[PCR-SSOP (sequence specific oligonucleotide probe) method].
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DNA Probes, HLA↗
Biomedical subjects
T Sasazuki
Publications and source records attributed to T Sasazuki.
[Familial cancer and oncogenic factors].
The recent remarkable progress in molecular biology has revealed that various kinds of genetic alteration occur in cancers. Recently, many genes that cause hereditary cancer have been identified. For example, hMSH2 and hMLH1, which are known as DNA mismatch repair genes have been found to cause HNPCC (hereditary non-poliposis colorectal cancer). Mutation of RET oncogene has been recognized in the families of MEN (multiple endocrine neoplasia) type II. Mutations of the tumor suppressor genes are the most common changes in the genes of familial cancer. BRCA1 and BRCA2 are tumor suppressor genes that have recently been identified as familial breast and ovarian cancer, familial breast cancer genes. This paper reviewed the hereditary cancer families in which genetic alterations have been revealed and the recent progress in mapping and cloning of familial breast cancer candidate genes which have not been identified.
Breast Neoplasms↗
HLA class I polymorphism and the susceptibility to malignant melanoma.
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Disease Susceptibility↗
Differences in MHC class I self peptide repertoires among HLA-A2 subtypes.
To investigate how single amino acid substitutions in MHC class I molecules affect differences in peptide repertoires, we eluted and sequenced the naturally processed peptides from three HLA-A2 subtypes (HLA-A*0204, -A*0206, and -A*0207) that differ by a single amino acid residue substitution each with HLA-A*0201 at the floor of the binding groove. Allele-specific peptide motifs for each HLA-A2 subtype substantially differed from that of HLA-A*0201 in the dominant anchor residues. The relative signal intensities for 18 self peptides, determined by mass spectrometry, precisely reflected these peptide motifs. Some overlapping peptides were isolated from both HLA-A*0201 and a single HLA-A2 variant, but no peptide was ubiquitously found across all variants. To rationalize the differences in peptide motifs, possible conformations of each allele were computer modeled by energy minimization calculations based on the reported crystal structure of HLA-A*0201. According to our models, the differences in peptide motifs could be explained by substituted-residue-driven conformational changes for each MHC-peptide complex. These results demonstrate the fine differences between HLA-A2 subtype self peptide repertoires and contribute to the prediction of antigenic peptides.
Amino Acid Sequence↗
Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis.
The growth of solid tumors in vivo beyond 1-2 mm in diameter requires induction and maintenance of an angiogenic response. This can occur through the release of various angiogenic growth factors from tumor cells. One such factor is vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a secreted and specific mitogen for vascular endothelial cells. We show that one of the most commonly encountered genetic changes detected in human cancer, i.e., expression of mutant ras oncogenes, is associated with marked up-regulation of VEGF/VPF in transformed epithelial cells. Thus, elevation of the levels of both VEGF/VPF mRNA and secreted functional protein were detected in human and rodent tumor cell lines expressing mutant K-ras or H-ras oncogenes, respectively. Genetic disruption of the mutant K-ras allele in human colon carcinoma cells was associated with a reduction in VEGF/VPF activity. Furthermore, pharmacological disruption of mutant RAS protein function in H-ras transformed rat intestinal epithelial cells by treatment with L-739,749 (a protein farnesyltransferase inhibitor) caused a significant suppression of VEGF/VPF. The results suggest that dominantly acting ras oncogenes may contribute to the growth of solid tumors in vivo not only by a direct effect on tumor cell proliferation but also indirectly, i.e., by facilitating tumor angiogenesis. Hence, pharmacologically targeting mutant ras oncogenes could conceivably suppress solid tumor growth in vivo, in part, by inhibiting tumor-induced angiogenesis.
Alkyl and Aryl Transferases↗
Characterization of T cell epitopes restricted by HLA-DP9 in streptococcal M12 protein.
