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Willy A Flegel

Publications and source records attributed to Willy A Flegel.

16 recordsLinked to original sources
PubMed2006

The D category VI type 4 allele is prevalent in the Spanish population.

BACKGROUND: The D category VI (DVI) is one of the clinically most important partial D. Three different molecular structures causing the DVI phenotype have been described. STUDY DESIGN AND METHODS: To determine the molecular basis of the DVI phenotype in the Spanish population, 20 DVI samples, previously detected in serologic screening, were examined by polymerase chain reaction with RHD exon-specific primers. Unexpected findings were further pursued by cDNA nucleotide sequencing. RESULTS: A novel pattern of RHD exon amplification was detected, which did not correspond to any of the previously described molecular structures. The cDNA sequence led to the identification of the new hybrid RHD-Ce(3-5)-D allele. The origin of exon 2 is undeterminable, because the 5' breakpoint was located within a region of RHD and RHCE identical sequence, which encompasses this exon. Sequencing of intron 5 allowed the 3' breakpoint to be mapped between the sixth and seventh polymorphic sites. Serologically, the hybrid protein has a D epitope expression pattern identical to the previously described DVI phenotypes and an antigen density slightly lower than DVI type 3. The new DVI variant is linked to the DCe haplotype and expresses the low-incidence BARC antigen. CONCLUSION: A novel structure causing the DVI phenotype, here named DVI type 4, has been characterized. This novel structure is the most frequent cause of DVI in Spain.

Base Sequence↗
PubMed2005

Random survey for RHD alleles among D+ European persons.

BACKGROUND: RHD alleles are considered more variable in African persons than in European persons. A systematic survey, however, was lacking among D+ European persons at the molecular level, precluding any definite frequency estimate. STUDY DESIGN AND METHODS: A random survey was performed among 500 ccDee, 250 CcDee, and 250 ccDEe blood donors in southwestern Germany. They were tested by polymerase chain reaction with sequence-specific priming (PCR-SSP) for up to 12 single-nucleotide polymorphisms representative for the most frequent RHD alleles among European persons. The RHD exon 5 nucleotide sequence was also tested in all 1000 samples. The nucleotide sequence of the 10 RHD exons was checked in all samples with aberrant exon 5 or positive PCR-SSP procedures. RESULTS: By PCR-SSP, 15 aberrant RHD alleles were found among the 500 ccDee, 2 among the 250 CcDee, and none among the ccDEe samples. One of these was the novel RHD(F223V, E233Q, T379M) allele dubbed DAU-5. Weak D type 4 was detected more frequently than expected, whereas the population frequencies of the other RHD alleles conformed to published estimates. Nucleotide sequencing of RHD exon 5 further revealed three novel alleles RHD(G212G), RHD(R234W), and RHD(V245L), dubbed DUC-1, DQC, and DUC-2. CONCLUSION: In a limited screen at the molecular level among 1000 random D+ donors in southwestern Germany, 20 donors were found carrying aberrant RHD alleles. Four of these alleles were new and likely sporadic. An estimate was derived of the variety that may be encountered in genotyping approaches, and it was concluded that even within the European population the variety of RHD alleles may be larger than anticipated.

Alleles↗
PubMed2005

Weak D type 1.1 exemplifies another complexity in weak D genotyping.

BACKGROUND: Weak D expression is caused by a large number of RHD alleles. Increasingly recommendations for D+ or D- transfusions are based on polymerase chain reaction (PCR) identification of certain RHD alleles. Possible sources of error are rare D variants that are inadvertently carrying known polymorphisms of frequent weak D types. STUDY DESIGN AND METHODS: Weak D donors were checked by direct column agglutination. In donors with unusually weak expression of D, the molecular weak D type was determined by weak D PCR and nucleotide sequencing. The serologic profile of a weak D type 1 variant was determined by agglutination serology and flow cytometry. RESULTS: Several donors in whom direct agglutination barely revealed any D expression were shown to carry the new RHD(L18V,V270G) allele dubbed weak D type 1.1. Initially, such donors had been mistyped as weak D type 1 by PCR. In a systematic study, weak D type 1.1 was shown to be present in 7 of 23 donors with very weak D expression who all lived in a restricted area of Northern Germany. Although weak D type 1.1 was typed D- or barely D+ by direct agglutination, it was easily detected by antiglobulin technique and was shown to carry about 600 antigens D per red blood cell. CONCLUSION: The observation of weak D type 1.1 with its distinct phenotype pinpointed to two general problems of current RHD genotyping strategies: Mistyping of alleles with additional mutations and striking geographic variation of the allele distributions.

ABO Blood-Group System↗
PubMed2005

Partial D, weak D types, and novel RHD alleles among 33,864 multiethnic patients: implications for anti-D alloimmunization and prevention.

