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1.
Tissue Antigens ; 71(1): 85-6, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17999654

ABSTRACT

The novel allele human leukocyte antigen (HLA)-A*030105 differs from HLA-A*030101 by a synonymous nucleotide exchange at codon 87 in exon 2 (G-->A).


Subject(s)
Alleles , HLA-A Antigens/genetics , Haplotypes/genetics , Sequence Analysis, DNA , Codon/genetics , Exons/genetics , HLA-A3 Antigen , Humans , Molecular Sequence Data , Point Mutation
2.
Tissue Antigens ; 70(4): 335-7, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17767557

ABSTRACT

Exon 3 of DRB1 is known to be polymorphic, but thought to be conserved within allelic groups. This implies that exon 3 polymorphisms would not need to be considered in evolutionary studies or clinical settings when assessing immunogenicity of allelic mismatches in stem cell transplantation. To further assess this, we determined the sequences of DRB1 exon 3 by hemizygote amplification and direct sequencing on 55 selected DNA samples containing 42 DRB1 alleles for which no exon 3 sequence data were previously available. The data confirmed the high degree of overall sequence conservation. The DRB4- and DRB5-associated alleles were completely conserved within their DRB1 groups. However, it could be shown that exon 3 is more diverse than previously expected. Multiple allelic differences within each group of DRB3-associated DRB1 alleles were found, without identifying unique group-related sequence motifs differentiating between these groups. For DRB1*1402 and DRB1*1406, it could be shown that they originated from DRB1*0302. In several samples previously typed as DRB1*1401, a novel DRB1 allele was identified: DRB1*1454. Thus, from a clinical viewpoint, the availability of exon 3 sequence information may be useful for optimizing typing as well as matching strategies. Additionally, it will allow for more detailed evolutionary studies, further elucidating the origin of alleles and the mechanisms driving sequence diversification.


Subject(s)
Exons/genetics , HLA-DR Antigens/genetics , Alleles , Base Sequence , Conserved Sequence , HLA-DRB1 Chains , Humans , Molecular Sequence Data , Polymorphism, Genetic
3.
Tissue Antigens ; 57(5): 440-6, 2001 May.
Article in English | MEDLINE | ID: mdl-11556968

ABSTRACT

HLA antigens are associated with psoriasis vulgaris across populations with different ethnic background. We have previously shown that in Caucasians this association is primarily based on the class I alleles of the extended HLA haplotype 57.1 (EH57.1/I), HLA-Cw6-HLA-B57. However, it remained unclear whether HLA-Cw6 itself or a closely linked locus predisposes to the disease. An interesting candidate for this presumed locus is corneodesmosin, which is exclusively synthesized in keratinocytes. The corneodesmosin gene locus (CDSN) is only 160 kb telomeric to HLA-C and tightly associated with psoriasis. In order to find out whether EH57.1/I or a corneodesmosin variant are the susceptibility determinants on 6p, HLA class I alleles and single-nucleotide polymorphisms (SNPs) of corneodesmosin were investigated at the sequence level and analyzed by comparative association tests. Transmission disequilibrium tests (TDT) were performed in 52 nuclear families, of which 36 were fully informative for a joint comparison of HLA and CDSN with regard to association to psoriasis. The extended TDT according to Wilson was employed to test for locus interaction. Using the HLA haplotype EH57.1/I and the CDSN haplotype formed by three intragenic variant sites at nt=619 (T), 1236 (T), and 1243 (C), we obtained the best resolution of parental transmission to index cases in the trio families. On direct comparison of the contributions of the HLA and the CDSN haplotypes, there was a markedly stronger association of the corneodesmosin TTC haplotype, which is not apparent in single locus analysis. We show furthermore that there is no higher order interaction between psoriasis, HLA, and CDSN. This lack of three-locus interaction is suggestive of two independent genetic contributions to psoriasis within the major histocompatibility complex (MHC).


