Hemoglobin (Hb) Sabine is an unstable Hb variant that causes hemolytic anemia in heterozygous state, with inclusion bodies in the red blood cells (RBC). This hemoglobin is the result of a point mutation at codon 91(CTG)(CCG) of the beta-globin gene. We report, for the first time in South America, the identification of Hb Sabine in a nine-month-old female baby, referred to our laboratory bearing a severe hemolytic anemia. We emphasize the need for the correct characterization of this unstable hemoglobin mainly for therapeutic purposes and for genetic counseling.Fil: Perez, Susana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Bragós, Irma. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Raviola, Mariana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Pratti, Arianna. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Detarsio, Germán. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Zirone, Sandra. Instituto Davoli; ArgentinaFil: Voss, María Eda. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Verón, Luciano. Instituto Davoli; ArgentinaFil: Acosta, Irma. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Ojeda, Mara Jorgelina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentin

Acosta, Irma

Bragós, Irma

Detarsio, Germán

Ojeda, Mara Jorgelina

Perez, Susana

Pratti, Arianna

Raviola, Mariana

Verón, Luciano

Voss, María Eda

Zirone, Sandra

International Blood Research & Reviews

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

_____________________________________________________________________________________________________  *Corresponding author: E-mail: perezsusanamabel@gmail.com;   International Blood Research & Reviews 3(4): 166-170, 2015, Article no.IBRR.2015.019 ISSN: 2321–7219  SCIENCEDOMAIN international                                    www.sciencedomain.org   Severe Hemolytic Anemia Due to De novo Hemoglobin Sabine in an Argentinian Newborn.  First Case in South America  Susana Perez1*, Irma Bragós1, Mariana Raviola1, Arianna Pratti1,  Germán Detarsio1, Sandra Zirone2, Maria Eda Voss1, Luciano Verón2,  Irma Acosta1 and Mara Ojeda1   1Departamento de Bioquímica Clínica, Cátedra de Hematología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. Suipacha 531. Rosario. Santa Fe, Argentina. 2Departamento de Hematología, Instituto Davoli. Laprida 1061. Rosario. Santa Fe, Argentina.  Authors’ contributions   This work was carried out in collaboration between all authors. Authors SP and IB designed the study, wrote the protocol, wrote the first draft of the manuscript, managed the analyses of the study and managed the literature searches. Authors MR, MO, LV, IA and AP performed laboratory tests. Authors AP, GD, SZ and MEV managed the literature searches.  Article Information  DOI: 10.9734/IBRR/2015/18559 Editor(s): (1) Ricardo Forastiero, Department of Hematology, Favaloro University, Argentina. Reviewers: (1) Mathew Folaranmi Olaniyan, Department of Medical Laboratory Science, Achievers University, Owo,  Nigeria. (2) Vlachaki Efthymia, Department of Internal Medicine, Aristotle University, Greece. Complete Peer review History: http://www.sciencedomain.org/review-history.php?iid=1082&id=28&aid=9551    Received 28th April 2015  Accepted 14th May 2015 Published 2nd June 2015   ABSTRACT  Hemoglobin (Hb) Sabine is an unstable Hb variant that causes hemolytic anemia in heterozygous state, with inclusion bodies in the red blood cells (RBC). This hemoglobin is the result of a point mutation at codon 91(CTG)(CCG) of the beta-globin gene. We report, for the first time in South America, the identification of Hb Sabine in a nine-month-old female baby, referred to our laboratory bearing a severe hemolytic anemia. We emphasize the need for the correct characterization of this unstable hemoglobin mainly for therapeutic purposes and for genetic counseling.   