BACKGROUND: The D antigen includes category D, partial D, and weak D types, which are important because anti-D alloimmunization can occur in some but not all persons that express a variant RHD allele. At present, there is little prospective information on the prevalence of D variants among obstetric patients and potential transfusion recipients. STUDY DESIGN AND METHODS: The RHD alleles were prospectively examined in a large patient population identified on the basis of a difference in anti-D reactivity between two reagents. RESULTS: Fifty-five discrepancies (0.96% of D-) were noted among 33,864 ethnically diverse patients over 18 months, of which 54 represented mutated RHD alleles. Seven obstetric patients were assigned D- status based on serology; only 1 patient had a partial RHD allele. Ten of 25 (36%) obstetric patients and 4 of 6 (67%) female potential transfusion recipients of childbearing age or younger were assigned D+ status, and they expressed a D variant known to permit anti-D alloimmunization. In total 20 RHD alleles were identified including category, DVa or DVa-like alleles (n = 7), DAR (n = 8), and four novel RHD alleles including two new DAU alleles. CONCLUSION: Given the complexity of D antigen expression, it is concluded that some clinically important D variants identified by standard serologic analysis phenotype as D+ and are potentially at risk for the development of anti-D.

Adult↗
PubMed2005

SCER and SCAN: two novel high-prevalence antigens in the Scianna blood group system.

BACKGROUND: More than 20 years ago, two probands were described whose red blood cells (RBCs) typed Sc:1,-2,3. Their serum samples contained alloantibodies reactive with all RBCs tested except those of the Sc:-1,-2,-3 phenotype. Cloning of the Scianna gene allowed us to determine the molecular bases of these samples. STUDY DESIGN AND METHODS: In a collaborative effort, the two probands' samples and also two Sc:-1,-2,-3 samples were obtained from frozen storage. All 11 SC (ERMAP) exons and their flanking regions were sequenced. RESULTS: The two probands with antibodies to Scianna-related antigens were homozygous, respectively, for an ERMAP(R81Q) allele caused by a G to A substitution at nucleotide 242 in the ERMAP gene and for an ERMAP(H26Y,G35S) allele, in which the G35S substitution was caused by a G to A substitution at nucleotide 103. Two patients with the Sc:-1,-2,-3 phenotype both carried ERMAP(R332X) alleles caused by a C to T substitution at nucleotide 994 that differed at one nucleotide position in the noncoding region of exon 11. In eight samples carrying orphan low-prevalence antigens, no ERMAP variants were detected that could be implicated in Scianna antigen expression. CONCLUSION: SCER and SCAN expanded the Scianna blood group system to seven antigens, have been assigned the ISBT numbers 013.006 (Sc6) and 013.007 (Sc7), and were associated with ERMAP(R81Q) and ERMAP(G35S) proteins, respectively. ERMAP(R332X) is a second molecular basis for the Sc(null) phenotype. The eight low-prevalence antigens By, To(a), Pt(a), Re(a), Je(a), Li(a), SARA, and Sk(a) do not belong to the Scianna blood group system.

Alleles↗
PubMed2005

Genetic mechanisms of Rhesus box variation.

BACKGROUND: The RHD gene is flanked by two highly homologous DNA segments of approximately 9000 bp, the upstream and downstream Rhesus boxes. In haplotypes with an RHD deletion, the fusion of the two Rhesus boxes generates the single-hybrid Rhesus box, the detection of which has been applied for RHD zygosity determination. Aberrant Rhesus boxes can confound this application and appear to be frequent among African individuals. STUDY DESIGN AND METHODS: A total of 5850 bp of the upstream and of the downstream Rhesus boxes were sequenced in 18 samples that were representative for all four D clusters and of the hybrid Rhesus boxes in four samples that were mistyped in assays for the hybrid Rhesus box. RESULTS: The known differences between upstream and downstream Rhesus boxes were in part restricted to subsets of RHD alleles. Forty-six additional polymorphisms were detected and caused by single-nucleotide substitutions, short insertions, or deletions. Gene conversions were found in the upstream Rhesus boxes of RHDpsi, DAU-1, and DAU-3 and in the downstream Rhesus boxes of Ccdes, weak D type 4.1, type 4.2 (DAR), and DAU-0. Recombinations between haplotypes were likely in several alleles like DIII type 4. Four nonstandard hybrid Rhesus boxes were suggestive of multiple RHD deletion events. CONCLUSION: There is considerable variation of Rhesus box sequences associated with distinct RHD alleles. RHD zygosity diagnostics in African persons is best based on quantitative polymerase chain reaction or amplification of the full-length hybrid Rhesus box. Because aberrant Rhesus boxes were observed among European persons, use of more than one method for hybrid Rhesus box detection may even be advisable in European persons.

