Neonatal Jaundice and Bilirubin UDP-Glucuronosyl Transferase 1A1 Gene Polymorphism in Turkish Patients
Author for correspondence: Melih O. Babaoglu, Department of Pharmacology, Hacettepe University Faculty of Medicine, Sihhiye, Ankara, 06100, Turkey (fax +90 312 310 5312, e-mail firstname.lastname@example.org).
Abstract: Bilirubin uridine diphosphate-glucuronosyltransferase (B-UGT) is the rate-limiting enzyme for the conjugation of bilirubin with glucuronic acid in its excretion process into the bile. Variations in B-UGT gene (UGT-1A1) have been related to disorders characterised by hyperbilirubinaemia. The aim of this study was to investigate whether the number of thymine-adenine repeats in the promoter region of UGT-1A1 was related to non-physiologic hyperbilirubinemia of unexplained aetiology in Turkish newborns. These patients (n=106) were genotyped for their thymine-adenine repeat number in the promoter region of UGT-1A1, and were divided into two groups according to their bilirubin level. Forty-nine newborns with bilirubin levels higher than 17 mg/dl within the first ten days of life comprised the hyperbilirubinaemia group and 25 newborns with bilirubin levels higher than 10 mg/dl after fifteen days of life formed the prolonged jaundice group. Thirty-two newborns were included as healthy controls. The observed frequencies for the wild-type six repeat allele thymine-adenine (TA6) within each subject group were similar (P>0.05; 75.5%, 78.0% and 73.4%, respectively). Likewise, the distribution of TA6/6, TA6/7 and TA7/7 genotypes among three groups were similar. These results imply that the TA7 repeat allele of UGT1A1 (UGT1A1*28) is a common variant in the Turkish population. Our results do not suggest an association between thymine-adenine repeat polymorphism of UGT1A1 and hyperbilirubinaemia of unexplained aetiology or prolonged jaundice in Turkish neonates.
Hyperbilirubinaemia is a common finding during the neonatal age and sometimes may lead to severe consequences such as kernicterus. Up to a certain bilirubin level in serum, hyperbilirubinaemia is regarded as a physiological phenomenon (Watchko et al. 2002). Recent studies from diverse populations define the upper limit of normal total serum bilirubin levels in newborns as 17–18 mg/dl (Bhutani et al. 1999; Maisels 1999). The mechanism of hyperbilirubinaemia has not been fully explained. In neonates bilirubin formation is increased and hepatic bilirubin clearance is decreased (Watchko et al. 2002). Neonatal hyperbilirubinaemia higher than physiologic levels is prevalent among Turkish newborns. In a recent study, the incidence of non-physiologic, significant hyperbilirubinaemia in Turkish newborns was found to be about 10,5% to 25.3% (Sarici et al. 2004). In most cases no reason such as metabolic syndromes or haematological pathologies could explain the aetiology of the hyperbilirubinaemia.
Glucuronidation is a clearance mechanism for numerous dietary and environmental chemicals including bilirubin. The UDP-glucuronosyltransferases (UGTs) are a family of enzymes responsible for glucuronidation of many xenobiotics and endobiotics in the human body. Among these, UGT1A1 is the rate-limiting enzyme for the elimination of bilirubin (Bosma et al. 1994; Tukey & Strassburg 2000). Several UGT1A1 genetic polymorphisms with absent or very low UGT1A1 activity are associated with several non-haemolytic hyperbilirubinemic syndromes such as Gilbert's syndrome and Criggler-Najjar's syndrome type I and type II (Burchell & Hume 1999; Maruo et al. 1999). Homozygosity for a promoter region polymorphism resulting in seven repeats of thymine/adenine nucleotides (TA7 or UGT1A1*28) leads to a 70% reduction in UGT1A1 expression as compared to the six repeat (TA6) carriers (Bosma et al. 1995; Burchell & Hume 1999). In addition, some other genetic variants, such as five to eight thymine adenine repeats in the promoter or missense mutations in the coding region of UGT1A1, have been described as causes of hyperbilirubinaemic syndromes (Bancroft et al. 1998; Iolascon et al. 1998; Cappellini et al. 1999; Maruo et al. 1999).
The aim of this study was to investigate whether UGT1A1 TA7 repeat variant (UGT1A1*28) was a contributory factor in hyperbilirubinaemia of unexplained aetiology or prolonged jaundice in Turkish newborns.
