DNA Variants Involved in Regulation of Fetal Hemoglobin in Mexican Patients with Sickle Cell Disease. a Preliminary Analysis
Authors: Rizo-De La Torre Lourdes Del Carmen , Perea-Díaz Francisco Javier , Aquino Efrain , Venegas Martha , Hernández-Carbajal Carmela , Espinoza-Mata Laura Lucía , Ibarra-Cortés Bertha 
Published: November 2020

HbS (HBB:c.20A&gt;T, beta 6 Glu&gt;Val) is a frequent Hb variant in Mexico, in particular in some regions close to the Pacific and Atlantic Oceans, with carrier frequencies from 0.5 to 12.8%, therefore Sickle Cell Disease patients are often seen in those regions. It is well known that increased levels of Fetal Hemoglobin (HbF) ameliorates the clinical complications of sickle cell disease. Several genetic studies have identifiedBCL11A,HBS1L-MYB(intergenic region),HBG2andHBBP1genes, among others, to be involved in HbF regulation; DNA variants in theselociare associated to elevated HbF. The aim of present study was to analyze 15 variants in HbF regulatorylociin Mexican patients with Sickle Cell Disease.
About 10 mL of peripheral blood was collected in EDTA for hematological and molecular testing from 24 sickle cell anemia patients (S/S) and 15 sickle cell trait carriers (S/A) from Southern Mexico, 13 were from the state of Guerrero and 26 from Chiapas. All subjects voluntarily agreed to participate in this study and gave signed informed consent; underaged patients' consent was obtained from their parents; all procedures were performed according to the ethical principles of the Declaration of Helsinki.
Hemoglobin S genotype was determined by Sanger sequencing; DNA variants genotyping was performed by qPCR using commercial Taqman probes for the following variants: inBCL11Ars11886868, rs4671393, rs7557939, rs1427407, rs766432, rs6706648, rs7599488; inHBS1L-MYBrs7776054, rs28384513, rs9399137, rs4895441, rs9402686, rs1320963, inHBG2rs7482144 and inHBBP1rs10128556. Hematological and clinical data were analyzed by IBM SPSS v24®.
A total of 39 sickle cell patients were studied, twenty-one patients were male and 18 were female (53.8% and 46.2% respectively). Thirty-two were pediatric age patients and 7 adults. Anemia was observed in all S/S patients, eight had severe anemia (&lt;8 g/dL); on the other hand, only 5/15 S/A patients had anemia (10 - 12 g/dL); HbF over 5% was observed in 23/24 S/S patients and in 13/15 S/A had HbF from 2% - 5%. Nineteen S/S patients were treated with hydroxyurea and presented less severe phenotype and more elevated HbF, however, the statistical analysis showed no significant differences (12.9%vs8.9%p=0.248) (Table 1).
Genotype and allele frequencies are displayed in Table 2. All minor alleles were observed in frequencies over 0.05, the mo2st commonly observed minor allele wasBCL11Ars1427407 (0.69), and the less frequently observed wasHBBP1rs101028556 (0.08). The allele frequencies of four HbF regulating variants were significant different from those reported for Mexican ancestry population (MXL) in 1000 Genome database (rs11886868 C&gt;T, rs4671393 A&gt;G, rs7599488 C&gt;T and rs10128556 C&gt;T), however, the analyzed sample in this study is undersized and is not representative of the global Mexican population. No differences were observed when comparing allele frequencies from GuerrerovsChiapas patients, nor S/SvsS/A. The correlation analysis of minor allele count (MAC) and HbF demonstrated no association when comparing it to HbF levels. Likewise, the analysis was performed onBCL11AandHBS1L-MYBvariants and showed no significant correlations. There was also no correlation between MAC and the other hematological parameters (RBC, Hb, PCV, MCV and MCH).
We report the first study of HbF regulating DNA variants in Mexican sickle cell disease patients. The preliminary analysis of HbFvsminor allele count showed no relation of total MAC and increased HbF, neither with the variants ofBCL11AorHBS1L-MYB.

Disclosures
No relevant conflicts of interest to declare.