Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
1.
Eur J Surg Oncol ; 29(6): 491-6, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12875854

ABSTRACT

AIMS: Loss of heterozygosity (LOH) may vary almost randomly within a colorectal tumour due to the heterogeneous morphologic character of these tumours. Despite this, as a rule, single biopsies are the source of genetic material used in studies of markers important for prognosis, clinical behaviour of the disease, or susceptibility of specific tumours to different treatment modalities. METHODS: To evaluate the importance of intratumoural variation for the results of analysis of LOH and point mutations in colorectal cancer and to determine the frequency of genetic alterations in different types of pre-neoplastic areas of the tumours, 36 consecutively operated patients with colorectal cancer were studied. After fixation, specimens were mounted on large slides containing the whole tumour. The specimens were sub classified into different areas defined as normal tissue, normal tissue closely adjacent the tumour mass, adenoma, dysplasia and invasive cancer cells. These areas were dissected and subjected to DNA extraction. RESULTS: The extracted genomic DNA was studied for LOH at chromosome 5q, 17p, and 18q and for k-ras mutations. Overall, a correlation between the intratumoural degree of neoplastic progression and the frequency of LOH and k-ras mutations was seen. These correlations were significant (p<0.008) except for dysplasia/adenomatous tissue versus invasive cancer. Microsatellite instability was found in 9% of the tumours, all except one in invasive parts of the tumours. CONCLUSIONS: This study demonstrates a statistical correlation between intratumoural differences in neoplastic degree of dedifferentiation and genetic instability in terms of LOH and point mutations of the k-ras gene in colorectal carcinoma. The importance of a careful dissection in order to localise the region with the highest probability of genetic aberrations and multiple biopsing must not be neglected. The observation that the prevalence of k-ras mutations and LOH are correlated to the degree of dedifferentiation within a colorectal tumour is in line with the concept that selected cell clones are responsible for the neoplastic progression of the tumour.


Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , DNA, Neoplasm/analysis , Genetic Markers , Genetic Variation , Precancerous Conditions/genetics , Adenoma/surgery , Aged , Aged, 80 and over , Chromosomes, Human, Pair 17/genetics , Chromosomes, Human, Pair 18/genetics , Chromosomes, Human, Pair 5/genetics , Colorectal Neoplasms/surgery , Electrophoresis, Gel, Two-Dimensional , Female , Genes, ras , Genetic Predisposition to Disease , Humans , Loss of Heterozygosity , Male , Microsatellite Repeats , Middle Aged , Point Mutation , Polymerase Chain Reaction , Precancerous Conditions/surgery , Temperature
3.
Bone Marrow Transplant ; 28(12): 1161-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11803361

ABSTRACT

Allogeneic stem cell transplantation (ASCT) has proved to have an important immune-mediated anti-tumour effect in patients with haematologic malignancies. There is also evidence of such an effect in patients with malignant tumours. We studied this effect of ASCT in a patient with colorectal cancer. A 77-year-old man having a primarily resected colonic cancer with disseminated lymph node involvement received ASCT from his HLA-identical sibling as the only treatment. Mixed haematopoietic chimerism was monitored using PCR-amplification of variable number tandem repeats and tumour size, assessed by repeated CT scans. Recipient leucocytes were gradually replaced by donor cells for 1 month. Continuous resolution of lymph node metastases was seen together with clinical graft-versus-host disease (GVHD). The patient died of pneumonia and cardiac insufficiency 4 months after transplantation. At autopsy, most of the metastases were necrotic, with few remaining tumour cells. Clinical and histopathological postmortem results showed a graft-versus-colorectal cancer effect.


