Sickle cell anemia (SCA) is a monogenic disorder caused by the homozygosity of a missense mutation (Glu6Val, rs334) in the β-globin gene (HBB), resulting in HbS polymerization upon deoxygenation.1 Although SCA is a Mendelian disorder, the clinical course of the disease can be very heterogeneous. For instance, some subjects present most of the clinical and laboratory sub-phenotypes of the disease, wherefore they require hospitalization, whereas others show only mild symptoms and are followed during routine appointments.2 The reasons that account for the pathophysiological heterogeneity of SCA are not entirely elucidated and studies suggest that this variability may be dictated by genetic variants.3–6

The most well-established genetic modifier of SCA is the persistence of high fetal hemoglobin (HbF, α2γ2) levels into adulthood.7,8 While healthy individuals produce less than 1% of HbF, these values can range between 1% and 30% in SCA patients.1 High levels of HbF lead to a reduction in HbS concentration inside the red blood cells, preventing HbS polymerization and, subsequently, the formation of sickle cells.9 Variabilities in HbF concentrations can be traced back to the β-globin gene (HBB) cluster haplotypes,10 whereby the first genetic variant associated with increased HbF was the Senegal haplotype (rs7482144). In 10 to 50% of patients with SCA and persistence of high HbF levels into adulthood, genetic variants are also found in the BCL11A and MYB genes, which encode hematopoietic transcription factors.1,4 The latter gene is extensively associated with intergenic single nucleotide polymorphisms (SNPs) located on chromosome 6q23, also known as the HMIP2 (HBS1L-MYB intergenic polymorphism (HMIP) block 2). Recently, the downregulation of a long noncoding RNA (HMI-LNCRNA) transcribed from the enhancer region of MYB was associated with a significant increase in HbF levels.11 Additionally, a link between polymorphisms on HMIP2 and high levels of HbF was demonstrated, while their impact on stroke development, which can be a major complication in SCA, has also been reported.12 In contrast to HMIP2, the influence of the HMIP1 on HbF levels and SCA clinical course is still largely unknown. Hence, we evaluated the HMIP1 C-839A (rs9376092), which presented strong linkage disequilibrium with other variants in SCA patients and correlated these findings with clinical and laboratory data.

Sustained postnatal expression of fetal hemoglobin is the main modulator of sickle cell anemia severity.9 The levels of HbF in SCA patients vary considerably, but appear to be constant after the age of 5 years.13,14 The HbF inhibits intracellular HbS polymerization and higher HbF levels are associated with reduced morbidity and mortality in patients with SCA.8 Hence, pharmacological induction of HbF is a logical approach to treat patients with SCA. Although hydroxycarbamide was shown to induce HbF in sickle cell disease over 30 years ago15 and its use is strongly encouraged due to its proven effectiveness,16,17 HbF induction can vary widely among patients on a similar daily dose.18–21 Considering the critical role of HbF in sickle cell anemia, it is pivotal to identify genetic modulators that impact HbF levels, in order to improve therapeutic strategies involving HbF induction.4,22,23

The intergenic region of HBS1L-MYB contains regulatory elements for MYB, which plays an important role in erythropoiesis.24 It is already well established that the overexpression/downregulation of MYB is associated with the inhibition/increment of HBG in adult erythroid cells.25,26 Therefore, the presence of polymorphisms in the HBS1L-MYB intergenic region, such as the rs9376092,27 affects the GATA transcription factor and thereby also MYB expression and HbF levels.27–30 Although polymorphisms in the HBS1L-MYB intergenic region have been associated with erythroid cell parameters, as red blood cells counts and mean corpuscular volume31,32 and HbF levels of patients with β-hemoglobinopathies,4,12,27,29,30 our results did not reveal any link between the HMIP1 C-839A (rs9376092) and clinical or laboratory features of SCA patients. To the best of our knowledge, our work is the first attempt to evaluate the impact of the HMIP1 C-893A (rs9376092) in a SCA context. However, we must bear in mind that only 3% of the patients enrolled in our study (9 out of 299) presented the HMIP1 C-839A AA genotype and that this low frequency may underestimate the real impact of this polymorphism on the HbF modulation.

The impact of HMIP2 polymorphisms on HbF levels appears to be more considerable in Brazilian patients, which is probably due to their genetic admixture,12 while further studies are still required to support this theory. In fact, this genetic admixture can also be the cause for the lack of association seen in our study. In Pernambuco and Rio de Janeiro, European admixture is marked in the general population33,34 and in SCA patients.35 Likewise, high admixture rates (65%) in sickle cell patients measured at the HBS1L-MYB locus was previously demonstrated.12 In this context, European chromosomes 6, in which the HMIP1 rs9376092-A is also more frequent, could have a large impact on our findings. Additionally, on European chromosomes, HMIP1 high-HbF variants are in a negative association with HMIP2 high-HbF variants.36 Thus, patients carrying the HMIP1 rs9376092-A are more likely to also carry the low-HbF genotype at HMIP2 as rs66650371 3bp insertion,37,38 negating the effect of the HMIP1 variant. Therefore, genetic admixture, in combination with the specific linkage disequilibrium at HBS1L-MYB, could be another reason for the lack of association seen in our study.

High levels of fetal hemoglobin ameliorate the clinical outcome of patients with β-hemoglobinopathies and its induction has been a major focus of studies over the last decades. Although we demonstrated a lack of association between the HMIP1 C-893A (rs9376092) in two Brazilian cohorts, these results indicate that a set of environmental and genetic biomarkers can predict disease severity and that this prediction may vary for each population. Naturally, identifying these modulators is important for patients with β-hemoglobinopathies to develop new strategies for alternative therapies to improve the clinical outcome of SCA patients.

D.A.P-M. and I.F.D. performed experiments, analyzed and interpreted data and drafted the manuscript. E.B-J. and J.L.C-S. performed experiments, updated the clinical data and reviewed the manuscript. I.W. updated the clinical data and reviewed the manuscript. A.S.A., C.L.L., C.R.B-D. and M.A.B. recruited patients, updated the clinical data and reviewed the manuscript. A.R.L-A. analyzed and interpreted data, performed statistical analyses and reviewed the manuscript. D.A.P-M., J.L.C-S., and A.R.L-A. conceived and designed the study. A.R.L-A. gave the final approval of the version to be submitted.

The authors declare no conflicts of interest.