Recessive Variants in PIGG Cause a Motor Neuropathy with Variable Conduction Block, Childhood Tremor, and Febrile Seizures: Expanding the Phenotype.

Biallelic variants in phosphatidylinositol glycan anchor biosynthesis, class G (PIGG) cause hypotonia, intellectual disability, seizures, and cerebellar features. We present 8 patients from 6 families with a childhood-onset motor neuropathy and neurophysiology demonstrating variable motor conduction block and temporal dispersion. All individuals had a childhood onset tremor, 5 of 8 had cerebellar involvement, and 6 of 8 had childhood febrile seizures. All individuals have biallelic PIGG variants, including the previously reported pathogenic variant Trp505*, plus 6 novel variants. Null enzyme activity is demonstrated via PIGO/PIGG double knockout system for Val339Gly and Gly19Glu, and residual activity for Trp505* due to read-through. Emm negative blood group status was confirmed in 1 family. PIGG should be considered in unsolved motor neuropathy. ANN NEUROL 2024.

Disease severity in subsequent pregnancies with RhD immunization: A nationwide cohort.

BACKGROUND AND OBJECTIVES: To evaluate the severity of haemolytic disease of the foetus and newborn (HDFN) in subsequent pregnancies with RhD immunization and to identify predictive factors for severe disease. MATERIALS AND METHODS: Nationwide prospective cohort study, including all pregnant women with RhD antibodies. All women with at least two pregnancies with RhD antibodies and RhD-positive foetuses were selected. The main outcome measure was the severity of HDFN in the first and subsequent pregnancy at risk. A subgroup analysis was performed for the group of women where RhD antibodies developed after giving birth to an RhD-positive child and thus after receiving anti-D at least twice (group A) or during the first pregnancy at risk for immunization (group B). RESULTS: Sixty-two RhD immunized women with a total of 150 RhD-positive children were included. The severity of HDFN increased for the whole group significantly in the subsequent pregnancy (p < 0.001), although it remained equal or even decreased in 44% of women. When antibodies were already detected at first trimester screening in the first immunized pregnancy, after giving birth to an RhD-positive child (group A), severe HDFN in the next pregnancy was uncommon (22%). Especially when no therapy or only non-intensive phototherapy was indicated during the first immunized pregnancy (6%) or if the antibody-dependent cell-mediated cytotoxicity result remained <10%. Contrarily, women with a negative first trimester screening and RhD antibodies detected later during the first pregnancy of an RhD-positive child (group B), often before they had ever received anti-D prophylaxis, were most prone for severe disease in a subsequent pregnancy (48%). CONCLUSION: RhD-mediated HDFN in a subsequent pregnancy is generally more severe than in the first pregnancy at risk and can be estimated using moment of antibody detection and severity in the first immunized pregnancy. Women developing antibodies in their first pregnancy of an RhD-positive child are at highest risk of severe disease in the next pregnancy.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Glycine 236 in the Lower Hinge Region of Human IgG1 Differentiates FcγR from Complement Effector Function.

Abs of the IgG isotype mediate effector functions like Ab-dependent cellular cytotoxicity and Ab-dependent cellular phagocytosis by Fc interactions with FcγRs and complement-dependent cytotoxicity upon IgG-Fc binding to C1q. In this study, we describe the crucial role of the highly conserved dual glycines at position 236-237 in the lower hinge region of human IgG, including the lack of one glycine as found in IgG2. We found several permutations in this region that either silence or largely abrogate FcγR binding and downstream FcγR effector functions, as demonstrated by surface plasmon resonance, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity assays. Although the binding regions of FcγRs and C1q on the IgG-Fc largely overlap, IgG1 with a deletion of G236 only silences FcγR-mediated effector functions without affecting C1q-binding or activation. Several mutations resulted in only residual FcγRI binding with differing affinities that are either complement competent or silenced. Interestingly, we also found that IgG2, naturally only binding FcγRIIa, gains binding to FcγRI and FcγRIIIa after insertion of G236, highlighting the crucial importance of G236 in IgG for FcγR interaction. These mutants may become invaluable tools for FcγR-related research as well as for therapeutic purposes in which only complement-mediated functions are required without the involvement of FcγR.

Reduction of anti-K-mediated hemolytic disease of newborns after the introduction of a matched transfusion policy: A nation-wide policy change evaluation study in the Netherlands.