Subject(s)
Colonic Neoplasms/therapy , Graft vs Tumor Effect , Hematopoietic Stem Cell Transplantation/adverse effects , Aged , Chimera , Colonic Neoplasms/pathology , Humans , Male , Tomography, X-Ray Computed , Transplantation, Homologous
4.
Cancer ; 88(12): 2661-7, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10870047

ABSTRACT

BACKGROUND: Loss of heterozygosity (LOH) at 17p and 18q in colorectal carcinoma has been depicted as a potential prognostic marker for the disease. However, conclusions vary among reports, and evidence of clinically useful genetic prognostic markers is still lacking. As a rule, single biopsies are used. In this study, the authors hypothesized that an important cause of earlier contradictory results was the heterogeneity of colorectal neoplasms. METHODS: In this study, DNA originating in each quadrant of tumors from 64 patients with colorectal carcinoma was analyzed. Microsatellite markers for chromosome 18q and 17p were amplified by polymerase chain reaction and automatically analyzed. RESULTS: The authors found that, regardless of stage, LOH and non-LOH in both 17p and 18q varied among biopsies within the tumors in a random fashion. LOH in 18q was detected in all 4 quadrants in 22% and in 1 of 4, 2 of 4, or 3 of 4 quadrants in 56% of the tumors, whereas 22% of the tumors were homogeneously without LOH in 18q. LOH 17p was distributed similarly throughout the tumors and was present in 1 of 4, 2 of 4, or 3 of 4 of the quadrants in 44%. The authors also reexamined a subset of tumors by subdividing one biopsy from each into four. Analysis of the microsatellite markers then yielded identical results. No correlation between the degree of LOH status and patient survival was observed. CONCLUSIONS: LOH status within a colorectal tumor is extensively heterogeneous. However, it is more homologous on a lower macroscopic level. For relevant genetic analysis, multiple biopsies and DNA sampling preceded by careful morphologic examination must be standard in the preparation of DNA.


Subject(s)
Biomarkers, Tumor/analysis , Colorectal Neoplasms/genetics , DNA, Neoplasm/genetics , Loss of Heterozygosity/genetics , Adult , Aged , Aged, 80 and over , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 18 , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Female , Humans , Male , Microsatellite Repeats , Middle Aged , Neoplasm Staging , Prognosis , Reproducibility of Results , Specimen Handling , Survival Rate
5.
Transplantation ; 69(10): 2043-8, 2000 May 27.
Article in English | MEDLINE | ID: mdl-10852594

ABSTRACT

BACKGROUND: In histocompatibility mismatched experimental animals, a combination of T-cell-depleted autologous and allogeneic marrow may induce mixed chimerism and tolerance. Patients with large primary liver tumors have a poor outcome. We investigated whether it were possible to induce mixed chimerism and obtain an antitumor effect in a patient with a large primary liver cancer after combined liver and bone marrow transplantation (BMT). METHODS: A 46-year-old female with a primary non resectable liver cancer received a liver transplant from a cadaveric donor. Subsequently, she was conditioned with 4x2 Gy of total lymphoid irradiation, 120 mg/kg cyclophosphamide, and 7.5 Gy total body irradiation. Twelve days after liver transplantation, she received T-cell-depleted autologous:cadaveric 5/6 antigen HLA-mismatched marrow in a proportion of CD34+ cells of 0.5:3.0x10(6)/kg. Chimerism status was determined with polymerase chain reaction amplification of variable number tandem repeats from DNA obtained from CD3+, CD19+, and CD45+ magnetic-bead-separated cells. RESULTS: The early posttransplant period was uneventful; liver function was normal and the hematopoietic engraftment of donor and recipient origin was prompt. Alpha-fetoprotein levels dropped from 440 to 35 microg/l. One month after marrow transplantation, donor T-cells decreased markedly. Monoclonal antibody OKT-3 and 10(5)/kg donor T-cells were given. One month later, the patient developed diarrhea and abdominal pain. A colonoscopy showed moderate gastrointestinal acute graft-versus-host disease and a Cryptosporidium infection. Three months after BMT, she became a complete donor chimera. Chimera cells showed little, if any, reactivity in mixed lymphocyte cultures to recipient and donor cells, but reacted to third party. Five months after BMT, she developed progressive Aspergillus fumigatus pneumonia and died. No tumor was found at the autopsy. CONCLUSION: We obtained mixed donor-recipient hematopoietic chimerism without severe acute graft-versus-host-disease, after combined T-cell depleted autologous and allogeneic BMT and a transplantation of a liver from an HLA-mismatched cadaveric donor. Additional donor T-cells enhanced donor bone marrow engraftment, but rejected the autograft. On the basis of this first attempt, further clinical studies are warranted.


