Christian Rittner

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.

Tumor necrosis factor alpha promoter polymorphism at position -238 is associated with chronic active hepatitis C infection

Tumor necrosis factor α (TNF‐α) is involved in the pathogenesis of chronic hepatitis C virus infection. The gene for TNF‐α is encoded in the major histocompatibility locus (MHC). Two polymorphisms at positions ‐308 and ‐238 in the TNF‐α promoter region might influence TNF‐α expression. These promoter polymorphisms have been linked previously to a number of infectious diseases. TNF‐α promoter polymorphisms at positions ‐238 and ‐308 were studied by DNA sequencing and sequence‐specific oligonucleotide hybridization in 82 individuals with chronic hepatitis C and 99 control subjects. Subjects had been HLA class I and class II typed in a previous study. The frequency of the TNF238.2 promoter allele was significantly higher in the hepatitis C group (18.7%) compared to the controls (3.5%; P < 0.0001; pcorr < 0.009). No significant differences in the frequency of the TNF308.2 allele were observed between patients and controls. The increased frequency of the TNF238.2 allele could not be explained by linkage disequilibrium to HLA‐B or ‐DR genes. These findings show an association between the TNF238.2 promoter variant and chronic active hepatitis C. They suggest that this polymorphism or a linked gene may be a host factor contributing to the development of chronic active hepatitis C. J. Med. Virol. 54:173–177, 1998.

HLA-DRB1*1301 AND *1302 protect against chronic hepatitis B

BACKGROUND/AIMS The outcome of acute hepatitis B infection may be influenced by host factors like the major histocompatibility complex (MHC). We have investigated MHC class I and class II antigens in patients with chronic hepatitis B compared to a healthy control population. To confirm the findings of this first study we performed a second study in a group of subjects who had spontaneously recovered from acute hepatitis B infection. METHODS Frequencies of MHC class I and class II antigens were analyzed in patients with chronic hepatitis B virus infection and in control subjects. MHC class I typing was done by standard microlymphocytotoxicity assays. DRB1 and DQA1 genotypes were determined by polymerase chain reaction based typing methods. RESULTS In the first study the class II allele HLA-DRB1*1301-02 was found in 4 of 70 subjects with chronic hepatitis B virus infection (5.7%) compared to 27 of 101 healthy controls (26.7%, relative risk 0.17; p=0.001; p(corr)=0.025). This protective effect of the DRB1*1301-02 allele was confirmed in the second study. Eight of 24 patients (33.3%) who cleared hepatitis B virus spontaneously were positive for DRB1*1301-02 (relative risk of developing chronic infection compared to chronic hepatitis B subjects 0.12; p=0.004). Subtyping confirmed that 1301 and 1302 were both decreased in frequency in patients with chronic hepatitis B. CONCLUSIONS The MHC class II allele DRB1* 1301-02 is associated with protection from chronic hepatitis B in Caucasian patients.

Differential genetic determination of immune responsiveness to hepatitis B surface antigen and to hepatitis A virus: a vaccination study in twins

BACKGROUND The course of viral hepatitis is thought to be affected by genetic host variability and, in particular, by genes of the major histocompatibility locus. Hepatitis A and B vaccination is a useful model to study the effect of host factors on the immune response to viral antigens. We aimed to assess the heritability of the HBsAg (anti-HBs) and anti-hepatitis A virus (anti-HAV) immune response and to estimate the effect of the HLA-DRB1 locus and other genetic loci unlinked to HLA. METHODS We did an open prospective study and vaccinated 202 twin pairs with a combined recombinant HBsAg/inactivated hepatitis A vaccine. We measured antibodies to HBsAg and HAV and determined HLA-DRB1* alleles. Heritability was calculated based on variance of antibody response within pairs. Model-fitting analyses were done to analyse genetic and environmental components of vaccine responses. FINDINGS Anti-HBs and anti-HAV showed heritabilities of 0.61 (95% CI 0.41 to 0.81) and 0.36 (-0.02 to 0.73), respectively. For the anti-HBs immune response, 60% of the phenotypic variance was explained by additive genetic and 40% by non-shared environmental effects. The heritability of the HBsAg vaccine response accounted for by the DRB1* locus was estimated to be 0.25, leaving the remaining heritability of 0.36 to other gene loci. INTERPRETATION Genetic factors have a strong effect on the immune response to HBsAg. Although genes encoded within the MHC are important for this immune response, more than half the heritability is determined outside this complex. Identification of these genes will help us to understand regulation of immune responses to viral proteins.

