Association of Hemoglobin E-Saskatoon with Hemoglobin S: Report of the First Case Found in Brazil

Hemoglobin (Hb) E-Saskatoon was first described in 1967 by Vella et al. [1] in a Canadian woman of Scottish and Dutch origin. Since then, this variant has been found in other countries such as Greece, Scotland, Spain, and Turkey [2,3,4,5,6]. Hb E-Saskatoon has its name because it was first identified in Saskatoon, Canada, as well as for its electrophoretic similarity with Hb E. This rare variant is the result of a mutation at codon 22 of the β-globin chain, which causes the change of glutamic acid to lysine. This substitution results in a change in molecular charge without affecting its stability, solubility, or functional properties [7,8].The discovery of Hb S by Pauling et al. [9] in 1949 was the first demonstration that the production of an abnormal protein could be the cause of a genetic disorder [9]. This Hb also appears due to a point mutation in the β-globin chain where glutamic acid is converted to valine at codon 6. Hb S presents a different stability and solubility, and when it is in the oxygenated form polymerization of its structure occurs, thus causing deformation and hardening of the erythrocyte membrane, called sickle cell [10]. There are several associations between Hb S and other Hb variants producing different clinical histories that range from asymptomatic to severe hemolytic anemias. Sickle cell anemia (Hb SS) is the most serious form of the disease.The first account of the association between Hb E-Saskatoon and Hb S was described in Greece in a 4-year-old girl. The blood films showed microcytosis, hypochromia, and anisocytosis. Electrophoresis showed the presence of Hb variants with electrophoretic properties of Hb E and Hb S. High-performance liquid chromatography (HPLC) showed the presence of 45.7% of Hb S and 42.6% of an Hb variant which, after molecular analyses, was identified as Hb E-Saskatoon [11].Due to the high degree of miscegenation in Brazil, the incidence of abnormal Hb genes is high and varies according to ethnic groups and environmental factors [12]. Identifying these Hb variants is important for the diagnosis of anemias and for genetic counseling, which is enriching for those who receive it and helps them to make conscious reproductive decisions [13].On this basis, the objective of this paper was to present an account of the first case in Brazil of the identification of compound heterozygosity for Hb E-Saskatoon and Hb S in a girl from a town in Rio Grande do Sul screened in the State Newborn Screening Reference Center, and to further characterize the pattern of Hb E-Saskatoon in the several diagnostic methods.A dried blood spot from a baby of a family from the northeast region of the State of Rio Grande do Sul, in the south of Brazil, was analyzed in the newborn screening laboratory. Samples of peripheral blood from the patient's entire family were collected and stored in tubes with anticoagulant EDTA. The techniques were carried out in the Newborn Screening Reference Center of Rio Grande do Sul and in the Hemoglobin Laboratory of the School of Pharmacy of the Federal University of Rio Grande do Sul (UFRGS). The methods used to identify the Hb variant were: HPLC (Bio-Rad VARIANT™ - Beta Thal Short Program and Sickle Cell Short Program), isoelectric focusing (IEF) (Resolve Hemoglobin Kit; PerkinElmer), and polymerase chain reaction (PCR). For the latter, genomic DNA was extracted from leukocytes of the peripheral blood and the β-globin gene was amplified using primers P1 and P5, with posterior purification and direct sequencing of nucleotides, as described by Kimura et al. [14].The family studied consisted of a female baby (10 months old), her mother (23 years old), her father (age not determined), and her brother (3 years old), all of whom live in Fontoura Xavier in the State of Rio Grande do Sul, Brazil.The newborn screening tests performed in dry blood samples on filter paper showed compound heterozygosity for Hb S and an Hb variant with a retention time (RT) suggestive of Hb D on HPLC and an isoelectric point (pI) suggestive of Hb E on IEF. When the baby and the family were called again for confirmatory tests for the presence of Hb variants, a survey of the Hb variants on filter paper and total blood was performed. The tests results and the Hb identified are shown in table 1.Figure 1 shows the HPLC (Bio-Rad VARIANT) values for Hb F, Hb A2, Hb S, and an unusual peak of 37.3% - RT 4.28, as well as IEF of the baby displaying Hb S and an unusual band pI of 7.60.Following PCR of the β-globin gene, it was verified that the patient had a compound heterozygosity for Hb S (β-globin gene in codon 6 ′GAG > GTG or glutamic acid - valine) and for Hb E-Saskatoon (β-globin gene codon 22 ′GAA > AAA or glutamic acid - lysine). The same genotype was found in the patient's brother. The father was identified as a carrier of Hb E-Saskatoon and the mother as a carrier of Hb S (fig. 2).In the literature there are accounts of an Hb variant with electrophoretic mobility very similar to that of Hb E [15,16,17,18]. It usually has fully normal hematological parameters and slow electrophoretic mobility, with a high prevalence in Southeast Asia. Additional HPLC analyses showed that this Hb variant was eluted after Hb A2, while Hb E could not be separated from Hb A2 by the same procedure. In the end the presence of Hb E-Saskatoon was characterized.In the only report in the literature of an association between Hb S and E-Saskatoon, at the time of reporting the girl was healthy, with normal development, and asymptomatic [11]. To date, the two affected children in Rio Grande do Sul are asymptomatic, but additional studies are being conducted to better understand the association of Hb in our midst.Therefore, the difficulty to identify rare Hb variants by common laboratory procedures is evident. Our results demonstrated that Hb complex profiles could be properly characterized using complementary diagnostic tools not used in everyday laboratory practice. It is necessary to associate classical electrophoretic methods with chromatographic analyses, and molecular analysis is often crucial as previously described in the characterization of the genotype of these Hb variants, thus enabling a precise diagnosis of its expression. The correct diagnosis is essential to establish the prognosis, treatment strategies, and genetic counseling.

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