The Evaluation of the Genetic Variation Types of the Uridine Diphosphate Glucuronosyl Transferase 1A1 Gene by Next-Generation Sequencing and Their Effects on Bilirubin Levels in Obese Children

Abstract

Background and Objectives:

Obesity is a major nutritional problem with an increasing prevalence among children and adolescents. The uridine-diphosphate-glucuronosyl-transferase1A1 (UGT1A1) gene encodes the UDP-glucuronosyl transferase enzyme, converting the toxic form of bilirubin to a soluble, nontoxic form. There are yet to be studies on the evaluation of the UGT1A1 variant types detected by next-generation sequencing (NGS) and their effects on bilirubin levels in nonsyndromic obese children.

Methods:

Forty-five children with body mass index (BMI) >95 percentile (p) constituted the obesity group and fourteen healthy children with BMI <85p constituted the control group. Anthropometric, clinical features, and biochemical parameters were evaluated. Furthermore, the UGT1A1 gene was sequenced by NGS.

Results:

The obese patients had lower total, direct, and indirect bilirubin levels (p = 0.422, 0.026, and 0.568, respectively). In addition, obese patients had more genetic variations in the UGT1A1 gene compared with the control group (62.2% and 50%, respectively). We found that children with variations had higher total direct and indirect bilirubin levels compared with those without variation (p = 0.016, 0.028, and 0.015, respectively). Children diagnosed with obesity in the first two years of their life had fewer genetic variations and lower total bilirubin levels (p = 0.000 and 0.013, respectively).

Conclusions:

It is assumed that bilirubin can be protective against many chronic diseases. Although bilirubin levels are found to be lower in obese children compared with the control group, some variations in the UGT1A1 gene may be supported by raising bilirubin. We suggest that high bilirubin levels caused by those UGT1A1 variations may be protective against obesity and its many negative effects.

Introduction

Obesity is a major nutritional problem today, with ever-increasing numbers among children and adolescents. The problem is particularly prevalent in developed countries. Globally, 38 million children under the age of five were overweight or obese in 2019, according to the World Health Organization (Gow et al., 2020).

While obesity is not fully understood, it has been demonstrated that obesity occurs when energy intake exceeds energy expenditure. This imbalance is caused by several etiological factors (Markwald et al., 2013). Obesity attributed to genetics varies from 6% to 85% (Yang et al., 2007). There has been limited evidence of genetic variations associated with obesity in spite of the prediction of high-degree inheritance and molecular genetic studies. The genetic component of obesity was identified through twin studies, which indicated that obesity and adiposity are inherited conditions (da Fonseca et al., 2017; Xia and Grant, 2013).

The uridine-diphosphate-glucuronosyl-transferase1A1 (UGT1A1) gene consists of eight exons and is located on the long (q) arm of chromosome 2 at position 37.1. This gene encodes a UDP-glucuronosyltransferase enzyme that plays a role in the glucuronidation pathway. The protein encoded by the UGT1A1 gene is also known as the bilirubin uridine diphosphate glucuronosyltransferase (bilirubin-UGT) enzyme, which is the only enzyme that has the function of bilirubin glucuronidation. This enzyme converts the toxic form of bilirubin (unconjugated bilirubin) to a soluble, nontoxic form (conjugated bilirubin), so it can be excreted from the body (Bellarosa et al., 2021). The relationship between bilirubin levels, UGT1A1 gene variants, and the risk of Gilbert syndrome and nonalcoholic fatty liver disease has been previously investigated (Bellarosa et al., 2021). In addition, it has been emphasized that bilirubin is involved in the pathogenesis of obesity, metabolic syndrome, and related complications. However, its mechanism has not been fully explained (Bellarosa et al., 2021; Lin et al., 2009a). As abdominal obesity increases, the bilirubin levels decrease, and each 1% decrease in a person’s weight results in a linear increase in serum bilirubin concentrations (Bellarosa et al., 2021). There is, therefore, a possibility that bilirubin may be related to obesity (Vítek 2012; Andersson et al., 2009).

Developments in next-generation sequencing (NGS) technologies provide significant contributions to the diagnosis of different diseases and a better understanding of their etiopathogenesis (Yavaş et al., 2024; Dogan et al., 2024; Karagün et al., 2020; Doğan et al., 2022a; Doğan et al., 2022b). There are few studies evaluating the UGT1A1gene by NGS and/or effects of detected variant types on bilirubin levels in nonsyndromic obese children. In the current study, we aimed to reveal the genetic variation types of the UGT1A1 gene and their effects on bilirubin levels in obese children.

