Novel mutations in KLF1 encoding the In(Lu) phenotype reflect a diversity of clinical presentations.

BACKGROUND: Mutation in the KLF1 gene is the cause of the In(Lu) (Inhibitor of Lutheran) Lu(a-b-) phenotype and more than 60 alleles have been associated with this phenotype. Here we describe findings from investigation of seven cases: six presenting with a Lu(a-b-) phenotype including the historical index case and one referred from a patient with chronic anemia. STUDY DESIGN AND METHODS: Serologic testing was by standard methods. DNA testing included amplification and sequencing of KLF1 and LU coding regions. A StuI polymerase chain reaction-restriction fragment length polymorphism was designed to target c.304T>C in KLF1. RESULTS: Five different KLF1 alleles were identified. Three are new: KLF1*90A (p.Trp30Ter), KLF*911A (p.Thr304Lys), and KLF1*304C,318G (p. Ser102Pro, Tyr106Ter) present in two unrelated individuals. Two, including the index case, had c.954dupG (p.Arg319Glufs*34), that is, KLF1*BGM06. The child with unexplained anemia had c.973G>A (p.Glu325Lys), associated with congenital dyserythropoietic anemia. The common c.304T>C was found in two of the seven samples investigated and in 60 of 100 blood donors. CONCLUSION: Mutations in KLF1 are pleiotropic and although most are benign, others are associated with hematologic abnormalities. We report three new KLF1 alleles associated with benign In(Lu) and document both the molecular basis of the original In(Lu) phenotype using a frozen sample stored for more than 50 years and the cause of unexplained anemia in a child. We also confirm previous observations that c.304C (p.102Pro) is not, by itself, associated with an In(Lu) phenotype in donors self-identified as U.S. minorities.

Mutations of the KLF1 gene detected in Japanese with the In(Lu) phenotype.

BACKGROUND: In(Lu) is characterized by a reduced expression of antigens in the Lutheran blood group system as well as other blood group antigens. Mutations of the erythroid transcription factor, KLF1, have been reported to cause the In(Lu) phenotype, and we investigated Japanese In(Lu) to estimate the prevalence of the phenotype and KLF1 polymorphism. STUDY DESIGN AND METHODS: Blood samples were screened by monoclonal anti-CD44 and the In(Lu) phenotype was confirmed by tube tests including adsorption and elution tests using anti-Lua and anti-Lub . KLF1, LU, and A4GALT genes were analyzed by polymerase chain reaction and sequencing. RESULTS: We identified 100 of 481,322 blood donors (0.02%), and the previously characterized 20 donors, who had the In(Lu) phenotype with the LUB/LUB genotype. A total of 100 of the 120 In(Lu) individuals had mutant KLF1 alleles, and we identified 13 known and 21 novel alleles. The mutant KLF1 alleles with c.947G>A (p.Cys316Tyr), c.862A>G (p.Lys288Glu), or c.968C>G (p.Ser323Trp) were major in the In(Lu) individuals. The P1 antigen of 29 In(Lu) (two P1 /P1 , 27 P1 /P2 ) showed significantly weakened expression by hemagglutination. CONCLUSIONS: The prevalence of the In(Lu) phenotype in the Japanese population was 0.02%, and we identified 13 known and 21 novel KLF1 alleles. The KLF1 mutations cause the reduced expression of the P1 antigen.

Erythroid Adhesion Molecules in Sickle Cell Anaemia Infants: Insights Into Early Pathophysiology.

Sickle cell anaemia (SCA) results from a single mutation in the ß globin gene. It is seldom symptomatic in the first semester of life. We analysed the expression pattern of 9 adhesion molecules on red blood cells, in a cohort of 54 SCA and 17 non-SCA very young infants of comparable age (median 144 days, 81-196). Haemoglobin F (HbF) level was unsurprisingly elevated in SCA infants (41.2% ± 11.2) and 2-4 fold higher than in non-SCA infants, yet SCA infants presented significantly decreased Hb level and increased reticulocytosis. Cytometry analysis evidenced a specific expression profile on reticulocytes of SCA infants, with notably an increased expression of the adhesion molecules Lu/BCAM, ICAM-4 and LFA-3, both in percentage of positive cells and in surface density. No significant difference was found on mature red cells. Our findings demonstrate the very early onset of reticulocyte membrane modifications in SCA asymptomatic infants and allow an insight into the first pathological changes with the release of stress reticulocytes expressing a distinctive profile of adhesion molecules.

Antibody screening in multitransfused patients: a prerequisite before each transfusion.