Africa↗
PubMed2005

Histo-blood group antigens as allo- and autoantigens.

The science of blood groups has made giant steps forward during the last decade. Blood-group typing of red blood cells (RBCs) is performed on more than 15 million samples per year in Europe, today much less often for forensic reasons than for clinical purposes such as transfusion and organ transplantation. Specific monoclonal antibodies are used with interpretation on the basis of RBC agglutination patterns, and mass genotyping may well be on its way to becoming a routine procedure. The discovery that most blood group systems, whose antigens are by definition found on RBCs, are also expressed in multiple other tissues has sparked the interest of transplantation medicine in immunohematology beyond the HLA system. The one and only "histo-blood group" (HBG) system that is routinely considered in transplantation medicine is ABO, because ABO antigen-incompatible donor/recipient constellations are preferably avoided. However, other HBG systems may also play a role, thus far underestimated. This paper is an up-to-date analysis of the importance of HBG systems in the alloimmunity of transplantation and autoimmune events, such as hemolytic anemia.

ABO Blood-Group System↗
PubMed2003

RHD allele distribution in Africans of Mali.

BACKGROUND: Aberrant and non-functional RHD alleles are much more frequent in Africans than in Europeans. The DAU cluster of RHD alleles exemplifies that the alleles frequent in Africans have evaded recognition until recently. A comprehensive survey of RHD alleles in any African population was lacking. RESULTS: We surveyed the molecular structure and frequency of RHD alleles in Mali (West Africa) by evaluating 116 haplotypes. Only 69% could be attributed to standard RHD (55%) or the RHD deletion (14%). The aberrant RHD allele DAU-0 was predicted for 19%, RHDPsi for 7% and Ccdes for 4% of all haplotypes. DAU-3 and the new RHD allele RHD(L207F), dubbed DMA, were found in one haplotype each. A PCR-RFLP for the detection of the hybrid Rhesus box diagnostic for the RHD deletion in Europeans was false positive in 9 individuals, including all carriers of RHDPsi. Including two silent mutations and the RHD deletion, a total of 9 alleles could be differentiated. CONCLUSION: Besides standard RHD and the RHD deletion, DAU-0, RHDPsi and Ccdes are major alleles in Mali. Our survey proved that the most frequent alleles of West Africans have been recognized allowing to devise reliable genotyping and phenotyping strategies.

Alleles↗
PubMed2003

The RHCE allele ceRT: D epitope 6 expression does not require D-specific amino acids.

BACKGROUND: False-positive D typing in patients may lead to anti-D immunization caused by D+ transfusions or by omission of anti-D prophylaxis. Known causes of such errors are RhCE variants carrying RhD-specific amino acids and cold agglutinin activity of some frequently used monoclonal anti-D. STUDY DESIGN AND METHODS: The molecular basis of eight samples referred because of "false-positive" reactions with some commercial monoclonal anti-D was investigated by PCR and nucleotide sequencing from genomic DNA. PCR with sequence-specific priming was developed to specifically detect the underlying aberrant RHCE allele. The D epitope profile of the allele was determined by serology. RESULTS: The aberrant reactivity of the samples was caused by the RHCE allele RHCE(R154T) that occurred in a cde haplotype. The phenotype dubbed ceRT expressed the important D epitope 6, which is the target epitope of most monoclonal anti-D used in routine typing. DISCUSSION: The characterization of ceRT demonstrated a previously unknown mechanism of antigen D expression that does not require any D-specific amino acid. At least for some D epitopes, D-like structures may be mimicked by RhCE proteins carrying amino acid substitutions not representative for RhD.

Acetyltransferases↗
PubMed2003

Antibodies to high-frequency antigens may decrease the quality of transfusion support: an observational study.

BACKGROUND: There is only little information on the transfusion support of patients with antibodies to high-frequency RBC antigens. STUDY DESIGN AND METHODS: In cooperation with reference laboratories and transfusion services in Austria, Germany, and Switzerland, the transfusion support provided to hospitalized patients identified as having such antibodies was reviewed during a 20-month period. RESULTS: A total of 52 patients with antibodies to high-frequency antigens were treated in hospitals. Twenty-two of them received 104 units of antigen-negative RBCs. In 23 cases, a deviation from the standard transfusion policy (e.g., transfusion of antigen-incompatible units) occurred. The use of frozen or fresh units varied amongst the different countries but did not affect the rate of deviation from protocol. About 20 percent of all units were supplied internationally. Four antibody specificities, anti-Kpb, anti-Vel, anti-Lub, and anti-Yta, were identified in two-thirds of the patients. CONCLUSION: This survey indicated that transfusion support was unsatisfactory in about one-third of the hospitalized patients with antibodies to high-frequency antigens. Maintaining a rapidly accessible stock of just four types of rare blood units would ensure adequate transfusion support for most of these patients.