Materials and Methods

This study is a cross-sectional, prospective study including 59 children aged 6–17 years who applied at the Pediatrics Outpatient Department between June 1, 2019 and September 1, 2019. Oral and written information were given to all patients. Only volunteers whose parents signed the informed consent form were included in the study. The Local Ethics Committee of the University Medical Faculty approved the study (Approval No: 2019/120). The children’s age, gender, type of delivery, week of gestation, birth weight, and age at diagnosis of obesity in a professional health institution, breastfeeding duration, and status of using infant formula were recorded.

After taking off the children’s top layers of clothes and their shoes, the same nurses measured the height and weight of the 59 children participating in the study using the same weight scale and height meter. The height and weight measurements of each child were evaluated according to Neyzi standards. The body mass index (BMI) of each child was calculated using the formula (BMI = weight/height² [kg/m²]) and their percentiles were determined (Neyzi et al., 2015). Forty-five children with BMI >95p constituted the obesity group and fourteen healthy children with BMI <85p constituted the control group.

For both groups, fasting blood sugar level (mg/dL), HbA1c (%), insulin (uIU/mL), alanine transaminase (ALT) (U/L), aspartate transaminase (U/L), total bilirubin (mg/dL), direct bilirubin (mg/dL), indirect bilirubin (mg/dL), gamma-glutamyl transferase (U/L), C reactive protein (mg/dL), total cholesterol (mg/dL), triglycerides (mg/dL), high-density lipoprotein (mg/dL), low-density lipoprotein (mg/dL), uric acid (mg/dL), freeT4 (ng/mL), and thyroid stimulating hormone (uIU/mL) values were measured and abdominal ultrasonography (USG) was performed to determine whether there was hepatosteatosis.

The total DNA was isolated from 200 μL peripheral blood samples of the patients using the Magnesia 16 Complete Blood Genomic DNA Isolation Kit-102 (Anatolia Diagnostics and Biotechnology Products Inc.) and stored at −20°C until the next step. Polymerase chain reaction (PCR) pools generated before the NGS reaction were purified using the NucleoFast 96 PCR (MACHEREY-NAGEL GmbH) kit.

Subsequently, the quantification of the PCR products was standardized on NanoDrop 1000 (Thermo Fisher Scientific Inc.), with the samples of 0.2 ng/μL. Standardized samples were prepared for NGS using the Nextera XT sample preparation kit from Illumina, and the UGT1A1 gene was sequenced using the NGS (MISEQ-Illumina) method.

Children in the obesity and healthy groups with at least one of the following criteria were excluded from the study:

Having acute infection, having any chronic diseases (cardiovascular, gastrointestinal, neurological, etc.), smokers, those with type 1 or type 2 diabetes mellitus, having any hereditary diseases, inflammatory diseases, infectious diseases, those with a history of drug use that may cause obesity (steroids, antipsychotics, etc.), having any endocrine pathology leading to secondary obesity (Cushing’s syndrome, hypothyroidism, etc.), and obesity-related syndrome (including Prader-Willi and Laurence-Moon syndromes).

Statistical Analyses

All data were evaluated through the SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) statistical software. The Shapiro–Wilk test was used to detect whether the data were normally distributed or not. As we found that the data were not normally distributed (p < 0.05), nonparametric tests were used for statistical analysis. The Mann–Whitney U test was used for double comparison. Moreover, crosstab analysis (chi-square test) was used to examine the relationship between two categorical variables. Risk variables (odds ratio [OR]) and 95% confidence intervals (95% CI) of these values were calculated by logistic regression analysis for variables with significant differences between the groups. The statistical significance level (p value) was accepted as p ≤ 0.05 in all tests.

Results

Forty-five (76.3%) obese patients and fourteen (23.7%) control subjects were included in the study. There was no significant difference between obese patients and the control group regarding gender, age at admission, gestational week, and birth weight (p > 0.05).

While the HbA1c value was 5.3 ± 0.4% in the obesity group, it was 5.5 ± 0.2% in the control group (p > 0.05). Insulin and ALT values were significantly higher in the obesity group compared with the control group (p = 0.012; p = 0.007, respectively). Although total direct and indirect bilirubin levels were within normal limits, the values were lower in obese children than in the control group (p = 0.422; p = 0.026; p = 0.026, respectively). The demographic, clinical characteristics, and laboratory test results of the obese patients and control subjects are given in Table 1.