Materials and Methods
Participants. A total of 106 newborns were included in the study. The study group consisted of newborns that were admitted to Hacettepe University Ihsan Dogramaci Paediatrics Hospital with the diagnosis of hyperbilurinaemia. Newborns with known risk factors, such as severe congenital malformation, sepsis, birth asphyxia, maternal diabetes, polycythemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, cephalohaematoma, dehydration, hypothyroidism or haemolysis for any reason were excluded from the study. Forty-nine newborns with bilirubin levels above physiologic levels (higher than 17 mg/dl) within the first ten days of life formed the hyperbilirubinaemia group. Twenty-five newborns with bilirubin levels higher than 10 mg/dl after fifteen days of life formed the prolonged jaundice group. The control group consisted of 32 newborns without visible jaundice within ten days of life.
The study was approved by the Ethics Committee of Hacettepe University Faculty of Medicine and conducted in accordance with the Declaration of Helsinki.
Genotyping procedure. DNA was isolated using QIAamp DNA blood kit (Qiagen, Hilden, Germany). In order to analyse thymine adenine repeat polymorphism in the promoter of the UGT1A1, an amplification product is generated by polymerase chain reaction (PCR) and sequencing. The sequence of the forward and reverse primers were 5′-AAA TTC CAG CCA GTT CAA CTG TTG TT-3′ and 5-CTG CTG GAT GGC CCC AAG-3′, respectively (Qiagen, Hilden, Germany). PCR was performed in a total volume of 50 μl containing 25 ng of template DNA, 0.2 mM of each deoxynucleotides, 2 mM MgCl2, 0.4 μM of each primer and 1 unit Taq polymerase (Qiagen, Hilden, Germany). Amplification was performed using a PE9700 thermal cycler (PerkinElmer Inc, MA, USA) (MJ Research Inc., Waltham, MA, USA) for 34 cycles, consisting of denaturation at 94 ° for 45 sec., annealing at 62 ° for 45 sec. and extension at 72 ° for 60 sec. between the initial denaturation at 94 ° for 2 min. and a final extension at 72 ° for 1 min. Twenty-five ng of the PCR product was subsequently sequenced to determine the number of thymine adenine repeats by using the same primers and BigDyeTM Terminator mix (Applied Biosystems, CA, USA). The amplification procedure involved 25 cycles of denaturation at 96 ° for 30 sec., annealing at 60 ° for 4 min. after an incubation at 96 ° for 2 min.
Statistical analysis. All the averages are given as mean±standard errors of the means. Weights and gestational ages of the groups were compared with one-way Anova. Chi-square test was used to determine whether there was a difference in sex distribution and in genotype distribution between groups. The differences were accepted as statistically significant when P-value was smaller than 0.05.
Demographical characteristics of the three groups according to their clinical diagnoses are shown in table 1. The mean gestational ages and the birth weights of the groups were similar. There was no difference between groups regarding to feeding patterns and gender. Out of 106 newborns, 98 newborns were exclusively breast-fed while 9 newborns received formula two to three times a day in addition to breast milk.
Table 1. Demographic characteristics of newborns enrolled in the study.
|Number of patients (n)||49||25||32|
|Gestational age (weeks, mean±S.D.)||38.5±1.5||38.3±1.7||38.9±1.2||>0.05|
|Birth weight (g, mean±S.D.)||3273±420||3162±489||3326±354||>0.05|
|Feeding pattern (breast milk/breast milk+formula)||47/2 ||22/3 ||28/4 ||>0.05|
The observed frequencies of the wild type TA6 allele within each subject group were 75.5% (95% confidence interval, CI95%=72.3–83.6%), 78.0% (CI95%=71.3–79.6%) and 73.4% (CI95%=68.0–78.7%). The overall prevalence of TA7 variant allele in our study population was 24.5% (CI95%=21.6–27.3%). The frequencies for TA6 and TA7 alleles in hyperbilirubinemia and prolonged jaundice groups were similar to those of the control group (P>0.05).
The genotypic distribution of the subjects within each group is given in table 2. The distribution of TA6/7 and TA7/7 genotypes the newborns with hyperbilirubinaemia or prolonged jaundice and in the control group were also similar (chi-square=0.68, P=0.95).