Subject(s)
Bone Marrow Transplantation , Carcinoma, Hepatocellular/therapy , Liver Neoplasms/therapy , Liver Transplantation/physiology , Bone Marrow Transplantation/immunology , Cadaver , Carcinoma, Hepatocellular/surgery , Combined Modality Therapy , Female , Graft vs Host Disease , Humans , Immunosuppression Therapy , Liver Neoplasms/surgery , Liver Transplantation/immunology , Lymphocyte Culture Test, Mixed , Middle Aged , Time Factors , Tissue Donors , Tissue and Organ Harvesting/methods , Transplantation Chimera , Transplantation, Autologous , Transplantation, Homologous , Whole-Body Irradiation , alpha-Fetoproteins/analysis
6.
Bone Marrow Transplant ; 25(8): 843-51, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10808205

ABSTRACT

One of the major problems after allogeneic bone marrow transplantation (BMT) is a high frequency of leukemia relapse. We have prospectively studied the presence of donor- and recipient-derived chimeric cells in bone marrow recipients with pre-B cell acute lymphoblastic leukemia (pre-B-ALL). The chimeric status of BMT recipients was compared to minimal residual disease (MRD) detection by analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TcR) genes. Post-transplant blood and bone marrow samples from 12 patients with pre-B-ALL were studied. Five patients showed mixed chimerism (MC) in the CD19-positive cell fraction. Four of them have relapsed to date. The remaining patient with MC in the B cell lineage was also MRD positive in the same samples. All seven patients with donor chimerism in the B cell fraction remain in clinical remission (P = 0.01). In samples from all five patients having MC in the B cell lineage, the patient-specific IgH or TcR rearrangement was also detected. In three of four patients who relapsed, MC in the B cell lineage was seen more than 2.5 months prior to morphologically verified relapse. The results of this comparison suggest that routinely performed MC analysis of the affected cell lineage may facilitate post-BMT monitoring and rapid therapeutic decisions in transplanted patients with pre-B-ALL.


Subject(s)
B-Lymphocytes/cytology , Bone Marrow Transplantation , Neoplasm, Residual/blood , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/blood , Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood , Transplantation Chimera/genetics , Adolescent , Adult , Antigens, CD19/blood , B-Lymphocytes/immunology , Biomarkers , Cell Lineage , Child , Child, Preschool , DNA/blood , DNA Primers , Female , Genes, T-Cell Receptor delta , Humans , Immunoglobulin Heavy Chains/genetics , Immunomagnetic Separation , Male , Minisatellite Repeats , Neoplasm, Residual/genetics , Prospective Studies , Recurrence , Sensitivity and Specificity , Sequence Analysis, DNA
7.
Leukemia ; 14(2): 247-54, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10673741

ABSTRACT

Following allogeneic stem cell transplantation (SCT), we studied the presence of donor and recipient derived cells within the CD19+ B cell fraction, in patients with B cell chronic lymphocytic leukemia (CLL). The chimeric status of the six patients studied was further investigated with minimal residual disease (MRD) detection, by sequencing and using patient-specific primers derived from junctional regions of clonally rearranged immunoglobulin heavy-chain (IgH) receptor genes. To date, five of six patients are alive with a median follow-up time of 24 months (range 15-60) post-SCT. All patients experienced acute and chronic graft-versus-host disease and responded clinically to SCT. All patients were MRD positive after SCT, which correlated to mixed chimerism within the CD19+ cell fraction in all samples except one (25/26). High levels of tumor necrosis factor-alpha (TNF-alpha) and soluble interleukin-2 receptor (sIL-2R) indicated advanced disease, and patients with increased levels pre- and post-SCT were also those with the most long-lasting PCR-detectable MRD post-SCT. Hence, a high tumor burden pre-SCT may reflect the long duration of detectable MRD in patients with B-CLL after SCT. A durable anti-leukemic effect was probably important in these patients.


