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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 2  |  Page : 76-81

Association between UGT-1A1 gene GLY71Arg polymorphism and severe unexplained indirect hyperbilirubinemia among neonates


1 Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
3 Department of Public Health and Community Medicine, Zagazig University, Zagazig, Egypt

Date of Web Publication11-Dec-2018

Correspondence Address:
Wesam A Mokhtar
Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig 44111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AJOP.AJOP_18_18

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  Abstract 


Background Neonatal jaundice is considered the most prevailing clinical health problem among neonates. Numerous etiological factors are responsible for the development of pathological neonatal jaundice. Almost half of the cases have no well-identified risk factor suggesting an underlying genetic risk.
Aim We aimed at investigating the relation between uridyl-diphosphate-glucuronosyltransferase 1A1 (UGT-1A1) gene Gly71Arg (G71R) polymorphism and the occurrence of unexplained severe indirect hyperbilirubinemia among Egyptian neonates.
Patients and methods A case–control study was conducted on 81 term neonates presented with serum total bilirubin greater than or equal to 17 mg/dl with no identified underlying cause. Eighty-one age-matched and sex-matched term neonates without clinical jaundice were taken as controls. All neonates (cases and controls) were genotyped for the presence of UGT-1A1 gene G71R polymorphism using PCR restriction fragment length polymorphism. Neonatal cases were classified according to serum total bilirubin into four classes of severity: significant, severe, extreme, and hazardous hyperbilirubinemia.
Results Genotype distribution frequency for G71R polymorphism was in accordance with Hardy–Weinberg equilibrium among controls but not among cases. There was significant increase in G71R A/G 50 (61.7%), A/A six (7.4%) genotypes, and A 62 (38.3%) allele distribution among cases with significant increase in estimated risk of unexplained hyperbilirubinemia with odds ratio (95% confidence interval) and P value of 8.6 (4.14–18.14) and P=0.000, 15.5 (1.78–136.2) and P=0.001, and 5.28 (2.92–9.57) and P=0.000, respectively when compared with controls. Significantly higher levels of total bilirubin among jaundiced neonates were observed with the A/A genotype followed by te A/G genotype as compared with the G/G wild genotype (P=0.000). Moreover, a significant association was found between the distribution of G71R genotypes and severity of jaundice.
Conclusion UTG-1A1 gene G71R A/G, A/A genotypes, and A allele were associated with significant increased risk of severe unexplained indirect hyperbilirubinemia among Egyptian neonates.

Keywords: Egyptian, G71R polymorphism, neonates, severe jaundice, unexplained hyperbilirubinemia, UTG-1A1 gene


How to cite this article:
Mokhtar WA, Allam RM, Zidan N, Hamed MS. Association between UGT-1A1 gene GLY71Arg polymorphism and severe unexplained indirect hyperbilirubinemia among neonates. Alex J Pediatr 2018;31:76-81

How to cite this URL:
Mokhtar WA, Allam RM, Zidan N, Hamed MS. Association between UGT-1A1 gene GLY71Arg polymorphism and severe unexplained indirect hyperbilirubinemia among neonates. Alex J Pediatr [serial online] 2018 [cited 2019 Mar 23];31:76-81. Available from: http://www.ajp.eg.net/text.asp?2018/31/2/76/247301




  Introduction Top


Neonatal jaundice is considered the most prevailing clinical health problem affecting about 60% of term and 80% of preterm babies throughout the first week of life [1]. There are numerous underlying etiological factors responsible for the development of pathological neonatal hyperbilirubinemia. Hemolytic diseases of the newborn, polycythemia, increased enterohepatic circulation, and inadequate breastfeeding are considered the most common underlying causes. However, in many neonatal cases, no well-identified risk factors have been detected suggesting the presence of an underlying genetic risk [2].

Despite the majority of cases being mostly benign, severe untreated unconjugated hyperbilirubinemia with a high-toxic bilirubin level can lead to an increased risk of disabling and permanent neurological, intellectual, and developmental sequelae, called ‘bilirubin encephalopathy’ or ‘kernicterus’ [3].

