New methodology for HLA class II oligonucleotide typing using polymerase chain reaction (PCR) amplification

Background: Numerous studies have shown a link between genes involved in the immune response and infertility and miscarriage. The most significant associations have been established for the cytokine genes (IL1B, IL6, IL10, IL18), chemokine genes (CXCL9, CXCL10, CXCL11), and genes of the major histocompatibility complex HLA II class (DQA1, DQB1, DRB1). HLA genes are associated with celiac disease, a genetically determined autoimmune disorder, where male and female reproduction impairment is one of the symptoms. Aim: To assess the prevalence of polymorphic variants of the immune response genes (HLA: DQA1 DQB1, DRB1; TNF, IL10, CXCL10) in patients with reproduction disorders. Materials and methods: This pilot study involved assessment of the following gene polymorphisms: IL10 (rs1800872), TNF (rs1800629), CXCL10 (rs4386624), and HLA class II (DQA1, DQB1, DRB1) in couples (n = 220) with reproduction disorders (infertility and miscarriage). Genotyping was performed by real-time polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The genotypes and alleles population data were used for comparison with the studied variants of the genes IL10 (rs1800872), TNF (rs1800629), and CXCL10 (rs4386624). Differences in the prevalence of alleles and genotypes were assessed by χ2 test. The differences were considered significant at p < 0.05. Haplotype diversity was calculated by the Arlequin software, version 3.5.x. Results: Compared to the populational data, there was significant re-distribution of the genotypes and alleles to the TNF gene (rs1800629) variant in men with impaired reproductive functions. No differences were found for other gene variants studied. The frequency of HLA class II gene (DQA1, DQB1, DRB1) haplotypes associated with celiac disease (DQ2 and DQ8) in the study sample was 23.8%. Conclusion: The results indicate the important role of genes associated with celiac disease in the development of reproduction disorders.

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Applications of the Polymerase Chain Reaction to HLA Class II Typing

Vox Sanguinis ◽

10.1111/j.1423-0410.1992.tb02491.x ◽

1992 ◽

Vol 63 (2) ◽

pp. 81-89 ◽

Cited By ~ 7

Author(s):

Jeffrey L. Bidwell

Keyword(s):

Polymerase Chain Reaction ◽

Class Ii ◽

Hla Class Ii ◽

Chain Reaction ◽

Polymerase Chain

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Nested Polymerase Chain Reaction With Sequence-Specific Primers Typing for HLA-A, -B, and -C Alleles: Detection of Microchimerism in DR-Matched Individuals

Blood ◽

10.1182/blood.v94.4.1471 ◽

1999 ◽

Vol 94 (4) ◽

pp. 1471-1477 ◽

Cited By ~ 16

Author(s):

Anthony S. Carter ◽

Lucia Cerundolo ◽

Mike Bunce ◽

Dicken D.H. Koo ◽

Kenneth I. Welsh ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Nested Pcr ◽

Solid Organ Transplantation ◽

Class Ii ◽

Hla Class Ii ◽

Solid Organ ◽

Chain Reaction ◽

Specific Primers ◽

Polymerase Chain ◽

Hla Class Ii Alleles

Abstract It is widely accepted that donor leukocytes survive within the recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect the donor-derived material. The techniques used to detect donor-derived DNA within recipient peripheral blood rely largely on major histocompatibility complex class II polymorphism. We and others have shown that the sensitivity of polymerase chain reaction with sequence-specific primers (PCR-SSP) typing for HLA class II alleles can be increased 100-fold by the addition of a primary amplification step (nested PCR-SSP). We have now extended this technique to encompass typing for HLA class I alleles, thereby adding flexibility to microchimerism testing by enabling testing of recipients HLA-DR matched with their donors. However, the high level of sensitivity achieved with the technique (1:100,000) leads to a concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in the development of our nested PCR-SSP typing system for HLA class II alleles. We describe here how it is possible to compensate for these anomalies by including multiple testing of a pretransfusion sample that acts as a specificity control, establishing a rigorous baseline for subsequent analysis.