Interaction of the HLA-DP9 (DPA1*0201/DPB1*0901) molecule and M protein of serotype 12 (SS95/12) streptococci, a main component of the streptococcal cell wall Ag, has been investigated to decipher peptide-binding capacity and T cell activation in the context of the HLA-DP molecule. Seven antigenic peptides (amino acids 19-25) restricted by the HLA-DP9 molecule were identified in M12 protein, using M12 protein- or peptide-specific T cell lines from naturally exposed individuals. The binding affinity of each peptide to the HLA-DP9 molecule was measured by fluorescence intensity of biotinylated peptides bound to L cell transfectants expressing HLA-DP9, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to the HLA-DP9 molecule was inhibited by an excess amount of corresponding nonbiotinylated peptides and other nonbiotinylated peptides, indicating that the peptides were bound to the HLA-DP9 molecule at a single binding site. Seven synthetic peptides containing the T cell epitopes restricted by the HLA-DP9 molecule had high binding affinity to the HLA-DP9 molecule. Comparison of the amino acid sequences of truncated analogues that could bind to the HLA-DP9 molecule and/or activate T cells suggested an HLA-DP9-specific binding motif, composed of a positively charged residue (R or K) at position 1, a hydrophobic residue (A, G, or L) at position 6, and another hydrophobic residue (L or V) at position 9. Analysis of single amino acid-substituted analogues suggested that the positively charged amino acid in the motif served as a key anchor residue for binding to the HLA-DP9 molecule, which differs from the binding motif to the HLA-DR molecules.
Amino Acid Sequence↗
HLA-A and -DRB4 genes in controlling the susceptibility to Hashimoto's thyroiditis.
HLA-linked genetic factors involved in the pathogenesis of HT were studied in 71 patients with HT by serologic typing for HLA-A, -B, -C, -DR, and -DQ specificities and by DNA typing for HLA-DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, AND -DPB1 genes using the PCR-SSOP method. Typing results demonstrated significant positive associations of HT with HLA-A2 and -DRB4*0101 (DR53) (p < 0.01, RR = 2.03, EF = 0.61 and p < 0.0001, RR = 4.48, EF = 0.69, respectively). Although HLA-DR8, -DRB1*0403, -DQA*03, and -DQB1*0303 were statistically more prevalent in the patient group than in the controls, these associations were presumably due to the strong linkage disequilibria of these alleles with HLA-A2 or -DRB4*0101 in the Japanese population. Ninety-seven percent of the patients (69 out of 71) were positive for HLA-A2 or -DRB4*0101 compared to 79% in controls (RR = 8.7, p < 0.0005). The combination of HLA-A2 and -DRB4*0101 showed higher OR of risk for HT (OR = 12.8) than HLA-A2 (OR = 7.3) or DRB4*0101 (OR = 7.5) alone. These observations suggest that at least two loci, HLA-A and HLA-DRB4 together, may control the susceptibility to HT. On the other hand, the frequency of DQA1*0102 was significantly decreased in the patient group, suggesting that DQA1*0102 might confer resistance to HT.
Genetic Predisposition to Disease↗
DNA typing of HLA class II genes in Japanese patients with dilated cardiomyopathy.
HLA class II genes (DRB, DQA, DQB, DPA, and DPB) were typed at the DNA level using polymerase chain reaction/sequence-specific oligonucleotide probe analysis in 78 unrelated patients with DCM and 336 unrelated healthy controls to elucidate the HLA alleles or HLA haplotypes associated with DCM. The frequencies of DRB1*1401 (15.4% v 4.5%, RR = 3.90, P < 0.0005, Pc < 0.03), DQB1*0503 (14.1% v 5.4%, RR = 2.93, P < 0.007) and DRB1*1401-DQB1*0503 haplotype (11.5% v 1.5%, RR = 8.24, P < 0.00001, Pc < 0.01) were increased in the DCM patients. The frequency of HLA-DRB1*1101 (9.0% v 3.0%, RR = 3.26, P < 0.02) also was increased in the patients. In addition, the frequencies of DQB1*0604 and DPB1*0401 were increased in the DRB1*1401 and DRB1*1101 negative patients. In contrast, the frequencies of DQB1*0303 (19.2% v 30.7%, RR = 0.55, P < 0.05) and DRB1*0901-DQB1*0303 haplotype (16.7% v 29.8%, RR = 0.49, P < 0.02) were decreased in the DCM group. Disease susceptibility to DCM in the Japanese population, thus, may be controlled in part by a gene (or genes) in close linkage disequilibrium with DRB1*1401-DQB1*0503, DRB1*1101-DQB1*0301, and DQB1*0604-DPB1*0401 haplotypes, while the resistance to DCM may be associated with the DRB1*0901-DQB1*0303 haplotype.
Adolescent↗
Significance of the HLA-DQB matching in one-haplotype identical kidney transplant pairs and the matching analysis by the polymerase chain reaction (PCR)--heteroduplex--polymorphism method.
