Universidade Federal de São Paulo, Discipline Hematol & Transfus Med, BR-04023092 São Paulo, BrazilUniversidade Federal de São Paulo, Discipline Hematol & Transfus Med, BR-04023092 São Paulo, BrazilWeb of Scienc

Bordin, Jose Orlando

Boturão-Neto, Edmir

Chiba, Akemi Kuroda

Figueiredo, Maria Stella

Vicari, Perla

Haematologica

Boturão-Neto, Edmir [UNIFESP]

Chiba, Akemi Kuroda [UNIFESP]

Vicari, Perla [UNIFESP]

Figueiredo, Maria Stella [UNIFESP]

Bordin, Jose Orlando [UNIFESP]

Repositório Institucional UNIFESP

Letters to the Editor| 1408 | haematologica | 2008; 93(9)by the –148C>T mutation, nor by any other putativemutation linked to this change. The –148C>T mutationin the erythroid-specific promoter of PKLR should, there-fore, be considered a benign polymorphism. The basis forthe lowered PK activity in the 3 individuals presentedhere thus remains to be established.Karen M.K. de Vooght, Richard van Wijk,Annet C. van Wesel, Wouter W. van SolingeDepartment of Clinical Chemistry and Haematology,Laboratory for Red Blood Cell Research, University Medical CenterUtrecht, Utrecht, The NetherlandsKeywords: PKLR, promoter mutation, transcriptional regulation,pyruvate kinase deficiency.Correspondence: Wouter W. van Solinge, Department of ClinicalChemistry and Haematology, Laboratory for Red Blood CellResearch, University Medical Center Utrecht, Postbus 85500,3508 GA, Utrecht, The Netherlands. Phone: international+31.88.7557604. Fax: international +31.88.7555418. E-mail: wsolinge@umcutrecht.nlCitation: de Vooght KMK, van Wijk R, van Wesel AC, vanSolinge WW. Characterization of the –148C>T promoter polymor-phism in PKLR. Haematologica 2008; 93:1407-1408.doi: 10.3324/haematol.12328References1. Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valentini G.Pyruvate kinase deficiency: The genotype-phenotypeassociation. Blood Rev 2007;12:12.2. Van Wijk R, Van Solinge WW, Nerlov C, Beutler E,Gelbart T, Rijksen G, et al. Disruption of a novel regula-tory element in the erythroid-specific promoter of thehuman PKLR gene causes severe pyruvate kinase defi-ciency. Blood 2003;101:1596-602.3. Manco L, Abade A. Pyruvate kinase deficiency: preva-lence of the 1456C-->T mutation in the Portuguese pop-ulation. Clin Genet 2001;60:472-3.4. Pissard S, Max-Audit I, Skopinski L, Vasson A, Vivien P,Bimet C, et al. Pyruvate kinase deficiency in France: a 3-year study reveals 27 new mutations. Br J Haematol2006;133:683-9.5. Wingender E, Chen X, Hehl R, Karas H, Liebich I, MatysV, et al. TRANSFAC: an integrated system for geneexpression regulation. Nucleic Acids Res 2000;28:316-9.6. Knight JC. Functional implications of genetic variation innon-coding DNA for disease susceptibility and gene reg-ulation. Clin Sci (Lond) 2003;104:493-501.7. de Vooght KMK, van Wijk R, Ploos van Amstel HK, vanSolinge WW. Characterization of the -16C>G sequencevariation in the promoters of both HBG1 and HBG2:Convergent evolution of the human γ-globin genes.Blood Cells Mol Dis 2007;39:70-4.8. Morse EE, Jilani F, Brassel J. Acquired pyruvate kinasedeficiency. Ann Clin Lab Sci 1977;7:399-404.9. Lacronique V, Lopez S, Miquerol L, Porteu A, Kahn A,Raymondjean M. Identification and functional character-ization of an erythroid-specific enhancer in the L-typepyruvate kinase gene. J Biol Chem 1995;270:14989-97.10. van Wijk R, van Wesel AC, Thomas AA, Rijksen G, vanSolinge WW. Ex vivo analysis of aberrant splicinginduced by two donor site mutations in PKLR of a patientwith severe pyruvate kinase deficiency. Br J Haematol2004;125:253-63.Molecular studies reveal a concordant KELgenotyping between patients withhemoglobinopathies and blood donors in São Paulo City, Brazil Kell is the most important blood group system afterABO and Rh because all frequently occurring Kell-specif-ic antibodies must be considered clinically significant. Thehighly immunogenic