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Hematology, Transfusion and Cell Therapy

Print version ISSN 2531-1379On-line version ISSN 2531-1387

Hematol., Transfus. Cell Ther. vol.41 no.4 São Paulo Oct./Dec. 2019  Epub Nov 25, 2019

http://dx.doi.org/10.1016/j.htct.2019.02.002 

Case Reports

Interaction between hemoglobin S and N-Baltimore: a case report in Pernambuco, Brazil

Thais Helena Chaves Batistaa 

Marcondes José de Vasconcelos Costa Sobreiraa 

Rodrigo Marcionilo de Santanaa 

Gabriela da Silva Arcanjoa 

Camila Mabel de Albuquerque Ramosa 

Dulcinéia Martins Albuquerqueb 

Aderson da Silva Araujoc 

Ana Cláudia Mendeonça dos Anjosc 

Fernando Ferreira Costab 

Antonio Roberto Lucena-Araujoa 

Marcos André Cavalcanti Bezerraa  * 
http://orcid.org/0000-0002-0887-7793

aUniversidade Federal de Pernambuco (UFPE), Recife, PE, Brazil

bUniversidade de Campinas (Unicamp), Campinas, SP, Brazil

cFundação de Hematologia e Hemoterapia de Pernambuco (HEMOPE), Recife, PE, Brazil


Introduction

Mutations in the globin genes that lead to amino acid substitutions in the globin chains result in hemoglobin (Hb) variants, and among these, hemoglobin S (Hb S) is the most well-known and frequent member worldwide, particularly in Brazil.1 Another variant is the Hb N-Baltimore (also known as Hb Jenkins, Hopkins-1, N-Memphis, and Kenwood), first described in 1958 by Smith and Torbert in an African-American family.2 This variant results from a mutation in codon 95 of the β globin chain, replacing the lysine amino acid for glutamic acid, which promotes a faster electrophoretic mobility of Hb N-Baltimore, in contrast with Hb A in alkaline pH.3 Using electrophoresis of hemoglobin at alkaline pH, high-performance liquid chromatography (HPLC) and sequencing procedures, we noticed an extremely rare interaction between the variant Hb S and Hb N-Baltimore in a patient admitted to a reference center in northeastern Brazil.

Methods

Blood samples were collected, with EDTA as the anticoagulant, from the child and family members after obtaining their informed consent. Hematological parameters were measured using a Coulter STKS (Coulter Electronics, Hialeah, FL, USA) and the erythrocyte morphology was analyzed. Biochemical parameters quantifications were performed by the Cobas C501 analyzer (Roche Diagnostics (®), Meylan, France). The methodological procedures applied included screening tests and validation for the diagnosis of hemoglobinopathy. Electrophoresis of hemoglobin at alkaline pH in cellulose acetate was executed, following which the Hb profile was investigated by cation-exchange high-performance liquid chromatography (Variant II™, Bio-Rad Laboratories, Hercules, CA, USA), as per the manufacturer's instructions. To confirm the presence of Hb S, the solubility test was executed.4 Sequencing procedures were performed on the ABI PRISM® 3500 Genetic Analyzer for identification of the unknown Hb.

Case report

A 2-month-old female child was admitted to the Hospital of Hematology and Hemotherapy Foundation of Pernambuco in July 2015, after variant hemoglobin was identified during the newborn screening test. The peripheral blood analysis showed normocytic/normochromic anemia (Table 1). The electrophoresis of hemoglobin at alkaline pH revealed migration of a slower band than hemoglobin A (Hb A), which is suggestive of Hb S-like and was confirmed by a solubility test, and a faster band than Hb A, co-eluent to Hb J (Figure 1A). The quantification by cationic exchange HPLC, showed 56% of fetal hemoglobin (Hb F), 13% of Hb S, 2% of Hb A2, and 29% of unknown Hb (so-called Hb X), with a retention time of 1.58 min (Figure 1B). Screening for hemoglobin variants through sequencing of the β globin gene revealed in heterozygosis a β95 Lys>Glu; HBB: c.286A>G, characterizing Hb N-Baltimore, along with the mutation β6 Glu>Val HBB: c.20A>T, corresponding to Hb S (Figure 1D). Further molecular analyses of the parents, both of African descent, showed the presence of Hb S in heterozygosis in the father, and Hb N-Baltimore, also in heterozygosis, in the mother.

Table 1 Summary of hematologic and biochemical data of the patient and his family members. 