Sixty-five living related kidney transplant pairs were analyzed for matching at HLA class II loci by the polymerase chain reaction (PCR)--sequence specific oligonucleotide probe (SSOP) method. The retrospective HLA matching study revealed that there were many early graft loss cases despite the DQB compatibility, contrary to our expectation. There were 54 DRB1 one-mismatched cases, in which 7 of the 11 (64%) DQB zero-mismatched cases had lost their grafts, while the graft loss cases were only 10 of the 43 (23%) DQB one-mismatched pairs (P value = 0.0006). The DQB matching of these cases was studied in detail, because the PCR-SSOP methods are based on the detection of sequence polymorphisms in a relatively narrow range, i.e., recognized sequences by SSOPs. The PCR--heteroduplex--polymorphism analysis method was developed to analyze the polymorphism in exon 2 of the DQB1 gene. However, all the pairs proved to be compatible for the DQB, demonstrating that the DQB compatibility was associated with a harmful influence on the graft outcome. These observations suggested that the DQB1 incompatibility might exert the low responsiveness to HLA haplo-identical allogeneic transplants.
Base Sequence↗
Early-onset pauciarticular juvenile chronic arthritis is associated with a mutation in the Y-box of the HLA-DQA1 promoter.
Early-onset pauciarticular juvenile chronic arthritis (EOPA-JCA) has associations with different alleles of the MHC region (HLA-A2, DR5, 6, 8, DQA1*0401, *0501, *0601 and DPB1*0201). All susceptible DQA1 alleles carry an exclusive sequence motif. MHC-class II gene expression is controlled by 5' flanking upstream regulatory regions (URR). A hypervariable region in the promoter region of the HLA-DQA1 gene (-240 and -200 base pairs upstream) defines ten different QAP (DQA1-Promoter) alleles, which are associated with certain DQA1-alleles. The Y-Box in the DQA1 promoter (YC-Box -125 to -115 upstream from the ATG) differs from the consensus sequence (-123 A for G) of all other MHC class II Y-Boxes, resulting in a lower affinity to the NF-Y transcription factor and in a reduced expression of DQA1. A second substitution in the Y-Box of QAP 4.1 and 4.2 (-119 A for G) is found in the promoter alleles of the DQA1-alleles (DQA1*0401, *0501, *0601) and is strongly associated with susceptibility to EOPA-JCA.
Age of Onset↗
HLA antigens in Japanese patients with psoriatic arthritis.
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Alleles↗
The role of HLA class II antigens in the induction of cytotoxic T lymphocytes.
Investigation of pairs of unrelated persons mismatched for a particular HLA-DQB1 or -DPB1 gene on the induction of cytotoxic T lymphocytes (CTL) revealed that HLA-DQ and HLA-DP antigens provided a slight proliferative stimulus which was, however, sufficient for the generation of CTL. Monomorphic anti-DQ and anti-DP monoclonal antibodies abrogated the induction of cytotoxic response. The results indicate that the HLA-DQ and HLA-DP antigens play a similar role to HLA-DR specificities in clinical bone marrow transplantation.
Female↗
Clinical characteristics of subacute thyroiditis classified according to human leukocyte antigen typing.
We investigated human leukocyte antigen (HLA) class I and class II antigens in 56 Japanese patients with subacute thyroiditis (SAT) who visited our out-patient clinic between 1988 and 1990. We found SAT to be associated with not only HLA-B35 (40 patients; P < 0.000001; relative risk, 18.02), but also with HLA-B67 antigens (9 patients; P < 0.00001; relative risk, 11.20). No heterozygotes of HLA-B35 or HLA-B67 were found in any of the 56 patients with SAT. Either HLA-B35 or HLA-B67 antigen is found in 87% of patients with SAT. When season of onset and clinical course of SAT were compared in the 49 patients with HLA-B35-positive SAT (B35-SAT) and HLA-B67-positive SAT (B67-SAT), we were able to identify certain characteristics: 1) B67-SAT often followed the course from transient thyrotoxicosis to a hypothyroid phase to a euthyroid phase [6 of 9 B67-SAT (67%) vs. 10 of 40 B35-SAT (25%); P < 0.05]; and 2) B67-SAT occurred mostly during the summer or autumn and at a higher rate than did B35-SAR [8 of 9 B67-SAT (89%) vs. 17 of 40 B35-SAT (43%)], whereas B35-SAT occurred throughout the year. We conclude that there are at least two types of SAT that can be classified by association with either HLA-B35 or HLA-B67 antigens.
Acute Disease↗
Polymerase chain reaction-based HLA-A genotyping and its application to matching in kidney transplantation.
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Base Sequence↗
Human CD4-major histocompatibility complex class II (DQw6) transgenic mice in an endogenous CD4/CD8-deficient background: reconstitution of phenotype and human-restricted function.