Kell antigens are usually involved inred cell (RBC) alloimmunization that can cause eitherhemolytic transfusion reactions or perinatal hemolyticdisease. Patients with hemoglobinopathies such as sicklecell disease (SCD) require frequent blood transfusions.Therefore, they often become alloimmunized and pro-duce antibodies to low-prevalence Kell antigens, especial-ly KEL1.1-4 The Kell low-prevalence antigens areexpressed due to single-nucleotide polymorphisms in theKEL exons and some of them show ethnic or racial speci-ficity. KEL1 is present in 9% of Caucasians and 2% ofthose of African descent, KEL6 is expressed in 19.5% ofAfricans and in less than 0.01% of white Caucasians,while KEL3 is found in 2.3% in Caucasians and is rareamong Africans.1 The incidence of alloimmunization,although multifactorial in etiology, is particularly high inSCD patients when compared with other multitransfusedpatients, primarily due to the disparity of phenotypingpattern distribution which is partly caused by ethnic orracial differences between the blood donor populationand the SCD patient population.2,4 Extended antigen-matched RBC transfusion has been recommended forreducing the incidence of alloimmunization in patientswith SCD. This practice adds greatly to the cost of provid-ing blood components to these patients and can cause dif-ficulties in finding RBC units for all patients.2,3 In order toevaluate differences in KEL genotyping distributionbetween blood donors and patients with SCD, and thebenefit of providing extended antigen-matched RBCtransfusion in multitransfused patients, we investigatedthe frequencies of different KEL genotypes in patientswith hemoglobinopathies and blood donors in São PauloCity, the largest city in Brazil. The Ethics CommitteeBoard approved the study.Aliquots of EDTA-anticoagulant peripheral venousblood were collected from 108 Brazilian patients ofAfrican descent with hemoglobinopathies and 205 ran-domized blood donors. Genomic DNA was isolated fromwhole blood using a commercial kit (QIAamp DNA Bloodmini kit, Qiagen, Hilden, Germany). Employing poly-merase chain reaction–restriction fragment length poly-morphism (PCR-RFLP) technique, we performed theKEL*1,2,  KEL*3,4,21 and KEL*6,7 genotyping as previous-ly described.5-7 The differences between the KEL genotypefrequencies in the two groups were analyzed by Fischer’sexact test. p values of 0.05 were considered statisticallysignificant. The results of the frequencies for KEL*1,2,KEL*3,4,21 and KEL*6,7 polymorphisms are summarizedin Table 1. The KEL*1/2 genotype was found in 6.5%(7/108) of patients with hemoglobinopathies and in 6.3%(13/205) of blood donors. The KEL*3/4 genotype wasobserved in only 1.0% (2/205) of blood donors. TheKEL*6/7 genotype was found in 6.5% (7/108) of patientswith hemoglobinopathies and in 4.4% (9/205) of blooddonors. We also found 1 (0.5%) donor with the KEL*6/6genotype. No KEL*21 allele, KEL*1/1, or KEL*3/3 geno-types were identified among the 313 subjects. There wasno statistically significant difference between the frequen-cies of KEL*1/2, KEL*3/4 and KEL*6/7 genotypes seen inLetters to the Editorhaematologica | 2008; 93(9) | 1409 |patients with hemoglobinopathies compared to thoseobserved in blood donors.Patients with hemoglobinopathies commonly requireRBC transfusion to manage clinical complications such asanemia, acute chest syndrome, stroke, and splenic seques-tration. Overall, RBC alloimmunization in transfusedpatients with SCD has a reported incidence of up to 40%.Alloantibodies against Kell antigens, especially anti-KEL1,have been observed as one of the most frequent antibod-ies in multitransfused SCD patients. Anti-KEL1 alloimmu-nization has a reported incidence of 28.1% up to 76.9%among alloimmunized SCD patients in North America andEurope,2-4,8,9 however, there are few data regarding the