Subject(s)
Graft vs Host Disease/metabolism , Hematopoietic Stem Cell Transplantation , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Adult , Bone Marrow/metabolism , Female , Graft vs Host Disease/blood , Hematopoietic Stem Cell Transplantation/methods , Humans , Immunoglobulins/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Male , Middle Aged , Neoplasm, Residual/prevention & control , Polymerase Chain Reaction , Receptors, Interleukin-2/blood , Sensitivity and Specificity , Sequence Analysis, DNA , Transplantation, Homologous , Treatment Outcome , Tumor Necrosis Factor-alpha/metabolism
8.
Eur J Immunogenet ; 24(3): 191-199, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9226125

ABSTRACT

Results from 360 HLA-DR and -DQ 'low-resolution' typings with polymerase chain reaction sequence-specific primers (PCR-SSP), performed by nine laboratories, were analysed for their overall utility in routinely defining the HLA-DR1-DR18, DR51-DR53 and DQ1-DQ9 specificities in less than 2.5 h. Thirty EDTA blood samples and 10 DNA samples were distributed and analysed by each laboratory. DNA was extracted using a rapid bromide salt extraction protocol. Complete HLA-DR and -DQ typings were performed, three by three, on pre-aliquoted 96-tube PCR trays. When compared with reference typing, 351/360 (98%) correct DR typings were obtained, whereas 320/360 (89%) of the DQ phenotypes were correctly assigned. The time for three complete HLA-DR and -DQ 'low-resolution' typings, including DNA extraction, ranged from 2.0 h to 2.3 h. Unfortunately, an unusually high level of PCR amplification failures was observed (3%), probably due to diffusion and a significant volume loss from some of the pre-aliquoted primer mixes. Consequently, only 52% of the typings were without any amplification failure, and 0-2 amplification failures where found in 88% of the PCR-SSP typings performed. The number of HLA-DR-DQ retypings needed was 7 and 8%, respectively, reflecting the low number of typings where allelic identification was directly affected by the relatively high level of amplification failures in this study. Thus, a 91-98% success rate of correctly identified HLA-DR and -DQ alleles could be maintained, even under suboptimal typing conditions.


Subject(s)
Genes, MHC Class II/genetics , HLA-DQ Antigens/analysis , HLA-DR Antigens/analysis , Histocompatibility Testing/methods , Alleles , DNA/isolation & purification , DNA Primers , Electrophoresis, Agar Gel , HLA-DQ Antigens/genetics , HLA-DR Antigens/genetics , Humans , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sensitivity and Specificity , Specimen Handling
9.
Clin Immunol Immunopathol ; 77(2): 185-92, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7586726

ABSTRACT

Screening of close relatives of Swedish patients with selective immunoglobulin A deficiency (IgAD) and common variable immunodeficiency (CVID) for serum immunoglobulin levels has identified the positive family history of IgAD/CVID as the most significant risk factor for developing the disease. The relative risk for siblings of patients with IgAD was estimated to be approximately 50. In 12 of 34 Swedish multiplex families identified in the study, both IgAD and CVID occurred, usually CVID in the parental generation and IgAD in the subsequent generation. This proportion was much higher than expected by chance and strongly suggests that the two clinically discernible disorders represent an allelic condition, reflecting a variable expressivity of a common defect. In 27 multiplex families the disorders segregated as an autosomal dominant trait, affecting at least two generations. A high relative risk for siblings, permanent phenotype, low number of phenocopies, and common population prevalence, which makes it possible to obtain a sufficient sample size, make these immunoglobin deficiencies amenable to genetic linkage analysis. In a pilot multicenter linkage study involving 16 multiplex families with dominant transmission of IgAD/CVID, we have attempted to confirm previously reported genetic linkage of the disease susceptibility to the major histocompatibility complex (MHC) region. Using both parametric and nonparametric linkage analyses with a set of microsatellite markers at and flanking the MHC region, no evidence for linkage was found. In accordance with these results, no evidence for linkage to the MHC region was obtained by analyzing previously published segregation data at the MHC region in multiplex families with IgAD/CVID in more than one generation.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Common Variable Immunodeficiency/genetics , IgA Deficiency/genetics , Common Variable Immunodeficiency/epidemiology , Common Variable Immunodeficiency/immunology , Family , Genetic Linkage , IgA Deficiency/epidemiology , IgA Deficiency/immunology , Sweden/epidemiology
11.
J Invest Dermatol ; 100(3): 271-4, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8095057

