Early Complication in Sickle Cell Anemia Children due to A(TA)nTAA Polymorphism at the Promoter of UGT1A1 Gene

Aim. To determine the implication of the polymorphism, namely, A(TA)nTAA of UGT1A1 in lithogenesis for the first time in Tunisia among sickle cell anemia (SCA) children patients. Material and Methods. Our study was performed in 2010 and it involved 76 subjects chosen as control group characterized with normal hemoglobin status and presence of cholelithiasis and 102 SCA pediatric patients among whom 52 have cholelithiasis. We analyzed the polymorphism A(TA)nTAA at the UGT1A1 promoter and the relationships between the various A(TA)nTAA genotypes and alleles and bilirubin levels and occurrence of cholelithiasis. Results and Discussion. The repartition of genotypes found according to serum bilirubin level shows a significant association between genotypes carrying variant (TA)7 and hyperbilirubinemia (P < 0.05). We demonstrated the association of two genotypes with gallstones formation among SCA children patients: (TA)7/(TA)7 and (TA)7/(TA)8 with P = 8.1 × 10−8 and P = 0.01, respectively. (TA)7 and (TA)8 allele variants act as a risk factor for early gallstones formation in SCA patients with P = 5.8 × 10−9 and P = 0.01, respectively. As for the control group only the genotype (TA)7/(TA)7 presented a risk factor for gallstones formation. Conclusion. The novelty of this report is that it is the first time that a similar study was made on the Tunisian children sickle cell population and that the results show a clear association of (TA)7 variant in early gallstones formation in Tunisian SCA children. Interestingly our findings highlighted the association of (TA)8 variant as well, which was not found in previous studies.


Introduction
SCA is a heterogeneous monogenic disease due to a single mutation A/T at the sixth codon of the -globin gene ( S) [1]. The clinical complications arising from sickle cell disease include vasoo-cclusive crisis and its outcomes [1]. As a result of chronic hemolysis, hyperbilirubinemia is often observed leading to the formation of pigment cholelithiasis which could be busted by the presence of UGT1A1 defects. Indeed UGT1A1 gene encodes the uridine diphosphate glucuronosyltransferase 1A1, enzyme responsible for bilirubin glucuronidation [2]. The UGT1A1 gene is located in chromosome 2q37 [3]. Various UGT1A1 gene defects and polymorphisms have been described so far at the origin of reduced enzyme activity [4]. Among these, a variation in the number of TA repeat at the A(TA) 6 TAA nucleotide sequence in the promoter region, considered as the wild type. In fact, the addition of an extra (TA) at this sequence leads to a variant A(TA) 7 TAA which was described to cause reduced glucuronidation and hyperbilirubinemia associated with the Gilbert syndrome [2,5]. This variation at the promoter seems to interfere with binding of the transcription factor IID which is responsible for the transcription of UGT1A1 gene. In fact, The A(TA) TAA element is the binding site for transcription factor IID, which is one of the factors responsible for the initiation of transcription and the presence of this longer A(TA) TAA element in the promoter region of the gene for bilirubin UDP-glucuronosyltransferase 1 resulting in reduced expression of bilirubin-UGT1 (30% of normal) and hence causing unconjugated hyperbilirubinemia [3]. Studies of a 68 Disease Markers possible association between polymorphisms of candidate genes related to the modulation of clinical complications of SCA have shown that sickle cell patients who carry the variation (TA) 7 are favorable for gallstone formation [4][5][6][7][8][9][10][11]. Besides, other studies have shown the correlation of cholelithiasis and A(TA) 7 TAA variant of UGT1A1 promoter with chronic hemolytic diseases such as thalassemia minor, which represent a risk factor for cholelithiasis and the Gilbert mutation further increases this risk [12][13][14][15][16]. The prevalence of cholelithiasis observed in SCA children is about 30% reported for different ethnical groups (United States, Guadeloupe) [17,18].
In this paper, we intend to study the impact of A(TA) TAA variation at the UGT1A1 gene promoter on hyperbilirubinemia and on the occurrence of cholelithiasis for the first time among SCA Tunisian children. SCA is the second sickle cell hemoglobinopathy after -thalassemia in Tunisia, representing a real public health problem. The average frequency of the trait in our country is 1.89% [19]. The organization of care of sickle cell disease children in Tunisia is the National Center of Bone Marrow Transplantation.

2.1.
Subjects. 76 subjects with cholelithiasis and 102 sickle cell patients were involved in this study performed in 2010. Patients were selected on the basis of homozygosity forglobin gene from National Center of Bone Marrow Transplantation, Tunis, Tunisia. All SCA patients are children (less than 16 years old) and were characterized by hyperbilirubinemia and 52 of them have cholelithiasis.