Keywords: Abnormal hemoglobin (Hb); Hb sabine; unstable hemoglobin. Case Study     Perez et al.; IBRR, 3(4): 166-170, 2015; Article no.IBRR.2015.019    167  1. INTRODUCTION  Up to date, there have been described more than 140 unstable hemoglobins [1], related to hemolytic anemia with different clinical expression. The mutations that cause destabilization of the tetrameric structure are the most frequent cause of hemolytic anemia. Proline introduction in the alpha helix, beyond the third residue, distorts the hemoglobin structure and cause instability. In general, these undergo increased oxidation of the heme with the formation of met Hb and further degradation leading to precipitated Hb and other products within the circulating red cells. [2] In Hb Sabine, as in many of the other unstable hemoglobins, the substitution is a neutral one [β91(F7)Leu-+Pro], so no alteration in charge of the globin chain would be expected. The diminished anodal mobility of these hemoglobins is probably due to loss of heme, an event to which they are peculiarly prone [3]. Hb Sabine is an unstable β chain variant which causes moderately severe hemolytic anemia and has been reported in a few unrelated patients, none of them from South America till date [4-8]. In this paper, we report a new de novo case of Hb Sabine in a nine-month-old female baby. The mutation was identified by DNA sequencing following amplification by polymerase chain reaction (PCR). The proband’s father is of German origin and her mother is of Spanish descent. The girl has no familial history of anemia. Paternity was confirmed through studies of DNA polymorphism (STRs loci).  1.1 Case Report  The child was admitted to the Service of Pediatrics because of a moderately severe anemia, pallor and a subicteric condition. Her facies indicates expansion of haemopoietic tissue in the skull bones, particularly in the frontal and parietal bones. She had never been transfused. At the age of seven months, she suffered a hemolytic episode in the course of an infection.  Hematological data were obtained with a Sysmex KX21 blood counter. The blood film showed microcytosis, polychromasia, target cells, coarse basophilic stippling, and 2 nucleated erythroid progenitors per 100 white cells. The Hb A2 was measured by elution post electrophoresis at alkaline pH, and Hb F according to the method described by Betke et al. [9].  Isopropanol test (Carrell & Kay) [10] was performed. Cellulose acetate electrophoresis at alkaline pH and globin chains electrophoresis at alkaline pH were carried out using standard methods.  Sickling test was negative. The isopropanol test was positive indicating the presence of an unstable Hb. Cellulose acetate (pH 8.4) electrophoresis detected an additional Hb fraction, between Hb A and Hb A2. (Fig. 1) Globin chains electrophoresis did not show the presence of an abnormal chain.      Fig. 1. Hemoglobin electrophoresis at alkaline pH Hb A Hb X 8% Hb A2 AA control Hb Sabine Trait ß thal. The patient’s hematological and biochemical data are shown in Table 1 and hematological and hemoglobin composition data of parents are shown in Table 2.  DNA was extracted from peripheral blood samples as previously described Polymerase chain reaction (PCR) was carried out and then the coding regions of the βglobin genes were sequenced. It was performed using a Big Dye Terminators Ready Reaction Kit  Table 1. Hematological and biochemical data of the patient Parameters Hb A/X/A2 (%) Hb (g/L) RBC (10l2/L) MCV (fL) MCH (pg) Reticulocytes (%) A2 (%) Hb F (%) Isopropanol Test Sickling Test Total serum bilirrubina (umol/L) LDH U/I Ferritina (ug/dl Iron (ug/dl) TIBC (ug/dl)  Table 2. Hematological and hemoglobin composition data of parents  Subjet Hb g/L RBC (10l2/L) MCV (fL)Father 152 4.8 91.7Mother 134 4.27 88 Fig. 2. Sequence of the -globin gene: CTGPerez et al.; IBRR, 3(4): 166-170, 2015; Article no.IBRR.2015.0 168  [11]. - and