Table 1.

Demographic and Clinical Characteristics and Laboratory Test Results of Obesity and Control Groups

	Obesity group (min–max)	Control group (min–max)	Z	p
	n: 45	n: 14
Gender (female; male)	22 (48.9%); 23 (51.1%)	7 (50%); 7 (50%)		0.942
Age at admission (months)	142.9 ± 38.8 (76–215)	134.6 ± 39.2 (77–208)	−0.330	0.742
Gestational week	39.3 ± 1.6 (32–41)	39.6 ± 0.8 (38–40)	−0.402	0.687
Weight at delivery (gr)	3365.3 ± 544.3	3359.2 ± 136.0	−0.590	0.555
Type of delivery (C/S; NVD)	28 (62.2%); 17 (37.8%)	3 (21.4%); 11 (78.6%)		0.018*
Breastfeeding (months)	16.5 ± 5.2	15.2 ± 11.3		0.621
Infant formula (yes; no)	18 (40%); 27 (60%)	6 (42.9%); 8 (57.1%)		0.849
Height at admission (SD)	0.91 ± 1.11 (−2.0 to 2.91)	0.03 ± 0.90 (−1.69 to 1.68)	−2.539	0.011*
BMI at admission (kg/m2)	29.9 ± 5.7 (17–43.70)	17.9 ± 3.3 (14.10–25.30)	−5.239	0.000**
BMI at admission (SD)	2.73 ± 0.61 (2.01–4.20)	−0.31 ± 0.86 (−1.81 to 1.46)	−5.613	0.000**
Fasting blood glucose level (mg/dL)	90.4 ± 9.6 (72.10–118)	99.6 ± 11.3 (84.20–122.70)	−2.744	0.006**
HbA1c (%)	5.3 ± 0.4 (4.30–6.30)	5.2 ± 0.2 (4.90–5.58)	−0.842	0.400
Insulin (uIU/mL)	14.9 ± 7.3 (4.77–36.87)	9.3 ± 6.3 (2.70–19.77)	−2.512	0.012*
ALT (U/L)	24.1 ± 13.5 (9.65–62.40)	15.7 ± 5.2 (8.84–25.90)	−2.700	0.007**
AST (U/L)	25.4 ± 9.0 (0.39–53.40)	27.0 ± 7.1 (20.16–39.46)	−0.900	0.368
Total bilirubin (mg/dL)	0.50 ± 0.19 (0.26–1)	0.60 ± 0.33 (0.22–1.32)	−0.802	0.422
Direct bilirubin (mg/dL)	0.09 ± 0.03 (0.04–0.15)	0.12 ± 0.56 (0.06–0.25)	−2.234	0.026*
Indirect bilirubin (mg/dL)	0.42 ± 0.18 (0.22–1.01)	0.47 ± 0.28 (0.13–1.07)	−0.571	0.568
GGT (U/L)	18.8 ± 7.4 (6–46)	13.0 ± 4.9 (9–25.50)	−3.405	0.001**
CRP (mg/dL)	0.45 ± 0.28 (0.10–1.63)	1.05 ± 2.18 (0.10–7.73)	−1.663	0.096
Total cholesterol (mg/dL)	170.01 ± 29.74 (90–239.30)	133.21 ± 23.94 (92.20–171.40)	−3.707	0.000**
Triglycerides (mg/dL)	137.61 ± 76.69 (41–342)	62.71 ± 21.59 (28.78–102.19)	−3.982	0.000**
HDL (mg/dL)	47.05 ± 11.65 (30.70–72.40)	47.48 ± 12.24 (26.31–68.50)	−0.241	0.810
LDL (mg/dL)	94.44 ± 22.57 (47.80–157)	72.61 ± 12.95 (46.90–96.60)	−3.525	0.000**
Uric acid (mg/dL)	5.29 ± 1.27 (3.14–9.43)	4.05 ± 1.32 (2.20–6.42)	−2.771	0.006**
fT4 (ng/mL)	0.86 ± 0.13 (0.56–1.18)	0.84 ± 0.15 (0.53–1.14)	−0.294	0.769
TSH (uIU/mL)	2.82 ± 1.20 (1.32–6.75)	2.49 ± 1.08 (0.87–4.84)	−0.232	0.817
Hepatosteatosis (yes; no)	16 (35.5%); 29 (64.5%)	0 (0%); 14 (100%)		0.009**

^* Mann–Whitney U test.

