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ABSTRACT: The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34(+) cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis.
PLoS ONE 01/2012; 7(6):e38356. · 4.09 Impact Factor
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ABSTRACT: Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.
Blood 04/2011; 118(1):182-91. · 9.90 Impact Factor
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ABSTRACT: To examine the feasibility of fetal RHD genotyping at 11-13 weeks' gestation from analysis of circulating cell-free fetal DNA (ccffDNA) in the plasma of RhD negative pregnant women using a high-throughput robotic technique.
Stored plasma (0.5 ml) from 591 RhD negative women was used for extraction of ccffDNA by a robotic technique. Real-time quantitative polymerase chain reaction (PCR) with probes for exons 5 and 7 of the RHD gene was then used to determine the fetal RHD genotype, which was compared to the neonatal RhD phenotype.
In total there were 502 (85.7%) cases with a conclusive result and 84 (14.3%) with an inconclusive result. The prenatal test predicted that the fetus was RhD positive in 332 cases and in all of these the prediction was correct, giving a positive predictive value of 100% (95% CI 96.8-100). The test predicted that the fetus was RhD negative in 170 cases and in 164 of these the prediction was correct, giving a negative predictive value for RhD positive fetuses of 96.5% (95% CI 93.7-99.2).
The findings demonstrate the feasibility and accuracy of non-invasive fetal RHD genotyping at 11-13 weeks with a high-throughput technique.
Fetal Diagnosis and Therapy 01/2011; 29(4):301-6. · 1.05 Impact Factor
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ABSTRACT: Identification of the molecular basis of the D polymorphism of the Rh blood group system in the 1990s made it possible to predict D phenotype from DNA. The most valuable application of this has been the determination of fetal D type in pregnant D-negative women with anti-D. Knowledge of fetal D type reveals whether the fetus is at risk of hemolytic disease of the fetus and newborn so that the pregnancy can be managed appropriately. Noninvasive fetal D typing for D-negative pregnant women with anti-D, performed on the small quantity of fetal DNA present in the blood of pregnant women, is now routine practice in several European countries. Noninvasive fetal blood grouping for C, c, E, and K also may be provided as a routine service for alloimmunized pregnant women. In many countries, all D-negative pregnant women are offered anti-D prophylaxis antenatally, yet in a predominantly Caucasian population, about 38% will be carrying a D-negative fetus and will receive the treatment unnecessarily. Large-scale trials to ascertain the accuracy of high-throughput, automated methods suggest that fetal D screening of all D-negative pregnant women is feasible, and it is likely that fetal D screening in D-negative pregnant women will be policy in some European countries within the next few years.
Clinics in laboratory medicine 06/2010; 30(2):431-42. · 1.17 Impact Factor
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ABSTRACT: The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation, high levels of enucleation (80-90%) and low levels of cell death (<10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available.
Haematologica 04/2010; 95(9):1594-8. · 6.42 Impact Factor
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ABSTRACT: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex.
We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis.
We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship.
We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.
Haematologica 02/2010; 95(8):1278-86. · 6.42 Impact Factor
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ABSTRACT: Dantu, a previously undescribed low-incidence red cell antigen, is inherited as a Mendelian dominant character. The Dantu antigen is associated with very weak s antigen, protease resistant N antigen and either very weak or no U antigen. Two of the propositi had previously been shown to have an unusual hybrid MNSs sialoglycoprotein, and it is probably this which carries these unusual N, s and U antigens as well as the Dantu antigen. A study of the family of one propositus suggests, by conventional genetics, that Dantu is not controlled by the MNSs locus; a possible explanation is given. Several examples of anti-Dantu are known, one was found to cause a positive direct antiglobulin reaction on neonatal red cells.
Vox Sanguinis 03/2009; 46(6):377 - 386. · 2.86 Impact Factor
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ABSTRACT: Red cells carrying the low-frequency MNS antigen M8 reacted with the only example of anti-DANE, an antibody which had previously defined the GP.Dane (Mi.IX) phenotype. Furthermore, Mg + cells reacted with the original anti-Mur (serum of Mrs. Murrell), but with none of 14 other anti-Mur. Therefore, M8 + cells carry both DANE antigen and an atypical Mur antigen. Immunoblotting of membranes from Mg + cells with anti-M, and with eluates prepared from anti-Mg and Mrs. Murrell's serum demonstrated a glycophorin A (GPA) molecule whose mobility was increased by an apparent Mr of about 3,000 presumably due to the loss of the three O-glycans known to be absent from Mg -active GPA.