Parameters Proband Proband Reference (1-3 years) Father Mother Reference (male adult) Reference (female adult)
Age 2 months 3 years 25 years 26 years
RBC (×106/µL) 3.25 4.69 4.0-5.1 5.24 3.67 4.5-6.1 4.0-5.4
Hb (g/dL) 9.7 12.2 10.6-13.0 15.3 12.1 12.8-16.3 11.3-14.5
Hct (%) 29.5 36.3 33.0-41.0 44.6 35.7 40-54 36-48
MCV (fL) 90.0 77.4 75.0-90.0 85.1 97.3 77-92 77-92
MCH (pg) 29.8 26.0 25.0-29.0 29.2 33.0 27-29 27-29
MCHC (g/dL) 32.8 33.6 30.0-35.0 34.3 33.9 30-35 30-35
RDW (%) 14.1 13.4 10.0-15.0 13.0 11.4 10-15 10-15
WBC (×103/µL) 8.0 6.4 5.0-15.0 4.2 3.6 4.0-11.0 4.0-11.0
Platelets (×103/µL) 206 401 140-450 176 320 140-450 140-450
Ret (%) NA 0.2 0.5-1.5 0.3 3.1 0.5-1.5 0.5-1.5
SF (ng/mL) NA 55.5 7.0-140 157.4 109.8 30-400 13-150
SI (µg/dL) NA 66.0 37-145 62.0 96.0 59-158 37-145
TIBC (µg/dL) NA 295.0 250-425 269 271 250-425 250-425
TS (%) NA 22.4 20-50 23.0 35.4 20-50 20-50
TB (mg/dL) NA 0.12 ≤1.2 0.45 0.34 ≤1.2 ≤1.2
IB (mg/dL) NA 0.06 ≤0.8 0.26 0.17 ≤0.8 ≤0.8
LDH (U/L) NA 260.0 120-300 184 140 135-225 135-214

RBC: red blood cells; Hb: hemoglobin; Hct: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW: red cell distribution width; WBC: white blood cells; Ret: reticulocyte count; SF: serum ferritin; SI: serum iron; TIBC: total iron binding capacity; TS: transferrin saturation; TB: total bilirubin; IB: indirect bilirubin; LDH: lactate dehydrogenase; NA: not available.

Figure 1 (A) Electrophoresis of hemoglobins at pH 8.0. 1: Hb SS, 2: Hb AA, 3: Hb SF, 4: Hb AJ (mother), 5: Hb AS (father), 6: Hb SJF↑ (patient at two months), 7: Hb AS. (B) HPLC of the patient at two months showing Hb F, unknown Hb, Hb A2 and Hb S. (C) HPLC of the patient at three years showing Hb F, unknown Hb, Hb A2 and Hb S. (D) Electropherogram of the β globin gene. Sequencing of the β globin gene revealed a mutation in the codon 95 (β95 Lys>Glu; HBB: c.286A>G) in heterozygosis, characterizing the Hb N-Baltimore variant, as well as a mutation in codon 6 (β6 Glu>Val HBB: c.20A>T) corresponding to Hb S. 

Three years later, subsequent HPLC showed 7% of Hb F, 32.9% of Hb S, 2.8% of Hb A2 and 57.3% of Hb N-Baltimore, with a retention time of 1.58 min (Figure 1C). Hematoscopy revealed erythrocyte morphology within the parameters of normality and the biochemical data was normal (Table 1). The child was asymptomatic, weighing 14 kg, and measuring 100 cm, with eutrophic nutritional status (BMI percentile = 50), blood pressure at 105/71 and spleen not palpable, as well as dullness to percussion over the left intercostal Traube's space. To date, the child is annually followed up by the sickle cell disease (SCD) clinical program, but no SCD prophylactic therapy was indicated, as the patient is asymptomatic.