^** Chi-square test p < 0.05.

Bold data indicate p < 0.05.

C/S, cesarean section; NVD, normal vaginal delivery; BMI, body mass index; ALT, alanine transaminase; AST, aspartate transaminase; GGT, gamma-glutamyl transferase; CRP, C reactive protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TSH, thyroid stimulating hormone; min–max, minimum–maximum; SD, standard deviation.

Total direct and indirect bilirubin levels of the obese children with UGT1A1 variations were significantly higher compared with those without any variations (p = 0.016; p = 0.028; p = 0.015, respectively) (Table 2).

Table 2.

Comparison of Total, Direct, and Indirect Bilirubin Levels and Status of UGT1A1 Variation in Obesity Group

	Obesity group (n = 45)
	UGT1A1 variation (+)	UGT1A1 variation (−)	Z	p
	(n = 28)	(n = 17)
Total bilirubin	0.58 ± 0.27	0.40 ± 0.06	−2.414	0.016*
Direct bilirubin	0.10 ± 0.03	0.08 ± 0.02	−2.201	0.028*
Indirect bilirubin	0.48 ± 0.21	0.32 ± 0.05	−2.437	0.015*

^* Mann–Whitney U test.

Bold data indicate p < 0.05.

UGT1A1, uridin diphosphate glucronyl transferase 1A1.

There were 17 (37.8%) obese children whose onset of obesity was within the first two years of life (early-onset group) and 28 children who were diagnosed with obesity at any age after the first two years of life (late-onset group). Total bilirubin levels were significantly higher in the late onset obesity group (p = 0.013). When we compared the groups using percentiles and chi-square tests in relation to genetic variations, we found that the rate of genetic variations was significantly higher in the late-onset group (p = 0.000) (Table 3). Since genetic variations in the UGT1A1 gene were more frequent in the late-onset obesity group, we evaluated the relationship between the genetic variations and the age at onset of obesity and found that the risk of late-onset of obesity was three times higher in those with genetic variations (3.42, 95% CI, 0.15–5.11 p = 0.000).

Table 3.

Comparison of Total Bilirubin, Direct Bilirubin, and UGT1A1 Variations Between the Obesity Groups with Onsets Before and After 2 Years of Age

	Obesity onset before 2 years of age (n: 17)	Obesity onset after 2 years of age (n: 28)	p
Total bilirubin (mg/dL)	0.42 ± 0.09	0.56 ± 0.24	0.013*
Direct bilirubin (mg/dL)	0.081 ± 0.001	0.096 ± 0.03	0.010*
UGT1A1 variation	7 (38.9%)	21 (77.8%)	0.000**

^* Mann–Whitney U test.

^** Chi-square test p < 0.05.

Bold data indicate p < 0.05.

UGT1A1, uridin diphosphate glucronyl transferase 1A1.

Discussion

Owing to major problems caused by obesity, studies have expanded to investigate the genetic traces of obesity in the human genome map. To the best of our knowledge, there are no studies that have investigated the whole exome sequencing of the UGT1A1 gene in obese individuals using NGS methods. Therefore, our study is the first on this subject.

The main outcome of our study is that obese children with UGT1A1 variations have higher bilirubin levels than those who do not have a variation.

In recent years, there have been many studies about bilirubin. It is thought that bilirubin may be an antioxidant (Wu et al., 2011). The previous studies demonstrated the protective effect of high bilirubin on coronary and peripheral arterial diseases, ischemic diseases, strokes, cardiovascular diseases, insulin resistance, and multiple sclerosis (MS) (Vítek 2012; Jain et al., 2021; Mahabadi et al., 2014; Wang et al., 2020a; Lin et al., 2009b). Furthermore, it has also been suggested that there is an inverse relationship between high bilirubin concentration and HbA1c level, hyperinsulinemia, and urinary albumin excretion (Wu et al., 2011; Nishimura et al., 2015; Choi et al., 2012). The only study that investigated the UGT1A1*28 genetic alteration in young obese patients was performed by Belo et al., in 2014. In this study, it was shown that body fat percentage was significantly associated with UGT1A1*28 gene polymorphism, even though no significant association was established between obese and healthy children in terms of bilirubin levels (Belo et al., 2014). In our study, in line with the literature, we found that the total bilirubin levels decreased as the patient’s weight increased. However, this decrease was not significant. Considering that low bilirubin levels have been associated with many chronic and metabolic diseases in the literature, our results show that there may be a relationship between low bilirubin levels and obesity.

