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

RESULTS

UGT1A1 (TA)n Promoter Genotyping as a Diagnostic Tool for Gilbert Syndrome. The GS patient group consisted of 32 males (62.75%) and 19 females (37.25%) and median age was 16 (range 3 to 19 years). The UGT1A1 (TA)n promoter genotypes detected in the control and GS patients’ groups were TA 6/6, TA 6/7, TA 7/7 and TA 7/8. The same UGT1A1 (TA)n promoter genotypes were identified using PCR amplification followed by acrylamide electrophoresis stained with Ag-nitrate and/or using fragment length analysis methodology. In order to validate both methods, we used reference samples previously determined by DNA sequencing. The detection of the number of TA repeats in UGT1A1 promoter using PCR amplification followed by 15.0% acrylamide electrophoresis stained with Ag-nitrate [Figure 1(a)] and fragment analysis [Figure 1(b)] are presented in Figure 1. Also, sequence of a TA 6/6 and TA 7/7 promoter repeats, used as controls in electrophoresis runs, are presented in Figure 1 [Figure 1(c) TA 6/6, and Figure 1(d) TA 7/7]. The distribution of UGT1A1 (TA)n promoter genotypes in pediatric GS patients are given in Table 2. The UGT1A1 (TA)n promoter genotype distribution in pediatric GS patients was as follows: TA 6/6 (3.92%), TA 6/7 (15.87%), TA 7/7 (76.47%) and TA 7/8 (3.92%). Due to small number of carriers of 6/6 and 7/8 UGT1A1 TA promoter genotypes, for further analysis groups of 6/6 TA UGT1A1 and 6/7 TA UGT1A1 promoter genotypes were considered as one, non-risk GS genotypes, as well as groups of 7/7 TA and 7/8 TA UGT1A1 promoter genotypes, risk GS genotypes. Carriers of risk GS genotypes had more than 21-fold higher odds for developing GS than carriers of non-risk GS genotypes, OR = 21.5 (9.0-51.6), p <10–14 (Fisher’s exact test), pointing out the importance of molecular genetic testing in GS diagnostic pipeline. The levels of total and direct bilirubin were measured at diagnosis and also after hypocaloric and phenobarbitone tests in pediatric GS patients (Table 3). The total and direct bilirubin levels did not follow normal distribution (Smirnof-Kolmogorov and Shapiro-Wilk tests, p <0.05). The difference between levels of unconjugated bilirubin at diagnosis in pediatric GS patients were statistically significant between the non-risk GS genotype carriers and risk GS genotype carriers (Mann-Whitney test, p = 0.079). 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 in pediatric GS patients. This increase was not UGT1A1 (TA)n promoter genotyperelated (Mann-Whitney test, p = 0.409). After a 3-day phenobarbitone test, levels of total and conjugated bilirubin were also measured in pediatric GS patients. The unconjugated serum bilirubin fracture decreased 27.4% in GS risk and 6.4% in GS non-risk genotype carriers. The difference of a decrease of unconjugated bilirubin levels after the phenobarbitone test was statistically significant when the pediatric GS patients carrying GS risk were compared to GS non-risk genotypes (Mann- Whitney test, p = 0.040) (Figure 2). UGT1A1 TA 7/7 and 7/8 variants were detected in 41 of 51 of our diagnosed GS patients. Diagnostic value of the UGT1A1 (TA)n marker was 80.0%. In order to elucidate the genetic basis of hyperbilirubinemia in the remaining 20.0% GS-positive patients, identified with UGT1A1 TA 6/6 and TA 6/7 repeats, we extended the UGT1A1 genetic testing. The UGT1A1 coding region with nearby intronic regions were sequenced. In only one patient with 6/7 TA repeats in promoter region of the UGT1A1 gene, two single nucleotide variants were found: NM_000463.2 c.997-82T>C (intron 2, position 602 of 683) and c.1084+12G>A (intron 3, position 12 of 283). Population Pharmacogenetic Potential of UGT1A1 (TA)n Promoter Genotypes in Serbia. The healthy control group had 67 pediatric individuals and 32 adults (median age 12.75, range 1.25 to 83 years). It consisted of 63 males (63.0%) and 37 females (37.0%). The UGT1A1 (TA)n promoter genotypes detected in the control group were TA 6/6, TA 6/7 and TA 7/7. The distribution of UGT1A1 (TA)n promoter genotypes in healthy control group in Serbia are given in Table 2. The frequencies of UGT1A1 (TA)n promoter genotypes in the healthy control group in Serbia were: TA 6/6 37.0%, TA 6/7 47.0% and TA 7/7 16.0%. Consequently, the frequency of the UGT1A1*28 allele is 40.0% in Serbia. The UGT1A1 (TA)n promoter genotypes detected in the control group were in Hardy-Weinberg equilibrium (χ2 test, p = 0.87; exact test, p = 1). The differences in the distribution of UGT1A1 (TA)n promoter genotypes between the control and GS patients’ groups were statistically significant (Fisher test, p <0.001).



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