Mutations clustered in exon 5 of the p53 gene in primary nasopharyngeal carcinomas from southeastern Asia.

Mutations in the p53 tumor suppressor gene play an important role in the development of many common human malignancies. In nasopharyngeal carcinomas (NPC), p53 gene mutations were not detected in primary tumors, with one exception for a primary tumor displaying a p53 mutation at codon 280, whereas p53 mutations were identified in some metastatic and nude mouse-passaged NPC specimens. In the present report, 41 NPC primary tumors of the undifferentiated carcinoma nasopharyngeal type (UCNT; 21 from Hong Kong and 20 from Guangxi, southeastern China) were studied. Four point mutations that result in amino acid substitutions were identified by PCR amplification of exons 2-9 and direct DNA sequencing, combined with PCR-single-strand conformation polymorphism analysis. The 4 mutations detected were clustered within the DNA stretch from codon 175 to 177. Our data, taken together with those of others, suggest that mutation in p53 may occur in NPC at various points during tumorigenesis. Alternative mechanisms of p53 inactivation in NPC are also possible.

A possible linkage of HLA-DRB haplotypes with Tiopronin intolerance in rheumatoid arthritis.

To investigate the relationship between HLA class II genotypes and toxic intolerance during treatment with Tiopronin, a slow-acting drug used in the treatment of rheumatoid arthritis (RA), we studied 40 patients who were divided into two groups: a group of 22 patients without side effects and a group of 18 patients with intolerance to Tiopronin. The PCR-RFLP method was used to determine the HLA-DR, DQ and DP genotypes. The patients in the two groups had similar genetic backgrounds with an expected high frequency of DRI and DR4 alleles. However, DR1/DR4 heterozygosity was significantly increased in patients with intolerance (p = 0.03, Odds Ratio = 10.5). In addition, one intolerant patient had a DR1/DR7 genotype which shared DRw53 (DRB4*0101) with DR1/DR4. Furthermore, two subtypes of DR5, DRB1*1102 and DRB1*1201, were increased among intolerant patients (11.1% vs 0%, p = 0.03, OR = 13.97). In total, DR1/DRw53 heterozygotes, DRB1*1102 and DRB1*1201 represented 61.1% of intolerant patients. Therefore, a detailed HLA class II typing might be useful before RA treatment by Tiopronin to predict and avoid toxic side effects in the patients with increased risk. Further investigation is currently underway.

HLA class II alleles in isocyanate-induced asthma.

Studying genetic factors that control human immune responsiveness may further our understanding of specific types of asthma in which the role of immune factors is uncertain to date. HLA Class II gene products are involved in the control of immune responses. Therefore, we investigated whether HLA Class II genetic markers contribute to susceptibility or resistance to isocyanate-induced asthma (IAA) in exposed workers. We collected venous blood samples from two groups of unrelated white adults: (1) patients with isocyanate-induced asthma documented by a positive inhalation challenge; and (2) exposed individuals with no history of IAA. The second exon of DQA1, DQB1, DPB1, and DRB genes was selectively amplified by the polymerase chain reaction (PCR) method. HLA typing was carried out by the PCR-RFLP method, which allowed discrimination of most HLA DQA1, DQB1, DPB1, and DRB alleles. No significant difference was found in the distribution of DPB1 alleles between patients and control subjects. Allele DQB1*0503 and allelic combination DQB1*0201/0301 were associated with susceptibility to the disease. Conversely, allele DQB1*0501 and the DQA1*0101-DQB1*0501-DR1 haplotype conferred significant protection to exposed healthy control subjects. Our results are consistent with the hypothesis that immune mechanisms are involved in isocyanate-induced asthma and that specific genetic factors may increase or decrease the risk of developing IAA in exposed workers.

Homogeneous phase pyrophosphate (PPi) measurement (H3PIM). A non-radioactive, quantitative detection system for nucleic acid specific hybridization methodologies including gene amplification.

A one-step, non-radioactive, homogeneous phase revelation system designed to detect and quantify nucleic acid hybridization is described. The principle of the procedure, termed homogeneous phase pyrophosphate (PPi) measurement (H3PIM), is to detect and quantify the release of PPi from nucleotides, which occurs stoichiometrically when nucleic acids are synthesized. The method does not require any special reagents before the H3PIM revelation step. H3PIM is particularly adapted to quantitative measurement of gene amplification or cDNA gene expression using PCR, but can also be used following random priming or simultaneous multi-step nucleic acid amplification. This rapid, sensitive, liquid phase procedure permits the design of low-cost, fully automated devices for gene detection and quantification.

HLA-DP distribution in Shanghai Chinese--a study by polymerase chain reaction--restriction fragment length polymorphism.

One hundred and four normal unrelated Chinese were typed for HLA-DPA1 and DPB1 alleles by polymerase chain reaction-restriction fragment length polymorphism. Increased frequencies of HLA-DPA1*0201 and DPB1*0501 were found in this Chinese population as compared with those detected in Caucasoids and blacks.

A simple nonradioactive method of DNA typing for subsets of HLA-DR4: prevalence data on HLA-DR4 subsets in three diabetic population groups.