We investigated the relationship between UGT1A1 variations and nonsyndromic childhood obesity, and we found that 28 (62.2%) children with obesity had genetic variations, while 7 (50%) children in the control group had genetic variations. In the literature, it has been reported that 11G>A (Gly71Arg), one of the most common variations in the coding region of the UGT1A1 gene, causes neonatal hyperbilirubinemia (Wang et al., 2020b). In our study, we found that the 11G>A (Gly71Arg) variation in the UGT1A1 gene was present only in obese children. Furthermore, when we divided the children with obesity into two groups, with or without UGT1A1 variations, the total direct and indirect bilirubin levels of obese children carrying genetic variations were significantly higher than those who did not carry a variation (p < 0.05). Perhaps this and similar variations protect hyperbilirubinemia. Considering the protective effects of hyperbilirubinemia, people with high levels provided through variation can be more fortunate in life. However, additional studies with more participants should be conducted to clarify the clinical and biochemical effects of these variations on obese patients.

In our study, we showed a statistically significant relationship between the UGT1A1 variations and the age of onset of obesity. As far as we know, breast milk exerts a protective effect against childhood obesity in the first 1000 days of life (Rito et al., 2019). We accepted the age of onset of obesity as two years of age because the baby is separated from the mother during this period and starts to be fed without breast milk (supplementary foods and formulas are replaced by table foods and the baby starts physical activities such as walking and running). The rate of UGT1A1 variation in the late obesity group, which developed obesity after two years of age, is two times higher compared with that of those with early onset obesity (38.9% vs 77.8%). We also showed a significant relationship between the late onset obesity and UGT1A1 variations (OR: 3.42, 95% CI: 0.15–5.11, p = 0.000) and this relationship also included bilirubin levels. In light of this information, we can suggest that the protective effect of lower bilirubin levels in obesity owing to lesser UGT1A1 variations may be lower in the early-onset obesity group. If this assumption is supported by additional studies with wider participation, it could be suggested that obese patients who have developed obesity in the first two years of life should be followed-up more carefully and those anti-obesity strategies should be started in the first year of life.

Conclusion and Suggestions for Future Studies

To conclude, obese patients had lower bilirubin levels and more genetic variations in the UGT1A1 gene than the control group. When we divided the obese children into two groups based on the presence and absence of the UGT1A1 gene variation, we found that children with variations had higher bilirubin levels than those without variations. Children diagnosed with obesity in the first two years of life had fewer genetic variations and lower bilirubin levels. In other words, assuming that variations in the UGT1A1 gene create bilirubin’s protective effect against obesity, we believe that bilirubin levels may have increased as a result of variations in obese patients. Perhaps with a new adaptation, the human body is trying to take advantage of bilirubin’s protective effect by increasing its presence. The lower bilirubin levels owing to fewer genetic variations in patients diagnosed with obesity under two years of age make children of that age group more vulnerable to obesity and its complications. We think that humans can protect themselves from obesity and its many negative effects with higher bilirubin levels, which are inversely correlated with many chronic diseases. So, we should investigate whether the variations in the UGT1A1 gene can be used to have information on chronic diseases of humanity such as obesity, MS, cardiovascular risk, and diabetes; and, whether the treatment strategies (transfer of the UGT1A1 gene carrying a variation that may cause hyperbilirubinemia, etc.) can be developed to eliminate the problems caused by these diseases related to the variations in this gene.

Our study has some limitations

First, the maternal factors could not be completely evaluated. Second, the number of children in the healthy control and obesity groups was low owing to financial concerns. Last, the daily eating frequencies and exercise habits of the children were not investigated, and other genes related to obesity were not evaluated.

Authors’ Contributions

M.A.: Conceptualization, software, resources, writing—review and editing. Ö.K.: Conceptualization, methodology, software, writing—review and editing. R.E.: Formal analysis, data curation. K.K.: Methodology, supervision.

Author Disclosure Statement

The authors have no financial or competing interests in relation to this work.

Funding Information

This work was supported by Duzce University, Project No: DUBAP2018.04.03.797.