BACKGROUND: During pregnancy, maternal red blood cell (RBC) antibodies can lead to life-threatening fetal hemolysis and anemia. Women can become immunized by a pregnancy or an unmatched transfusion. Our aim was to quantify the effect of a nationwide K-matched transfusion policy for women of childbearing age potential to prevent K-immunization in pregnancy. STUDY DESIGN AND METHODS: In this nation-wide policy change evaluation study we determined the occurrence of RBC antibodies before and after introduction of a K-matched transfusion policy and evaluated the cause K alloimmunization 10 years after introduction of this measure. K-matched transfusion for females under 45 years of age is advised in the Dutch transfusion guideline since 2004. We used laboratory data from pregnancies with RBC antibodies identified in the period 1999-2018 obtained as part of a population-based screening program in the Netherlands. RESULTS: Tests of 36 286 pregnancies produced a positive antibody screening result which concerned anti-K in 1550 pregnancies. The occurrence of anti-K decreased from 67.9 to 20.2 per 100 000 pregnancies. The relative risk reduction was 0.70 which largely exceeded the relative risk reduction of 0.27 for antibodies against RBC antigens for which no preventive matching is required. The number of pregnancies at risk for anti-K-mediated disease decreased from 9.7 to 4.2 per 100 000 pregnancies. CONCLUSIONS: A K-matched transfusion policy is associated with a major decrease in a number of pregnant women with anti-K and pregnancies at risk for anti-K-mediated disease. A relatively simple measure is now shown to impact prevention of hemolytic disease in the fetus and newborn.

Functional Attributes of Antibodies, Effector Cells, and Target Cells Affecting NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity.

Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell-cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell-mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell-mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa-Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.

Multiplex blood group typing by cellular surface plasmon resonance imaging.

BACKGROUND: Blood-group typing of donors and patients is essential to avoid incompatible transfusions. Transfusion of incompatible RBCs may result in alloimmunization complicating future transfusions or in the presence of antibodies in adverse reactions. With more than 300 blood group antigens identified, it is difficult to provide fully compatible blood. Currently, standard practice is to match for the most immunogenic antigens. While the current agglutination-based RBC-typing methods are reliable for testing a selected number of antigens, they are not easily adaptable for high-throughput multiplex blood typing beyond the current standard. STUDY DESIGN AND METHODS: Surface plasmon resonance (SPR) is a label-free method to follow molecular-and, very recently, also cellular-interactions in real time. Demonstration of binding of RBCs to blood group antigen-specific antibodies by SPR has already been achieved. Here, we demonstrate the generation of an SPR array equipped with clinically relevant blood group antibodies (A, B, and Rh blood groups). To validate this method, we blindly compared typing of 946 blood donors with results of current diagnostic agglutination-based methods. RESULTS: RBC typing was achieved by monitoring RBC binding to blood group-specific antibodies on the sensor simultaneously within 5 minutes per sample. Regeneration of the chip was robust, allowing for typing of at least 100 samples. The typing results gave a 100% match with classical serology with all antibodies tested besides anti-E/e monoclonals, which gave inconsistent results due to low antibody specificity. CONCLUSION: This study demonstrates that SPR-based RBC typing for multiple antigens can be realized simultaneously with high-quality antibodies, enabling reduced hands-on time and possibly improving cost efficiency.

Development of a recombinant anti-Vel immunoglobulin M to identify Vel-negative donors.

BACKGROUND: Alloimmunization against the high-frequency Vel blood group antigen may result in transfusion reactions or hemolytic disease of fetus and newborn. Patients with anti-Vel alloantibodies require Vel-negative blood but Vel-negative individuals are rare (1:4000). Identification of Vel-negative donors ensures availability of Vel-negative blood; however, accurate Vel blood group typing is difficult due to variable Vel antigen expression and limited availability of anti-Vel typing sera. We report the production of a recombinant anti-Vel that also identifies weak Vel expression. STUDY DESIGN AND METHODS: A recombinant anti-Vel monoclonal antibody was produced by cloning the variable regions from an anti-Vel-specific B cell isolated from an alloimmunized patient into a vector harboring the constant regions of immunoglobulin (Ig)G1-kappa or IgM-kappa. Antibody Vel specificity was tested by reactivity to SMIM1-transfected HEK293T cells and by testing various red blood cells (RBCs) of donors with normal, weak, or no Vel expression. High-throughput donor screening applicability was tested using an automated blood group analyzer. RESULTS: A Vel-specific IgM class antibody was produced. The antibody was able to distinguish between Vel-negative and very weak Vel antigen-expressing RBCs by direct agglutination and in high-throughput settings using a fully automated blood group analyzer and performed better than currently used human anti-Vel sera. High-throughput screening of 13,288 blood donations identified three new Vel-negative donors. CONCLUSION: We generated a directly agglutinating recombinant anti-Vel IgM, M3F5S-IgM, functional in manual, automated agglutination assays and flow cytometry settings. This IgM anti-Vel will improve diagnostics by facilitating the identification of Vel-negative blood donors.

