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R Blasczyk

Publications and source records attributed to R Blasczyk.

At least 73 records · Page 4Linked to original sources
PubMed1997

Increased diversity within the HLA-A*66 group: implications for matching in unrelated bone marrow transplantation.

We have identified a new A*66 allele (A*6603) in three related individuals, an Arabic patient suffering from acute myeloid leukemia and two of her relatives. The A*66 alleles differ in three amino acid residues at positions 70, 90 and 163. The closer relationship between A*6602 and A*6603, which only differ at amino acid 70, replacing GLN with HIS, suggests that the alloreactive potential in this mismatch combination is lower than in all other mismatched A*66 donor-recipient combinations, which exhibit two (A*6601 versus A*6602) and three (A*6601 versus A*6603) differences at the pivotal positions, respectively. This emphasizes the potential role of the A*66 subtypes in bone marrow transplantation with alternative donors. For that reasons, allelic subtyping should be considered in donor-recipient matching to identify the kind of disparity.

Amino Acid Sequence↗
PubMed1997

Sequencing of HLA class I genes based on the conserved diversity of the noncoding regions: sequencing-based typing of the HLA-A gene.

We present a sequencing-based typing strategy for the HLA-A locus that is generally applicable to all HLA class I genes. Sequencing-based typing is the method of choice for matching in unrelated bone marrow transplantation on the allelic level. We determined the noncoding sequences of all serological antigens and most of their subtypes and discovered a remarkably conserved diversity characterized by polymorphic sequence motifs. In this study we took advantage of this diversity we uncovered in the 5' flanking region, 5' untranslated region and in the introns 1, 2 and 3, which was related to serological families. We established 12 primer mixes for setting up a PCR-based template preparation. Our strategy is based on the separate amplification of haplotypes and therefore defines the cis/trans linkage of polymorphic sequence motifs. This allowed individual sequencing of the haplotypes in all samples heterozygous for the broad antigens as well as the complete analysis of the polymorphic exons 2 and 3. All templates included the 2nd intron which was used as a priming site for the gene-specific 5' and 3' universal sequencing primers regardless of the amplified haplotypes. The independent sequencing of the haplotypes allows the application of the dye terminator cycle sequencing technique, which is less time-consuming and less-laborious than dye primer chemistry. The lack of heterozygous positions essentially facilitates on the one hand the data analysis and on the other hand the detection of new alleles. Sequencing is only required in one direction due to the absence of peak shift problems. The results will remain unambiguous regardless of a growing HLA sequence data bank since this sequencing technique defines the cis/trans linkage of sequence motifs in more than 95% of the cases.

Base Sequence↗
PubMed1997

Fluorotyping of HLA-C: differential detection of amplicons by sequence-specific priming and fluorogenic probing.

Conventional PCR-SSP, which is based on an agarose gel-based read-out, has the disadvantages of time-consuming post-PCR steps and low potential for automation. The aim of our study was to sort out these drawbacks by establishing a fluorescence-based PCR-SSP system for HLA-C. The assay relies on the sequence-specific identification of amplicons with individually labeled probes that are cleaved during successful PCR by the 5'-3' exonuclease activity of the Taq-DNA Polymerase. The oligonucleotides are labeled with a unique and spectrally resolvable fluorescent reporter dye at the 5' terminus (FAM or TET) and a common quencher dye at the 3' terminus (TAMRA). In case of amplification, the reporter escapes from the quenching control caused by the physical separation of the dyes, resulting in a significant increase of the reporter fluorescence. This allows simultaneous and differential detection of the specific HLA (FAM) and internal control (TET) product. The HLA-C fluorotyping information is based on the individual reporter fluorescence released by 18 PCR primer mixes. Using this method, we analyzed 145 samples previously typed with conventional PCR-SSP and found a concordance rate of 100%. Furthermore, fluorotyping revealed quantitative results that may indicate the presence of homozygosity by high signal intensities. This provided extra protection not to miss new alleles which are not amplified by the current primer mixes. These features as well as the capability of high sample throughput and the possibility of automation makes fluorotyping an attractive tool for PCR-based HLA typing.

DNA Primers↗
PubMed1997

The nature of polymorphism of the HLA class I non-coding regions and their contribution to the diversification of HLA.