RBCalloimmunization rate in South American SCD patients.Table 2 summarizes the results of some studies evaluatingRBC alloimmunization in patients with hemoglo-binopathies. We have reported an overall RBC alloimmu-nization rate of 12.9% (11/85) in SCD Brazilian patients,and anti-KEL1 specific alloimmunization rate of 18.2%(2/11) among such subjects.10 A larger Brazilian studydescribed an overall RBC alloimmunization rate of 9.9%(82/828) and anti-KEL1 specific alloimmunization frequen-cy of 14.6% (12/82) among alloimmunized SCD patients,11suggesting a lower KEL alloimmunization risk for patientsin Brazil.A frequently recommended strategy for reducing theincidence of RBC alloimmunization in patients with SCDis to provide RBC transfusion matched for ABO, Rh (D),and additional blood group antigens, ranging from limitedmatching, i.e., C, E, and K antigens, to extended matchingfor as many as 8 additional antigens, i.e., C, c, E, e, K, S,Fya, Fyb, and Jkb.2,3 Besides the benefit of the phenotypematching protocols that would prevent alloimmunizationin 52% to 70.8% of all immunized patients, these strate-gies may also diminish the risk of delayed hemolytic trans-fusion reactions, hyperhemolysis syndrome, and autoanti-body formation. On the other hand, these protocolsdemand high cost, need access to uncommon RBCs phe-notypes, and do not totally avoid the risk of alloimmuniza-tion, or the occurrence of delayed hemolytic transfusionreactions or hyperhemolysis syndrome.2,3Molecular techniques have been used in transfusionmedicine as an alternative tool to serological methods inorder to investigate patients who have been recently trans-fused, or when antibody reagents, such as anti-Jsa and anti-Jsb, are scarce or not commercially available.1 Using molec-ular studies, we demonstrated that the frequency of theKEL*1/2 (6.5%) and KEL*6/7 (6.5%) genotypes amongBrazilian patients with hemoglobinopathies differed fromthose usually observed in subjects of African descent.Moreover, the frequency of the KEL*1/2 (6.3%) andKEL*6/7 (4.4%) genotypes in blood donors differed fromthose to be expected in a Caucasian population or in sub-jects of African descent, but quite similar to the resultsobserved in our patients with hemoglobinopathies. Thesefindings suggest a high rate of racial admixture in Brazilianpatients of African descent with hemoglobinopathies, aswell as in Brazilian blood donors, and possibly explain thelower risk of Kell alloimmunization seen in Brazilian SCDpatients.10,11In conclusion, the observed concordant KEL genotypingdonor-recipient population suggests that Brazilian patientsare at a lower risk of Kell alloimmunization than thatreported in studies from North America and Europe wherethe donor population is predominantly formed by whiteCaucasians. In addition, our data indicate that the require-ment for extended antigen-matched RBC transfusion inmultitransfused patients may not be cost-effective inBrazil.Edmir Boturão-Neto, Akemi K. Chiba, Perla Vicari,Maria S. Figueiredo, José O. BordinDiscipline of Hematology and Transfusion Medicine, FederalUniversity of São Paulo, São Paulo, SP, BrazilFunding: this study was partially supported by CNPq (Process nº474995/03-8) and FAPESP (Process n° 05/55237-9).Key words: KEL genotyping, sickle cell disease, transfusionreactions, alloimmunizationCorrespondence: José O. Bordin, MD, PhD, Disciplina deHematologia e Hemoterapia, Universidade Federal de São Paulo,Rua Botucatu, 740 São Paulo, 04023-092, SP, Brazil.Phone: international +55.11.55791550. Fax: international+55.11.55718806. E-mail: jobordin@hemato.epm.brCitation: Boturão-Neto E, Chiba AK, Vicari P, Figueiredo MS,Bordin JO. Molecular studies reveal a concordant KEL genotypingbetween patients with hemoglobinopathies and blood donors in SãoPaulo City, Brazil. Haematologica 2008. 