Acute hemolytic transfusion reaction due to a warm reactive anti-A1.

BACKGROUND: Anti-A1 are regularly observed by reverse testing and are generally considered clinically irrelevant. For compatibility testing and the selection of blood, we use the type-and-screen (T&S) strategy, in which ABO confirmation of patients with a definitive blood group is performed by forward grouping only. Because anti-A1 seem clinically irrelevant, it is our policy to provide group A blood in patients with an anti-A1 . STUDY DESIGN AND METHODS: This is a case report of a 96-year-old woman who died shortly after transfusion of blood group A red blood cells (RBCs). She was known to have blood group A2 with an anti-A1 and the absence of other RBC antibodies. Directly after starting transfusion, acute dyspnea was observed, while other clinical signs for a transfusion reaction were absent. In the laboratory, indications for a severe hemolytic transfusion reaction (HTR) triggered serologic investigations and complement deposition experiments. RESULTS: Analyses revealed that the anti-A1 was present as a high-titer IgM class immunoglobulin that induced complement deposition on A1 RBCs. The anti-A1 reacted in a wide temperature amplitude up to 37°C with A1 RBCs, while weak agglutination was observed with A2 RBCs at room temperature. CONCLUSION: A pretransfusion detectable anti-A1 caused a severe HTR that, in view of the rapid onset of clinical symptoms and concomitant deterioration, contributed to the death of the patient. Considering its clinical significance in this case, we encourage an unambiguous procedure for patients with an anti-A1 , especially when T&S is used for donor RBC selection.

Antigen specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn.

Haemolytic disease of the fetus and newborn (HDFN) is a severe disease in which fetal red blood cells (RBC) are destroyed by maternal anti-RBC IgG alloantibodies. HDFN is most often caused by anti-D but may also occur due to anti-K, -c- or -E. We recently found N-linked glycosylation of anti-D to be skewed towards low fucosylation, thereby increasing the affinity to IgG-Fc receptor IIIa and IIIb, which correlated with HDFN disease severity. Here, we analysed 230 pregnant women with anti-c, -E or -K alloantibodies from a prospective screening cohort and investigated the type of Fc-tail glycosylation of these antibodies in relation to the trigger of immunisation and pregnancy outcome. Anti-c, -E and -K show - independent of the event that had led to immunisation - a different kind of Fc-glycosylation compared to that of the total IgG fraction, but with less pronounced differences compared to anti-D. High Fc-galactosylation and sialylation of anti-c correlated with HDFN disease severity, while low anti-K Fc-fucosylation correlated with severe fetal anaemia. IgG-Fc glycosylation of anti-RBC antibodies is shaped depending on the antigen. These features influence their clinical potency and may therefore be used to predict severity and identify those needing treatment.

Frequency and characterization of known and novel RHD variant alleles in 37 782 Dutch D-negative pregnant women.

To guide anti-D prophylaxis, Dutch D- pregnant women are offered a quantitative fetal-RHD-genotyping assay to determine the RHD status of their fetus. This allowed us to determine the frequency of different maternal RHD variants in 37 782 serologically D- pregnant women. A variant allele is present in at least 0·96% of Dutch D- pregnant women The D- serology could be confirmed after further serological testing in only 54% of these women, which emphasizes the potential relevance of genotyping of blood donors. 43 different RHD variant alleles were detected, including 15 novel alleles (11 null-, 2 partial D- and 2 DEL-alleles). Of those novel null alleles, one allele contained a single missense mutation (RHD*443C>G) and one allele had a single amino acid deletion (RHD*424_426del). The D- phenotype was confirmed by transduction of human D- erythroblasts, consolidating that, for the first time, a single amino acid change or deletion causes the D- phenotype. Transduction also confirmed the phenotypes for the two new variant DEL-alleles (RHD*721A>C and RHD*884T>C) and the novel partial RHD*492C>A allele. Notably, in three additional cases the DEL phenotype was observed but sequencing of the coding sequence, flanking introns and promoter region revealed an apparently wild-type RHD allele without mutations.

Fetal RHD genotyping after bone marrow transplantation.

