Compound heterozygosity induces a rare Gerbich-negative phenotype in an immunized blood donor.

BACKGROUND: Antibodies to Gerbich blood group antigens are exceedingly rare and can cause moderate transfusion reactions. Several deletional variants of the GE-gene, that harbors long sequence repeats, enable alloimmunization and formation of naturally occurring antibodies. SUBJECT AND METHODS: A female blood donor and soldier of the German Army without history of pregnancy or transfusion showed an antibody reactive with all test cells except for GE:-2-3 RBC. Thus, anti-Ge2 was suspected. Molecular analysis including fragment length specific PCR, Sanger sequencing and NGS should reveal the molecular background of the deficiency. Segregation of the variant alleles should be demonstrated by family analysis. RESULTS: Compound heterozygosity for GYPC exon 2 (GE*01.-02) and exon 3 (GE*01.-03) deletion was detected in the donor and her sister. The mother had one exon 3 amplicon of reduced length, while the father heterozygously exhibited a truncated GYPC exon 2. NGS clearly demonstrated reduced coverages within the deletional fragments within each family member. The donor and her sister showed the complete absence of a 640 bp fragment. DISCUSSION AND CONCLUSION: Rare GE deletion variants can induce naturally occurring anti-Ge2 in Caucasians. Because of an enhanced risk of injury as soldier autologous RBC of the donor were cryopreserved. The donor and her sibling can give blood for each other because of identical ABO, Rh, and K antigen blood types.

Characterization of the novel HLA-DPB1*1219:01 allele by next-generation sequencing.

The new allele differs from DPB1*296:01 by a c.292G>A substitution in exon 2.

Platelet CD36 deficiency is present in 2.6% of Arabian individuals and can cause NAIT and platelet refractoriness.

BACKGROUND: CD36 isoantibodies are capable of inducing neonatal alloimmune thrombocytopenia (NAIT) and platelet refractoriness. As to now the CD36 type I deficiency has been reported in East Asian and African individuals. However, it is virtually unknown in Caucasians. The aim of this study was to display the prevalence of the CD36 deficiency within parts of the Arabian population in Germany. Secondly, we are presenting the case of a newborn suffering from NAIT which was induced by CD36 antibody. METHODS: Platelet (p) CD36 was determined by flow cytometry on 1328 samples mainly from individuals of Arabian origin and a family with a neonate affected by NAIT. DNA sequencing was performed on all pCD36-negative samples. RESULTS: Thirty-five (2.64%) of all donor samples were pCD36 negative, 19 (1.43%) had a weak expression. Including only individuals from the Arabian peninsula, frequencies were 3.39% and 1.75%, respectively. CD36 type I deficiency on both platelets and monocytes combined with a CD36 isoantibody were detected in the mother of the NAIT baby. The baby was successfully transfused with two HPA-unselected platelet concentrates. In case of need, two platelet units with a weak pCD36 expression were on hand. A total of 45 different CD36 mutations were detected within pCD36-negative individuals, some being homozygous, most of them only present on one allele. CONCLUSION: The CD36-negative phenotype is present in a significant number of individuals of Arabian origin and enables CD36 isoimmunization in NAIT or refractoriness. Blood transfusion services should be aware of such cases.

Multicenter Study on Differential Human Neutrophil Antigen 2 Expression and Underlying Molecular Mechanisms.

BACKGROUND: The human neutrophil antigen 2 (HNA-2), which is expressed on CD177, is undetectable in 3-5% of the normal population. Exposure of these HNA-2null individuals to HNA-2-positive cells can cause immunization and pro-duction of HNA-2 antibodies, which can induce immune neutropenia and transfusion-related acute lung injury. In HNA-2-positive individuals, neutrophils are divided into a CD177pos. and a CD177neg. subpopulation. The molecular background of HNA-2 deficiency and the bimodal expression pattern, however, are not completely decoded. STUDY DESIGN: An international collaboration was conducted on the genetic analysis of HNA-2-phenotyped blood samples, including HNA-2-deficient individuals, mothers, and the respective children with neonatal immune neutropenia and regular blood donors. RESULTS: From a total of 54 HNA-2null individuals, 43 were homozygous for the CD177 *787A>T substitution. Six carried the CD177 *c.1291G>A single nucleotide polymorphism. All HNA-2-positive samples with >40% CD177pos. neutrophils carried the *787A wild-type allele, whereas a lower rate of CD177pos. neutrophils was preferentially associated with *c.787AT heterozygosity. Interestingly, only the *c.787A allele sequence was detected in complementary DNA (cDNA) sequence analysis carried out on all *c.787AT heterozygous individuals. However, cDNA analysis after sorting of CD177pos. and CD177neg. neutrophil subsets from HNA-2-positive individuals showed identical sequences, which makes regulatory elements within the promoter unlikely to affect CD177 gene transcription in different CD177 neutrophil subsets. CONCLUSION: This comprehensive study clearly demonstrates the impact of single nucleotide polymorphisms on the expression of HNA-2 on the neutrophil surface but challenges the hypothesis of regulatory epigenetic effects being implicated in the bimodal CD177 expression pattern.

