Chester A. Alper

Genetic Prediction of Nonresponse to Hepatitis B Vaccine

In previous studies of the antibody response to hepatitis B vaccine in 598 subjects who received a full course of vaccination, we observed a bimodal response, with about 14 percent producing less than approximately 1000 radioimmunoassay (RIA) units. An analysis of the major histocompatibility complex (MHC) HLA and complement types of 20 of the subjects with the lowest responses indicated a greater-than-expected number of homozygotes for the extended or fixed MHC haplotype [HLA-B8, SC01, DR3]. This finding suggested that the lack of a normal response was a recessive MHC-linked trait. In this study, we prospectively vaccinated five homozygotes and nine heterozygotes for this haplotype in the expectation that the homozygotes would produce much lower levels of antibody than the heterozygotes. When the antibody response was assessed two months after the third injection, four of the five homozygotes had produced very low levels (approximately 1000 units or less) of antibody (mean, 467 RIA units; range, less than 8 to 1266), whereas all nine heterozygotes produced more than 2500 RIA units (mean, 15,608; range, 2655 to 28,900) (P less than 0.01). We conclude that the usual response to hepatitis B surface antigen is due to the presence of a dominant immune-response gene in the MHC and that a low response is due to the absence of such a gene and the presence on both chromosomes of MHC haplotypes (such as [HLA-B8, SC01, DR3]) that indicate such a response.

Increased Susceptibility to Infection Associated with Abnormalities of Complement-Mediated Functions and of the Third Component of Complement (C3)

Abstract In a patient with Klinefelters syndrome and lifelong increased susceptibility to infection, no abnormalities were found in humoral antibody production, cellular immunity or leukocyte function. In contrast, the patients serum complement-mediated functions were grossly deficient. The concentrations of serum complement components were normal except for that of C3 (β1C-globulin), which was less than one-third normal. The bulk of this was in the form of the inactive conversion product, C3b, at all times that the patients serum or plasma was examined over a two-year period. Addition of small amounts of normal serum, but not purified C3, to the patients serum improved all complement-mediated functions in vitro. This disorder, which may represent an inborn deficiency of a protein necessary for C3 stability in vivo and in vitro, is detected by the lowered serum C3 concentration and a positive non-gamma (C3) Coombs antiglobulin test.

HOMOZYGOUS DEFICIENCY OF C3 IN A PATIENT WITH REPEATED INFECTIONS

Abstract A patient with striking susceptibility to infection by pyogenic organisms was found to have 1/1000th or less of the normal serum concentration of C3. Investigation of members of her family revealed many individuals, including her mother and father, with C3 levels approximately half-normal, and it seems that she is homozygous for C3 deficiency.

Selective Gamma-G Globulin Deficiencies in Patients with Recurrent Pyogenic Infections

Abstract Three patients with lifelong susceptibility to pyogenic infections and progressive pulmonary disease were found to have selective deficiencies in their γG globulins. The serum of the first patient contained abnormally low concentrations of γG1, γG2 and γG4; the serum of the second patient was deficient in γG1, γG2 and γG3, and the third patient lacked γG1 and γG2. The serum concentrations of the other immunoglobulins were either normal or elevated. None of these abnormalities could be shown to have a genetic basis. Gamma-globulin replacement therapy prevented recurrence of infection in these patients.

Statement on the Nomenclature of Human C4 Allotypes

As a collaborative work of three laboratories the polymorphism of the canine fourth complement component (C4) was studied in a total of 131 unrelated dogs from different breeds and mongrels. Using high voltage electrophoresis followed by an immunoblotting technique, we detected eight distinct variants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of canine C4 showed an additional heterogeneity of the α and γ chains which resulted in a total of 11 variants in the population studied. So that more precise information concerning the respective C4 allotypes will be available, a nomenclature is proposed designating not only the migration pattern of the C4 variants in agarose gels but also the heterogeneity of the C4 chains observed in SDS-PAGE.

Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants.

Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease.

Nonresponsiveness to hepatitis B vaccine in health care workers. Results of revaccination and genetic typings.