Subject(s)
Glycoproteins/genetics , HLA-B Antigens/genetics , HLA-C Antigens/genetics , Psoriasis/genetics , Alleles , Child , Female , Genetic Markers/genetics , Genetic Predisposition to Disease/genetics , Germany/epidemiology , Haplotypes/genetics , Humans , Intercellular Signaling Peptides and Proteins , Male , Polymorphism, Single Nucleotide/genetics , Risk Factors
4.
Tissue Antigens ; 58(4): 250-4, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11782276

ABSTRACT

In fluorescence-based sequence-specific primed polymerase chain reaction (PCR), referred to as fluorotyping for HLA typing, a major problem is the overlap of the emission spectra of the single dyes. In order to increase the robustness of the previously described HLA-DRB1,3,4,5 low-resolution fluorotyping method, we have constructed two probes quenched by the non-fluorescent acceptor dye DABCYL. The HLA-DRB-specific probe was labeled with FAM, and the internal control probe with TAMRA, respectively, as reporter fluorescent dyes. TAMRA was replaced by DABCYL as a quencher, which led to increased robustness and better discrimination between negative and positive amplification results. ROX was used as a reference to normalize the fluorescence of the reporter dyes. Moreover, as FAM and TAMRA differ strongly by their emission maxima, HLA-DRB-specific and internal control amplification could be clearly distinguished. To further automate data analysis, the software of the TaqMan system 7700 was supplemented by an EXCEL-based calculation table, which directly took over the data. Using modified fluorotyping chemistry and automated data analysis, a total of 201 DNA samples was typed correctly. In summary, HLA-DRB fluorotyping by dark quenching and automated analysis proved to be a robust and reliable tool for research and routine purposes.


Subject(s)
HLA Antigens/classification , Polymerase Chain Reaction/methods , p-Dimethylaminoazobenzene/analogs & derivatives , Electronic Data Processing , Fluorescent Dyes , HLA Antigens/analysis , HLA-DR Antigens/genetics , HLA-DRB1 Chains , HLA-DRB3 Chains , HLA-DRB4 Chains , HLA-DRB5 Chains , Humans , Rhodamines/chemistry , p-Dimethylaminoazobenzene/chemistry
5.
Tissue Antigens ; 56(6): 553-5, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11169247

ABSTRACT

We here describe two new DRB alleles (DRB1*0317 and DRB3*0210) which were detected by polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and confirmed by direct sequencing. The exon 2 sequence of DRB1*0317 is the result of an intergenic recombination event yielding a hybrid allele between 5'DRB1 and 3' DRB3 sequences with the recombination breakpoint located between codons 39-51. The new DRB3*0210 allele is closest related to DRB3*02021 except for a single nucleotide position at codon 51. Here, the sequence AGG at codon 51 which was a group-specific motif for all DRB3*02 alleles described previously, is replaced by the DRB consensus sequence ACG.


Subject(s)
HLA-DR Antigens/genetics , Alleles , HLA-DRB1 Chains , HLA-DRB3 Chains , Histocompatibility Testing , Humans , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain Reaction/methods , Sequence Homology, Nucleic Acid
6.
Tissue Antigens ; 53(3): 301-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10203025

ABSTRACT

Similar to our recently described HLA-A and -C fluorotyping strategies, the aim of this study was to develop a sequence-specific primed polymerase chain reaction (PCR-SSP)-based fluorotyping method for HLA-DRB. Applying the fluorogenic 5' nuclease assay, it is possible to increase the sample throughput rate by abolishing all labor-intensive post-amplification steps. Additionally, problems related to contamination are eliminated. The method relies on the 5'-3' exonuclease activity of the Taq-DNA Polymerase which cleaves a target-specific and individually labelled fluorogenic probe during successful PCR. Different labelled probes specific for different targets can be applied in a single PCR, allowing independent detection of the specific HLA and the internal control product. The probe used to detect the HLA-DRB specific amplicons was labeled at its 5' end with FAM as the reporter and further 3' with TAMRA as the quencher. The probe hybridized within the 2nd exon to a conserved region which was covered by all primer mixes. In case of amplification, the cleavage of the fluorogenic probe led to an interruption of the TAMRA-mediated quenching effect and generated a significant increase of the FAM fluorescence. The HLA-DRB fluorotyping information was based on the FAM fluorescence released by 24 individual primer mixes. A TET-TAMRA-labelled probe was used to indicate amplification of the internal control sequence in each PCR reaction. So far, 170 PCR typed clinical samples representing all serologically defined HLA-DRB specificities were analyzed using this fluorotyping method. The results were 100% concordant with those obtained by conventional agarose gel detection.


Subject(s)
HLA-DR Antigens/genetics , Polymerase Chain Reaction/methods , Alleles , HLA-DR Antigens/chemistry , HLA-DRB1 Chains , HLA-DRB3 Chains , HLA-DRB4 Chains , HLA-DRB5 Chains , Histocompatibility Testing/methods , Humans
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