Amino Acid Polymorphisms in Hla Class II Differentiate Between Thyroid and Polyglandular Autoimmunity.

CONTEXT: The structure of the human leucocyte antigen (HLA) peptide-binding clefts strongly contributes to monoglandular and polyglandular autoimmunity (AP). OBJECTIVE: To investigate the impact of amino acid polymorphisms on the peptide-binding interactions within HLA class II and its association with AP. DESIGN: Immunogenetic study. SETTING: Tertiary referral center for autoimmune endocrine diseases. SUBJECTS: 587 subjects with AP, autoimmune thyroid disease (AITD), type 1 diabetes (T1D), and healthy unrelated controls were typed for HLA class II. METHODS: Amino acids within the peptide binding cleft that are encoded by HLA class II exon 2 were listed for all codon positions in all subjects. Overall comparisons between disease and control groups with respect to allele distribution at a given locus were performed by assembling rare alleles applying an exact Freeman Halton contingency table test with Monte-Carlo P values based on 150 000 samples. RESULTS: The Monte Carlo exact Fisher test demonstrated marked differences in all 3 loci, DQA1, DQB1, and DRB1 (P < .0001) between AP and both AITD and controls, as well as between AP type II (Addison's disease as a major endocrine component) and AP type III (T1D + AITD). Differences were also noted between AP and T1D pertaining to the DRB1 allele (P < .041). Seven amino acid positions, DRB1-13, DRB1-26, DRB1-71, DRB1-74, DQA1-47, DQA1-56, and DQB1-57, significantly contributed to AP. Five positions in DQA1 (11, 47, 50, 56, and 69) completely correlated (P < .0001). CONCLUSION: Amino acid polymorphisms within HLA class II exon 2 mediate the AP risk and differentiate between thyroid and polyglandular autoimmunity.

Sex Alters the MHC Class I HLA-A Association With Polyglandular Autoimmunity.

CONTEXT: The major histocompatibility complex (MHC) strongly contributes to the development of polyglandular autoimmunity (PGA). OBJECTIVE: To evaluate the impact of sex on human leukocyte antigen (HLA) association with PGA for the first time. DESIGN: Cross-sectional immunogenetic study. SETTING: Academic tertiary referral Orphan Disease Center for PGA (ORPHA 282196) and immunogenetics laboratory. SUBJECTS: Patients (158) with coexistent type 1 diabetes and autoimmune thyroid disease (adult type 3 PGA, ORPHA 227982) and 479 unrelated healthy controls. INTERVENTIONS: All 637 white subjects were typed for HLA-A, -B, -DRB1, -DQA1, and -DQB1 alleles at a two-field level. MAIN OUTCOME MEASURES: Modification of the gene-disease association by sex. RESULTS: MHC class I HLA-A association was sex related to both the total white adult type 3 PGA collective (n = 158, P = 0.0065), as well as in PGA patients with autoimmune Hashimoto thyroiditis (n = 91, P = 0.010). Compared with HLA-A*02:01, A*11:01 was over-represented in male patients, yet under-represented in women (OR 1.49, 95% CI 0.55 to 3.88 vs 0.42, 0.12 to 1.17). A*24:02 was under-represented in male but not in female patients (OR 0.37, 95% CI 0.11 to 1.04 vs 1.19, 0.65 to 2.15). With the exclusion of the five most frequent alleles (A*01:01, A*02:01, A*03:01, A*11:01, and A*24:02), the sum of all other identified alleles was under-represented in male patients (OR 0.37, 0.18 to 0.72, P = 0.0046). The strong MHC HLA-B association with PGA (P < 0.0001) was not sex related (P = 0.55). Furthermore, no interaction with sex was observed for the MHC class II HLA-DRB1, -DQA1, and -DQB1 alleles. CONCLUSION: MHC class I HLA-A association with type 3 PGA is significantly affected by sex.

The AQP1 mutation c.601delG causes the Co-negative phenotype in four patients belonging to the Romani (Gypsy) ethnic group.