Table 2. The distribution of UGT1A1 genotypes according to their thymine adenine (TA) repeat number in the promoter across the groups with hyperbilirubinemia and prolonged jaundice and in the control.
|TA6/6||29 (59.2; 52.4–65.9)||15 (60.0; 50.5–69.4)||18 (56.3; 47.8–64.7)|
|TA6/7||16 (32.6; 26.1–39.0)|| 9 (36.0; 26.7–45.2)||11 (34.4; 26.3–42.4)|
|TA7/7||4 (8.2; 4.4–11.9) ||1 (4.0; 0.2–7.7) ||3 (9.3; 4.3–14.2) |
|Total||49 (100) ||25 (100) ||32 (100) |
In this study, we investigated the number of thymine adenine repeats in the promoter region of UGT1A1 in a group of Turkish neonates with hyperbilirubinaemia of unknown aetiology. Polymorphic expression of various UGT isoforms and their influence in systemic disposition of endobiotics and xenobiotics are active areas of investigation. We sought to determine if UGT1A1 TA7 repeat allele (UGT1A1*28) could be an underlying cause for the development of unconjugated hyperbilirubinaemia. Our results showed that the TA7 variant, which is the predominant mutation in Caucasians, was also prevalent in Turkish population with an allele frequency of 24.5%. The frequency of subjects with TA7/7 genotype was about 7.5% (CI95%=5.4–10.5%). The observed rate for TA7 homozygosity was similar to those values reported from several other Caucasian populations (∼10%) and higher than rates found in East Asian populations (1 to 3%) (Aono et al. 1995; Maruo et al. 2000; Tukey & Strassburg 2000).
There is some evidence in the literature suggesting a possible relationship between the activity of UGT1A1 and neonatal jaundice. Among Caucasians with Gilbert's syndrome, a homozygous thymine adenine insertion or deletion in the TATA box upstream from the 1A1 exon was found to be important in the aetiology of the disorder (Bosma et al. 1995; Burchell & Hume 1999). The serum bilirubin levels of individuals homozygous or even heterozygous for seven repeats have been found to be higher than those with wild type six repeats in Gilbert's syndrome (Bosma et al. 1995). Furthermore, Kaplan et al. (1997) reported that G6PD-deficient neonates with the heterozygous or homozygous thymine adenine repeat promoter polymorphism had a higher incidence of hyperbilirubinemia than corresponding controls. We found no relationship between the presence of thymine adenine repeat polymorphism in the promoter region of UGT1A1 gene and the occurrence of hyperbilirubinemia or prolonged jaundice in Turkish neonates (table 2). The distribution of TA6/7 and TA7/7 genotypes across the two pathological groups and the control group were similar and the small difference in favour of the control group did not lead to a statistical significance.
A recent study conducted in Turkish neonates also found no association between UGT1A1 polymorphism and neonatal hyperbilirubinaemia (Ulgenalp et al. 2003). There are some differences between the findings of our study and the study by Ulgenalp et al. (2003). We found that in all three groups about 60% of neonates were homozygote for the wild-type allele (table 2), whereas Ulgenalp et al. (2003) reported lower frequency of homozygotes (around 45% in their three subgroups, range of CI95%'s=32.1–54.8, P>0.05). Likewise, 34.0% (CI95%=29.5–38.4%) of the neonates in the present study had TA6/7 genotype while Ulgenalp et al. (2003) reported this ratio to be 46.3% (CI95%=41.4–50.5%, P<0.05). The difference in patient selection criteria might be the reason for observing differences in the distribution of UGT1A1 genotypes between the two studies. The differences between two studies may also be explained by regional differences since the participants in the present study were collected from Central Anatolia while the previous study was conducted in the Western part of the country. The findings of these two studies together suggest no relation between neonatal hyperbilirubinaemia and UGT1A1*28 polymorphism.
Maruo et al. (2000) previously reported that defects of UGT1A1 could be the underlying causes of the prolonged unconjugated hyperbilirubinaemia associated with breast milk. On the other hand, a study by Bertini et al. (2001) concluded that breast feeding was not associated with the increased frequency of neonatal jaundice in Italian neonates. Most of the neonates in our study were fed with breast milk alone and we could not determine any impact of breast feeding on development of hyperbilirubinaemia.
It has previously been shown that among individuals without clinically diagnosed Gilbert's syndrome who were homozygotes for UGT1A1 polymorphisms, only males had significant elevations in serum bilirubin levels (Bosma et al. 1995). In agreement with that study, all five hyperbilirubinaemic cases homozygous for TA7 variant in our study were also male. However, it would not be possible to reach a conclusion with such a small sample without further evaluating the effect of gender in larger subject homozygote for the TA7 mutation in further investigations.
In conclusion, this study demonstrates that UGT1A1TA7 repeat variation (UGT1A1*28) is prevalent in Turkish population. Our findings do not suggest an association between the TA7 polymorphism of UGT1A1 hyperbilirubinaemia or prolonged jaundice in Turkish neonates.
This study was supported by a grant from the Scientific and Technical Research Council of Turkey (SBAG-COST B15-2356).