HLA DRw8 and complement C4 deficiency as risk factors in primary biliary cirrhosis.

HLA class I, II, and III alleles were investigated in 25 consecutive unrelated German patients with primary biliary cirrhosis and in two families with two primary biliary cirrhosis patients in each. In primary biliary cirrhosis patients, HLA class I antigens did not differ significantly from in health controls. For HLA class II antigens, a highly significant increase of HLA DRw8 was found in patients with primary biliary cirrhosis compared with controls. Thirty-six percent vs. 3.6% were DRw8 positive [relative risk = 15.28; P (corrected) = 0.00013]. The genetic typing of HLA class III alleles revealed an increased incidence for C4AQ0 alleles [72% vs. 34.5%, relative risk = 4.89: P (corrected) = 0.0056]. A highly significant proportion of primary biliary cirrhosis patients carrying both DRw8 and C4A-Q0 alleles (relative risk = 183.75; P = 9.7 x 10(-7)) were found. In one family, a mother and her daughter had primary biliary cirrhosis, both sharing the major histocompatibility complex haplotype HLA-A1, -B8, -DR3, -C4AQ0B1. In the other family, two sisters with primary biliary cirrhosis shared the major histocompatibility complex haplotype HLA-A24, -B8, -DRw8, -C4A4B2. These studies contribute to the further elucidation of the immunogenetic background of primary biliary cirrhosis.

MHC class II genes influence the susceptibility to chronic active hepatitis C.

BACKGROUND/AIMS Chronic hepatitis C develops in more than 70% of hepatitis C virus infected subjects. Viral factors influence the disease course, but little is known about the importance of host factors. METHODS Frequencies of major histocompatibility complex (MHC) class I and class II antigens were analyzed in two groups of patients with chronic hepatitis C virus infection and in control subjects. MHC class I typing was done by standard microlymphocytotoxicity assays. DRB1 and DQA1 genotyping was done by PCR based typing methods. RESULTS DRB1*0301 was found in 26 of 75 patients with chronic hepatitis C virus infection (34.7%) and in 12 of 101 control subjects (11.9%) (relative risk 3.9; p < 0.001). Homozygosity for this allele appeared to confer a stronger risk. In contrast, DRB1*1301 was detected in three subjects with persistent infection (4.0%) compared to 21 control subjects (20.8%) (relative risk 0.2; p < 0.008). This allele was linked with DQA1*0103, which was found in 10 patients (13.3%) compared to 34 control subjects (33.7%) (relative risk 0.31; p < 0.003). An even stronger protective effect was provided by the presence of DRB1*1301 and DQA1*0103 (relative risk 0.08; p < 0.005). These findings were confirmed in a second group of chronic hepatitis C virus infected patients. CONCLUSIONS The MHC class II allele DRB1*0301 appears to predispose to progression to chronic active hepatitis C, whereas the class II alleles DRB1*1301 and DQA1*0103 appear to provide protection against chronic active infection with hepatitis C virus.

Genetic basis of human complement C4A deficiency. Detection of a point mutation leading to nonexpression.

The fourth component of the human complement system (C4) is coded for by two genes, C4A and C4B, located within the MHC. Null alleles of C4 (C4Q0) are defined by the absence of C4 protein in plasma. These null alleles are due either to large gene deletions or to nonexpression of the respective genes. In a previous study, evidence was obtained for nonexpressed defective genes at the C4A locus, and for gene conversion at the C4B locus. To further characterize the molecular basis of these non-expressed C4A genes, we selected nine pairs of PCR primers from flanking genomic intron sequences to amplify all 41 exons from individuals with a defective C4A gene. The amplified products were subjected to single-stranded conformation polymorphism (SSCP) analysis to detect possible mutations. PCR products exhibiting a variation in the SSCP pattern were sequenced directly. In 10 of 12 individuals studied, we detected a 2-bp insertion in exon 29 leading to nonexpression due to the creation of a termination codon, which was observed in linkage to the haplotype HLA-B60-DR6 in seven cases. In one of the other two individuals without this mutation, evidence was obtained for gene conversion to the C4B isotype. The genetic basis of C4A nonexpression in the second individual is not yet known and will be subject to further analysis.