Antibody Specificity↗
PubMed2002

DNB: a partial D with anti-D frequent in Central Europe.

To improve routine D typing and define transfusion strategy, it is important to establish the frequency of partial D alleles and their susceptibility to anti-D alloimmunization due to transfusion or pregnancy. We identified the partial D DNB that was caused by an RHD(G355S) allele associated with a CDe haplotype and whose phenotype presented a normal D in routine typing. The antigen density was about 6000 D antigens per red blood cell, and the Rhesus index was 0.02. Five anti-D immunization events with allo-anti-D titers up to 128 were observed. Twelve carriers of DNB were whites of Central Europe; the only Danish proband had Austrian ancestry. DNB was the most frequent partial D recognized so far in whites, occurring with frequencies of up to 1:292 in Switzerland. DNB was the underlying partial D phenotype in a relevant fraction of anti-D immunizations occurring in whites.

Alleles↗
PubMed2002

Scianna antigens including Rd are expressed by ERMAP.

The Scianna blood group encompasses the high-frequency antigens Sc1 and Sc3 and the low-frequency antigen Sc2. Another low-frequency antigen Rd (Radin) was suggested to belong to the Scianna blood group. The molecular basis of the Scianna blood group was unknown. The erythrocyte membrane-associated protein (ERMAP) shared the genomic location, protein product size, and localization to the red blood cell (RBC) membrane surface with Scianna. The ERMAP gene was sequenced in probands with known Scianna and Radin phenotypes. In a Sc:-1,-2 proband, only an ERMAP allele with a 2-bp deletion in exon 3 causing a frameshift could be detected. A Sc:-1,2 proband was homozygous for the ERMAP(Gly57Arg) allele. An Rd(+) proband was heterozygous for the ERMAP(Pro60Ala) allele. Polymerase chain reaction with sequence-specific priming (PCR-SSP) systems was developed to detect the Sc2 and Rd alleles of the ERMAP gene. The 2 alleles occurred with about 1% and less than 1% frequency in the population, which was compatible with the frequency of the Sc2 and Rd antigens known in whites. Two Sc2(+) and one Rd(+) samples that were found by genotyping were confirmed by serology. The antigens of the Scianna blood group include Rd and are expressed by the human ERMAP protein. Sc2 is caused by an ERMAP(Gly57Arg) allele and Rd by an ERMAP(Pro60Ala) allele. Scianna is the last of the previously characterized protein-based blood group systems whose molecular basis was discerned. Hence, the phenotype prediction by genotyping became possible for all human blood group systems encoded by proteins.

Antigens, Surface↗
PubMed2002

The DAU allele cluster of the RHD gene.

Variant D occurs frequently in Africans. However, considerably less RHD alleles have been described in this population compared with Europeans. We characterized 5 new RHD alleles, dubbed DAU-0 to DAU-4, that shared a T379M substitution and occurred in a cDe haplotype. DAU-1 to DAU-4 were detected in Africans with partial D phenotypes. They harbored one and 2 additional missense mutations, respectively, dispersed throughout the RhD protein. An anti-D immunization was found in DAU-3. DAU-0 carrying T379M only was detected by screening European blood donors and expressed a normal D phenotype. Within the phylogeny of the RHD alleles, DAU formed an independent allele cluster, separate from the DIVa, weak D type 4, and Eurasian D clusters. The characterization of the RH phylogeny provided a framework for future studies on RH alleles. The identification of the DAU alleles increased the number of known partial D alleles in Africans considerably. DAU alleles may be a major cause of antigen D variability and anti-D immunization in patients of African descent.

Africa↗
PubMed2002

Molecular biology of partial D and weak D: implications for blood bank practice.

Two genes, RHD and RHCE, encode the antigens of the RH blood group system. The clinically most important antigen D is determined by the presence of a functional and grossly normal RHD gene. About 18% of Europeans do not express an antigen D, most often but not always caused by the RHD gene deletion. Rhesus negative phenotypes in Africans are caused by the RHD gene deletion, the RHD pseudogene RHD psi, and the Cde(s) allele. About 1% of Europeans carry RHD alleles with aberrant structures encoding for diminished D-immunoreactivity. In Africans the frequency of aberrant RHD alleles is much higher. Aberrant RHD alleles encode partial D, some of which were dubbed D categories, and weak D. Since we defined the molecular basis of the RHD deletion, a specific detection of heterozygous carriers became feasible.

Alleles↗