Clinical Events
Analyzed. Liver/biliary ultrasound scans were performed annually to detect cholelithiasis only in SCA patients over the age of three years. Cholelithiasis was diagnosed for all patients on the basis of echodense images within the gallbladder with acoustic shadowing or gravitational change in position.

Laboratory Methods.
Diagnosis of sickle cell patient is performed using cation-exchange high-performance liquid chromatography (HPLC) (D10 Biorad) and further confirmation by means of molecular diagnosis by restriction fragment length polymorphism (RFLP) using DdeI as previously described by Bachir 2000 [20]. Biochemical data were averaged for each patient in steady state (at least three values). We determined total and fetal hemoglobin (HbF) concentrations (D10, Biorad) and reticulocyte count and other hematologic parameters using (ABX pentra 60c+). Total unconjugated and conjugated bilirubin concentrations in serum were determined by a standardized colorimetric procedure (Cobras Integra, Meylan, France).

A(TA) TAA Genotyping.
Genomic DNA was isolated from white blood cells of total blood using standard method (phenol/chloroform). A(TA) TAA sequences were genotyped by polymerase chain reaction (PCR) using a couple of primers, namely, TAF: 5 -TCGTCCTTCTTCCTCTCTGG-3 and TAR: 5 -TCCTGCTCCTGCCAGAGGTT-3 . Polymerase chain reaction was performed in 25 L reaction volumes containing 100 ng of genomic DNA, 0.2 mmol/L of each dNTP, 50 mmol/L KCl, 15 mmol/L Tris-HCl PH 8.0, 2.5 mmol/L MgCl 2 , 0.5 U AmpliTaq polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA), and 10 pmol of each forward and reverse primers. The PCR cycling conditions included an initial denaturation of 10 min at 96 ∘ C followed by 35 cycles of 96 ∘ C for 30 s, annealing at 58 ∘ C for 30 s, and extension at 72 ∘ C for 1 min. The run was ended by a final extension at 72 ∘ C for 7 min.
PCR products were then purified and doubly sequenced (forward and reverse) by ABI PRISM Big Dye Terminator on Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) and an ABI 310 DNA sequencer (PEApplied Biosystems, Foster City, USA).

Data Analysis.
The sample of SCA patients was divided into two groups according to the presence or absence of cholelithiasis. 76 patients with normal hemoglobin (AA) and presented cholelithiasis were enrolled in the analysis. We compared demographic and hematological and clinical data between the groups of patients. As for A(TA) TAA polymorphism genetic differences between the groups were evaluated. We defined two intervals of total bilirubin levels. The first includes total bilirubin value <35 mol/L which is the critical value of total bilirubin associated with the Gilbert syndrome. The second interval includes bilirubin value higher than the cutting point 35 mol/L. We investigated the relationships between genotypes found and these intervals.

Statistical Analysis.
The demographic and hematologic data are normally distributed, so we used means and standard deviations. The bilirubin data are not normally distributed, so we used medians. For each variable (demographic, hematological, and biochemical) differences between cases and controls were evaluated applying the t-test or the nonparametric Mann-Whitney test as appropriate using SPSS (version 18). The Hardy-Weinberg equilibrium was tested using the software package Arlequin (version 3.01). Genetic differences between cases and controls were evaluated applying exact tests to genotypic or allelic contingency tables using compare 2 (version 1.02). The relationships between genotypes found and total bilirubin level were evaluated applying Fisher's exact test using compare 2 (version 1.02). We calculated values for the entire tests and Fisher's exact test and chi-squared test were used as appropriate.

Demographic, Hematological, and Biochemical Analysis.
The distribution of each continuous variable was performed using the nonparametric Mann-Whitney test. Our results show that there is no significant difference between the two groups of SCA patient according to the presence or the absence of cholelithiasis ( > 0.05), whereas, the comparison of total conjugated and unconjugated bilirubin concentrations between the two groups of SS children patients shows a significant difference with < 0.05. Our findings show a significant difference between SCA patients and patients with cholelithiasis considered as control group with < 0.05 (Table 1).  Table 2. The comparison of these genotypes according to the number of (TA) reported that the genotypes (TA) 7 /(TA) 7 and (TA) 7 /(TA) 8 were significantly associated with SCA patients with gallstones ( < 0.05). Our results show a significant association between genotype (TA) 7 /(TA) 7 and gallstones in the control group. Moreover, (TA) 7 and (TA) 8 allelic variants are found to be associated with gallstones in SCA children patients ( < 0.05) Table 2. Association is not found in the control group.