Twenty-eight health care workers who had a poor antibody response when initially vaccinated with hepatitis B vaccine were revaccinated with three additional 20-microgram doses. Eight of the twenty nonresponders, who had levels of antibody to hepatitis B surface antigen (anti-HBs) of less than 8 estimated radioimmunoassay (RIA) units, and all 8 of the hyporesponders, who had anti-HBs levels of 8 or 16 RIA units, attained anti-HBs levels of 36 RIA units or more after revaccination. Tests for HLA-A, B, C, and DR; for complement proteins C2, C4A, C4B, and BF; and for the erythrocyte enzyme glyoxalase I were done in 17 nonresponders and 3 hyporesponders. Nine (45%) had HLA-DR7 and 8 (40%) had HLA-DR3, compared with an expected rate of 23% in the general population. At least one of two extended haplotypes (B44, DR7, FC31 or B8, DR3, SCO1) were detected in 6 of the 9 who did not respond to revaccination, compared with 2 of 11 who responded to a second course of vaccine. Poor responders to vaccine may benefit from revaccination, and genetic factors may modulate the immune response to vaccination.

HLA-DQB1*0602 Is Associated With Dominant Protection From Diabetes Even Among Islet Cell Antibody–Positive First-Degree Relatives of Patients with IDDM

HLA-DQB1 alleles confer susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). We investigated whether the susceptibility alleles DQB1*0302 and DQB1*0201 affect progression to diabetes among islet cell antibody–positive (ICA+) first-degree relatives of IDDM patients and whether the protective allele DQB1*0602 can be found and is still protective among such relatives. We human leukocyte antigen–typed and periodically tested β-cell function (first-phase insulin release [FPIR] during the intravenous glucose tolerance test) in 72 ICA+ relatives, of whom 30 became diabetic on follow-up (longest follow-up 12 years); 54 (75%) relatives carried DQB1*0302 and/or DQB1*0201. The frequency of DQB1*0302 and DQB1*0201 and of the high-risk genotype DQB1*0302/DQB1*0201 did not differ significantly between diabetic relatives and those remaining nondiabetic. On follow-up, progression to IDDM was not statistically different for relatives with or without the DQB1*0302/DQB1*0201 genotype. However, those relatives with the DQB1*0302/DQB1*0201 genotype had a tendency to develop diabetes at an earlier age (log-rank P = 0.02). We found DQB1*0602 in 8 of 72 (11.1%) ICA+ relatives. Relatives with DQB1*0602 did not develop diabetes or show any decline of FPIR versus 28 of 64 DQB1*0602– relatives who developed IDDM (log-rank P = 0.006; Wilcoxons P = 0.02). The protective allele DQB1*0602 is found in ICA+ relatives who have minimal risk of progression to IDDM. Therefore, DQB1*0602 is associated with protection from IDDM both in population studies and among relatives with evidence of autoimmunity who should not enter prevention trials.

Prognostically significant heterogeneity of cytoplasmic islet cell antibodies in relatives of patients with type I diabetes.

A significant proportion of relatives of patients with insulin-dependent (type I) diabetes with high titers of cytoplasmic islet cell autoantibodies (ICAs) do not progress to overt diabetes with up to 8 yr of follow-up. This may reflect that follow-up of such relatives has not been long enough to observe diabetes, that despite expression of identical ICAs, some relatives will not progress to diabetes; or that there is heterogeneity in what is identified as ICA. We identified a subset of ICA that was restricted in its species (not reacting with mouse islets) and cell-type reactivity within islets (β-cell specific). Only one of eight relatives whose sera had the restricted pattern of reactivity progressed to overt diabetes, and on sequential evaluation, all but the one relative who progressed to diabetes have maintained normal first-phase insulin secretion to intravenous glucose. In contrast, by life-table analysis, 70% of relatives expressing nonrestricted ICA became diabetic within 5 yr of follow-up (1 of 8 vs. 16 of 25 diabetic at last follow-up, P < 0.02). Moreover, preliminary data suggest a significant association of the human leukocyte antigen DQB1*0602 allele of DR2 haplotypes with the restricted ICA pattern (4 of 5 DQB1*0602 restricted vs. 0 nonrestricted ICA, P = 0.006). We propose that expression of a genetically determined restricted ICA pattern confers a markedly lower risk for progression to diabetes. Our studies suggest that relatives with restricted ICA may comprise most ICA+ relatives who do not develop diabetes or abnormal first-phase insulin secretion on follow-up of >5 yr and that expression of such autoantibodies may be associated with the ‘protective’ DQB1*0602 allele.

Serum Complement ‘Supergenes' of the Major Histocompatibility Complex in Man (Complotypes)

Abstract. The loci for the complement proteins C2 and BF, and the two loci for C4 arc 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, complotypes may also be of importance in screening programs for transplantation.