Hemoglobin, 30 (3):401–403, (2006)Copyright © Taylor & Francis Group, LLCISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260600755930

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LHEM0363-02691532-432XHemoglobin, Vol. 30, No. 3, May 2006: pp. 0–0Hemoglobin

SHORT COMMUNICATION

Hb SOUTHAMPTON [b106(G8)Leu®PRO, CTG®CCG] IN AN

ARGENTINEAN BOY

Hb Southampton in an Argentinean BoyS. Eandi Eberle et al.

Silvia Eandi Eberle,1 Nélida I. Noguera,2 Gabriela Sciuccati,1 Mariana

Bonduel,1 Lilian Díaz,1 Raquel Staciuk,1 and Aurora Feliu-Torres1

1Servicio de Hematología-Oncología, Hospital de Pediatría “Prof. Dr. Juan P. Garrahan”, Buenos Aires, Argentina2Departamento Bioquímica Clinica, Cátedra de Hematología, Facultad de Ciencias Bioquímica y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina

� Hb Southampton (also known as Hb Casper) is characterized by the substitution of a leucineresidue for a proline at codon b106 (CTG®CCG). This mutation breaks the G helix and severelydistorts the tertiary structure of the molecule, producing an unstable hemoglobin (Hb) and severehemolysis. We identified this hemoglobinopathy in a young patient with severe hemolytic anemiaand hepatosplenomegaly.

Keywords Unstable hemoglobin (Hb), Abnormal Hb, Hemolytic anemia

In the past two decades, new molecular methods have increased ourunderstanding of hemoglobin (Hb) variants. Out of 900 variants, more than25% have been designated as unstable (1,2). Hb Southampton (also knownas Hb Casper) [β106(G8)Leu→Pro, CTG→CCG] was reported by Hyde et al.in 1972, by Koler et al. in 1973 and by Didkowskii et al. in 1976 (3–5).

We observed the same variant in a 4-year-old boy, referred to our labora-tory because of hemolytic anemia and hepatosplenomegaly. The patientwas born at term after a normal pregnancy and delivery. No abnormalitieswere detected during the neonatal period. He had a history of multiple epi-sodes of pallor, jaundice and dark urine related to infections since he was

Received 1 February 2006, accepted 2 March 2006.Address correspondence to Dr. Aurora Feliu-Torres, Servicio de Hematología-Oncología, Hospital de

Pediatría “Prof. Dr. Juan P. Garrahan”, Combate de los Pozos 1881, (C1245AAM), Buenos Aires, Argentina;Tel.: +54-11-4308-4300, Ext: 1301 & 1597; Fax: +54-11-4308-5325; E-mail: afeliutorres@yahoo.com;afeliu@garrahan.gov.ar

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402 S. Eandi Eberle et al.

18 months old, receiving several red blood cell (RBC) transfusions. Onadmission at our Institution, his physical examination revealed a peculiarfacial appearance with bossing of the skull, hypertrophy of the maxilla,prominent malar eminences and a mongoloid slant of the eyes, generalizedicterus, pallor and marked hepatosplenomegaly.

Hematological data were obtained with a Coulter Counter Model JT(Coulter Corporation, Hialeah, FL, USA). Hb A2 was measured by anionexchange chromatography (6) and Hb F according to the method describedby Betke et al. (7). Isopropanol and heat stability tests, Heinz bodies stain, cel-lulose acetate electrophoresis at alkaline pH and citrate agar electrophoresisat pH 6.0, were carried out using standard methods (8).

DNA was extracted from peripheral blood samples by standardmethods. The coding regions of the β-globin gene were amplified in twosegments, and polymerase chain reaction (PCR) amplification was accom-plished according to conditions already described (9). Hematologicaldata of the patient and his mother are shown in Table 1. The putativefather was excluded by molecular analysis using GenePrint® STR Systems(Promega Corporation, Madison, WI, USA). Severe anisocytosis, hypo-chromia and microcytosis with prominent basophilic stippling were foundin the proband. He had reticulocytosis (46%); unconjugated bilirubin544 μmol/L (normal range 51–170 μmol/L); total bilirubin 748 μmol/L(normal range 68–238 μmol/L); haptoglobin <5 mg/dL (normal range70–378 mg/dL); LDH 1279 UI/L (normal range 137–415 UI/L). Thebrilliant cresyl blue technique showed inclusion bodies in the RBCs. Theisopropanol and the heat tests were positive, indicating the presence of anunstable Hb.