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Nonradioactive HLA class II typing using polymerase chain reaction and digoxigenin-11-2′-3′-dideoxyuridinetriphosphate-labeled oligonucleotide probes

Human Immunology ◽

10.1016/0198-8859(91)90042-8 ◽

1991 ◽

Vol 31 (1) ◽

pp. 7-13 ◽

Cited By ~ 59

Author(s):

C. Nevinny-Stickel ◽

M.d.l.P. Bettinotti ◽

A. Andreas ◽

M. Hinzpeter ◽

K. Mühlegger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Class Ii ◽

Hla Class Ii ◽

Oligonucleotide Probes ◽

Chain Reaction ◽

Polymerase Chain

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Polymerase chain reaction based human leucocyte antigen genotyping for the investigation of suspected gastrointestinal biopsy contamination

Gut ◽

10.1136/gut.45.2.259 ◽

1999 ◽

Vol 45 (2) ◽

pp. 259-263 ◽

Cited By ~ 4

Author(s):

A C Bateman ◽

S J Turner ◽

J M Theaker ◽

B F Warren ◽

W M Howell

Keyword(s):

Polymerase Chain Reaction ◽

Human Leucocyte Antigen ◽

Class Ii ◽

Hla Class Ii ◽

Chain Reaction ◽

Reference Tissue ◽

Biopsy Material ◽

Biopsy Specimens ◽

Gastrointestinal Pathology ◽

Polymerase Chain

BACKGROUNDMislabelling or contamination of surgical specimens may lead to diagnostic inaccuracy, particularly within gastrointestinal pathology when multiple small mucosal biopsy specimens are commonly taken, and where a tiny fragment of foreign tissue may be indistinguishable from true biopsy material using histological assessment alone.AIMSTo assess the utility of polymerase chain reaction (PCR) based human leucocyte antigen (HLA) genotyping techniques for the investigation of potentially mislabelled or contaminated gastrointestinal biopsy specimens.PATIENTSTen cases (28 samples) in which mislabelling or contamination was suspected, comprising four upper gastrointestinal tract biopsies and six colonoscopic biopsy series.METHODSDirect and nested PCR-sequence specific primer (SSP) based HLA class II genotyping was performed on DNA extracted from formalin fixed and paraffin wax embedded tissue (23 samples) or peripheral blood leucocytes (five samples).RESULTSA full HLA-DRB1 genotype was determined in all 28 samples. In seven cases the HLA-DRB1 genotype of the putative contaminant was different to that of the corresponding reference tissue, confirming different individual origins for the contaminant and reference material. In one case the contaminant tissue was shown to possess the same HLA-DRB1 alleles as a second patient (probable source). In the remaining three cases the same HLA-DRB1 alleles were detected within the potential contaminant and reference tissues.CONCLUSIONSPCR based HLA class II genotyping is a valuable tool for investigating potential contamination or mislabelling within gastrointestinal biopsy specimens and this report has confirmed contamination in seven of ten cases studied.

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Nested Polymerase Chain Reaction With Sequence-Specific Primers Typing for HLA-A, -B, and -C Alleles: Detection of Microchimerism in DR-Matched Individuals

Blood ◽

10.1182/blood.v94.4.1471.416k10_1471_1477 ◽

1999 ◽

Vol 94 (4) ◽

pp. 1471-1477

Author(s):

Anthony S. Carter ◽

Lucia Cerundolo ◽

Mike Bunce ◽

Dicken D.H. Koo ◽

Kenneth I. Welsh ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Nested Pcr ◽

Solid Organ Transplantation ◽

Class Ii ◽

Hla Class Ii ◽

Solid Organ ◽

Chain Reaction ◽

Specific Primers ◽

Polymerase Chain ◽

Hla Class Ii Alleles

It is widely accepted that donor leukocytes survive within the recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect the donor-derived material. The techniques used to detect donor-derived DNA within recipient peripheral blood rely largely on major histocompatibility complex class II polymorphism. We and others have shown that the sensitivity of polymerase chain reaction with sequence-specific primers (PCR-SSP) typing for HLA class II alleles can be increased 100-fold by the addition of a primary amplification step (nested PCR-SSP). We have now extended this technique to encompass typing for HLA class I alleles, thereby adding flexibility to microchimerism testing by enabling testing of recipients HLA-DR matched with their donors. However, the high level of sensitivity achieved with the technique (1:100,000) leads to a concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in the development of our nested PCR-SSP typing system for HLA class II alleles. We describe here how it is possible to compensate for these anomalies by including multiple testing of a pretransfusion sample that acts as a specificity control, establishing a rigorous baseline for subsequent analysis.

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