Vox Sanguinis 03/2009; 66(3):237 - 241. · 2.86 Impact Factor
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ABSTRACT: Alloimmunization to the blood group antibody anti-RhD (anti-D) is the most common cause of hemolytic disease of the fetus and newborn. Knowledge of fetal D type in women with anti-D makes management of the pregnancy much easier and avoids unnecessary procedures in those women with a D-negative fetus. Fetal D typing can be performed by detection of an RHD gene in cell-free DNA in the plasma of D-negative pregnant women. The technology involves real-time quantitative polymerase chain reactions targeting exons 4, 5, and 10 of RHD, with the exons 4 and 10 tests performed as a multiplex. Testing for SRY in multiplex with the RHD exon 5 test provides an internal control for the presence of fetal DNA when the fetus is male. Fetal D typing has become the standard of care in England in pregnant women with a significant level of anti-D.
Methods in molecular biology (Clifton, N.J.) 02/2009; 496:143-57.
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ABSTRACT: Fetuses of women with alloantibodies to RhD (D) are at risk from hemolytic disease of the fetus and newborn, but only if the fetal red cells are D-positive. In such pregnancies, it is beneficial to determine fetal D type, as this will affect the management of the pregnancy. It is possible to predict, with a high level of accuracy, fetal blood group phenotypes from genotyping tests on fetal DNA. The best source is the small quantity of fetal DNA in the blood of pregnant women, as this avoids the requirement for invasive procedures of amniocentesis or chorionic villus sampling (CVS). Many laboratories worldwide now provide noninvasive fetal D genotyping as a routine service for alloimmunized women, and some also test for c, E, C and K.In many countries, anti-D immunoglobulin injections are offered to D-negative pregnant women, to reduce the chances of prenatal immunization, even though up to 40% of these women will have a D-negative fetus. High-throughput, noninvasive fetal D genotyping technologies are being developed so that unnecessary treatment of pregnant women can be avoided. Trials suggest that fetal D typing of all D-negative pregnant women is feasible and should become common practice in the near future.
Prenatal Diagnosis 12/2008; 29(2):101-7. · 2.11 Impact Factor
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Vanja Karamatic Crew,
Joyce Poole,
Shannon Long,
Nicole Warke,
Connie Colavecchia,
Nicholas Burton,
Marilyn Moulds,
Gloria Schlanser,
Laura Wilson,
Ghislain Noumsi,
Joann M Moulds,
John J Moulds, Geoff Daniels
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ABSTRACT: MER2 (RAPH1), the only antigen of the RAPH blood group system, is located on the tetraspanin CD151. Only four examples of alloanti-MER2 are known. We report here two new examples of alloanti-MER2, in women of Pakistani and Turkish origin, one of whom showed signs of a hemolytic transfusion reaction (HTR) after transfusion of 3 units of red cells (RBCs).
Standard serologic methods were used. A monocyte monolayer assay (MMA) was used to assess the potential clinical significance of one of the antibodies. All exons and flanking intronic sequences of CD151 were amplified and sequenced. A homology model for CD151 second extracellular loop (EC2) was constructed based on the crystal structure of CD81.
RBCs of both patients did not react with alloanti-MER2, and neither of their antibodies reacted with MER2-negative RBCs. The MMA results suggested that the antibody that appeared to have caused an HTR had the potential to be clinically significant. Both patients were homozygous for a 511C>T mutation in CD151 encoding an Arg171Cys change. This change did not result in any significant structural rearrangement in the protein model.
Two MER2-negative patients with anti-MER2 are homozygous for the same novel mutation encoding an amino acid substitution in the EC2 of CD151. One of the antibodies may have been responsible for an HTR, and crossmatch-compatible RBCs should be recommended for transfusion to patients with anti-MER2.
