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

INTRODUCTION

Uridin diphospho-glucuronosyl transferases (UGT) are a family of enzymes responsible for glucuronidation of numerous endobiotics, xenobiotics and drugs. Glucuronidation is a process of biotransformation of substrate to water soluble, mainly non-toxic products ready for excretion [1]. The human UGT superfamily is divided into four families: UGT1A, UGT2, UGT3 and UGT8 [2]. The UGT1A family is encoded by the UGT1A gene complex located on chromosome 2q37. This gene complex at the 5 region has 13 variable exons, linked to four common exons at the 3 region of the UGT1A gene. Each of the 13 5 exons has its own TATA promoter elements. Four of 13 first exons are pseudoexons, and nine exons are viable, independently transcribed as the first exon. Those nine different first exons with highly conserved regions from exon 2 to exon 5, generate nine different transcripts (UGT1A1, UGT1A3 through UGT1A10). Each transcript has identical 3 ends and a unique 5 end. Variable first exon provides substrate specificity, while highly conserved common exons contain sites for interactions with UDP glucuronic acid as common substrate [3]. The UGT1A1 gene is expressed in the liver, colon, intestine and stomach [4,5]. The UGT1A1 enzyme has a main role in glucuronidation of bilirubin. There are no alternative metabolic pathways for efficiently detoxification and elimination of bilirubin. Currently, more than 130 UGT1A1 variants have been reported as a cause of Gilbert syndrome (GS), Crigler-Najjar syndrome type 1 and Crigler- Najjar syndrome type 2 [6]. Allelic variations were found in both the exonic and promoter sequences [7]. The most common variants detected in the UGT1A1 gene are different numbers of TA repeats in its promoter, rs8175347 [8,9]. Wild-type UGT1A1 contains six TA repeats [A(TA)6TAA] in its promoter region. UGT1A1*28, UGT1A1*37 and UGT1A1 *36 variants have seven, eight and five TA repeats, respectively. Transcriptional activity of the UGT1A1 gene and, consequently, the activity of the UGT1A1 enzyme, depends on the number of TA repeats. A higher number of TA repeats causes decreased enzyme activity. Heterozygous status of UGT1A1*28 results in 25.0% decreased enzyme activity and homozygous status of this variant reduced transcription activity by 70.0% [7]. This leads to GS, a mild form of intermittent unconjugated hyperbilirubinemia, that lacks hemolysis or hepatocellular injury. Therefore, six TA repeats in the UGT1A1 gene promoter is considered as non-risk GS allele and seven or eight TA repeats are responsible for development of GS (risk GS allele). Gilbert syndrome is a benign hereditary condition, commonly presented in 3.0-13.0% in the general population. Gilbert syndrome is inherited in an autosomalrecessive manner, but several cases have been reported as dominantly inherited [10,11]. Gilbert syndrome is rarely clinically manifested until before puberty. Males are more often diagnosed than females, two-to-seven times, because there is a bigger bilirubin load per kilogram body weight in males, and/or androgen steroid hormone suppresses hepatic bilirubin clearance [12,13]. The UGT1A1*28 and UGT1A1*37 variants have been shown to contribute to hyperbilirubinemia and increased likelihood of gallstone formation in several inherited hemolytic conditions [14]. They are also associated with increased risk of breast cancer, neonatal hyperbilirubinemia and the risk of developing gallstones in cystic fibrosis. Shorter erythrocyte life and damage in transport of unconjugated bilirubin further contributes to development of GS. Gilbert syndrome does not require therapy as it is considered to be a benign condition. However, fasting, excessive physical stress, febrile conditions and hormone changes during menstrual cycles additionally potentiate hyperbilirubinemia and could be treated with phenobarbitone which stimulates the UGT1A1 enzyme activity [15]. Sometimes, for cosmetic purposes, GS could be treated to moderate the subicterus. The complete or nearly complete lack of ability of glucuronidation of bilirubin is not compatible with life. Genetic variations within exons 2 to 5 of the UGT1A1 gene locus, resulting in total absence of bilirubin glucuronide formation, cause Crigler-Najjar syndrome type 1 [8]. Also, some genetic variations in introns resulting in frameshifts and a premature stop codon have been described. Lack of UGT1A1 activity leads to a high level of bilirubin shortly after birth (20.0-50.0 mg/dL), causing bilirubin encephalopathy, kernicterus and death. Crigler-Najjar syndrome type 1 could be managed by repeated exchange transfusions, long-term phototherapy, plasmapheresis, hemoperfusion and finally, the only successful therapy is liver transplantation. Several single nucleotide variations in the UGT1A1 gene have been reported to cause substitution of a single amino acid and reduce enzyme activity to less than 10.0% of normal activity, but total bilirubin levels do not exceed 20.0 mg/dL. This condition is recognized as a Crigler-Najjar syndrome type 2 [15]. In this case, optimal therapy is phenobarbital that stimulates UGT1A1 activity. Taking into account that a number of medications are metabolized by the UGT1A1 enzyme, UGT1A1 (TA)n promoter variants are recognized as clinically relevant pharmacogenetic markers [16]. The presence of seven or eight TA repeats in the promoter of the UGT1A1 gene could severely impact the ability to metabolize certain medications, including antineoplastic drugs used in oncology: irinotecan (solid tumors, lymphoma and colorectal cancer treatment), belinostat (peripheral T-cell lymphoma treatment), epirubicin (breast cancer treatment), 5-fluorouracil (colorectal cancer treatment), axitinib and busulfan (various hematological and non hematological cancers), atazanavir and ritonavir (HIV infections treatment) and widely used analgesic/antipyretic drug acetaminophen (paracetamol). Alterations in hepatic metabolism may result in life-threatening toxicities in various organ systems [17,18]. Recommendations for pharmacogenomic testing for several of these drugs are given and have already been applied in routine clinical practice [19]. The aim of this study was to analyze if UGT1A1 (TA)n promoter variants can be efficient diagnostic tools for easier and faster diagnosis of GS, by using accurate and cost-effective methodology for detection of number of TA repeats in the UGT1A1 promoter. Correlation of UGT1A1 (TA)n promoter genotypes, risk GS and non-risk GS, with levels of unconjugated bilirubin at diagnosis and after hypocaloric diet and phenobarbitone tests in pediatric GS patients will be used for assessment of diagnostic value of UGT1A1 (TA)n promoter variants genotyping. Additionally, given that the UGT1A1 enzyme is very important for metabolism of various drugs and that, in the case of presence of defined UGT1A1 (TA)n promoter variants, numerous drugs can present unwanted side effects or toxicities, the aim of this study was to assess pharmacogenetic potential of those variants in Serbia. The results will point out if pharmacogenetic testing should be performed in Serbia before administration of medications whose metabolism is UGT1A1 (TA)n genotype-dependent.



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