ABSTRACT

The increased concordance rate of nickel sensitivity in monozygotic compared to dizygotic twins indicates a genetic causal component. We have previously described an association in nickel-sensitive subjects with an HLA-DQA restriction fragment length polymorphism (RFLP) (4.5-kb TaqI band, DQA1*0501). The purpose of the present study was to investigate if our previous finding could be confirmed in an independent study, and also to investigate the distribution of HLA class II alleles in chromium- and cobalt-sensitive individuals. Using TaqI- or MspI-digested DNA and DQA, DQB, DRB, DPA and DPB cDNA probes alleles were defined by RFLP analysis. The association with the DQA1*0501 allele was not confirmed in the new group of 37 nickel-sensitive subjects (compared to 150 new controls), nor when the two groups of patients were combined. The distribution of HLA class II alleles and DR-DQ haplotypes were similar in the pooled group of 70 nickel-sensitive subjects and the combined control groups (n = 250). No significant changes in the distribution of HLA class II allele among the chromium- (n = 26) and/or cobalt- (n = 38) sensitive individuals were found. Our results indicate that it is unlikely that the tendency to develop metal sensitivity is associated with alleles of the HLA class II region.


Subject(s)
Chromium/adverse effects , Cobalt/adverse effects , Dermatitis, Allergic Contact/etiology , HLA-D Antigens/genetics , Nickel/adverse effects , Alleles , Dermatitis, Allergic Contact/genetics , Female , Genes, MHC Class II , HLA-DP Antigens/genetics , HLA-DQ Antigens/genetics , HLA-DR Antigens/genetics , Haplotypes , Humans , Male , Polymorphism, Restriction Fragment Length
12.
Gut ; 33(7): 942-6, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1644334

ABSTRACT

The familial predisposition to chronic inflammatory bowel disease and the increased concordance rate in monozygotic twins with Crohn's disease, suggest that genetic factors influence disease susceptibility. A 100% association with the supertypic HLA class II specificity DRw52a was recently described in white North American patients with primary sclerosing cholangitis, with or without concurrent ulcerative colitis. HLA class II alleles of the DR, DQ, and DP subregions were determined by genomic typing techniques in a large group of Swedish patients with ulcerative colitis or Crohn's disease as well as in a series of patients with primary sclerosing cholangitis. No statistically significant HLA class II association was observed in any of the investigated diseases or when the patients were subgrouped according to disease site or occurrence of extraintestinal manifestations, except an insignificant increase of the DRw17, DQw2 haplotype in patients with primary sclerosing cholangitis. The failure to confirm the well established DRw17 association in Swedish patients with sclerosing cholangitis probably represents a statistical type II error. Furthermore, this study did not verify the recently described strong DRw52a association in sclerosing cholangitis--52% of the patients were DRw52a positive compared with 28% of the controls (p less than 0.05, pc NS). This discrepancy was probably caused by different typing techniques. The DRw52a specificity was determined directly by hybridising HLA-DRB3 genes, group specifically amplified by the polymerase chain reaction, with an allele specific oligonucleotide probe, whereas in the previously mentioned study DRw52a was assigned by indirect serological criteria, which overestimate the frequency of this allele.


Subject(s)
Cholangitis, Sclerosing/genetics , Colitis, Ulcerative/genetics , Crohn Disease/genetics , Genes, MHC Class II/genetics , HLA-DR Antigens/genetics , Blotting, Southern , Cholangitis, Sclerosing/immunology , Colitis, Ulcerative/immunology , Crohn Disease/immunology , Disease Susceptibility , HLA-DR Serological Subtypes , Histocompatibility Testing , Humans , Sweden
13.
Hum Immunol ; 34(1): 64-74, 1992 May.
Article in English | MEDLINE | ID: mdl-1356957

