Search PubMed⌕ Search

Biomedical subjects

Willy A Flegel

Publications and source records attributed to Willy A Flegel.

At least 19 recordsLinked to original sources
PubMed2006

Tissue distribution of blood group membrane proteins beyond red cells: evidence from cDNA libraries.

The proteins of blood group systems are expressed on red blood cells (RBC) by definition. We searched nucleotide databases of human expressed sequence tags (EST) to collate the distribution of 22 distinct membrane proteins in cells and tissues other than RBC. The documented blood group genes are: MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, John-Milton-Hagen and Gill. The genes were grouped according to their overall and their relative expression in embryo and adults. We describe the distribution of EST in cells, tissues and cell lines with a focus on non-RBC tissues.

ABO Blood-Group System↗
PubMed2006

How I manage donors and patients with a weak D phenotype.

PURPOSE OF REVIEW: Since the adoption of molecular blood-group typing, the considerable heterogeneity of the serologic entities weak D and DEL at the molecular level has come to light. I offer an approach to the management of donors and patients expressing D antigen weakly and carrying any of the various molecular types of weak D and DEL. RECENT FINDINGS: More than 50 distinct weak D alleles have been described. An internet-based survey of anti-D immunizations occurring in D-positive transfusion recipients reveals that no allo-anti-D has been observed in patients carrying prevalent weak D types. Allo-immunizations are documented for weak D types 4.2 (also known as DAR), 11 and 15. Anti-D immunizations have been reported in D-negative persons transfused with weak D and DEL red blood cells. SUMMARY: Patients carrying any of the prevalent weak D types 1, 2, 3 or 4.1 are not prone to allo-anti-D immunization and may safely be transfused with D-positive red blood cells. Pregnant women with these weak D types need not receive RhIg. We should pay attention to weak D- or DEL-positive blood units that are labelled D-negative. The clinical benefit of removing DEL blood units from our supply of D-negative red blood cell units should be determined.

Amino Acid Substitution↗
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↗
PubMed2006

The RHCE allele ceSL: the second example for D antigen expression without D-specific amino acids.

BACKGROUND: The example of ceRT proved that the expression of some D epitopes does not require D-specific amino acids. This allele denoted as RHce(R154T) caused the "false-positive" reactions that were observed in ccddee blood donors who typed positive for the D antigen with some monoclonal anti-D. No other example exposing a similar molecular mechanism was known. STUDY DESIGN AND METHODS: Eleven donor and 1 patient ccddee samples were collected in Switzerland that typed "false-positive" with some monoclonal anti-D in bromelain technique. Their RHCE alleles were determined by nucleotide sequencing from genomic DNA and by a polymerase chain reaction with sequence-specific priming. The D epitope profile was compared to ceRT. The population frequencies were estimated in Switzerland and Germany by serology or at the molecular level, respectively. RESULTS: The "false-positive" reactions were caused by the RHCE allele RHce(S122L) occurring in the cde haplotype. Its ceSL phenotype expressed few D epitopes that belonged to the D epitope 6 group. The frequency of ceSL among D- donors was about 1:675 in the region of Bern, Switzerland. No ceSL donors were found elsewhere in Switzerland or in southwestern Germany. CONCLUSION: ceSL represented the second molecular mechanism for D antigen expression without any D-specific amino acids. ceSL and ceRT were useful to delineate the molecular mechanisms of D expression by RhCE proteins carrying amino acids not representative for the RhD proteins. The ceSL population frequencies differed significantly among three Swiss and German populations.

Alleles↗
PubMed2006

The RHCE allele ceCF: the molecular basis of Crawford (RH43).

BACKGROUND: The Crawford antigen (RH43) was described in 1980. It occurred in African American people, as a low-prevalence Rhesus antigen, who were also VS+. STUDY DESIGN AND METHODS: Twelve blood samples were analyzed because of inquiries into discrepant reactions in routine anti-D typing. The RHCE alleles were determined by nucleotide sequencing from genomic DNA. The D epitope profile was determined with 60 monoclonal anti-D. The population frequency was estimated in four major US regional blood centers. RESULTS: The novel RHce(W16C, Q233E, L245V) allele, dubbed ceCF, was found to be occurring in the cde haplotype as cause of the reactivity with the immunoglobulin M anti-D GAMA401. The ceCF phenotype expressed few D epitopes resembling but not matching the reaction patterns observed with other RhCE variants, like R0 (Har), ceRT, and ceSL. The frequency of the ceCF phenotype was 0.056 percent among African American persons and 0.007 percent in the general US population. CONCLUSION: The novel RHce(W16C, Q233E, L245V) allele, which is a variant of the known ce(s) allele, RHce(W16C, L245V), occurs in a haplotype with the RHD deletion and represents the molecular basis of the Crawford antigen.

Black or African American↗
PubMed2006

Outliers in RhD membrane integration are explained by variant RH haplotypes.