Transfusion 09/2008; 48(9):1912-6. · 3.22 Impact Factor
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ABSTRACT: Tn polyagglutination results from inactivating mutations in C1GALT1C1, an X-borne gene encoding a core 1 beta3-galactosyltransferase-specific molecular chaperone (cosmc) required for the functioning of T-synthase (beta 1,3-galactosyltransferase), a glycosyltransferase essential for the correct biosynthesis of O-glycans. This study found novel inactivating mutations (Glu152Lys, Ser193Pro and Met1Ile) in the coding sequence of C1GALT1C1 in three Tn positive individuals and a complete lack of C1GALT1C1 cDNA expression was observed in an additional Tn positive individual. In addition, expression of ST6GALNAC1, which encodes (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1, 3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 and gives rise to sialyl-Tn antigen, was present at comparable levels in normal and Tn-positive human erythroblasts. Expression studies of wild-type and Tn positive C1GALT1C1 cDNA in the Jurkat cell line confirmed that the amino acid substitutions observed in Tn are inactivating. Analysis of the transcriptome of cultured normal and Tn positive erythroblasts revealed numerous differences in gene expression. Reduced transcript levels for fatty acid binding protein 5 (FABP5) and plexin D1 (PLXND1), and increased levels for aquaporin 3 (AQP3) were confirmed by quantitative real-time polymerase chain reaction. These data show that alteration of O-glycan structures resulting from T-synthase deficiency is accompanied by altered expression of a wide variety of genes in erythroid cells.
British Journal of Haematology 07/2008; 142(4):657-67. · 4.94 Impact Factor
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Vanja Karamatic Crew,
Joyce Poole,
Shannon Long,
Nicole Warke,
Connie Colavecchia,
Nicholas Burton,
Marilyn Moulds,
Gloria Schlanser,
Laura Wilson,
Ghislain Noumsi,
Joann M. Moulds,
John J. Moulds, Geoff Daniels
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ABSTRACT: BACKGROUND: MER2 (RAPH1), the only antigen of the RAPH blood group system, is located on the tetraspanin CD151. Only four examples of alloanti-MER2 are known. We report here two new examples of alloanti-MER2, in women of Pakistani and Turkish origin, one of whom showed signs of a hemolytic transfusion reaction (HTR) after transfusion of 3 units of red cells (RBCs).STUDY DESIGN AND METHODS: Standard serologic methods were used. A monocyte monolayer assay (MMA) was used to assess the potential clinical significance of one of the antibodies. All exons and flanking intronic sequences of CD151 were amplified and sequenced. A homology model for CD151 second extracellular loop (EC2) was constructed based on the crystal structure of CD81.RESULTS: RBCs of both patients did not react with alloanti-MER2, and neither of their antibodies reacted with MER2-negative RBCs. The MMA results suggested that the antibody that appeared to have caused an HTR had the potential to be clinically significant. Both patients were homozygous for a 511C>T mutation in CD151 encoding an Arg171Cys change. This change did not result in any significant structural rearrangement in the protein model.CONCLUSIONS: Two MER2-negative patients with anti-MER2 are homozygous for the same novel mutation encoding an amino acid substitution in the EC2 of CD151. One of the antibodies may have been responsible for an HTR, and crossmatch-compatible RBCs should be recommended for transfusion to patients with anti-MER2.
Transfusion 06/2008; 48(9):1912 - 1916. · 3.22 Impact Factor
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ABSTRACT: To assess the feasibility of applying a high throughput method, with an automated robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin.
Prospective comparison of fetal RHD genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood.
Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory.
Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit.
Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result.
Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping.
High throughput RHD genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of anti-RhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.
BMJ (Clinical research ed.). 05/2008; 336(7648):816-8.
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ABSTRACT: Alloimmunization against the fetal Kell (KEL1) blood group antigen is gaining importance relative to the Rhesus problem and is the second most important cause of hemolytic disease of the fetus and newborn. Molecular diagnosis for fetal KEL1, which currently involves invasive procedures, is routinely done for accessing whether a fetus is at risk. Here we developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based single allele-based extension reaction (SABER) to examine the fetal KEL1 gene from KEL1-negative pregnant women using cell-free fetal DNA in maternal plasma.
Thirty-two maternal plasma samples taken at the second and third trimesters of gestation (median: 21.5 weeks) were examined with MALDI-TOF MS-based SABER. The results were confirmed by serological tests on cord blood or polymerase chain reaction (PCR) typing on amniocyte-derived fetal DNA.