To reconstitute the human immune system in mice, transgenic mice expressing human CD4 and human major histocompatibility complex (MHC) class II (DQw6) molecules in an endogenous CD4- and CD8-deficient background (mCD4/8-/-), after homologous recombination, have been generated. We report that expression of human CD4 molecule in mCD4/8-/- mice rescues thymocyte development and completely restores the T cell compartment in peripheral lymphoid organs. Upon vesicular stomatitis virus (VSV) challenge, the reconstituted mature T cell population effectively provide T help to B cells in immunoglobulin class switching from IgM to specific IgG-neutralizing antibodies. Human CD4+DQw6+ double transgenic mice are tolerant to DQw6 and the DQw6 molecule functions in antigen presentation, effectively generating a human MHC class II-restricted T cell response to streptococcal M6C2 peptide. These data show that both the hCD4 and DQw6 molecules are functional in mCD4/8-/- mice, fully and stably reconstituting this limb of the human immune system in mice. This animal model provides a powerful in vivo tool to dissect the human CD4-human class II MHC interaction, especially its role in human autoimmune diseases, superantigen-mediated diseases, and acquired immunodeficiency syndrome (AIDS).
Animals↗
Functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecule in antigen recognition by T cells in HLA-DR and DQ transgenic mice.
Studies in vitro have suggested that a species barrier exists in functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecules. However, whether mouse CD4+ T cells restricted by HLA class II molecules are generated in HLA class II transgenic mice and respond to peptide antigens across this barrier has remained unclear. In an analysis of T cell responses to synthetic peptides in mice transgenic for HLA-DR51 and -DQ6, we found that DR51 and DQ6 transgenic mice acquired significant T cell response to influenza hemagglutinin-derived peptide 307-319 (HA 307) and Streptococcus pyogenes M12 protein-derived peptide 347-397 (M6C2), respectively. Inhibition studies with several monoclonal antibodies showed that transgenic HLA class II molecules presented these peptides to mouse CD4+ T cells. Furthermore, T cell lines specific for HA 307 or M6C2 obtained from the transgenic mice could respond to the peptide in the context of relevant HLA class II molecules expressed on mouse L cell transfectants that lack the expression of mouse MHC class II. These findings indicate that interaction between HLA class II and mouse CD4 molecules is sufficient for provoking peptide-specific HLA class II-restricted T cell responses in HLA class II transgenic mice.
Amino Acid Sequence↗
DCC gene alteration in human endometrial carcinomas.
The present study was undertaken to define the gene(s) of importance on the long arm of chromosome 18 (chromosome 18q) in endometrial carcinomas. We analyzed loss of heterozygosity (LOH) at 3 loci on chromosome 18q and DCC gene expression by the reverse-transcriptase/polymerase chain reaction (RT-PCR) method. Among 61 tumors that were informative, 16 (26%), estimated to be a minimum number, showed allelic losses at one or more chromosome 18q loci. Deletions in these tumors possibly involved the region within or near the chromosome 18q 21.3 band where the DCC gene was localized. Moreover, the incidence of altered DCC mRNA expression was high in these tumors. Appropriate transcription was lost in 5 of 7 (71%) carcinoma cell lines in addition to 14 of 28 (50%) surgically resected tumors. Histopathological differentiation and clinical stage of disease were not related to LOH frequency or to DCC mRNA expression. These results suggest that the target for allelic loss on chromosome 18q seen in endometrial carcinomas is the DCC gene, and that inactivation of this gene may be critical for the development of most endometrial carcinomas.
Alleles↗
Possible gene dose effect of a mutant cardiac beta-myosin heavy chain gene on the clinical expression of familial hypertrophic cardiomyopathy.
We have examined for a mutation in the cardiac beta myosin heavy chain gene from Japanese patients with familial hypertrophic cardiomyopathy. A missense mutation due to a G to A transition in codon 935, leading to a replacement of Glu with Lys, was found in one patient. Family members of this patient were then examined. It was revealed that both the proband and his elder brother, who was also a symptomatic patient, were homozygous for the mutation. The proband eventually died of intractable heart failure, and his brother died suddenly in their thirties. On the other hand, his parents, who were first cousins and heterozygous for the mutation, had cardiac hypertrophy without clinical symptoms. His elder sister was also heterozygous for the mutation, however, she did not manifest with cardiac hypertrophy. These observations suggest a gene-dose-like effect of the mutant cardiac beta myosin heavy chain gene on the clinical manifestation of familial hypertrophic cardiomyopathy.
Adenine↗