicted by their gene frequencies in much the same way as alleles of the Rh and MNS systems. These units are termed complotypes. There are 14 complotypes with frequencies in excess of 1% in our study population of normal sixth chromosomes from Caucasians. Since they are also intimately associated with HLA-DR, comploytypes may also be of importance in screening programs for transplantation.

The MHC in human bone marrow allotransplantation.

In this chapter, we have considered the theoretical and practical background of bone marrow transplantation. The immune response and its regulation by genes within the major histocompatibility complex, particularly of the I region of the mouse and of the HLA-D/DR region in man, is of central importance in both graft acceptance (rejection) and graft-versus-host disease. Methods which are available for typing alleles at the HLA-A, -C, -B, -DR and complotype (BF, C2, C4A, C4B) loci, have been considered in detail. The extent to which recombination affects specific alleles on haplotypes within families is discussed, as is the occurrence of linkage disequilibrium and extended haplotypes in populations of unrelated individuals. Because the HLA-DR and complotype region in man is thought to be critical for the success of bone marrow transplantation, methods for typing of HLA-D by both the HTC and PLT approaches have been examined. Although HLA-D/DR assignments are easily made in normal subjects, they are ambiguous in about 50 per cent of candidates for bone marrow transplantation, including, particularly, patients with aplastic anaemia, leukaemia, and severe combined immunodeficiency. In this setting, it is particularly important to obtain additional information by modification of HLA-D typing procedures and through complotype and GLO allele determinations in all family members. Finally, we can hope that there will be an increased possibility of using non-family donors through methods for removing cytotoxic T cells from donor marrow and through the identification, in the general population, of individuals who are genotypically similar or identical to the recipient. In this regard, the recognition that some 30 per cent of chromosome 6 in caucasians (50 per cent of individuals) bear extended haplotypes, which include a relatively fixed set of alleles particularly in the HLA-B, -DR, complotype and GLO regions, offers considerable promise.

Extended HLA/complement allele haplotypes: evidence for T/t-like complex in man.

The chromosomal distribution of alleles for HLA-A,-B,-C, and -DR and the serum complement protein alleles of factor B and C2 and C4 was studied in normal Caucasian families. Eight combinations of HLA-B, DR, BF, C2, C4A, and C4B markers were found to occur in haplotypes at frequencies significantly higher than expected. In these combinations, which were defined as extended major histocompatibility complex haplotypes, HLA-A showed limited variation. A possible mechanism for the maintenance of extended haplotypes are human analogs of murine t mutants which are characterized by crossover suppression and male transmission bias. One human 6p haplotype, HLA-B8, DR3, SCO1, GLO 2, was found to be transmitted from males to 83% of their offspring. The same haplotype with GLO 1 had no transmission bias. It is suggested that this GLO 2-marked chromosome is a human analog of a murine t mutant.

Genetic polymorphism of serum complement components in the chimpanzee.

Significant polymorphism of serum complement components Bf, C2, C3, C6, and C8 in the chimpanzee has been demonstrated. The data are consistent with the hypothesis that C2 and Bf are closely linked to ChLA and argue against close linkage of ChLA to C3 or to C8, as in man. In addition, a blank allele for C6 and C6 deficiency was detected in several chimps.

The location of C2, C4, and BF relative to HLA-B and HLA-D.

The loci for HLA-A,B,C,D, and DR are known to be closely linked to the structural loci for the complement components C2, BF, and the duplicated loci for C4, C4A and C4B. Conflicting evidence has been presented for the order of these genes. However, new techniques have made possible identification of markers in the HLA-D and C4 region for nearly all identified haplotypes. In our population we have confirmed five HLA-B-D crossovers and in each case informative allotypes of C2, BF, or C4A and C4B segregated with HLA-D or DR suggesting that the loci for these proteins lie close to HLA-D and DR. These findings may be of importance for resolving problems encountered in the assignment of HLA-D alleles.

Mapping of the structural gene for the second component of complement with respect to the human major histocompatibility complex.

Families have been HLA typed, and allotypes of the second component of complement and properdin factor B determined. The lod score for the C2 structural gene and HLA-B from the study of 11 families and 55 informative meioses was 14.39 at maximum likelihood estimate of the recombination fraction of .02. This is related to other estimates of the distance between these two genes. The relative kinetic activities of the C2 allotypes were studied and no differences were demonstrated. No crossovers between Bf and C2 were observed.

Evidence for a silent or null gene in hereditary C2 deficiency.

Three generations of a family with hereditary C2 deficiency were studied, Six members heterozygous for C2 deficiency were identified by serum C2 levels that were approximately 50% of normal C2 values and the identity was supported by HLA analysis. All six members with low C2 levels had only a single electrophoretic variant. Two of four children did not have the variant found in the parent from whom they inherited the partial C2 deficiency. It is inferred that the low levels of C2 result from the inheritance of a silent or null gene, C2D allelic with the structural genes controlling the electrophoretic variants.