Discussion

The Hb N-Baltimore results from a mutation in codon 95 of the β globin chain, replacing the lysine amino acid for glutamic acid β95 Lys>Glu; HBB: c.286A>G, which promotes a faster electrophoretic mobility, compared to the Hb A in alkaline pH, but does not present a difference, as to migration at acidic pH. In addition, Hb N-Baltimore has normal oxygen affinity and stability and an excellent separation by IEF (isoelectric focusing).3,5

A previous study showed that the interaction of Hb N-Baltimore with Hb S in three children in France resulted in no alteration of clinical symptoms and normal hematologic and biochemical parameters.6 This variant was also described in association with β-thalassemia and Hb C, and although Hb N-Baltimore accelerates Hb C crystallization and contributes to abnormalities in erythrocyte morphology, this combination leads to a mild clinical course in the carriers.7-9 Although it has no clinical relevance, little is known about the interaction between Hb N-Baltimore and Hb S.6 Thus, this report is the first to elucidate this interaction in South America. In the case we studied, the child is clinically healthy 3 years after diagnosis. Nevertheless, owing to the short clinical follow-up of the patient, long-term complications cannot be predicted. The clinically healthy status of the patient may lie in the protective effect of Hb F, as it prevents the Hb S polymerization; however, later analyses showed a decreasing percentage of Hb F and higher levels of the Hb N-Baltimore/Hb S ratio, which may presuppose a dominant effect of Hb N-Baltimore over the Hb S. This finding can be explained by the high stability of the Hb N-Baltimore,10 which presents similar biochemical features to Hb A, including the affinity for alpha globin chains.11 For this reason, possible intermolecular cooperation between the Hb S and Hb N-Baltimore variants should be further explored.

In summary, we herein describe a rare case of interaction between Hb N-Baltimore and Hb S in a health carrier, which was identified through electrophoretic analysis, HPLC and sequencing methodology. The association of these methodological procedures is useful in the identification of rare variant hemoglobins and aids in the avoidance of erroneous diagnoses, thus leading to an appropriate clinical approach. In addition, the study of these interactions enhances the knowledge on the pathophysiology of sickle cell disease.

References

1 Arduini GA, Rodrigues LP, De Marqui AB. Mortality by sickle cell disease in Brazil. Rev Bras Hematol Hemoter. 2017;39(1):52-6. [ Links ]

2 Smith EW, Torbert JV. Study of two abnormal hemoglobins with evidence for a new genetic locus for hemoglobin formation. Bull Johns Hopkins Hosp. 1958;102:38-45. [ Links ]

3 Bonini-Domingos CR, Da Silveira EL, Viana-Baracioli LM, Canali AA. Hemoglobin N-Baltimore characterization in a blood donor of São José do Rio Preto, SP, Brazil. J Bras Patol Med. 2003;39(1):41-4. [ Links ]

4 Dacie JV, Lewis SM. Practical haematology. Edinburgh: Churchill Livingstone; 1995.5 [ Links ]

5 Huisman TH, Carver MF, Efremov GD. A syllabus of human hemoglobin variants. The Sickle Cell Anemia Foundation; 1996. [ Links ]

6 Fabritius H, Lacroix H, Le Corroller Y. Clinical hematological and biochemical pattern of three SN Baltimore hemoglobin carriers. Med Trop (Marte). 1981;41(2):147-50. [ Links ]

7 Schneider RG, Hightower B, Hosty TS, Ryder H, Tomlin G, Atkins R, et al. Abnormal hemoglobins in a quarter million people. Blood. 1976;48(5):629-37. [ Links ]

8 Johnson C, Powars D, Schroeder WA. A case with both hemoglobins C and N-Baltimore. Acta Haematol. 1976;56:183-8. [ Links ]

9 Hirsch RE, Witkowska HE, Shafer F, Lin MJ, Balazs TC, Bookchin RM, et al. HbC compound heterozygotes [HbC/Hb Riyadh and HbC/Hb N-Baltimore] with opposing effects upon HbC crystallization. Br J Haematol. 1997;97(2):259-65. [ Links ]

10 Ballas SK, Park DK. Biosynthetic evidence for stability of Hb N-Baltimore. Hemoglobin. 1985;9(5):489-94. [ Links ]

11 Abraham EC, Huisman TH. Differences in affinity of variant β chains for α chains: a possible explanation for the variation in the percentages of β chain variants in heterozygotes. Hemoglobin. 1977;1(8):861-73. [ Links ]

Received: September 21, 2018; Accepted: February 18, 2019; Published: May 12, 2019

*Corresponding author at: Department of Genetics, Federal University of Pernambuco, Av. Prof. Moraes Rego, 1235, Recife, PE 50670-901, Brazil. E-mail address: macbezerra.ufpe@gmail.com (M.A. Bezerra).

Conflicts of interest

The authors declare no conflicts of interest.

Author contributions

All authors read and approved the manuscript.

Creative Commons License This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivative License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited and the work is not changed in any way.