The sequence database of HLA class I genes is mainly derived from mRNA analysis. Little is known about the non-coding sequences of the different class I alleles. In this study we have determined the sequence of the 1st through 3rd introns of the majority of HLA-A and -B alleles. The few published sequences emerged to contain substantial errors. The introns turned out to be highly polymorphic with a variability of 14.6% in the 1st intron decreasing to 6.2% in the 3rd intron. Against all expectations, this variability is not characterised by random point mutations but by a highly systematic diversity reflecting the ancestral relationship of the HLA alleles. The variability is arrested on the level of the serological diversity. The striking conservation within each ancestral lineage suggests that point mutations have been negatively selected. This finding could be explained by the evolutionary pressure on base order, promoting the potential to extrude single-strand stem-loops from supercoiled duplex DNA, which is believed to be important for combination. Moreover, the GC content was found to be as high as 78% in the 1st and 2nd introns and 55% in the 3rd intron. These CpG islands are directly involved in the exchange of short stretches of DNA in unequal crossing-over events. Additionally, conversion between different class I sequences is facilitated by regions of strong homology, stabilizing the pairing of variable regions. All these observations indicate the potential of a substantial contribution of introns to the recombinational activity of class I genes. The exclusive clustering of CpG islands in the 1st and 2nd introns restricts the gene conversion events to the regions of the 2nd and 3rd exons and therefore protects the conservation of the 5 flanking region and the 3 part of the gene. Since there are less diversification forces acting on introns they may be more conserved in a trans-species manner than exons. Therefore, they could provide the answer for the controversy regarding intra- or trans-species evolution.

Alleles↗
PubMed1997

Refined mapping of the epilepsy susceptibility locus EJM1 on chromosome 6.

Juvenile myoclonic epilepsy (JME) is a genetically determined common subtype of idiopathic generalized epilepsy. Linkage to the HLA complex on chromosome 6p21.3 and an allelic association with HLA-DR13 and -DQB1 alleles suggest that a susceptibility locus for JME, designated as "EJM1," is located within or near the HLA region. However, further studies revealed controversial results, and genetic heterogeneity has been suspected. The present study was designed to evaluate the validity of the association and linkage findings and to refine the map position of EJM1. Our association analysis showed no significant difference of the frequency of HLA-DR13 carriers in 62 German JME patients compared with that in 77 German controls (X2 = 0.98, df = 1, p = 0.161, one-tailed). Multipoint linkage analysis with use of microsatellite markers from the chromosomal region 6p25-q13 in 29 German families of JME patients provided significant evidence that an epilepsy locus (EJM1) close to the HLA locus confers susceptibility to "idiopathic" generalized seizures (Zmax = 3.27 at theta max = 0.033 centromeric to the HLA-DQ locus), assuming an autosomal dominant mode of inheritance with 70% penetrance. Haplotype analyses revealed key recombinations in five families, which locate EJM1 to the centromeric side of the HLA-DQ locus. This study confirms a causative role of EJM1 in the pathogenesis of idiopathic generalized seizures in the majority of German families of JME patients and refines a candidate region of 10.1 cM in the chromosomal region 6p21 between the flanking loci HLA-DQ and D6S1019. A possible explanation for the current controversial results in families of different populations might be ethnic variation of interfering polygenic effects that could be permissive for heterogeneous susceptibility alleles.

Alleles↗
PubMed1997

The diversity of the HLA class I introns reflects the serological relationship of the coding regions.

The introduction of PCR-based HLA typing techniques has uncovered that the HLA system is much more variable than it has been expected from the conventional typing methods. More and more new alleles are detected which are not characterized by new sequence motifs, but by new combinations of already existing sequence motifs. This variability, reflecting the immunological necessity to have the greatest capacity for presenting antigenic peptides, is increasingly complicating DNA typing techniques based on the diversity of the coding region. We have determined the sequence of the 1st through 3rd intron of the majority of HLA-A and HLA-B alleles in 48 well-defined cell lines and 195 PCR-typed clinical samples. The few published sequences emerged to contain substantial errors. The introns turned out to be highly polymorphic. Besides extensive homologies, numerous locus- and group-specific sites could be identified. The most intriguing finding was that most of the polymorphic motifs were related to serological families. These sequence motifs were extremely beneficial for setting up PCR-based typing systems. In particular, sequencing-based typing strategies benefited from intron-restricted priming for amplification and sequencing by enabling complete analysis of the polymorphic exons. The determination of cis/trans linkages of sequence motifs was substantially facilitated. Apart from these advantages, the intron sequences were useful for evolutionary studies, delivering more insights into the genetic relationship between different alleles and the mechanisms involved in the development of the diversity of HLA. Moreover, the variability of the introns may provide a structural basis for the identification of regulatory elements acting on the level of transcription.