BACKGROUND: Fetal RHD genotyping allows targeted diagnostic testing, fetal surveillance, and eventually intrauterine treatment to D-alloimmunized pregnant women who carry an RHD+ fetus. However, false-positive and false-negative results of noninvasive prenatal fetal RHD genotyping have been described due to a variety of causes. In this case report we present two cases where noninvasive fetal RHD typing was complicated by a previous bone marrow transplantation (BMT). CASE REPORT: We describe two women with a history of allogeneic BMT in early childhood. Both were born D+ and received a transplant of their D- male sibling. Anti-D were detected during pregnancy in one of them. The biologic father of this pregnancy was D+. In both cases polymerase chain reaction procedures specific for RHD on maternal plasma DNA were positive whereas a D- neonate was born in one case (Case 1). CONCLUSION: False-positive results of noninvasive fetal RHD genotyping occur in D+ women transplanted with marrow of a D- donor, due to circulating cell-free DNA originating from nonhematopoietic tissue. The cases highlight that health care professionals and laboratories should be aware that allogeneic BMT can be a cause for false-positive results in fetal RHD genotyping with cell-free DNA in maternal plasma, and likewise the wrong fetal sex can be reported in the case of a male donor and a female fetus. Based on one of the cases we also recommend giving D- blood products to young female patients who receive a BMT of D- donors.

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D.

BACKGROUND: RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with FcγRIII, with less Fc-fucosylation strengthening the interaction. STUDY DESIGN AND METHODS: Anti-D IgG1 Fc-glycosylation patterns in 93 plasma samples from 28 male and 28 female Dutch HIDs and RhIG were analyzed with mass spectrometry. The Fc-glycosylation profiles of HIDs were evaluated with regard to their immunization history. RESULTS: HID sera demonstrated clearly lowered anti-D Fc-fucosylation compared to normal IgG fucosylation (93%); this was more pronounced for female than for male HIDs (47% vs. 65%, p = 0.001). RhIG preparations from seven manufacturers varied greatly in the level of Fc-fucosylation (56%-91%). The level of fucosylation slightly increased upon repeated immunization, although it remained fairly constant over time. The RhIG from the different manufacturers all demonstrated increased Fc-galactosylation (64%-82%) compared to total IgG (38%-51%). CONCLUSION: RhIG preparations vary in Fc-fucosylation and all demonstrate increased galactosylation. Despite not knowing the exact working mechanism, immunoprophylaxis could perhaps be optimized by selection of donors whose anti-D have low amounts of Fc-fucose, to increase the clearance activity of anti-D preparations, as well as high amounts of galactosylation, for anti-inflammatory effects. Implementing a biologic assay in the standardization of RhIG preparations might be considered.

Novel alleles at the Kell blood group locus that lead to Kell variant phenotype in the Dutch population.

BACKGROUND: Alloantibodies directed against antigens of the Kell blood group system are clinically significant. In the Netherlands, the KEL1 antigen is determined in all blood donors. In this study, after phenotyping of KEL:1-positive donors, genotyping analysis was conducted in KEL:1,-2 donors to identify possible KEL*02 variant alleles. STUDY DESIGN AND METHODS: A total of 407 donors with the KEL:1,-2 phenotype were genotyped for the KEL*01/02 polymorphism, followed by direct sequencing of the KEL gene if the KEL*02 allele was detected. Two K0 patients were also included. Transcript analysis was conducted in two probands with the KEL*02. M05 allele defined by a synonymous mutation (G573G). Flow cytometry analysis to determine the expression of Kell antigen was performed. RESULTS: Thirty KEL:1,-2 individuals (30/407, 7.4%) with discrepant KEL*01/02 genotype were identified. Seven novel alleles were identified: KEL*02(R86Q, R281W)mod, KEL*02(L133P)null, KEL*02(436delG)null, KEL*02(F418S)null, KEL*02(R492X)null, KEL*02(L611R)null, and KEL*02(R700X)null. Nine variant alleles described before were detected: KEL*02N.06, KEL*02N.15, KEL*02N.17, KEL*02N.19, KEL*02N.21, KEL*02M.02, KEL*02M.04, KEL*02M.05, and KEL*02(Q362K)mod. A transcript lacking Exon 16 was identified in two probands with the KEL*02M.05 allele as described before. Finally, flow cytometry analysis showed a decreased total Kell expression and a relatively increased KEL1 expression in individuals with the KEL:1,2null or KEL:1,2mod phenotype, compared to KEL:1,2 controls. CONCLUSION: In 7.4% of a group of tested KEL:1,-2 Dutch donors, a KEL*02null or KEL*02mod allele was found. A relatively increased KEL1 antigen expression in KEL:1,2null and KEL:1,2mod individuals suggest that the expression of Kell-XK complexes depends on the availability of the XK protein.

Impact of genetic variation in the SMIM1 gene on Vel expression levels.

