Microsatellite and Single Nucleotide Polymorphisms in the β-Globin Locus Control Region-Hypersensitive Site 2: Specificity of Tunisian β^SChromosomes

Abstract

The diversity of sickle cell disease severity is attributed to several cis acting factors, among them the single nucleotide polymorphisms (SNPs) and (AT) rich region in the β-locus control region (β-LCR). This contains five DNase I hypersensitive sites (HS) located 6 to 22 kb upstream to the ϵ gene. The most important of these is the HS2 (5' β-LCR-HS2), characterized by the presence of three different SNPs and a microsatellite region known to be in association with β(S) chromosomes in various populations. The aim of this study was to present the molecular investigation of the 5' β-LCR-HS2 site in normal and sickle cell disease individuals in order to determine if there is any correlation or specificity between these molecular markers, the β(S) Tunisian chromosomes and phenotypical expression of sickle cell disease. One hundred and twenty-four chromosomes from Tunisian individuals (49 β(S) carriers and 13 normal individuals) were screened by polymerase chain reaction (PCR) and sequencing for the polymorphic short tandem microsatellite repeats (AT)(X)N(12)(AT)(Y) and the three SNPs (rs7119428, rs9736333 and rs60240093) of the 5' β-LCR-HS2. Twelve configurations of the microsatellite motif were found with an ancestral configuration elaborated by ClustalW software. Normal and mutated alleles were observed at the homozygous and heterozygous states for the three SNPs. Correlation between microsatellites and SNPs suggests that mutant SNP alleles were mainly associated, in the homozygous sickle cell disease phenotype, with the (AT)(8)N(12)GT(AT)(7) configuration, whereas, normal SNP alleles were associated with the (AT)(X)N(12)(AT)(11) configurations in normal β(A) chromosomes. The correlation of these various configurations with Hb F expression was also investigated. The principal component analysis (PCA) showed the correlation between the homozygous sickle cell disease phenotype, mutated SNP alleles and the Benin microsatellite configuration (AT)(8)N(12)GT(AT)(7), which confirmed the specificity of this configuration to the β(S) chromosomes. In addition, the observed high level of Hb F (14.6%) could play a protective role against Hb S to justify the modulation of sickle cell disease severity within the Benin haplotype compared to the other haplotypes. This study highlights the fact that the β-LCR-HS2 could be a genetic marker to identify the ethnic Tunisian β(S) chromosomes and facilitate the molecular diagnosis of sickle cell disease.

Haplotype Map of Sickle Cell Anemia in Tunisia

“…In this study, we used the results previously published in the article by Ben Mustapha et al [13]. Figure 2 shows the existence of several variations in the 5′ β -LCR HS-2 for β S chromosomes and the L6 (AT) 8 N 12 GT(AT) 7 configuration was predominant in the studied sickle cell disease population with 62.11% of the total alleles.…”

“…The following framework polymorphisms were investigated by polymerase chain reaction (PCR) and direct sequencing: (AT) x N 12 (AT) y repeat configurations within the 5′ HS2 region of β -LCR site [12, 13], (TG) n  (CG) m configurations in the IVSII region of fetal globin gene ( G γ and A γ ) [14], and (AT) x T y repeat configuration in the 5′ region of β -globin gene [15] (Figure 1), using respective couples of primers as summarized in Table 1. Tunisian β S RFLP haplotypes (HR) described previously in Imen et al have been used in this study [10].…”

Structural and functional significance of microsatellites

“…Microsatellites can determine activity of the upstream gene regulation elements like Locus Control Region of the Beta-globin gene domain. The (AT)8(N)12GT(AT)7 configuration of microsatellite found in the Hypersensitive site of the structure is associated with a special form of sickle cells -Tunisian βs chromosomes [118]. In some gene clusters the changes in microsatellite structure drastically modify the transcription factor binding and gene switching [119].…”

Fetal Hemoglobin in Tunisian Sickle Cell Disease Patient: Relationship with Polymorphic Sequences Cis to the β-Globin Gene

“…Genomic DNA was isolated from peripheral blood leukocytes by phenol-chloroform extraction method [16]. The following framework polymorphisms were investigated by Polymerase chain reaction (PCR) followed by direct sequencing: (AT)xN12(AT)y repeat configurations within the 5 0 HS2 region of b-LCR site (rs72452842) [17,18], (TG)n (CG)m configurations in the IVSII region of fetal globin gene (Gc (rs141297974) and Ac (rs61080176)) [19] and (AT)xTy repeat configuration in the 5 0 region of bglobin gene (rs372547761) [20] (Fig. 1).…”