Cell Line↗
PubMed1997

Allele-specific PCR amplification of factor V Leiden to identify patients at risk for thromboembolism.

Resistance to activated protein C is the most common hereditary cause of thrombophilia and is significantly linked to factor V Leiden. We designed primers in order to identify factor V Leiden by allele-specific PCR amplification. Amplification specificity for factor V was ensured by a 3' primer located at the intron 9/exon 10 border of the gene. One sense and two antisense primers were used in two separate primer mixes specific for factor V ARG506 (wild-type) or factor V GLN506 (factor V Leiden). In each PCR reaction a pair of primers amplifying a fragment of the human growth hormone gene was included as an internal positive amplification control. The presence or absence of specific PCR amplification allowed definite allele assignment without the need for any postamplification specificity step. The internal positive control primers indicate a successful PCR amplification, allowing the assignment of homozygosity. In a prospective study 126 patients with thromboembolic events were analyzed using this technique and PCR-RFLP. The concordance between these methods was 100%. In 27 patients a heterozygous factor V GLN506 mutation was detected, whereas 1 patient with recurrent thromboembolism was homozygous. No false-positive or false-negative results were observed in the homozygous as well as heterozygous samples. Additionally, in 15 samples identified to carry the point mutation by allele-specific PCR amplification, automatic sequencing has confirmed the heterozygous or homozygous point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor V Leiden.

Alleles↗
PubMed1996

Discrimination of HLA-B27 alleles by group-specific amplification followed by solid-phase sequencing.

HLA-B27 is known to be highly associated with ankylosing spondylitis. Until now, nine B27 subtypes have been sequenced and may contribute in different fashions to ankylosing spondylitis. Additionally, the divergent subtypes may be of clinical importance in bone marrow transplantation with alternative donors. The purpose of this study was to determine the different subtypes of HLA-B27 by a direct sequencing approach. The typing strategy is based on a group-specific amplification of the second and third exon followed by automated fluorescence sequencing of the polymorphic regions. The extensive sharing of sequence motifs between the different B alleles made it impossible to specifically amplify the B27 group under the precondition of including all sequence variations necessary for a postamplification specificity step. Therefore, for setting up a direct sequencing approach of B27, co-amplified B alleles had to be taken into account. In order to get unambiguous sequencing chromatograms without any heterozygous positions, nested sequencing primers were used which selectively matched sequence motifs only present in the second and third exon of the amplified B27 alleles. This strategy allowed in all cases investigated a clear separation of the haplotypes, revealing unequivocal sequencing results. Using this method, we have investigated 93 B27-positive individuals. Sequencing identified the alleles B*2702, 2703, 2704, 2705, and 2707. B*2701, 2706, 2708, and 2709 were not represented in the population studied.

Alleles↗
PubMed1996

Sequence analysis of the 2nd intron revealed common sequence motifs providing the means for a unique sequencing based typing protocol of the HLA-A locus.

We here present a sequencing strategy for the HLA-A locus which is generally applicable for all HLA class I genes. The typing strategy is based on a group-specific amplification according to the serologically defined antigens. The PCR products carry the typing-relevant polymorphic regions of the 2nd and 3rd exon including the 2nd intron. The sequencing primers were designed to match conserved sequence motifs in the 2nd intron allowing a nested sequencing approach in 3' and 5' direction. These conserved regions were identified after sequence compilation of the 2nd intron of 143 clinical samples and 48 cell lines mostly from the 9th and 10th IHWC representing all serologically defined groups of alleles. This strategy allowed the use of only one 5' and one 3' sequencing primer regardless of the amplified allele. Therefore, it was possible to use dye terminator as well as dye primer sequencing chemistry. The amplification strategy allowed the separation of the haplotypes in almost all cases. Thus, an assignment of heterozygous positions requiring high sequencing quality was not necessary, allowing the application of Sequenase as well as TaqPolymerase as sequencing enzyme. Concerning the resolution of heterozygosity it is obvious that this approach is superior to a typing system using a single pair of generic primers followed by direct sequencing, since the latter technique is not capable of defining the cis/trans linkage of polymorphic sequences and, hence, cannot exclude the presence of unknown alleles.