We were able to detect the fetal KEL1 allele in 11 of the 13 KEL1-positive samples. No false positive results were scored. The paternal KEL1 allele could be correctly determined in 94% of cases (30/32).
Our results indicated that the MALDI-TOF MS-based SABER has been used successfully for the detection of the fetal KEL1 status with the accuracy of 94%. Further, large-scale study, such as multicenter study, can now be explored for clinical application.
Prenatal Diagnosis 04/2008; 28(3):203-8. · 2.11 Impact Factor
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ABSTRACT: When a pregnant woman has an antibody with the potential to cause hemolytic disease of the fetus and newborn, it is beneficial to determine whether her fetus has the corresponding antigen to assess risk. In many countries this is now done routinely for RhD, by testing cell-free fetal DNA in the maternal plasma. Similar tests for K, C, c, and E are reported.
Real-time quantitative polymerase chain reaction incorporating an allele-specific primer was developed for detecting the K allele of KEL and the C, c, and E alleles of RHCE. These methods were used to test DNA isolated from plasma of pregnant women with antibodies to K, C, c, or E. Accuracy of the tests was determined by comparing results with serologic tests performed on cord red blood cells (RBCs) after delivery or by molecular genotyping on DNA obtained from fetal cells.
The K test incorporated an allele-specific primer with two locked nucleic acids and a mismatch. In 70 tests, including 27 K+ fetuses, only one false-negative and no false-positive results were obtained. The C, c, and E tests, performed on 13, 44, and 46 samples, respectively, gave rise to no false results.
Reliable methods have been developed for predicting fetal K, C, c, and E phenotypes, by testing fetal DNA in the plasma samples of pregnant women whose RBCs lack the corresponding antigens. These methods are now being used routinely in a diagnostic service in the United Kingdom.
Transfusion 12/2007; 47(11):2126-33. · 3.22 Impact Factor
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ABSTRACT: To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA).
The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples.
Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series.
This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA.
Prenatal Diagnosis 11/2007; 27(10):932-7. · 2.11 Impact Factor
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10/2007: pages 207 - 224; , ISBN: 9780470987056
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ABSTRACT: Blood samples were referred over a 10-year period from five patients whose serum samples contained antibodies to unidentified high-incidence antigens. Three patients (A, B, C) were of Moroccan origin and their antibodies and red blood cells (RBCs) were mutually compatible, but incompatible with those of the other two patients (D, E), who were of Pakistani origin. The antibodies and RBCs of D and E were mutually compatible, but incompatible with those of Patients A, B, and C. All the antibodies were detected during pregnancy.
Serologic tests, including the use of enzyme-treated and chemically modified RBCs, suggested a relationship to CD44 (Indian blood group system). The monoclonal antibody immobilization of erythrocyte antigens (MAIEA) assay with monoclonal CD44 antibodies, immunoblotting of RBC membranes, and CD44 gene sequencing were carried out.
Positive reactions in the MAIEA assay confirmed that the patients' antibodies are directed at CD44. Immunoblotting with two of the antibodies gave positive reactions of identical size to monoclonal anti-CD44 and failed to react with the RBCs of a CD44-deficient patient. One of the antibodies reacted with purified CD44. Sequencing of Exons 1 to 5 of CD44 revealed 255C>G in Exon 3 for A, B, and C encoding H85Q and 488C>A in Exon 5 for D and E encoding T163K [corrected]
Two novel CD44 antigens of high incidence have been identified: IN3 (INFI) and IN4 (INJA) in the IN (Indian) blood group system. Lack of IN3 and IN4 results from homozygosity for mutations encoding H85Q and T163R in CD44, respectively.
Transfusion 08/2007; 47(7):1306-11. · 3.22 Impact Factor
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Neil D Avent,
Antonio Martinez,
Willy A Flegel,
Martin L Olsson,
Marion L Scott,
Núria Nogués,
Martin Písăcka, Geoff Daniels,
Ellen van der Schoot,
Eduardo Muñiz-Diaz, [......],
Inge von Zabern,
Elisa Jiménez,
Diego Tejedor,
Mónica López,
Emma Camacho,
Goedele Cheroutre,
Anita Hacker,
Pavel Jinoch,
Irena Svobodova,
Masja de Haas
Transfusion 08/2007; 47(1 Suppl):40S-6S. · 3.22 Impact Factor