93:1408-1410.doi:10.3324/haematol.12766.Table 2. Results of studies reporting RBC alloimmunization rateand anti-KEL1 specific alloimmunization frequency among alloim-munized SCD patients.Author Country n RBC Anti-KEL1alloimmunization frequency inrate alloimmunizedpatientsRosse et al.8 USA 1,814 18.6% (338/1,814) 28.1% (95/338)Vichinsky et al.4 USA 107 29.9% (32/107) 56.2% (18/32)Moreira et al.10 Brazil 85 12.9% (11/85) 18.2% (2/11)Aygun et al.2 USA 140 37.1% (52/140) 76.9% (40*/52)Castro et al.3 USA 351 39% (137/351) 38% (52/137)Murao and Viana11 Brazil 828 9.9% (82/828) 14.6% (12/82)Schonewille et al.9 Netherlands 1,778† NR 30% (541/1778)n: number of transfused patients; *Included anti-KEL7 (anti-Jsb);†Alloimmunized patients (not only SCD); NR: not reported.Table 1. Frequencies of KEL*1,2, KEL*3,4,21 and KEL*6,7 genotypes of patients with hemoglobinopathies and blood donors in Brazil.Group (n) KEL*1/2 KEL*2/2 KEL*3/4 KEL*4/4 KEL*6/6 KEL*6/7 KEL*7/7 n (%) n (%) n (%) n (%) n (%) n (%) n (%)Hemoglobinopathies† (108) 7 (6.5) 101 (93.5) 0 108 (100) 0 7 (6.5) 101 (93.5)Blood donors (205) 13 (6.3) 192 (93.7) 2 (1.0) 203 (99.0) 1 (0.5) 9 (4.4) 195 (95.1)p value > 0.999 0.547 0.430†HbSS:n= 79; HbSC:n=19; HbS-Thal:n= 6; HbCC:n= 4 Letters to the Editor| 1410 | haematologica | 2008; 93(9)References1. Lee S. The value of DNA analysis for antigens of the Kell andKx blood group systems. Transfusion 2007;47:32S-39S.2. Aygun B, Padmanabhan S, Paley C, Chandrasekaran V.Clinical significance of RBC alloantibodies and autoantibod-ies in sickle cell patients who received transfusions.Transfusion 2002;42:37-43.3. Castro O, Sandler SG, Houston-Yu P, Rana S. Predicting theeffect of transfusing only phenotype-matched RBCs topatients with sickle cell disease: theoretical and practicalimplications. Transfusion 2002;42:684-90. 4. Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams A,Lubin B. Alloimmunization in sickle cell anemia and transfu-sion of racially unmatched blood. N Engl J Med 1990;322:1617-21.5. Murphy MT, Fraser RH, Goddard JP. Development of a PCR-based diagnostic assay for the determination of KEL geno-type in donor blood samples. Transfus Med 1996;6:133-7.6. Lee S, Wu X, Son S, Naime D, Reid M, Okubo Y, et al. Pointmutations characterize KEL10, the KEL3, KEL4, and KEL21alleles, and the KEL17 and KEL11 alleles. Transfusion1996;36:490-4.7. Lee S, Wu X, Reid M, Redman C. Molecular basis of the K:6,-7 [Js(a+b-)] phenotype in the Kell blood group system.Transfusion 1995;35:822-5.8. Rosse WF, Gallagher D, Kinney TR, Castro O, Dosik H,Moohr J, et al. Transfusion and alloimmunization in sicklecell disease. Blood 1990; 76:1431-7.9. Schonewille H, van de Watering LMG, Loomans DSE, BrandA. Red blood cell alloantibodies after transfusion: factorsinfluencing incidence and specificity. Transfusion2006;46:250-6.10. Moreira Jr G, Bordin JO, Kuroda A, Kerbauy J. Red blood cellalloimmunization in sickle cell disease: the influence of racialand antigenic pattern differences between donors and recip-ients in Brazil. Am J Hematol 1996; 52:197-200.11. Murao M, Viana MB. Risk factors for alloimmunization bypatients with sickle cell disease. Braz J Med Biol Res 2005;38:675-82.Association of asymmetric dimethylarginine withsickle cell disease-related pulmonary hypertensionPulmonary hypertension (PHT) occurs in approximate-ly 30% of adult sickle cell patients and is associated witha high risk of early death. Hemolysis driven reductions innitric oxide (NO) bioavailability resulting from NO scav-enging by cell free hemoglobin and increased arginaseactivity are of importance in the pathophysiology of SCDrelated PHT.1Elevated plasma concentrations of asymmetricdimethylarginine (ADMA) contribute to limiting NObioavailability in SCD.2 ADMA and symmetric dimethy-larginine (SDMA) derive from the irreversible post-trans-lational methylation of arginine residues by