Gilbert syndrome (GS) is defined as a fluctuating or discrete elevation of unconjugated serum bilirubin where hemolysis, liver disease or other causes are excluded. Among the general population, Gilbert syndrome is the main cause of unconjugated hyperbilirubinemia  . A. Gilbert  described this syndrome as a “imple familial jaundice”. Thereafter, Meulengracht described Gilbert syndrome as “icterus intermittens juvenilis”  , as it is benign and intermittent episodic in character.
In Gilbert syndrome, the mild jaundice occurs as a result of inherited unconjugated hyperbilirubinemia, the most common human metabolic disorders which is caused by polymorphisms in UDP-glucuronosyltransferase 1 (UGT1A1). UGT is a member of super family of enzymes which catalyzes the glucuronidation of many substrates (both exo and endogenous) and UGT1A1 is also the isozyme that is able to conjugate bilirubin, the endogenous yellow pigment which comes from haeme catabolism  . Till to date, more than one hundred variants have been found in the UGT1A1 gene, among them UGT1A1*28, UGT1A1*6 and UGT1A1*27 are associated with the phenotype of Gilbert syndrome    . Out of those 3 variants, UGT1A1*28, UGT1A1*6 have been found to be commonly associated in Asian population  . The UGT1 gene expresses 9 functional UGT1A proteins, and UGT1A1 is the major protein that catalyzes the glucuronidation  . Variants of the UGT1A1 gene polymorphism results in the absence or reduction of UGT1A1 activity which may lead to unconjugated hyperbilirubinemia. The UGT1A1*28 polymorphism occurs as insertion of a thymine-adenine (TA) dinucleotide in the TATAA box of the promoter region and it results in genotype of A(TA)7TAA and A(TA)8TAA, the mutant variety which is opposite of the A(TA)6TAA the wild variety   . UGT1A1*28 polymorphism is classified depending on the number of TA repeats in 2 alleles: 1) TA6/TA6 (wild type), 2) TA6/TA7 (heterozygous mutant), or 3) TA7/TA7 (homozygous mutant).
The TA6/TA7 or TA7/TA7 mutants are associated with decreased enzyme activity and, which may account for increased irinotecan related toxicity  . UGT1A1 28 has been reported to cause approximately 70 per cent reduction of UGT1A enzyme activity  UGT1A1 28 has severe toxicities with irinotecan which is an anti-cancer drug compared to wild type UGT1A1 1 or heterozygous UGT1A1 28.
There is ethnic variability in UGT1A1*28 polymorphism. The prevalence of homozygous TA7/TA7 was significantly higher among African (12% - 27%) and in Caucasian (5% - 15%) but it is lower (1.2% - 5%) in South-east Asian (1.2% - 5%) and Pacific region     . The UGT1A1 gene TA7 variant is associated with increased bilirubin levels in normal individual  , or glucose-6-phosphate dehydrogenase (G6PD) deficiency  , or in β-thalassemia minor (heterozygous)  , and it is also associated with neonatal jaundice in G6PD deficiency  and hereditary spherocytosis  . Genotyping of polymorphisms in UGT1A1 gene is an important step to determine the etiology of free hyperbilirubinemia. Till now, distribution of Gilbert Syndrome (GS) from Bangladesh has not been reported. Therefore, we aimed to determine the genetic profile (prevalence of polymorphisms of UGT1A1) which causes the Gilbert syndrome among Bangladeshi population.
2. Materials and Methods
A total of 150 apparently healthy Bangladeshi children presented with minor diseases such as colds, diarrhea, or other minor illnesses were enrolled over a one-year period between January 2013 and December 2013 from the outpatient department of Bangabandhu Sheikh Mujib Medical University (BSMMU) and Central Hospital Limited, Dhaka, Bangladesh. We explained the objectives of the study to the legal guardians. After obtaining written informed consent from all participants, venous blood sample was obtained in Ethylene diamine tetra acetic acid (EDTA) tubes from each subject. After collection, blood sample were shifted to the University of Tsukuba, Japan and then genetic analysis was performed in the genetic lab of the University of Tsukuba. This study was approved by the ethical review committees of BSMMU, Central Hospital Limited, and the University of Tsukuba Hospital.
3. DNA Extraction and Genotyping
DNA was extracted using Genomic DNA Isolation Kit (QiAamp DNA Blood Mini Kit, Qiagen, Vealo, Netherlands) from 0.5 to 2 ml peripheral blood following the manufacturer’s instructions. Polymorphisms ofUGT1A1*6 (c.211G > A) was genotyped using the TaqMan Assay-on-Demand SNP Typing System (Applied Bio Systems, Foster City, CA, USA) following the manufacturer’s instructions. PCR was performed on a 384-well format with 5 ng of DNA each, and automatic allele calling was performed using ABI PRISM 7900HT data collection and analysis software, version 2.2.2 (Applied Biosystems). UGT1A1*28 (c.-53_-52TA) promoter repeat number polymorphism was determined by PCR method on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Descriptive statistics were performed by using SPSS statistics version 21.0 (IBM Corporation, NY, USA) and data were expressed as number, percentage, mean and standard deviation.
