UGT1A1 (TA)n PROMOTER GENOTYPE: DIAGNOSTIC AND POPULATION PHARMACOGENETIC MARKER IN SERBIA
Vukovic M, Radlovic N, Lekovic Z, Vucicevic K, Maric N, Kotur N, Gasic V, Ugrin M, Stojiljkovic M, Dokmanovic L, Zukic B, Pavlovic S
*Corresponding Author: Sonja Pavlovic, Ph.D., Laboratory for Molecular Biomedicine, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO BOX 23, 11010 Belgrade, Serbia. Tel: +38111-3976445. Fax: +38111-3975808. E-mail: sonya@sezampro.rs
page: 59

DISCUSSION

Detection of the number of TA repeats in the UGT1A1 promoter region using PCR amplification followed by acrylamide electrophoresis stained with Ag-nitrate and fragment length analysis methodology in pediatric GS patients and in a healthy control group showed completely the same results. Ten percent of the results were confirmed by DNA sequencing. Therefore, both methods used in our study can be considered as reliable and accurate. Acrylamide electrophoresis methodology is time-consuming but its main advantage is being cost-effective. Using samples previously validated for number of TA repeats (TA 6/6 and TA 7/7) as controls, makes this approach sufficiently reliable for diagnostic purposes. Fragment analysis is fast and accurate but quite expensive. Sequencing analysis is relatively expensive and time-consuming but the most reliable of all methods used in this study. For that reason positive control samples were validated using DNA sequencing. Numerous variants of the UGT1A1 gene, leading to elevated serum level of of unconjugated bilirubin and development of several syndromes or diseases characterized with different levels of severity, have been reported. Some of these genetic variants, detected in homozygous or heterozygous states, give inactive UGT1A1 alleles causing severe conditions, such as Crigler-Najjar type 1 or 2 syndromes [15]. The variants in UGT1A1 gene also cause prolonged unconjugated hyperbilirubinemia in the neonatal period, breast milk jaundice (BMJ) [15]. The mildest form of unconjugated hyperbilirubinemia is GS. The most significant UGT1A1 variant for development of GS is UGT1A1 *28, with seven TA repeats in the promoter region. Our current study determined UGT1A1 (TA)n promoter genotype distribution in pediatric GS patients in Serbia. As expected, 7/7 TA repeats in UGT1A1 promoter (UGT1A1 *28 genotype) were found in the majority of GS patients (76.47%), while the wild-type 6/6 TA promoter repeats (UGT1A1*1 genotype) was found in only two pediatric GS patients (3.92%). Gilbert syndrome non-risk UGT1A1 (TA)n promoter genotypes (TA 6/6 and TA 6/7) were detected in 20.0% of our GS patients. Our results have shown that efficacy of the UGT1A1 (TA)n promoter genotype variants as a diagnostic tool for GS is 80.0%, pointing out that genotyping of UGT1A1*28 is not enough of a reliable genetic test for GS, and that hypocaloric diet and phenobarbitone tests cannot be replaced by this genetic test. In an attempt to make the UGT1A1 genetic test more reliable, we decided to analyze coding and nearby intronic regions of the UGT1A1 gene in GS patients, carriers of non-risk GS alleles (TA 6/6 and TA 6/7). Surprisingly, only in one GS patient with TA 6/7 did we find additional variants, namely two intronic single nucleotide variants in a heterozygous state, both of uncertain significance for GS development. Therefore, despite our finding that carriers of the risk GS genotypes have more than 21-fold higher odds for developing GS than carriers of non-risk GS genotypes, genotyping of the UGT1A1 gene is not sufficiently reliable enough for diagnosis of GS. For the Romanian cohort of GS individuals, the results showed that the 7/7 TA promoter genotype was identified in 32.33% of all subjects, the 6/7 TA promoter genotype was the most prevalent (57.64%) and the 6/6 TA promoter genotype was detected in 7.36% of the GS patients [25]. Another study reported that the prevalence of the UGT1A1 *28 allele in Valencia reached 87.6% in the patients referred for GS [26]. Our results demonstrated a high concordance rate between clinical and genetic tests. Our current study showed that the difference between levels of unconjugated bilirubin at diagnosis were statistically significant between the non-risk GS carriers vs. risk GS genotype carriers. In other words, the level of unconjugated bilirubin was UGT1A1 (TA)n promoter genotype-related in pediatric GS patients at diagnosis. After hypocaloric diet, the mean level of unconjugated bilirubin was increased 2.91-fold in GS risk and 2.52- fold in GS non-risk genotype carriers. This increase was not UGT1A1 (TA)n promoter genotype-related. The 3-day hypocaloric diet test is still considered as one of the important diagnostic tools for GS. The variation in bilirubin levels after a diet is considered positive for GS when the level of unconjugated bilirubin increases by 100.0% over baseline. Nevertheless, a recent study revealed that the comparison between the results of the 3-day hypocaloric diet test and the genetic study of the UGT1A1 gene show a low association rate [26]. Our results also showed that, after a 3-day phenobarbitone test, levels of unconjugated serum bilirubin fracture decreased 27.4% in GS risk and 6.4% in GS non-risk genotype carriers in pediatric GS patients. The decrease in levels of unconjugated bilirubin after phenobarbitone test comparing to levels of bilirubin before the test was UGT1A1 (TA)n promoter genotype-related. It seems like the level of unconjugated