BACKGROUND: The Colton blood group antigens Co(a), Co(b) and Co3 are encoded by the AQP1 gene which produces a water channel forming integral protein. The extremely rare Co-deficiency enables immunisation against the Co3 isoantigen. MATERIALS AND METHODS: Four patients from different regions of Europe who belong to the ethnic minority of Romani (Gypsy) presented with irregular antibodies against a high frequency red blood cell antigen. Positive cross-matches with all red blood cells tested were reported. An Anti-Co3 antibody was identified as the cause of incompatibility in the four cases. The genetic background was determined by polymerase chain reaction typing with sequence-specific primers and by DNA sequencing. RESULTS: The Co(a-b-) phenotype was confirmed in the four patients despite the fact that genotyping revealed the CO*01 allele of the AQP1 gene. A homozygous AQP1 c.601delG mutation, leading to a frame shift and producing a premature stop in the next codon, was responsible for the Co-negative phenotype in all four cases. While one patient was successfully transfused with blood from his sibling with the identical mutation, another case, a baby affected by haemolytic disease of the newborn, recovered without transfusion. DISCUSSION: Despite the difficulties in undertaking a population study to determine the prevalence of this AQP1 c.601delG allele in the ethnic minority of Romani, the observations described in this report clearly suggest an accumulation of this mutation, which causes the Co(a-b-) phenotype, in Romani (Gypsy) patients. Further studies are necessary to prove such an accumulation.

Expression of the CTL2 transcript variants in human peripheral blood cells and human tissues.

BACKGROUND: The HNA-3a antigen is an important antibody target in the pathophysiology of transfusion-related acute lung injury (TRALI). It is encoded by the choline transporter-like protein 2 (CTL2) gene, which exists in the two transcript variants TV1 and TV2, differing in the upstream promoter and coding region. Only TV1 has been demonstrated to enable choline transport across the cell membrane. STUDY DESIGN AND METHODS: The aim of this study was to determine the CTL2 transcript pattern in human peripheral blood cells and tissues and its capacity to bind HNA-3a antibodies. RNA was isolated from human whole blood, isolated neutrophils, mononuclear blood cells, leukoreduced platelets, human lung, liver, and colon. After reverse transcription, the single-stranded cDNA was amplified using primer combinations specific for the respective transcript. Plasmids containing the entire CTL2 coding cDNA of the transcript variant TV1 or TV2 served as controls. HEK293T cells expressing both variants were used to determine the binding of HNA-3a antibodies. RESULTS: The shorter TV2 transcript was demonstrated in each RNA sample derived from human peripheral blood tested so far, as well as in human lung and liver, whereas the longer TV1 transcript was only detected in human lung and colon. TV1 and TV2 had the same binding capacity to HNA-3a antibodies. CONCLUSION: The expression of TV1 and TV2 is tissue and cell specific, with peripheral blood cells expressing only TV2. This does not affect binding of HNA-3a antibodies. Whether the unequal expression might be relevant in the pathogenesis of TRALI remains to be investigated.

HNA-1d: a new human neutrophil antigen located on Fcγ receptor IIIb associated with neonatal immune neutropenia.

BACKGROUND: Neonatal immune neutropenia (NIN) is a rare, but potentially life-threatening, disorder caused by maternal alloantibodies recognizing paternal neutrophil antigens on fetal cells. Alloantibodies directed against the human neutrophil alloantigen system (HNA)-1 located on Fcγ receptor IIIb (FcγRIIIb) are most frequently implicated in NIN. In this report, we describe two cases of NIN with alloantibodies against FcγRIIIb, which did not match one of the known HNA-1a, -1b, or -1c specificities, but define a new antigen, HNA-1d. STUDY DESIGN AND METHODS: Neutrophil-reactive antibodies were detected by agglutination, microscopic immunofluorescence, and monoclonal antibody (MoAb)-specific immobilization of neutrophil antigens (MAIGA) assay. For epitope mapping of FcγRIIIb-reactive antibodies, recombinant chimeric variants of FcγRIIIb were used in the MAIGA assay. Genotyping of FCGR3B was performed by allele-specific polymerase chain reaction. RESULTS: Both mothers were typed FCGR3B*01+, *02-, *03+. Antibody screening revealed the presence of alloantibodies reactive with FcγRIIIb encoded by FCGR3B*02, but not with FcγRIIIb encoded by FCGR3B*03. MAIGA with recombinant, partly chimeric FcγRIIIb variants demonstrated that the antigen recognized by maternal antibodies is characterized by two amino acids, Ala78 and Asp82. Among the FCGR3B alleles, the sequence Ala78---Asn82 is exclusively encoded by FCGR3B *02. CONCLUSION: A previously unrecognized second antigen, HNA-1d, is present on FcγRIIIb encoded by FCGR3B*02. This antigen is characterized by the sequence Ala78---Asn82. It appears that only individuals carrying the HNA-1c phenotype can form anti-HNA-1d alloantibodies. The HNA-1 system now consists of four antigens encoded by three alleles.