The molecular basis of eight DR4 subtypes resides in several nucleotide substitutions in the third hypervariable region of the DR beta 1 chain. The typing of DR4 subsets using the mixed lymphocyte culture (MLC) assay or allele-specific oligonucleotide hybridization is expensive, cumbersome, and requires the use of radioisotopes. We have therefore developed a rapid and safe procedure for subtyping DR4-alleles that involves selective amplification of the second exon of the DR4-DRBI gene followed by unambiguous subtype discrimination after digestion with five allele-specific endonucleases [polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)] and visualization of the polymorphic fragments with silver or ethidium bromide staining. Validity of this subtyping procedure was initially examined by the use of cell lines of known subtypes. Three groups of DR4 patients with insulin-dependent diabetes mellitus (IDDM) from Chinese, Tunisian, and Caucasian populations were subtyped and the prevalence of subtype associations with IDDM was compared.

Aspartic acid at position 57 of the HLA-DQ beta chain in insulin-dependent diabetes mellitus: an association with one DRw9-DQw9 subtype in the Chinese population.

Eighteen unrelated Chinese patients with insulin-dependent diabetes mellitus (IDDM) were analyzed for HLA Class II genes using a variety of molecular biological techniques including restriction fragment length polymorphism (RFLP), polymerase chain reaction with allele-specific oligonucleotides (PCR-ASO) and direct DNA sequencing. The high frequency of DR3/DR4 heterozygotes found in the Chinese with IDDM strengthens the importance of this combination of haplotypes in IDDM susceptibility since it is present in two genetically distant populations--Chinese and Caucasians. The frequency of DRw9, a rare allele in the Caucasian population, is much higher in the Chinese. Moreover, the DQ beta chain linkage of DRw9 was different in IDDM patients compared with control subjects. In contrast with previous results, codon 57 of the DQ beta chain was aspartic acid in DRw9 Chinese IDDM patients. Furthermore, one particular DRw9-DQw9 haplotype may be associated with IDDM susceptibility in the Chinese population.

Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles by enzymatic amplification and allele-specific restriction fragment length polymorphism.

A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size-separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory.

Association of type 1 diabetes mellitus with the HLA-DQA1*0301 allele in a Tunisian population.

HLA class II antigens are transmembrane glycosylated heterodimers composed of an alpha and a beta chain. Several of these chains are highly polymorphic. The structural bases of the polymorphism are nucleotide acid substitutions which are situated in the first domain (exon II) of alpha and beta genes. Specific sequences of these domains can be obtained by amplification of genomic DNA using the polymerase chain reaction. Polymorphic sites are recognized by restriction endonuclease treatment and separation of the DNA fragments by polyacrylamide gel electrophoresis. The resulting fragments of different lengths are used to identify different alleles. We used the above technique for typing the HLA-DQA1 alleles in 41 Tunisian diabetic patients. The frequency of DQA1*0301 was greatly increased compared with the control group. This was in agreement with previously published data in Caucasian and Japanese insulin-dependent diabetes mellitus (IDDM) patients, while the significant increase in the frequency of the DQA1*0501 allele was comparable with that of Caucasian IDDM patients but contrasted with a decrease in this allele in Japanese IDDM patients. Our results provide confirmation of the contribution of the DQA1*0301 allele to disease susceptibility in a Tunisian population.

Early biochemical events after MHC class II-mediated signaling on human B lymphocytes.

These studies examined the role of the MHC class II Ag in signal transduction using human B lymphocytes. Early events in signal transduction were considered including the intracellular calcium [Ca2+)i) flux, the activation of phospholipase C, and induction of protein phosphorylation. The (Ca2+)i was enhanced after incubation of B lymphocytes with several mAb anti-HLA class II and cross-linking with rabbit anti-mouse-F(ab')2. We have also demonstrated an enhancement of the (Ca2+)i in response to a suboptimal concentration of a monoclonal anti-IgM either in the presence of or after preincubation with a mAb anti-HLA class II. The activation of phospholipase C was assessed by measuring the generation of inositol phosphates in permeabilized B lymphocytes. mAb anti-HLA-class II of two different epitopes were used to demonstrate both the (Ca2+)i flux and the generation of inositol phosphates. Two-dimensional gel electrophoresis was used to investigate the phosphorylation pattern of resting B lymphocytes and the changes in the pattern after stimulation with soluble mAb anti-HLA-DR, immobilized mAb anti-HLA-DR, and PMA. In addition to the augmentation of phosphorylation observed with regard to phosphoproteins already present in resting B lymphocytes, new phosphorylations were observed after stimulation by any one of the reagents. Furthermore, stimulation by PMA did not result in an identical pattern to that observed after stimulation by mAb anti-HLA class II. An inhibition of the proliferative response to PMA was demonstrated after prestimulation of cells with immobilized mAb anti-HLA-DR, supporting the notion of a shared pathway of activation. In summary, these data demonstrate signal transduction via MHC class II Ag as assessed by three different measures of early events in human B lymphocyte activation and suggest that a protein kinase C pathway is at least partly involved.