protein argi-nine methyltransferases (PRMT) and are released as freeamino acids upon proteolysis. ADMA (but not SDMA)competitively inhibits NO synthase (NOS) enzymes,thereby limiting NO production. ADMA is degraded bydimethylarginine dimethylaminohydrolases (DDAH)whereas SDMA is mainly cleared renally.3 Elevated plas-ma ADMA concentrations occur in several forms of PHTand are associated to PHT outcome.4,5 We investigatedwhether ADMA concentrations are associated with PHTin SCD. Serum and EDTA plasma samples were available fromadult sickle cell patients consecutively screened for PHTwith echocardiography as previously reported.6 Mild andmoderate-severe PHT are defined as tricuspid regurgitantjet flow velocity (TRV) of 2.5-2.9 m/s and TRV≥3 m/srespectively, with pulmonary-artery pressures considerednormal in patients with trace or no tricuspid regurgita-tion (with TRV assigned 1.3 m/s).1 Plasma concentrationsof ADMA, SDMA, amino acids and serum soluble vascu-lar cell adhesion molecule-1 (sVCAM-1) levels weredetermined as previously described.2,7 For analysis, HbSSand HbSβ0-thalassemia patients were grouped together,as were HbSβ+-thalassemia and HbSC patients. p-values<0.05 were considered statistically significant (SPSS12.0.2, SPSS Inc, Chicago, IL, USA). The study was carriedout in accordance with the principles of the Declarationof Helsinki.Two out of 19 PHT patients had moderate-severe PHT.Hydroxyurea use did not differ between patients withand without PHT and no patients used anticoagulation,calcium antagonists, endothelin receptor blockers orsildenafil. Between group comparisons were only per-Table 1. Demographics and laboratory parameters in sickle cellpatients with and without pulmonary hypertension. HbSS (n=40)/ HbSC (n=16) / HbSβ0-thalassemia (n=6) HbSβ+-thalassemia (n=5) PHT– PHT+ p PHT– PHT+N 28 18 20 1Age (years) 33 (21-44) 28 (22-52) 0.80 29 (23-39) 41Male:female 6:22 5:13 10:10 0:1TRV (m/s) 2.0 (1.3-2.3) 2.7 (2.6-2.8) 2.1 (1.3-2.2) 2.6sPAP*(mmHg) 21 (12-28) 34 (32-43) 21 (12-25) 31Hb (mmol/L) 5.7 (5.0-6.2) 4.9 (4.2-5.9) 0.05 7.0 (6.6-7.6) 6.5HbF (%) 10.6 5.9 0.13 1.0 4(6.1-18.3) (2.2-14.1) (1.0-2.4)LDH (U/L) 369 575 0.02 231 261(300-515) (388-846) (202-359)GFR (mL/min)** 151 120 0.10 140 114(120-195) (66-172) (125-160)ADMA (µmol/L) 0.57 0.63 0.01 0.50 0.48(0.52-0.65) (0.58-0.79) (0.45-0.58)SDMA (µmol/L) 0.47 0.51 0.07 0.46 0.47(0.42-0.55) (0.47-0.83) (0.44-0.56)Arginine (µmol/L) 45 46 0.26 67 56(32-56) (41-62) (57-79)Ornithine (mmol/L) 56 56 0.41 56 44(42-66) (45-75) (49-62)Citrulline (mmol/L) 23 27 0.66 29 32(16-32) (20-32) (24-33)Proline (mmol/L) 208 209 0.84 197 257(162-257) (176-234) (148-247)Arginine/ornithine 0.84 0.93 0.45 1.14 1.1(0.66-1.0) (0.72-1.15) (0.86-1.21)Arginine/citrulline 1.87 1.98 0.84 2.1 1.5(1.66-2.49) (1.43-2.65) (1.6-2.3)Arginine/proline 0.23 0.25 0.41 0.30 0.19(0.18-0.31) (0.19-0.35) (0.21-0.37)sVCAM-1 (ng/mL) 1089 1542 0.007 851 971(801-1239) (1119-1880) (628-1011)Data are presented as medians with their corresponding inter quartile range.A p-value <0.05 is considered statistically significant. *Right ventricular systolicpressure was estimated based on the modified Bernoulli equation (1) andconsidered to be equal to the systolic pulmonary artery pressure (sPAP) inabsence of right ventricular outflow obstruction. **Glomerular filtration (GFR)rate calculated with Cockcroft and Gault-formula (males: creatinine clearance=1.23xweight x (140-age)/serum creatinine, females: creatinine clearance =1.03xweight x (140-age)/serum creatinine). 

Molecular studies reveal a concordant KEL genotyping between patients with hemoglobinopathies and blood donors in São Paulo City, Brazil

Molecular studies reveal a concordant KEL genotyping between patients with hemoglobinopathies and blood donors in São Paulo City, Brazil

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