Uridyl-diphosphate-glucuronyl-transferase-1A1 (UGT-1A1) enzyme, encoded by the UGT-1A1 gene on chromosome (2q37), is considered the main key liver enzyme involved in bilirubin conjugation and metabolism [4],[5]. In recent reports, various polymorphisms in the promoter and coding region of the UGT-1A1 gene can lead to decreased gene expression or decreased enzyme activity under normal expression [6],[7].

UGT-1A1 gene G211A (Gly71Arg) polymorphism, which leads to replacement of aminoacid glycine by aminoacid arginine at codon 71, was found to be responsible for low UGT-1A1 enzyme activity and increases the risk of neonatal unconjugated hyperbilirubinemia in different races and ethnicities [2],[8].

To our knowledge, there is few data about the frequency distribution of UGT-1A1 gene G71R polymorphism among Egyptian population.


  Aim Top


In this study, we performed genotypic analysis of a group of Egyptian neonates to find the relation between UGT-1A1 gene G71R polymorphism and risk of severe early neonatal unexplained indirect hyperbilirubinemia (UIHB).


  Patients and methods Top


A case–control study was conducted in the neonatal intensive care unit (NICU) at Zagazig University Children’s Hospital, Zagazig University, Egypt, in the period from April 2016 to April 2018. A total sample size of 162 newborns were enrolled in the study. Eighty-one term neonates with UIHB [serum total bilirubin (STB) ≥17 mg/dl] who presented within the first week after birth were taken as cases. Eighty-one sex-matched and age-matched term neonates with no clinical jaundice were taken as controls. Exclusion criteria were the following:
  1. Preterm neonates (gestational age of <37 weeks).
  2. Neonates who had conjugated hyperbilirubinemia, underlying hemolytic cause, birth asphyxia, cranial birth trauma, intracranial hemorrhage, sepsis, congenital infection, polycythemia, gastrointestinal malformations, and congenital hypothyroidism.
  3. Neonates with a history of maternal diseases during pregnancy including diabetes, hypertension, autoimmune disease, and renal insufficiency.


Both cases and controls were subjected to thorough history taking (including gender, gestational age, mode of delivery, order in family, history of previous siblings with unexplained jaundice, type of feeding) and detailed clinical examination. Neonatal cases were subjected to routine laboratory investigations for the diagnosis of neonatal hyperbilirubinemia as recommended by our NICU protocols and guidelines (including serum total and direct bilirubin level, complete blood count, reticulocyte count, blood grouping for the mother and the neonate, blood film, Coombs test, glucose-6-phosphate-dehydrogenase assay, and osmotic fragility test when needed, and sepsis screen and thyroid function test). The cases were classified according to the of level STB into four classes of severity [9]:
  1. Significant hyperbilirubinemia (STB of 17 to <20 mg/dl).
  2. Severe hyperbilirubinemia (STB of 20 to <25 mg/dl).
  3. Extreme hyperbilirubinemia (STB of 25 to <30 mg/dl).
  4. Hazardous hyperbilirubinemia (STB of >30 mg/dl).


Two cases with hazardous hyperbilirubinemia (with an STB of 33 and 35 mg/dl on day 4 and 5, respectively) presented with manifestations of bilirubin encephalopathy. Both cases and controls were genotyped for UGT-1A1 gene G71R polymorphism.

The current study was conducted in accordance with the ethical standards of the Helsinki Declaration of 1964 as revised in 2008 and approved by our ethics committee of Faculty of Medicine, Zagazig University, Egypt. An informed written consent was taken from parents of all participants in the study.

Methods

Blood sampling

Peripheral venous blood samples of 2 ml were collected from all studied groups and stored at −20°C until further analysis.

Analysis of Gly71Arg (rs 4148323) polymorphism

Genomic DNA extraction was done from whole blood using the commercially available G-spin TM total DNA extraction kit (iNtRON Biotechnology, Seongnam, Korea) as described in the user manual. The quality of the genomic DNA purity and concentration were determined spectrophotometrically at 260 and 280 nm. The purified genomic DNA was stored at −80°C until use. Gly71Arg mutation sequences were amplified using the following specific primers (Biosource Europe SA, Nivelles, Belgium) according to Huang et al. [10].