BACKGROUND: Variations in a multipass transmembrane protein may affect its membrane integration. To study this effect, the systematic molecular characterization of variant D antigen density is a suitable model. Unlike most other membrane proteins, the expression of the D antigen is often determined by a single allele, because it occurs frequently in hemizygous form. STUDY DESIGN AND METHODS: The D antigen density distribution of 530 CcDee, 475 ccDEe, and 514 ccDee random samples was established by flow cytometry. The molecular bases of samples with D antigen densities outside a bell-shaped peak was investigated. RESULTS: The antigen densities of 499 CcDee, 437 ccDEe, and 480 ccDee samples formed bell-shaped peaks. Three, 10, and 12 samples, respectively, had decreased antigen densities and carried variant RHD alleles. Weak D type 19, RHD(I204T); weak D type 20, RHD(F417S); and the partial D DYU (also known as DQC), RHD(R234W) were new RHD alleles. Twenty-eight CcDee, 28 ccDEe, and 22 ccDee samples had increased antigen densities; 53 of them lacked a hybrid Rhesus box and were thus predicted to be RHD homozygous. Eight ccDee samples were predicted to be heterozygous despite a large relative dose of RHD to RHCE alleles in quantitative polymerase chain reaction. One of these samples was further investigated and carried an RHD-CE hybrid transcript characteristic for a -D- haplotype. CONCLUSIONS: Unusual little and large RhD protein integration into the membrane could be traced to a host of distinct protein variants. Weak expression of D antigen was invariably associated with variant RHD alleles. Larger than normal D antigen density may often be caused by the presence of two D encoding alleles, which may be located in cis, and confounding zygosity testing that is solely based on gene copy number.

Alleles↗
PubMed2006

An easy RHD genotyping strategy for D- East Asian persons applied to Korean blood donors.

BACKGROUND: In East Asian populations RHD alleles are known to occur frequently among D- donors, requiring suitable genotyping strategies. The molecular basis of the "RHD(el)" allele previously reported in Taiwan to harbor a genomic 1013-bp deletion was questioned by several authors. STUDY DESIGN AND METHODS: The presence of the RHD gene was investigated in 126 random serologic D- blood donors from Gwangju, southwest Korea. Four donors who typed weakly positive for the D antigen were also analyzed. RH alleles were determined by polymerase chain reaction (PCR) with sequence-specific priming (PCR-SSP) or nucleotide sequencing. RESULTS: Seventy-five percent of the serologically D- samples lacked the RHD gene, 10 percent carried the hybrid RHD-CE(2-9)-D2 or RHD-CE(2-7)-D2 alleles, 13 percent represented the RHD(K409K), and 2 percent were weak D type 15 and type 17. Among the four donors typing weak D, two carried weak D type 15, one RHD(K409K), and one the novel weak D type 43. Critical molecular characteristics of RHD(K409K) and its population frequencies were indistinguishable to those reported for the RHDel allele. CONCLUSION: Korean RHD allele frequencies are comparable to Chinese and Japanese frequencies. It is concluded that the RHDel allele may actually not exist but is identical to RHD(K409K). A practical RHD genotyping strategy applicable to D- donors in all East Asian populations was devised. The strategy requires four PCR-SSP procedures only for RHD intron 4 and exon 7 as well as RHD(K409K) and non-RHD(K409K).

Base Sequence↗
PubMed2006

In-frame triplet deletions in RHD alter the D antigen phenotype.

BACKGROUND: The deletion of three adjacent nucleotides in an exon may cause the lack of a single amino acid, while the protein sequence remains otherwise unchanged. Only one such in-frame deletion is known in the two RH genes, represented by the RHCE allele ceBP expressing a "very weak e antigen." STUDY DESIGN AND METHODS: Blood donor samples were recognized because of discrepant results of D phenotyping. Six samples came from Switzerland and one from Northern Germany. The molecular structures were determined by genomic DNA nucleotide sequencing of RHD. RESULTS: Two different variant D antigens were explained by RHD alleles harboring one in-frame triplet deletion each. Both single-amino-acid deletions led to partial D phenotypes with weak D antigen expression. Because of their D category V-like phenotypes, the RHD(Arg229del) allele was dubbed DVL-1 and the RHD(Lys235del) allele DVL-2. These in-frame triplet deletions are located in GAGAA or GAAGA repeats of the RHD exon 5. CONCLUSION: Partial D may be caused by a single-amino-acid deletion in RhD. The altered RhD protein segments in DVL types are adjacent to the extracellular loop 4, which constitutes one of the most immunogenic parts of the D antigen. These RhD protein segments are also altered in all DV, which may explain the similarity in phenotype. At the nucleotide level, the triplet deletions may have resulted from replication slippage. A total of nine amino acid positions in an Rhesus protein may be affected by this mechanism.

Alleles↗
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↗