Life-long red blood cell (RBC) transfusions remain the main treatment for severe thalassemia. We hereby report a case of anti S and anti Lu(a) in a ß-thalassemia major patient detected incidentally on antibody screening. The patient was a known case of ß-thalassemia major and was on regular blood transfusion every 3 weeks from the institute from the age of 6 months. Subsequently, on one occasion, patient's crossmatch was compatible despite positive antibody screen using microcolumn gel technique. Autocontrol and direct antiglobulin test were negative on microcolumn gel. Anti S and anti Lu(a) antibodies were identified. Blood unit found compatible was negative for S and Lu(a) antigens. Antibody titers were 1:1 for both anti S and anti Lu(a) in AHG phase using tube technique and antibodies were of IgG type. Blood unit was transfused uneventfully to the patient. Donors were traced back (last three donations) and called for repeat blood sample testing for S and Lu(a) antigen. Two out of three donors were found to be S antigen positive and one out of these two was Lu(a) antigen positive. Anti S and anti Lu(a) antibodies were again identified on patient's subsequent visit for transfusion. The present case re-emphasize the importance of antibody screening at each visit in earlier detection of antibodies in multi transfused patients. Encouraging patients to receive transfusion from one center and dedicating donors could reduce alloimmunization rate but larger studies are required.

Molecular analysis of the rare in(Lu) blood type: toward decoding the phenotypic outcome of haploinsufficiency for the transcription factor KLF1.

KLF1 encodes an erythroid transcription factor, whose essential function in erythropoiesis has been demonstrated by extensive studies in mouse models. The first reported mutations in human KLF1 were found in individuals with a rare and asymptomatic blood type called In(Lu). Here, we show that KLF1 haploinsufficiency is responsible for the In(Lu) blood type, after redefining this peculiar blood type using flow cytometry to quantify the levels of BCAM and CD44 on red blood cells. We found 10 (seven novel) heterozygous KLF1 mutations responsible for the In(Lu) blood type. Although most were obligate loss-of-function mutations due to the truncation of the DNA-binding domain of KLF1, three were missense mutations that were located in its DNA-binding domain and impaired the transactivation capacity of KLF1 in vitro. We further showed that the levels of the hemoglobin variants HbF and HbA(2) were increased in the In(Lu) blood type, albeit differently. The levels of the membrane glycoproteins BCAM and CD44 were also differently reduced on In(Lu) red blood cells. This biochemical and genetic analysis of the In(Lu) blood type tackles the phenotypic outcome of haploinsufficiency for a transcription factor.

Role of the interaction between Lu/BCAM and the spectrin-based membrane skeleton in the increased adhesion of hereditary spherocytosis red cells to laminin.

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.

Erythroid cell adhesion molecules Lutheran and LW in health and disease.

The Lutheran and LW glycoproteins are blood group-active proteins found at the surface of human red cells. The Lutheran glycoprotein (Lu gp) is a member of the immunoglobulin superfamily (IgSF) that binds the extracellular matrix protein laminin, in particular, laminin isoforms containing the alpha 5 subunit. The LW glycoprotein (LW gp), also an IgSF member, has substantial sequence homology with the family of intercellular adhesion molecules (ICAMs). LW gp binds the integrin very late antigen-4 (VLA-4, alpha 4 beta 1) and alpha V-containing integrins. Studies on the expression of LW and Lu gps during erythropoiesis utilizing in vitro cultures of haemopoietic progenitor cells have shown that LW gp expression precedes that of Lu gp. These observations have led to the suggestion that LW gp on erythroblasts may interact with VLA-4 on macrophages to stabilize erythroblastic islands in normal bone marrow and that Lu gp may facilitate trafficking of more mature erythroid cells to the sinusoidal endothelium where alpha 5-containing laminins are known to be expressed. Levels of Lu gp and LW gp expression on sickle red cells are greater than on normal red cells and sickle red cells adhere to alpha 5-containing laminins. These data suggest that the Lu and LW molecules may contribute to the vaso-occlusive events associated with episodes of acute pain in sickle cell disease.

Transient loss of proteins carrying Kell and Lutheran red cell antigens during consecutive relapses of autoimmune thrombocytopenia.

A patient is described in whom two consecutive relapses of autoimmune thrombocytopenic purpura (AITP) were associated with loss of red cell antigens of the Kell and Lutheran blood group systems respectively. During the second relapse the glycoprotein CD44 and to a lesser extent the LW antigen were also depressed. Both relapses were associated with concomitant production of IgG antibody recognizing high-frequency determinants on the corresponding antigen-carrying protein. Blocking of antigen sites by these antibodies was not the cause of reduced antigen expression, because immunoblotting studies showed absence of Kell protein during the first relapse, and Lutheran protein during the second. On both occasions the red cell changes reverted to normal with disappearance of the antibody as the AITP entered remission. There was no evidence of clonal lymphocyte expansion as demonstrated using immunoglobulin JH and T cell receptor beta chain probes.

Autologous and allogeneic red cell survival studies in the presence of autoanti-AnWj.

Chromium survival studies were performed with AnWj-positive allogeneic blood in a patient with autoanti-AnWj. 99mTc-labeled autologous RBCs that had depressed AnWj expression had normal survival (77% [94.7% 51Cr equivalent]) at 24 hours, whereas 51Cr-labeled allogeneic AnWj-positive cells had 76 percent survival at 24 hours and 55 percent survival at 7 days. These studies suggest that the specificity of the autoantibody may have implications for transfusion therapy when the development of such autoantibodies is associated with decreased antigen expression on the patient's cells.