C4A deficiency and nonresponse to hepatitis B vaccination

BACKGROUND/AIMS Hepatitis B vaccination failure has been linked to the presence of certain human leukocyte antigen class II alleles. However, the functional background of these associations has remained unclear. Complement component C 4 is encoded within the major histocompatibility complex and is essential for classical pathway activation. METHODS Healthy individuals (n=4269) were vaccinated in a prospective trial with Engerix B. Nonresponse was classified as anti-HBs<10 U/l after the last vaccination. Seventy-three nonresponders (NR) (1.7%) were identified. For comparison 53 responders (R) (anti-HBs>10 IU/l) were drawn randomly from the same cohort. C4 allotyping was carried out by high-voltage agarose gel electrophoresis and C4alpha-chain typing using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C4 gene deletions (C4Del) were studied by Southern blot. RESULTS C4AQ0 alleles were observed in 45/73 (62%) NR compared to 17/53 (32%) R (P=0.001). C4ADel was observed in 24/73 (33%) NR and in 6/52 (12%) R (P=0.006). C4AQO alleles were present in 21/49 (43%) NR without C4Del compared to 10/46 (22%) in R without C4Del (P=0.031). In a logistic regression with DRB1*0301, DRB1*07, DRB1*1301 and C4AQ0 all except for DRB1*0301 showed a significant association. CONCLUSIONS C4AQ0 shows a DRB1*0301 independent association with vaccine failure. C4AQ0 alleles probably contribute to inefficient complement activation and failure of B cells to secrete anti-HBs.

The Influence of Major Histocompatibility Complex Class II Genes and T-Cell Vβ Repertoire on Response to Immunization with HBsAg

Nonresponsiveness to HBsAg vaccination is observed in 5-10% of vaccine recipients and is possibly caused by a defect in the T helper cell compartment. The immune response to HBsAg is influenced by genes of the major histocompatibility complex. We have investigated MHC class I and class II antigens in 53 adult responders and 73 nonresponders. Results obtained in this first study were tested in a second study with 56 responders and 62 nonresponders from an infant vaccination trial. In addition, the peripheral Vbeta-chain T-cell receptor repertoire was investigated using monoclonal antibodies and flow-cytometry in 26 adult responders and 38 nonresponders. As previously reported, nonresponsiveness to HBsAg vaccination was associated with DRB1*3 and DRB1*7. In addition, DRB1*13 was significantly increased among vaccine responders (35.2% vs 5.4%;p < 0.0001) suggesting an immune response promoting effect for this allele whereas the closely related allele DRB1*14 was associated with nonresponse in the infant study. There was no evidence for a hole in the T cell receptor Vbeta repertoire. In conclusion, in agreement with results obtained in mice there appears to be a hierarchy of DRB1* genes in the HBsAg immune response. The possible differential association of DRB1*13 and DRB1*14 may allow the identification of differences between responsiveness and nonresponsiveness to a few amino acid differences in the beta1-domain of the class II heterodimer.

Forensic mtDNA hair analysis excludes a dog from having caused a traffic accident

Abstract A dog was suspected of having caused a traffic accident. Three hair fragments were recovered from the damaged car and subjected to DNA sequence analysis of the canine mitochondrial D-loop control region. The results were compared to saliva and hair samples from the alleged dog, as well as to control hair samples from four unrelated dogs of different breeds. Two sequence types exhibiting five nucleotide differences in a 377 bp fragment were identified among the four controls. Whereas the evidence hair fragment was identical to the type 1 control sequence, the alleged dog shared the type 2 control sequence except for one position. Thus the dog could be excluded as the origin of the hair fragment. As canine mtDNA appears to exhibit only limited polymorphism, mitochondrial D-loop sequence comparison is currently only suitable for exclusions.