α-(Perkin-Elmer Cetus, Norwalk, CT, USA) in an ABI PRISM 310 sequencer (Perkin Alpha1 and 2 genes were normal, while sequencing of  gene revealed a CTG(AspHis) substitution at codon 91, corresponding to Hb Sabine (Fig. 2). sequence analysis of the ß-globinparents showed the absence of the Hb Sabine mutation. Paternity was confirmed by the study of nine short tandem repeats (STRs) and four variable-number tandem repeat (VNTRs) loci. Proband8 770 2.84 95.1 27.1 40 2.6 10  positivenegative35.2 (NR:5.71075 (NR:36084 (NR:24114 (80351 (280 MCH (pg) Reticulocytes (%) A2 (%) Hb F (%) 31.7 1.3 2.4 1  31.4 1.2 2.6 0.9    CCG (AspHis) substitution at codon 91 corresponding to Hb Sabine     19   -Elmer Cetus).   CCG DNA  gene of both    -17) -720) -120) -110) -330)   Hb X (%) 0 0     Perez et al.; IBRR, 3(4): 166-170, 2015; Article no.IBRR.2015.019    169  2. DISCUSSION  Numerous cases of hemolytic anemia have been described that are a consequence of the presence in the erythrocyte of unstable hemoglobins.  Most unstable hemoglobin variants in heterozygous state have a variable percentage of the variant. In our case it was 8% of total hemoglobin. This is a much smaller proportion than that found for most Hb variants, presumably because it is precipitated both in vivo and in vitro during manipulation in the experimental procedures. The erythrocyte containing Hb Sabine, have a markedly shortened survival as a consequence of impaired metabolism, probably due to dissociation and precipitation of abnormal beta polypeptide chains of Hb Sabine. Unstable Hbs are inherited in an autosomal dominant fashion, and virtually all affected individuals are heterozygous. The homozygous state for the unstable variants has not been found.  Our patient, as all Hb Sabine carriers reported so far had significantly increased Hb F levels (HbF:10%) Patients with higher levels of HbF as those refered by Pavlovic S el al. [8] and Panagoula Kollia et al. [12] one had an association with the Xmn I polymorphism at -158 Gγ and the other a compound heterozigocity with non-deletional hereditary persistence of fetal hemoglobin respectively.   Almost all reported patients suffered from severe hemolytic anemia and were splenectomized at early age.   Our patient that now is 17 months is been managed with folic acid and when she was 14 months received one transfusion. Splenectomy cannot be predicted now, it will depend on the transfusion regimen, hypersplenism and how she passes her early childhood. In Pediatrics is ideal to splenectomize after the age of 6.  Even though Hb Sabine is detectable by electrophoresis, its correct characterization requires more sophisticated methods, like high performance liquid chromatography, capillary electrophoresis or molecular analysis.  The so far published cases of Hb Sabine in 1969 [4], 1983 [5], 1992 [6] and 2008 [13] were all de novo presentations. The only inherited case reported in 2004, was the son of a Yugoslavian patient [8]. 3. CONCLUSION   We report this case to emphasize the need for the correct identification of this unstable hemoglobin, mainly for therapeutic purposes and for genetic counseling, especially in therapeutic cases [14-15].  These patients present mild to severe chronic hemolytic anemia that may be exacerbated by stress, especially infections, and treatment with oxidizing agents.  CONSENT   All authors declare that written informed consent was obtained from the patient's parents for publication of this case report.    ETHICAL APPROVAL   All authors hereby declare that all laboratoty tests have been examined and approved by the "Comité de Etica de la Facultad de Ciencias Bioquímicas" and have therefore been performed in accordance with the ethical standads laid down in the 1964 Declaration of Helsinki.    COMPETING INTERESTS  Authors have declared that no competing interests exist.  