Alleles↗
PubMed1996

Structural definition of the A*74 group: implications for matching in bone marrow transplantation with alternative donors.

We have identified two new A*74 alleles (A*7402 and 7403) in two unrelated individuals. A*7402 differs from A*7401 by a single amino acid substitution in the signal peptide and may be the result of a gene conversion event at the 3' end of exon 1. A*7403 differs from A*7401 by a single amino acid exchange in the alpha 1 domain and is most likely due to a point mutation in exon 2, since no HLA class I donor allele has been found. Since A*7402 appears to be the ancestor of the other two A*74 alleles, it is possible that A*7401 and 7403 have been created by successive point mutations. The sequences of the expressed proteins of A*7401 and 7402 are identical. The heavy chain sequence of A*7403 differs from these alleles at the crucial residue 79 which is located in the sequence stretch of the alpha 1 alpha-Helix where the Bw4/Bw6 determinants have been identified and which probably affects TCR interaction. This variation can therefore be expected to stimulate alloreactive T cells, graft rejection and graft versus host disease emphasizing the relevance for matching in bone marrow transplantation with alternative donors.

Alleles↗
PubMed1996

Analysis for recovery and loss of mononuclear cells and colony-forming units granulocyte-macrophage during ex vivo processing of autologous bone marrow.

During ex vivo processing of autologous bone marrow (BM) substantial loss of stem and progenitor cells should be avoided to achieve rapid and sustained hematopoietic reconstitution after high-dose radio-/chemotherapy. We processed 25 autologous BM grafts with the Fresenius AS104 cell separator for cryopreservation and we determined recoveries for mononuclear cells (MNC) and colonyforming units granulocyte-macrophage (CFU-GM) in the BM concentrates. To identify cell loss in BM fractions not cryopreserved, we investigated the MNC and CFU-GM content of BM fat and BM blood. MNC and CFU-GM recovery yielded a mean ( +/- SEM) of 42 +/- 12 and 54 +/- 20% in the BM concentrate. BM fat showed a mean loss of 7 +/- 5% for MNC and 4 +/- 3% for CFU-GM, BM blood 30 +/- 12% for MNC and 13 +/- 13% for CFU-GM, respectively. CFU-GM recovery was significantly higher in the BM concentrate of patients with hematologic malignancy (HM) compared with patients suffering from germ cell cancer (GCC): 66 +/- 21 vs. 43 +/- 12% (p < 0.02). Seventeen patients (7 GCC, 10 HM) underwent high-dose chemotherapy or radio-/chemotherapy and were autografted with 0.8 +/- 0.2 x 10(8) MNC/kg and 3.7 +/- 2.0 x 10(4) CFU-GM/kg. All patients achieved engraftment with neutrophils > 0.5 x 10(9)/l at a mean of 14 +/- 6 days. We conclude that: (1) ex vivo processing of autologous BM with a mean of recovery of 42% for MNC and 54% for CFU-GM in the BM concentrate can result in a cell population capable of sustained hematopoietic reconstitution, (2) CFU-GM recovery is significantly higher in patients with HM than in patients with GCC and (3) 37% MNC and 17% CFU-GM represent in fact cell losses recovered from BM fractions not cryopreserved (BM fat, BM blood). Furthermore, it is likely that MNC and CFU-GM not recovered from BM concentrate, BM fat and BM blood are cell losses related to the cell separator.

Adolescent↗
PubMed1996

Simple and rapid detection of factor V Leiden by allele-specific PCR amplification.

Resistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

Alleles↗
PubMed1995

A novel HLA-DR13 allele (DRB1*1314) identified by single-strand conformation polymorphism analysis and confirmed by direct sequencing.

A novel variant of the HLA-DR13 group is described. The new allele was found in a DR10, 13 heterozygous patient of Turkish origin, two HLA genotypically identical children of the patient typed as DR11,13, and one child typed as DR13,13. DR13 subtyping of the patient was initially performed by SSCP analysis of PCR-amplified DNA, using the 11th OHWS primers DRBAMP-3 and DRBAMP-B. Due to an unusual SSCP banding pattern, the PCR product was subjected to solid-phase sequencing. The sequence of the new allele, DRB1*1314, is different from that of DRB1*1307 by a single nucleotide substitution in codon 47, with T replacing consensus A. This results in a single amino acid change of tryptophan to phenylalanine in the first domain of the DR beta chain.

Anemia, Sickle Cell↗
PubMed1995

Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis.

A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.

Alleles↗