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vol. 6

Case report
The XmnI polymorphic site 5’ to the gene Gγ in a Brazilian patient with sickle cell anaemia – fetal haemoglobin concentration, haematology and clinical features

Édis Belini Júnior
Rodolfo D. Cançado
Claudia R.B. Domingos

Arch Med Sci 2010; 6, 5: 822-825
Online publish date: 2010/10/27
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Sickle cell anaemia (SCA) is a genetic disorder characterized by a multifaceted pathophysiology that involves changes in erythrocytes, vaso-occlusive processes, chronic inflammatory states, oxidative stress and endothelial dysfunction. Therefore, there is decreased blood flow and obstruction of the microcirculation, which leads to a variety of clinical complications [1].
The reasons for this heterogeneity are not fully understood; however, fetal haemoglobin (HbF) is the best-known and most frequently studied genetic regulator of SCA, because it inhibits HbS polymerization. The individual variation in HbF levels is probably one of the main factors contributing to the clinical heterogeneity observed in these patients [2].
We considered the genotype of SCA and the haplotypes of the beta globin gene cluster in 50 patients with clinical features of sickle cell disease from the Sa~o Paulo state, south-eastern Brazil. We found one patient with HbF levels above the expected and reported values. The presence of the XmnI polymorphic site in the 5' G gene influences HbF expression, and a few studies have been conducted to explain the genetic factors involved in HbF levels in genetically mixed populations, such as that of Brazil. We evaluated the XmnI site and correlated it with HbF values, haplotype and haematological data, and clinical features from a patient with SCA.

Case report

This study analyses a 20-year-old woman of African descent, diagnosed with SCA through electrophoresis tests. She was receiving clinical follow-up care at the Blood Centre of the Santa Casa Medical School in Sa~o Paulo, Brazil. She was not receiving hydroxyurea, or any other drug that induces the expression of HbF. She was not receiving regular blood transfusions, and the only medication in use was folic acid.
According to the haematological data of the patient, she presented with: RBC (2.6 × 106/µl), Hb (7.8 g/dl), Hct (24%), MCV (94.1 fl), MCH (28.9 pg), WBC (8.3 × 103 mm3), neutrophil (61.3%), lymphocyte (19.0%), monocyte (3.0%), eosinophil (2.2%), platelet (539 103 mm3), reticulocyte (10.8%).
The patient never had any chronic manifes­tations of sickle cell disease, such as leg ulcers, osteonecrosis, retinopathy, stroke, cholecystectomy or acute chest syndrome. But in the 3 years prior to our study, she had 5 painful crises with mild intensity that did not require hospitalization.
After the patient gave her consent to participate in this study, we collected blood samples and performed tests.
In order to characterize the genotype of sickle cell disease, we performed cytological tests, electrophoresis in alkaline pH, acid pH and neutral pH, high performance liquid chromatography (HPLC) (VARIANTTM, Bio-Rad Laboratories, CA, USA) and molecular analysis using PCR-RFLP. Next, the amplificated fragment was digested using the FastDigestTM DdeI restriction enzyme (Fermentas, ON, CA) [3]. Haplotypes were determined through the analysis of the following polymorphic restriction sites: G (HindIII), A (HindIII),  (HincII), 3’ (HincII) e 5’ (HinfI), following Sutton et al. [4].
The identification of the polymorphic XmnI site in G (rs7482144) was performed through PCR, followed by restriction analysis using the XmnI enzyme (New England Biolabs) [4].
The test results are shown in Table I. According to the genotype of sickle cell disease, the patient was homozygous for HbS, and she had not inherited the -thalassaemia gene. Her HbF concentration was 15.8%. Because the presence of the XmnI site was observed in only one allele, we investigated other polymorphic sites to verify whether the SNP patterns presented were related to haplotypes listed in the literature.
Based on these findings, we concluded that the patient was HbSS positive with the Bantu/atypical haplotype (+ – – + + –). The atypical allele has a combination of individual polymorphic sites that do not fit the classification described in the literature, 5’ G (XmnI) = +, G (HindIII) = –, A (HindIII) = –,  (HincII) = +, 3’  (HincII) = +, 5’ (HinfI) = –.


The association between the XmnI polymorphism (rs7482144) in the proximal promoter of the G-globin gene and HbF levels is well documented in sickle cell disease patients. In 1985, a polymorphism (C/T at position –158 of G-globin, later named XmnI-HBG2 or rs7482144), was identified and proven to promote the expression of G-chain and to contribute to HbF variability. Other studies in the same decade characterized SNP patterns in and around -globin cluster genes, and found the S chromosome to be associated with five major haplotypes originating from different geographical regions of Africa, the Middle East, and the Indian subcontinent [5].
Of the main haplotypes, the one carrying the Senegal and Arab-Indian characteristics exhibits the polymorphism XmnI, and this is strongly associated with increased levels of Hb F and, consequently, more minimal clinical manifestations, unlike the Bantu haplotype, which is associated with low levels of HbF and haematocrit, and which is clinically more severe [6].
In our study, the SCA patient carried a Bantu allele and an atypical allele (+ – – + + –). She also had an XmnI site, and an HbF concentration of 15.8%. Even with the low haematocrit values, the clinical complications were minimal when compared to other SCA patients and to those with the Bantu haplotype.
An increase in HbF has been associated with minimal clinical manifestations in SCA patients. Leg ulcers are less common in patients who have high levels of HbF [7]; the occurrence of acute chest syndrome and the frequency of painful crises are inversely proportional to HbF levels. Buchanan et al. [8] suggested an association between high HbF levels and a low frequency of leg ulcers. Powars and Hiti [9] and Nagel and Steinberg [10] showed that high levels of HbF were associated with milder SCA symptoms. They also found that a higher expression of HbF in adulthood improves the rates of morbidity and mortality in SCA patients [11]. These results support our observations, in which the patient has a high level of HbF and a milder phenotype, therefore requiring fewer blood transfusions.
We would like to highlight that the XmnI site's contribution to the variation in HbF levels in sickle cell patients is low when compared in isolation; however, Lettre et al. [12] showed that the rs7482144 polymorphism in homozygotes accounted for 2.2% of the variation in levels of HbF in 1275 SCA patients from the African American Cooperative Study of Sickle Cell Disease. However, the sum of five different SNPs – rs4671393 in the BCL11A gene; rs28384513, rs9399137, and rs4895441 in the intergenic region between HBSL1 and MYB on chromosome 6; and rs7482144 in the 5' gene G – explain 20% of the variation of HbF in SCA patients. In this study, it was not possible to evaluate the effect of the polymorphism in the group of patients in Brazil, because the Senegal and Arabic-Indian haplotypes are rare in the Brazilian population.
The polymorphism rs7482144 acts to increase the expression of HbF. Carriers who have the XmnI site also respond better to treatment with hydroxyurea [13], and this shows that new drugs can act in this region in the search for different therapies for the treatment of SCA.
In conclusions, the presence of the XmnI polymorphic site (rs7482144) in patients who are heterozygous for the 5' gene G was responsible for the increased expression of HbF. Although one of the alleles was of the Bantu haplotype, the clinical manifestation was minimal. These results reveal the importance of investigating factors that regulate HbF in sickle cell patients in a genetically mixed population such as that of Brazil.


Financial support was received from the Brazilian Ministry of Health (Grant MS 3072/2007) and the National Council for Scientific and Technological Development (CNPq), Brazil (Grant 132573/2008-3).


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