BACKGROUND: Serologic determination of the Vel- phenotype is challenging due to variable Vel expression levels. In this study we investigated the genetic basis for weak Vel expression levels and developed a high-throughput genotyping assay to detect Vel- donors. STUDY DESIGN AND METHODS: In 548 random Caucasian and 107 Vel+(w) donors genetic variation in the SMIM1 gene was studied and correlated to Vel expression levels. A total of 3366 Caucasian, 621 black, and 333 Chinese donors were screened with a high-throughput genotyping assay targeting the SMIM1*64_80del allele. RESULTS: The Vel+(w) phenotype is in most cases caused by the presence of one SMIM1 allele carrying the major allele of the rs1175550 SNP in combination with a SMIM1*64_80del allele or in few cases caused by the presence of the SMIM1*152T>A or SMIM1*152T>G allele. In approximately 6% of Vel+(w) donors genetic factors in SMIM1 could not explain the weak expression. We excluded the possibility that lack of expression of another blood group system was correlated with weak Vel expression levels. Furthermore, using a high-throughput Vel genotyping assay we detected two Caucasian Vel- donors. CONCLUSION: Weak Vel expression levels are caused by multiple genetic factors in SMIM1 and probably also by other genetic or environmental factors. Due to the variation in Vel expression levels, serologic determination of the Vel- phenotype is difficult and a genotyping assay targeting the c.64_80del deletion in SMIM1 should be used to screen donors for the Vel- phenotype.

Low anti-RhD IgG-Fc-fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn.

Haemolytic disease of the fetus and newborn (HDFN) may occur when maternal IgG antibodies against red blood cells (RBCs), often anti-RhD (anti-D) antibodies, cross the placenta and mediate the destruction of RBCs via phagocytic IgG-Fc-receptors (FcγR). Clinical severity is not strictly related to titre and is more accurately predicted by the diagnostically-applied monocyte-based antibody-dependent cellular cytotoxicity (ADCC), a sensitive test with relatively low specificity. This suggests that other factors are involved in the pathogenesis of HDFN. Binding of IgG to FcγR requires the N-linked glycan at position 297 in the IgG-Fc-region, consisting of several different glycoforms. We therefore systematically analysed IgG-derived glycopeptides by mass spectrometry from 70 anti-D IgG1 antibodies purified from the plasma of alloimmunized pregnant women. This revealed a variable decrease in Fc-fucosylation in the majority of anti-D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated, like in healthy individuals (>90%). The degree of anti-D fucosylation correlated significantly with CD16 (FcγRIIIa)-mediated ADCC, in agreement with increased affinity of defucosylated IgG to human FcγRIIIa. Additionally, low anti-D fucosylation correlated significantly with low fetal-neonatal haemoglobin levels, thus with increased haemolysis, suggesting IgG-fucosylation to be an important pathological feature in HDFN with diagnostic potential.

Molecular analysis of immunized Jr(a-) or Lan- patients and validation of a high-throughput genotyping assay to screen blood donors for Jr(a-) and Lan- phenotypes.

BACKGROUND: Individuals with anti-Jr(a) or anti-Lan are ideally transfused with rare Jr(a-) or Lan- red blood cells. We characterized mutations in Dutch Jr(a-) and Lan- individuals and developed a high-throughput genotyping assay to detect Jr(a-) and Lan- donors. STUDY DESIGN AND METHODS: Six Jr(a-) and seven Lan- persons, who all made anti-Jr(a) or anti-Lan, were sequenced for ABCG2 or ABCB6 and the copy number of ABCG2 and ABCB6 was determined. A total of 3366 Caucasian, 621 black, and 333 Chinese donors were screened with a high-throughput screening assay targeting frequently occurring mutations causing the Jr(a-) or Lan- phenotype. RESULTS: In the six tested Jr(a-) individuals previously described, c.376C > T, c.706C > T, and c.736C > T nonsense mutations in ABCG2 were detected. In the seven Lan- individuals 12 different mutations, of which 10 underlie the Lan- phenotype, were detected. No copy number variation was detected for ABCG2 and ABCB6. The high-throughput screening assay detected five Caucasian donors heterozygous for the c.706C > T or 736C > T mutation in ABCG2 and nine Caucasian donors heterozygous for the 574C > T mutation in ABCB6. No black or Chinese donors were found positive for a mutation. CONCLUSION: We describe eight new mutations in ABCB6 of which seven, including three missense mutations, underlie the Lan- phenotype and determine that a complete gene deletion of ABCG2 or ABCB6 is not responsible for the Jr(a-) or Lan- phenotype, respectively. The extended heterogeneity of mutations causing the Jr(a-) or Lan- phenotype in most populations makes genetic screening for the Jr(a-) and Lan- phenotype inefficient in those populations.