Polymorphism analysis was carried out by PCR amplification which was carried out in a total volume of 25 µl, 12.5 µl of 2× Dream TaqTM Green Master Mix (MBI Fermentas, St. Leon-Rot, Germany), and 25 pmol of each primer with cycling parameters as follows: denaturation at 94°C for 5 min, 35 cycles at 94°C for 30 s, annealing at 61°C for 30 s and 72°C for 60 s, and one final cycle of extension at 72°C for 7 min. Then, digestion of amplified PCR product of143-bp for Gly71Arg was carried out using four specific restriction endonuclease enzymes (New England Biolabs, Ipswich, Massachusetts, United States). They were incubated at 37°C for 4 h; then the digested products were separated in 2% agarose electrophoresis system and visualized with ethidium bromide staining under ultraviolet transillumination.

The digested fragments were expressed as G/G for wild allele that showed complete digestion and resulting in two bands of 119-bp and 24-bp, A/A for mutant allele that showed resistance to digestion and resulting in one band of 143-bp and A/G for heterozygote allele that showed partial digestion and resulting in three fragments 143 119 and 24 bp.

Statistical analysis

The collected data were computerized and statistically analyzed using the SPSS program (Statistical Package for Social Science) version 22.0, IBM Corp, Armonk, NY. Qualitative data were represented as frequencies and percentages. χ2-Test was used to compare between groups regarding qualitative variables with calculation of odds ratio as a risk estimate. Mann–Whitney and Kruskal–Wallis tests were used to compare between independent groups regarding quantitative variables that was not normally distributed and represented with median (interquartile range). Analysis was done at 5% level of significance.


  Results Top


[Table 1] represented the general basic characteristics of both cases (n=81) and controls (n=81) with no significant difference in general characterization between cases and controls. The cases were classified according to severity of hyperbilirubinemia into four severity classes: significant 26 (32.1%), severe 39 (48.2%), extreme 13 (16%), and hazardous three (3.7%) hyperbilirubinemia ([Table 2]). Genotype distribution frequencies for UGT-1A1 gene G71R polymorphism were in accordance with Hardy–Weinberg equilibrium among controls but not among cases. Significantly higher percentage of mutant heterozygous A/G genotype, homozygous A/A genotype, and A allele were found among cases as compared with controls with estimated odds ratios of 8.66, 15.6, and 5.28, respectively ([Table 3]). Regarding the peak total bilirubin level among cases, significantly higher levels of total bilirubin were detected with A/A genotype followed by A/G genotype as compared with G/G wild genotype (P=0.000). Moreover, a significant association was found between the distribution of G71R genotypes and severity of hyperbilirubinemia ([Table 4]).
Table 1 Distribution of the studied groups according to their baseline characteristics

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Table 2 Distribution of studied cases according to severity of hyperbilirubinemia

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Table 3 UGT-1A1 gene G71R genotypes and allele frequency distribution among cases and controls

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Table 4 Association between uridyl-diphosphate-glucuronosyltransferase-1A1 gene G71R genotypes and both total bilirubin level and severity of hyperbilirubinemia

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  Discussion Top


It has been well established that several multifactorial mechanisms are responsible for the occurrence of neonatal IHB. Increased bilirubin production, decreased hepatic uptake, defective conjugation, and increased enterohepatic circulation represent the most common causes of neonatal pathological jaundice [11],[12]. Almost half of all neonatal jaundiced cases have no identified underlying risk factor. In addition, the prevalence and severity of neonatal jaundice have greatly differed among different populations and ethnicities [13]. In the Asian population, the peak bilirubin levels in full-term neonates are double than that present among white and black populations, suggesting presence of underlying genetic factors involved in the pathogenesis of neonatal jaundice [2],[10]. In the present study, we investigated the association between UGT-1A1 gene G71R polymorphism and risk of severe UIHB among Egyptian term neonates. We found a significant increase in the frequency of G71R A/G (61.7%) and A/A (7.4%) genotypes and A allele (38.3%) among cases with significant increase in the risk of UIHB compared with controls.