bilirubin drops more in GS risk genotype carriers comparing to the GS non-risk ones when applying phenobarbitone. Although the fact that GS risk genotypes contribute to hyperbilirubinemia more than GS non-risk genotypes, the treatment with phenobarbitone abolishes those differences leading to normal values of unconjugated bilirubin. Population pharmacogenomic research has shown that the study of pharmacogenomic markers in various populations is of great importance. Comprehensive data repositories that record the prevalence of clinically relevant genomic variants in populations worldwide, including pharmacogenomic biomarkers, are valuable tools that can be exploited not only to develop guidelines for medical prioritization, but most importantly, to facilitate integration of pharmacogenomics into health care systems and to support preemptive pharmacogenomic testing [27]. UGT1A1 (TA)n promoter genotypes resulting in decreased function of the UGT1A1 enzyme are pharmacogenomic markers. Therefore, data on frequency of UGT1A1 (TA)n promoter genotypes in particular populations are useful. The frequency of the UGT1A1*28 allele varies among ethnicities, being the highest in those of African (43.0%) and European (39.0%) origin and lowest in those of Asian (16.0%) origin [28,29]. Our results showed that the frequency of the UGT1A1*28 allele in Serbia was 40.0%, which is in complete agreement with the data published for European populations. Our study revealed that in Serbia the frequencies of UGT1A1 TA 6/6, 6/7 and 7/7 promoter genotypes were as follows: 37.0, 47.0 and 16.0%, respectively. This finding was similar with the literature data for Caucasian populations. In the population of the Republic of Macedonia the frequencies of UGT1A1 6/6, 6/7 and 7/7 TA promoter genotypes were 50.0, 37.5.0 and 12.5% [30,31]. For healthy Croatian preschoolers, the frequencies of the UGT1A1 6/6, 6/7 and 7/7 TA promoter genotypes were 38.4, 47.9 and 9.8% [32]. These data were similar for neighboring Slovenians, with the frequencies of UGT1A1 (TA)n promoter genotypes as follows: 6/6 TA 38.1%, 6/7 TA 47.9%, 7/7 TA 13.6% [33,34]. Furthermore, in the Slovenian study, it was confirmed that subjects with the UGT1A1 TA 7/7 promoter genotype had the highest and subjects with the 6/6 TA promoter genotype the lowest total serum bilirubin levels. An Italian study analyzed UGT1A1 (TA)n promoter genotypes in healthy subjects and the following results were reported: 43.9% were 6/6 TA promoter genotype carriers, 39.8% were 6/7 TA promoter genotype carriers and 16.3% were TA 7/7 promoter genotype carriers. In the same study, the identified UGT1A1 (TA)n promoter genotypes were correlated to serum bilirubin concentrations. The serum bilirubin concentrations were the highest in the Italian subjects with 7/7 TA promoter genotype, intermediate in the subjects who were 6/7 TA carriers and the lowest in the subjects with 6/6 TA promoter genotype [35]. The unusually high frequency of UGT1A1 TA 7/7 promoter genotype was reported for general Valencian population, reaching 32.0% [26]. Our finding that in Serbia the frequency of pharmacogenomic relevant UGT1A1 promoter genotype (UGT1A1 *28/UGT1A1*28) is 16.0%, points out that the UGT1A1 (TA)n promoter genotyping could be recommended for preemptive testing in Serbia. It is clinically confirmed that carriers of UGT1A1 7/7 TA promoter genotypes treated with standard doses of irinotecan show an increased risk for developing hematological and/or digestive toxicities [36]. Keeping in mind that for some medications, such as atazanavir, the pharmacogenetic relevance of UGT1A1 promoter genotypes should be considered even for heterozygous carriers (UGT1A1*1/UGT1A1*28), pharmacogenetic testing of this marker is even more important [37]. Thus, for 63.0% of Serbian patients to be treated with atazanavir, preemptive genetic testing for UGT1A1*28 should be performed. UGT1A1 genotyping is clinically beneficial. Genetic test can contribute to the confirmation of diagnosis in patients with elevated serum unconjugated hyperbilirubinemia. However, genotyping of the UGT1A1 gene alone is not sufficient for diagnosis of GS, and cannot replace standard hypocaloric and phenobarbitone tests. Furthermore, UGT1A1 molecular genetic testing is important for individualization of therapy when drugs metabolized by UGT1A1 are administered. Enzyme functiondecreased individuals are at risk for developing serious adverse drug reactions. Finally, UGT1A1 genotyping can enable the participation of patients with unconjugated hyperbilirubinemia in clinical trials. High bilirubin levels disqualify these patients from many clinical trials. Atazanavir-associated hyperbilirubinemia has been described in GS patients receiving therapy for HIV [38] and in chronic myeloid leukemia treatment with nilotinib [39]. Hyperbilirubinemia has been reported in GS patients undergoing hepatitis C treatment [40]. However, if genetic testing shows that the patients are carriers of UGT1A1 GS risk genotypes, they should not be excluded from clinical trials despite increased bilirubin values but should be treated, taking into account UGT1A1 (TA)n promoter genotype-related higher bilirubin levels. Declaration of Interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Funding. This study was supported by Ministry of Education, Science and Technological Development, Republic of Serbia [Grant No. III41004].



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