Electrophoretic techniques detected no abnormal Hb, however,sequencing of the β gene showed a substitution of CTG→CCG at codon106, corresponding to Hb Southampton. This Hb is characterized by a sub-stitution of Leu→Pro at position 106(G8) of the β chain.

TABLE 1 Hematological Data

Parameters Proband Mother

Hb (g/dL) 6.4 13.4PCV (L/L) 0.217 0.400RBC (1012/L) 2.91 4.64MCV (fL) 74.7 86.1Reticulocytes (%) 46.0 1.5Hb A2 (%) 2.9 3.4Hb F (%) 7.0 0.8Inclusion bodies [+] [−]Isopropanol test [+] [−]Heat test [+] [−]

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Hb Southampton in an Argentinean Boy 403

Hb Southampton [β106(G8)Leu→Pro] was first described by Hyde et al.in 1972 (2). Here, the substitution of a proline residue for leucine at position106(G8) of the β chain interrupts the helical sequence and severely distortsthe tertiary structure of the molecule at a point where there is direct contactwith heme (10).

Hb Southampton is electrophoretically silent with standard procedures,although separation with isoelectric focusing (IEF) is possible. Molecularanalyses are mandatory for complete characterization of this variant. Wewere not able to ascertain if, in this case, there was a de novo mutation or adominant pattern of inheritance because the paternity test resulted in nopaternity, ruling out the putative father.

REFERENCES

1. Hardison RC, Chui DHK, Riemer CR, Miller W, Carver MFH, Molchanova TP, Efremov GD,Huisman THJ. Access to A Syllabus of Human Hemoglobin Variants (1996) via the world wide web.Hemoglobin 1998; 22(2):113–127 (http://globin.cse.psu.edu).

2. Huisman THJ, Carver MFH, Efremov GD. A Syllabus of Human Hemoglobin Variants (SecondEdition). Augusta: The Sickle Cell Anemia Foundation. 1998 (http://globin.cse.psu.edu).

3. Hyde RD, Hall MD, Wiltshire BG, Lehmann H. Haemoglobin Southampton, β106(G8)Leu→Pro:an unstable variant producing severe haemolysis. Lancet 1972; 2(7788):1170–1172.

4. Koler RD, Jones RT, Bigley RH, Litt M, Lovrien E, Brooks R, Lahey ME, Fowler R. HemoglobinCasper: β106 (G8) Leu→Pro; a contemporary mutation. Am J Med 1973; 55(3):549–558.

5. Didkowskii NA, Idel’son LI, Filippova AV, Lemann G. A new case of the unstable HemoglobinSouthampton-Casper (β106) (G8) leucine→proline. Probl Gematol Pereliv Krovi 1976; 21(6):48–50.

6. International Committee for Standardization in Haematology: recommendations for select meth-ods for quantitative estimation of Hb A2 and for Hb A2 reference preparation. Br J Haematol 1978;38(4):573–578.

7. Betke K, Marti HR, Schlicht I. Estimation of small percentages of foetal haemoglobin. Nature 1959;184(Suppl 24):1877–1878.

8. Dacie JV, Lewis SM. Practical Haematology, 9th ed. London: Churchill Livingstone. 2001.9. Noguera NI, Cardozo MA, González FA, Benavente C, Milani AC, Villega A. Hb Agenogi

[β90(F6)Glu→Lys] in an Argentinean girl. Hemoglobin 2002; 26(2):201–203.10. Perutz MF. Stereochemistry of cooperative effects in haemoglobin. Nature 1970; 228(273):726–739.

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