By reviewing similar reports from different ethnicities and populations, several studies were conducted on the Chinese population. Wu et al. [14] studied UGT-1A1 gene G71R polymorphism in neonatal jaundice among two ethnic groups in China (Han and Guangxi Heiyi Zhuang populations). They reported great significant association between G71R polymorphism and neonatal hyperbilrubinemia in both ethnic groups with significant increase in A/G (27%) and A/A (6.9%) genotypes and A allele (20.4%) frequency in cases compared with controls with significant increase in the risk of developing UIHB. In addition, Liu et al. [15], Guo et al. [16], Min et al. [17], and Yang et al. [3] also studied the same UGT-1A1 gene G71R polymorphism on the Chinese population and showed nearly similar results. In a meta-analysis conducted by Yu et al. [18] including a total of 32 eligible studies from different ethnicities and populations, they reported that the presence of UGT-1A1 gene G71R polymorphism (homozygous and heterozygous mutant genotypes) resulted in significant increase in the risk of neonatal UIHB. All previous results were matched with our results. Regarding Egyptian population, there was only one study conducted by Mohammed et al. [19] who investigated UGT-1A1 gene G71R genotype distribution on a small sample of jaundiced neonates (n=30) with exclusive breastfeeding. They concluded a significant increase in A/G (33.3%), A/A (3.3%) genotype, and A alleles (20%) among the cases compared with controls with significant increase in the risk of neonatal hyperbilirubinemia among breastfed term neonates. Despite the small sample size, their results were in agreement with our results.

In contrast, Narter et al. [20] conducted a case–control study to investigate UGT-1A1 gene G71R polymorphism among 109 Turkish neonates with peak total bilirubin (>15 mg/dl) in the first 10 days of life. They reported no significant difference in genotype distribution between cases and controls. Similarly, an Iranian study conducted by Dastgerdy et al. [21] showed no significant relation between the frequency of UGT-1A1 gene G71R polymorphism and development of hyperbilirubinemia in a small number of jaundiced Iranian term neonates. Among the Malaysian population, Sutomo et al. [22] also reported no significant contribution of UGT-1A1 gene G71R to the high incidence of neonatal jaundice among Javanese Indonesian and Malay-Malaysians. The previous Malaysian results were confirmed by another Malaysian study conducted by Yusoff et al. [23].Regarding severity of neonatal hyperbilirubinemia, we concluded that there was significant increase in peak total bilirubin level in jaundiced neonates carrying the A/A genotype followed by A/G genotype when compared with the G/G genotype. Also, we found significant increase in the frequency of A/G genotype (94.4%) among neonates with severe IHB, increased frequency of both A/G (76.9%), and A/A (23.1%) genotypes among those with extreme hyperbilirubinemia and all cases with hazardous hyperbilirubinemia were carrying the A/A genotype (100%).

In agreement with our results, Yang et al. [3] and Chou et al. [24] reported significant association between development of severe hyperbilirubinemia and both G71R A/G and A/A genotypes. Meanwhile, Huang et al. [10] reported significant higher bilirubin level (≥20 mg/dl) in Chinese neonates carrying only homozygous mutant (A/A genotype) of G71R polymorphism when compared with the wild genotype. Also, Youyou et al. [25] and Sato et al. [26] reported significant association between G71R A/G genotype and higher levels of total bilirubin.

In contrast Narter et al. [20] showed no significant influence of UGT-1A1 gene G71R polymorphism on the severity of neonatal jaundice.

The limitations of the current study were the lack of investigating the UGT-1A1 gene G71R genotype frequency distribution among neonates with physiological jaundice, mild and moderate pathological jaundice in order to detect the actual G71R genotype distribution in neonatal jaundiced populations.


  Conclusion Top


In the present study, we concluded that there were significant associations between UGT-1A1 gene G71R A/G, A/A genotypes and A allele and risk of severe UIHB among Egyptian neonates. There is a great need for larger prospective studies to consider UGT-1A1 gene G71R genotyping as a marker for predicting severity of unexplained early neonatal IHB to guard against the poor outcome of high-toxic bilirubin levels.

Acknowledgements

The authors acknowledge all the participants of this study for their unstinted cooperation.

The research was supported by the Zagazig Faculty of Medicine.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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