ABSTRACT

The clinical applicability of genomic HLA class II typing techniques has increased after the introduction of PCR-based typing strategies. In typing by PCR amplification using sequence-specific primers (PCR-SSP), amplification of specific alleles or groups of alleles is achieved, provided that the mismatch(es) of the SSP is located in the 3' end of the primer. Thus, the specificity of the typing system becomes part of the amplification step, which reduces the total typing time to a minimum by simplifying the postamplification processing of samples. The set of primers presented here identifies all of the alleles of the DR4 group, DRB1*0401-DRB1*0411, as well as the DRB1*07 and DRB1*0901 alleles. In the present study of DR4 alleles, PCR-SSP was compared with hybridization with sequence-specific oligonucleotide probes following group-specific PCR amplification (PCR-SSO). The two typing strategies gave completely concordant results in the 90 DR4-positive and the 32 DR4-negative individuals and cell lines studied. DR7,DQ9/DR9,DQ9 discrimination using PCR-SSP, was compared with MspI DQA RFLP typing, also with concordant results in the 33 DR7- and/or DR9-positive and 36 DR7- and DR9-negative individuals and cell lines tested. No false-negative or false-positive typing results were obtained. Genomic typing by PCR-SSP was performed in the overall time of 2 hours, including rapid DNA preparation, PCR amplification, postamplification processing, documentation, and interpretation of results. This makes the PCR-SSP strategy for HLA class II typing attractive not only in population- and disease-association studies, but also in routine clinical practice, including donor-recipient matching prior to cadaveric transplantation.


Subject(s)
Genes, MHC Class II , HLA-DR Antigens/genetics , Histocompatibility Antigens Class II/genetics , Polymerase Chain Reaction , Sequence Tagged Sites , Alleles , Base Sequence , Exons , HLA-DRB1 Chains , Heterozygote , Humans , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , Sensitivity and Specificity
14.
Tissue Antigens ; 39(5): 225-35, 1992 May.
Article in English | MEDLINE | ID: mdl-1357775

ABSTRACT

In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.


Subject(s)
Genes, MHC Class II , HLA-DR Antigens/genetics , Histocompatibility Testing/methods , Oligonucleotide Probes , Polymerase Chain Reaction , Transplantation Immunology , Alleles , Base Sequence , Cadaver , Humans , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , Reproducibility of Results , Sequence Alignment , Sequence Homology , Time Factors , Tissue Donors
15.
Lakartidningen ; 88(40): 3275-7, 1991 Oct 02.
Article in Swedish | MEDLINE | ID: mdl-1682538

ABSTRACT

All the biologically relevant HLA class II allelic variants can not be identified with conventional serological tissue typing techniques. During the past few years considerable advances have been made in HLA class II typing with molecular techniques now widely used in routine clinical tissue typing. The next few years are likely to see the development of tissue typing techniques based on the polymerase chain reaction (PCR), for use in acute transplantation. A new method for the rapid identification of genetic polymorphisms is described--allele-specific PCR amplification.


Subject(s)
HLA-D Antigens/genetics , Histocompatibility Testing/methods , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , HLA-D Antigens/immunology , Humans , Molecular Biology
16.
Tissue Antigens ; 37(5): 197-204, 1991 May.
Article in English | MEDLINE | ID: mdl-1685264

ABSTRACT

The two DR1-associated cellular specificities Dw1 and Dw20, as well as DR'Br' (Dw'BON'), cannot be unequivocally assigned by serological typing or restriction fragment length polymorphism (RFLP) analysis. We have developed and compared two polymerase chain reaction-based (PCR) typing methods for distinguishing these DRB1 alleles; allele-specific amplification of DRB1*01 alleles followed by an agarose gel electrophoresis detection step and group-specific DRB1*01 amplification followed by hybridization with sequence-specific oligonucleotide probes. The two typing strategies gave completely concordant results in the 33 DRB1*01-positive and the 46 DRB1*01-negative individuals and cell lines studied. No false-negative or false-positive typing results were obtained. All possible heterozygous combinations of the DRB1*0101-0103 alleles could be distinguished by both typing methods. DRB1*01 subtyping by allele-specific PCR amplification was performed in less than 3 hours, including PCR amplification, detection and interpretation steps. The technique will be a valuable complement to DR typing by serology and RFLP analysis. Allele-specific DRB1 amplifications or group-specific amplifications followed by directed allele-specific amplifications of DRB1 alleles, typing based on the absence or presence of amplified products, may well prove to be the technical innovation that will firmly establish PCR-based DR typing in routine clinical tissue typing.


Subject(s)
Alleles , HLA-DR Antigens/genetics , Histocompatibility Antigens Class II/genetics , Base Sequence , DNA/genetics , Electrophoresis, Agar Gel , Gene Amplification , HLA-DRB1 Chains , Heterozygote , Humans , Molecular Sequence Data , Oligonucleotides , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
SELECTION OF CITATIONS
SEARCH DETAIL
...