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
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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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