REFERENCES  1. Available:http://globin.bx.psu.edu//hbvar//menu.html Hb Var: a database of human     hemoglobin variants and thalassemias  (Available:http://globin.cse.psu.edu/hbvar/menu.htm) 2. Beutler E, Lichtman M, Coller B, Kipps T. Hemoglobinopathies associated with unstable hemoglobin, in Wlliams Hemalology (5th ed), edited by. Beutler E, Lichtman MA, Coller BS, Kipps TJ. New York, McGraw-Hill. 1995;650–654. 3. Ueda S, Schneider R. Brief Report: Rapid Differentiation of Polypeptide Chains of Hemoglobin by Cellulose Acetate Electrophoresis of Hemolylsates. Blood. 1969;34:230-235. 4. Schneider RG, Ueda S, Alperin J et al. Hemoglobin Sabine Beta 91 (F 7) Leu → Pro-An Unstable Variant Causing Severe Anemia with Inclusion Bodies. N Engl J Med. 1969;280:739-745. 5. Bogoevski P, Efremov GD, Kezic J, Lam H, Wilson JB, Huisman THJ. Hb Sabine or α2     Perez et al.; IBRR, 3(4): 166-170, 2015; Article no.IBRR.2015.019    170  β2 91(F7) Leu-Pro in a Yugoslavian boy. Hemoglobin. 1983;7:195-200. 6. Gasperini D, Galanello R, Melis MA, et al. Hemoglobin Sabine [β 91(F7) Leu Pro]: occurrence in a Sardinian individual with hemolytic anemia and inclusion bodies. Haematologica. 1992;77:381-3. 7. Hull D, Winter PC, McHale CM et al. Familial hemolytic anemia due to Hb Sabine (beta-91(F7)leu-to-pro) identified by polymerase chain reaction. Hemoglobin. 1998;22:263-266. 8. Pavlovic S, Kuzmanovic M, Urosevic J et al. Severe central nervous system thrombotic events in hemoglobin Sabine patient. Eur J Haematol. 2004;72:67-70. 9. Efremov GD, Huisman THF. Diagnótico   de laboratorio de hemoglobinopatías en Hemoglobinas anormales. En: Weatherall DJ. Clínica Hematológica 2/2. Barcelona: Salvat. 1976;A) 319-320 B) 322-323. 10. Carrell RW, Kay R. A simple method for the detection of unstable haemoglobins. Br J Haematol 1972;23:615-9.  11. Noguera N, Tallano C, Bragós I et al. Modified salting-out method for DNA isolation from newborn cord blood nucleated cells. J Clin Lab Analysis. 2000; 14(6);280-283. 12. Panagoula Kollia, Angelos Kalamaras, Christos Chassanidis, Maria Samara, Nikolaos K. Vamvakopoulos. Compound heterozygosity for the Cretan type of non-deletional hereditary persistence of fetal hemoglobin and β-thalassemia or Hb Sabine confirms the functional role of the Aγ −158 C>T mutation in γ-globin gene transcription. Blood Cells, Molecules, and Diseases 41. 2008;263–264. 13. Kollia P, Kalamaras A, Chassanidis C et al. Compound heterozygosity for the Cretan type of non-deletional hereditary persistence of fetal hemoglobin and β-thalassemia or Hb Sabine confirms the functional role of the Aγ−158 C>T mutation in γ-globin gene transcription. Blood Cells, Molecules, and Diseases. 2008;41:263–264. 14. Eandi Eberle S, Noguera N, Calvo K et al. Severe hemolytic anemia due to hemoglobin Hammersmith. Arch Argent Pediatr. 2009;107(4):347-352. 15. Zanotto MI, Calvo K, Schavartzmanb G et al. Hemolytic anemia due to hemoglobin Evans in an Argentinean family. Arch Argent Pediatr. 2010;108(6):e130-e133.  _________________________________________________________________________________ © 2015 Perez et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.           Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history.php?iid=1082&id=28&aid=9551  

English

Severe Hemolytic Anemia due to De novo Hemoglobin Sabine in an argentinian newborn: first case in South America

Susana Perez

Irma Bragós

Mariana Raviola

Arianna Pratti

Germán Detarsio

Sandra Zirone

Maria Voss

Luciano Verón

Irma Acosta

Mara Ojeda

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Severe Hemolytic Anemia Due to De novo Hemoglobin Sabine in an Argentinian Newborn. First Case in South America

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Severe Hemolytic Anemia due to De novo Hemoglobin Sabine in an argentinian newborn: first case in South America

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