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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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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