Table 1 shows that among the 150 study children, 86 (57.3%) were male and rest of them were female. Age of the study participants was 4.03 ± 2.05 (mean ± SD) years. Weight and height of the study participants was 15.86 ± 5.88 kg (mean ± SD) and 101.80 ± 16.50 cm (mean ± SD) respectively. Mid upper arm circumference (MUAC) of the study participants was 15.35 ± 2.62 (mean ± SD). Out of 150 blood sample, all samples were successfully genotyped for UGT1A1*6 and 149 were genotyped for UGT1A1*28. Table 2 shows that minor allele frequency of UGT1A1*28 are: wild/wild is 31.33% (47), wild/mutant is 48% (72), mutant/mutant 20% (30) and this table also shows that majority of minor allele frequency of UGT1A1*6 is wild/wild (94.66%), few of them is wild/mutant (4.66%) and only 1 is mutant/mutant. Overall minor allele frequency (MAF) were 0.442(44.2%) for UGT1A1*28 and 0.047(4.7%) for UGT1A1*6. Table 3 shows the comparison of allele frequencies of UGT1A1*28 & UGT1A1*6 in different ethnicity and found UGT1A1*28 is very high among Bangladeshi population in compare to other ethnicity but low in UGT1A1*6 in compare to other Asian population.
Table 1. Characteristics of the study participants.
Table 2. Frequency of minor allele.
Table 3. Comparison of Allele frequencies of UGT1A1*28 & UGT1A1*6 in other ethnicity.
UGT1A1*28 & *6 in other ethnicity; *Li Minmin et al. 2014, Agrawal SK et al. 2009; MAF data of Caucasian was collected from NCBI dbSNP database.
This is the first ever study where we evaluated the frequency of the main genetic determinant of Gilbert Syndrome (UGT1A1*28 and UGT1A1*6) among Bangladeshi population. Our result confirms that UGT1A1*28 polymorphism is a highly frequent condition among Bangladeshi population. Previously it was believed that, this is not a disease of Bangladeshi population, that’s why there was no interest to conduct research on Gilbert syndrome among Bangladeshi population. We believe this finding will completely change the perception level of clinician as well as researcher in Bangladesh. Now a day it is evident that there is an inter-individual and interethnic variation in glucuronidation  . There is a wide range of diversification among UGT family. This is best explained by the UGT1A1 gene, which is responsible for over 50 genetic lesions  . It has been reported that, among Caucasian the frequency of the UGT1A1*28 variant allele is 30% - 45%. Among African and Indian populations, the distribution is twice that of the 10% - 20% reported in East Asian populations      . In Uzbekistan, the incidence of UGT1A1*28 homozygotes was 9.3%, which was higher than that in the Japanese population was studied previously  . Other published reports shown that, UGT1A1*28 homozygotes is high in distribution in Europe (5% - 14.8%), Africa (5.9% - 17.9%), and India (12.8% - 19.3 %); and less frequent in Japan and East Asia and (0% - 5.9 %)  -  .
Individuals with Gilbert syndrome have reduced UGT activity due to UGT1A1gene polymorphism. This reduced UGT activity causes jaundice under the conditions such as illness, physical exertion or fasting. Study among Malaysian population concluded that Gilbert syndrome is associated with a homozygous (TA7) in Malaysian population. They found the levels of serum bilirubin in GS patients from 20 μM to 90 μM  . Due to decreased UGT1A1 enzyme activity, neonates with this syndrome have higher risk to develop neonatal jaundice results in further complications like Kernicterus and irreversible brain damage. In a recent study, higher polymorphism of the UGT1A1gene was found to be significantly associated with hyperbilirubinemia among North Indian neonates  . Another study concludes that, UGT1A1polymorphism is a risk factor for severe neonatal unconjugated hyperbilirubinemia among Malaysian population  . Moreover, UGT1A1*28 allele is associated with reduced levels of enzyme. Therefore modification of dosage of irinotecan should be done on the basis of patients’ UGT1A1 genotyping. Thereby, adverse effects of this drug can be avoided to some extent. The FDA in USA approved an amendment of the label for Camptosar (irinotecan hydrochloride) which is invariably used as chemotherapeutic agent for the treatment of colon cancer, which includes the warning to reduce the starting dose of irinotecan for UGT1A1*28 homozygous patients. For East Asian individuals, requires both UGT1A1*6 and UGT1A1*28 genotyping, since there is no linkage disequilibrium between the two polymorphisms  .
Our study found the evidence of very high prevalence of UGT1A1 polymorphism among Bangladeshi population. This newly discovered result might be the alert for the clinicians treating neonatal jaundice at children hospital in Bangladesh. It is well known that most of the children with Kernicterus have no underlying etiological identity to explain the hyperbilirubinemia. In fact, preventing a case of Kernicterus might be one of the most valuable strategies in all healthcare settings, resulting in a healthy baby rather than a child with a life-time of debilitating neurodevelopment handicaps. UGT1A1 polymorphism also has an implication in case of treatment of cancer. We recommend genotyping of UGT1A1 prior to management of neonatal jaundice as well as before treating by irinotecan chemotherapy as patients with UGT1A1 polymorphism having more adverse effects of this chemotherapeutic agent. Furthermore, study regarding the correlation between prevalence of polymorphism and hyperbilirubinemia is an urgent need.
We would like to express our thanks and gratitude to the University of Tsukuba, Bangabandhu Sheikh Mujib Medical University (BSMMU) and Central Hospital for their support. We would also like to thanks to the study participants for their voluntary contribution to this study. This work was supported by a grant from the Grant of National Center for Child Health and Development (25-2), Japan. We dedicate this article to the National Professor of Pediatrics, Bangladesh Dr. MR Khan.
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