Genetic variation of the HNA-3a encoding gene.

BACKGROUND: Antibodies against the human neutrophil alloantigen-3a (HNA-3a) play an important role in transfusion-related acute lung injury. The HNA-3a and -3b alloantigens result from a single-nucleotide exchange in the choline transporter-like protein 2 gene (CTL2). We sought for additional polymorphisms that might impair antibody binding to or genotyping of the HNA-3a or -3b antigens. STUDY DESIGN AND METHODS: CTL2-specific complementary DNA (cDNA) fragments were generated from 67 unrelated blood donors followed by DNA sequencing. Polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to test a higher number of donors for relevant new single-nucleotide polymorphisms (SNPs). The granulocyte agglutination test recommended for HNA-3a antibody detection was performed to check HNA-3a antibody binding to the products of the CTL-2 gene variants. RESULTS: Two new missense mutations were demonstrated in the CTL2 cDNA: a 537C>T* exchange leading to a Leu153Phe amino acid substitution and 988C>T variation predicting Thr301Met change. The inherited 537T variant is located in HNA-3a allele results impaired granulocyte agglutination by four of 14 antibodies tested while 988T remains nearly unaffected. CONCLUSIONS: The Leu153Phe exchange next to the HNA-3a/b defining amino acid position can impede the binding of HNA-3a alloantibodies. The HNA-3a genotyping by PCR-SSP might produce misleading results in HNA-3ab heterozygous individuals with the additional CTL2-537T variation of the HNA-3a antigen. These findings must account for the development of new screening assays.

Paired crossover study of two plateletpheresis systems concerning platelet product quality and donor comfort.

BACKGROUND: Two plateletpheresis cell separator systems were compared in a paired crossover study with respect to the product quality, the number of platelet (PLT) units per donation, and the donor comfort. STUDY DESIGN AND METHODS: Forty-four female and 47 male donors were distributed to three body weight groups. Double PLT units with 6 x 10(11) PLTs were collected from three Fenwal Amicus Crescendo (AC) and three CaridianBCT Trima Accel (TA) machines. Each donor made one donation on each randomly assigned system and answered a questionnaire on the subjective donor comfort. The answers were scored from 5 (best) to 1 (worst). RESULTS: Based on 182 donations, with 91 donations on AC and TA separators each, 179 runs resulted in double PLT units and three (2xAC, 1xTA) in single units. The white blood cell counts were below 1 x 10(6) in all but eight therapeutic units (8xTA; mean, 1.98 x 10(6)). The mean PLT yield (AC 6.00 x 10(11), TA 5.98 x 10(11)), the collection rate, and the PLT extraction coefficient did not significantly differ between the two devices. Differences of the donor comfort over all groups were only observed for the loudness of the instrument (4.63 AC vs. 4.24 TA, p < 0.001) and the subjective impression of the run time (4.24 AC vs. 4.48 TA, p < 0.05). Male donors greater than 88 kg preferred the TA instruments concerning the impact of the needle, run time, overall experience (p < 0.01 each), and willingness to donate on the same instrument again (p < 0.05). CONCLUSIONS: Only minor differences were observed despite the fact that the AC separators are run with two needles and the TA with one needle.

Variant FCGR3 genes: transcription and possible origins.

The Fc receptors for human immunoglobulin G (FcgammaR) IIIb are encoded by genes clustered on the long arm of chromosome 1 (band q21 --> 24) and exhibit allelic polymorphisms. Several rare FCGR3B sequences were identified in both white and black donors. However, the origins of these genomic variants are unknown and their transcription has not yet been investigated. Blood from a donor with known FCGR3 variants was used to extract DNA from peripheral blood CD34+ cells, CD19+ B-cells, neutrophils and buccal cells, after which FCGR3 gene sequencing was performed. Additionally, RNA samples from 5 Caucasian individuals containing known variant FCGR3 genes were reverse-transcribed to cDNA and the FCGR3 genes were sequenced. Our results showed that the frequencies of variant clones were higher in B-cell preparations than in CD34+ hematopoietic progenitor cells from peripheral blood and neutrophils. Very high variant frequencies were found in buccal cell-derived clones. Variant cDNA sequences were identified in three of five individuals with known FCGR3 variants. We conclude that FCGR3 gene variants are differentially transcribed between cell types and tissues, increasing the likelihood of the presence of variant FcgammaRIII receptors on the cell surface. The significance of the high number of variant clones in buccal cells, however, is unclear.