Discussion
In the current study we tested 102 SCA Tunisian children patients among whom 52 have cholelithiasis and 76 subjects were chosen as control group characterized with normal hemoglobin status and presence of cholelithiasis and analyzed the polymorphism at the promoter and the relationships between the various UGT1A1 promoter genotypes and alleles and bilirubin levels. In a previous study we were interested to determine the frequency of A(TA) TAA and    : index of significance, each < 0.05 is considered as significant.
Gly71Arg of UGT1A1 in a healthy population [21]. The polymorphism A(TA) TAA showed that genotype (TA) 7 /(TA) 7 described as being associated with Gilbert's syndrome was encountered in 11% of the population studied. This percentage is close to the value described in the Caucasian population, estimated at 10% [22,23]. Concerning the polymorphism Gly71Arg, our results show that the mutated allele is encountered in 15.7% of our studied population. This frequency differs greatly from that reported for Caucasians and Afro-Americans but it is similar to that perceived at the Japanese population [24][25][26][27][28]. All these results suggest that the Tunisian population appears to be heterogeneous for UGT1A1 gene mutation status. The heterogeneity of Tunisian population for SCA haplotypes has been reported previously by Imen et al., 2011 [4]. The authors have demonstrated the predominance of the Benin haplotype in SCA patients suggests that the S mutation present in Tunisia may have originated from the Benin region and was brought to Tunisia along the slave trade routes. However, they have reported the presence of another atypical haplotype that could be considered as specific to Tunisian chromosome S. In a previous study we have been interested in the implication of the polymorphism A(TA) TAA of UGT1A1 in occurrence of cholelithiasis among Tunisian patients with normal hemoglobin status. Our findings have showed that subjects with (TA) 7 or (TA) 8 variant in their genotypes are associated with high bilirubin level. Furthermore, we have demonstrated that (TA) 6 /(TA) 7 and (TA) 7 /(TA) 7 genotypes and (TA) 7 and (TA) 8 alleles were significantly associated with an increased risk of gallstone diseases = 0.0017, = 6.1 × 10 −6 , = 1.5 × 10 −6 , and = 0.025, respectively [29]. In this study, our results show that total bilirubin level increased with the genotypes (TA) 6 /(TA) 7 , (TA) 7 /(TA) 7 , and (TA) 7 /(TA) 8 ( = 7.1 × 10 −7 ; = 2.4 × 10 −16 ; and = 8.2 × 10 −7 ), respectively. In fact, the addition of an extra (TA) at the TATA box seems to interfere with binding of the transcription factor IID which is responsible for the transcription of UGT1A1 gene. This interference leads to the reduced expression of UGT1A1 and hence in the expression of bilirubin-UGT1 (30% of normal) [2]. Furthermore, our findings show a significant association between genotypes (TA) 7 /(TA) 7 and (TA) 7 /(TA) 8 [7], where they demonstrated that frequency of cholelithiasis was significantly higher in both adult and children patients with (TA) 7 /(TA) 7 and (TA) 7 /(TA) 8 genotypes compared to those with other genotypes. Our results are similar to those of previous studies concerning (TA) 7 variant which presents an excess risk for gallstone occurring in children patients with SCA and thalassemia (minor, intermedia, and 0 ) [4][5][6][7][8][9][10][11][12][13][14][15][16]. Outside of hemolytic disease, (TA) 7 variant has been reported by many studies to be associated with both hyperbilirubinemia and cholelithiasis [25,26,28,30,31]. Herein, we demonstrated the association of the genotype (TA) 7 /(TA) 7 with cholelithiasis in SCA patients and in the control group. Our study is the first findings about the implication of A(TA) TAA of UGT1A1 in lithogenesis among SCA children patients in Tunisia. Our data confirmed the role of (TA) 7 variant and highlighted the role of (TA) 8 in early gallstones formation; association is not found in previous studies. As future directions of our research, we will focus on the Gly71Arg polymorphism in the first exon of the UGT1A1 gene reported to be associated with the same phenotypes [2,25,27,32,33]. Also we will focus on other candidate genes which can be associated with both hyperbilirubinemia and cholelithiasis in SCA such as SLCO1BI and SLCO1A2 [34].

Conclusion
The novelty of this report is that it is the first time that a similar study was made on the Tunisian children sickle cell population and that the results show a clear association of (TA) 7 /(TA) 7 and (TA) 7 /(TA) 8 genotypes as well as the (TA) 7 allele with the cholelithiasis and hyperbilirubinemia. Interestingly our findings show the association of (TA) 8 allele with the cholelithiasis, association not described previously in other population.