FCGR3B gene frequencies and FCGR3 variants in a Chinese population from Zhejiang Province.

The human neutrophil antigens HNA-1a, -1b and -1c play an important role in immune neutropenia. The frequencies of the coding FCGR3B genes were determined in different populations. New FCGR3B variants were also found in some populations. This study investigated the FCGR3B gene frequencies and FCGR3 variants in a Chinese population compared with the results of Northern Germans and African Blacks (Uganda). Our results show that the gene frequencies in 413 healthy Chinese individuals from Zhejiang Province were 0.565 for FCGR3B*1, 0.430 for FCGR3B*2 and 0.00 for FCGR3B*3. The genotype frequency of FCGR3B(null) was 0.48% (2/413). Sequencing of FCGR3 revealed that in seven out of 19 Chinese individuals, cloned and sequenced DNA fragments that exhibited variants caused by single nucleotide exchanges at one or more of the polymorphic positions 141, 147, 227, 266 and 277 in exon 3 also existed in this Chinese population. From the present study, it is concluded that the FCGR3B*1 gene is more frequent in a Chinese population from Zhejiang Province than the FCGR3B*2 gene, and the FCGR3B*3 gene seems to be absent, which is in contrast to studies in the white populations. Gene variants caused by single nucleotide exchanges were found in addition to the well-known forms, but the reason for this remains unclear.

FCGR3 variants and expression of human neutrophil antigen-1a, -1b, and -1c in the populations of northern Germany and Uganda.

BACKGROUND: Human neutrophil antigen-1c (HNA-1c) (SH) has been described as the third alloantigen of the Fc receptor type IIIb (FcgammaRIIIb) for IgG beside the known alloantigens HNA-1a (NA1) and HNA-1b (NA2). Controversy exists on the assignment of the HNA-1c coding gene to the FCGR3B locus and on a possible linkage between the HNA-1c and HNA-1a coding genes. STUDY DESIGN AND METHODS: Two hundred sixty northern German blood donors and 43 individuals from Uganda were typed for FCGR3B*1 (NA1), FCGR3B*2 (NA2), and FCGR3B*3 (SH) by allele-specific PCR. In a subset of FCGR3B*3-positive probands, PCR-amplified FCGR3 fragments were subcloned and sequenced. Transmission of FCGR3B*3 was analyzed in family studies. A possible correlation with the FcgammaRIIIb alloantigen expression was investigated by flow cytometry. RESULTS: In the northern German population, FCGR3B*3 was found exclusively in individuals carrying FCGR3B*1 independent of the existence of FCGR3B*2 at a frequency of 5 percent. In the individuals from Uganda, each possible combination of FCGR3B*1, FCGR3B*2, and FCGR3B*3 was detected. FCGR3B*3 frequency was 34.9 percent. Within both populations, some individuals carried each of the three genotypes. DNA sequencing revealed new FCGR3 variants caused by single nucleotide exchanges at the typical polymorphic positions. In one individual, six different FCGR3 variants were detected. CONCLUSION: The coincidence of the three known FCGR3B alleles varies within the population of Germany and Uganda. Three simultaneous FCGR3B forms may be explained by two gene loci, but the basis of the high number of different variants in some individuals still remains unclear. Possible explanations may be a hypermutation mechanism or a number of FCGR3 higher than expected hitherto.

Transfusion-related acute lung injury caused by human leucocyte antigen class II antibody.

Fcgamma receptor (FcgammaR)-mediated destruction of immunoglobulin-coated red blood cells (RBCs) is the underlying mechanism of haemolytic disease of the newborn. Human leucocyte antigen (HLA) antibodies in vitro are able to block monocyte FcgammaRs and prevent phagocytosis. The intention was to demonstrate this effect in vivo upon a volunteer. Plasma containing a non-cytotoxic HLA-DR alloantibody was infused into the subject. The FcgammaR blockade was achieved and persisted for about 2.5 d, but, unexpectedly, a mild transfusion-related acute lung injury (TRALI) was also caused. Monocytes disappeared completely from the peripheral blood within the first hour after infusion and a mild pulmonary oedema was observed within 3-4 h. The subject recovered within 2 d.