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Neonatal hyperbilirubinemia is a common symptom in the neonatal period, with a complex pathogenesis. Recently, UGT1A1 gene mutations have been considered a risk factor for this ailment.

UGT1A1 is a key enzyme in the metabolism of bilirubin (1), and is encoded by the UGT1A1 gene. Ritter et al. (2) first discovered that UGT1A1 gene product shows glucuronosyltransferase activity. UGT1A1 is located at chromosome 2q37 (3), and includes five exons (exons 1–5). The promoter region is close to the upstream of the transcription start site of exon 1, including the TATA-box. Mutations of the UGT1A1 gene may cause decreased or abolished enzyme activity, leading to neonatal hyperbilirubinemia (4).

More than 100 types of UGT1A1 gene mutations have been identified so far. The mutational hotspots are mainly located in the promoter region and exon1 of UGT1A1 (5). According to the previous studies, UGT1A1 gene mutations and its relation to neonatal hyperbilirubinemia may differ among areas and ethnic groups. The (TA)7 insertion in the UGT1A1 promoter is the most common mutation in Caucasians, and appears to cause Gilbert’s syndrome (6,7). Meanwhile, the G71R substitution in exon 1 of the UGT1A1 gene is the most common mutation type described as a risk factor for neonatal hyperbilirubinemia in Asians (8,9).

There is a high incidence of neonatal hyperbilirubinemia in the Guangxi area, which is also a multiethnic region. Han is the largest ethnic group there, followed by Zhuang. Heiyi Zhuang is a major branch of the Zhuang ethnic group, and comprises people mainly living in Napo County, Guangxi, China. We hypothesized that UGT1A1 gene mutations may be related to neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations. So, we performed this study to test this hypothesis.

Results

Demographic Factors

Demographic characteristics of the cases and controls are shown in Table 1 . No differences between the two groups were found in gestational age, birth weight, gender, feeding pattern, and population.

Table 1 Demographic characteristics of newborns enrolled in the study
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UGT1A1 Polymorphisms

The following variations were observed in the promoter TATA-box: A(TA)6TAA/A(TA)6TAA, (TA)6/6; A(TA)6TAA/A(TA)7TAA, (TA)6/7; A(TA)7TAA/A(TA)7TAA, (TA)7/7. Other variations were at nucleotides 211 (G>A, heterozygous and homozygous) and 686 (C>A, heterozygous), in addition to a SNP (rs191471887, 189C>T, heterozygous) in the exon 1 of the UGT1A1 gene.

As shown in Table 2 , 211G>A was the most common mutation in this study. The incidence rates of homozygous variation (A/A), heterozygous variation (G/A), and wild type (G/G) at nucleotide 211 were 3.7, 27.1, and 66.1%, respectively, in the case group, and 0, 16.3, and 83.7%, respectively, in the control group. Meanwhile, (TA)7 insertion mutation was the second common mutation. The incidence rates of homozygous variation ((TA)7/7), heterozygous variation ((TA)6/7), and wild type ((TA)6/6) in the promoter TATA-box were 1.8, 17.4, and 80.7%, respectively, in the case group, and 0.5, 25.2, and 74.2%, respectively, in the control group.

Table 2 Odds ratios (ORs) and 95% confidence intervals (CIs) for neonatal hyperbilirubinemia with suspected factors
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Only one 686C>A (P229Q) heterozygous carrier and two 189C>T (rs191471887) heterozygous carriers were identified in the Han ethnic group. Therefore, statistical analysis was not possible.

Risk Factor of Neonatal Hyperbilirubinemia

Logistic regression analysis ( Table 2 ) showed that the genotypic distribution of UGT1A1 211G>A (G71R) differed significantly between the case and control groups. In addition, 211G>A homozygosity and heterozygosity were both significantly associated with neonatal hyperbilirubinemia. Moreover, the ORs of developing hyperbilirubinemia were even higher in neonates harboring homozygous 211G>A (34.23) compared with their heterozygous counterparts (2.10).

However, the distribution of (TA)7 insertion mutations was not significantly different between the two groups. In addition, no association was found between (TA)7 insertion mutation and neonatal hyperbilirubinemia, either in (TA)7/7 homozygous or (TA)6/7 heterozygous individuals.

Furthermore, breastfeeding was not a significant independent risk factor for neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations (OR = 0.65; 95% CI 0.41–1.01; P = 0.079).

Discussion

The exact pathogenesis of neonatal hyperbilirubinemia is not yet clear. Except for other possible risk factors such as congenital malformations, G6PD deficiency, ABO incompatibility, and low birth weight, UGT1A1 gene mutations were found to be closely implicated (10,11,12). This study examined the association of UGT1A1 gene mutations and hyperbilirubinemia in two ethnic subpopulations of China. For the 211G>A genotype, a strong association was found, with estimated ORs of 34 and 2.10 obtained in homozygous and heterozygous individuals, respectively. No association was found for the (TA)7 genotype, and our data indicated that breastfeeding is not an independent risk factor for neonatal hyperbilirubinemia.

The reported mutation types of the UGT1A1 gene include insertion, deletion, missense, nonsense, and synonymous mutations (12). In this study, (TA)7 insertion, 211G>A (G71R) and 686C>A (P229Q) missense, and the synonymous 189C>T mutations were detected; no nonsense mutation was found.

Among the four mutations, 211G>A was the most common as shown above, and resulted in a change of glycine to arginine at position 71 (G71R). The A allele frequency in the case group was 20.4%, which is higher than data from Turkish (4.3%) (13) and North India (0%) (14), but lower than values in Korean (32%) (15) and Japan (34%) (16), and close to our previous study (23.3%) (17). The frequency of the 211G>A mutation was significantly higher in hyperbilirubinemia infants than controls in this study, indicating its close relationship to neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations. The same conclusion was reached in most Asian countries such as Korea (15), Japan (16), and other regions of China (18). However, different findings have been reported in Caucasians and Africans (6). Studies in Turkey (19) and the United States (20) found no significant association between 211G>A and neonatal hyperbilirubinemia. In addition, the risk for neonatal hyperbilirubinemia in individuals carrying homozygous 211G>A was higher than in their heterozygous counterparts in this study. Our previous study showed that G71R homozygous subjects have higher incidence of bilirubin encephalopathy compared with wild-type and heterozygous individuals (17). Therefore, more attention should be paid to homozygous 211G>A subjects.

(TA)7 insertion mutations are common in Caucasians and Africans but rare in East Asians (8,21,22,23). Although (TA)7 insertion mutation was the second common mutation in this study, (TA)7/7 homozygous individuals were rare as well. In addition, logistic regression analysis showed that (TA)7 insertion mutation alone is not a risk factor for neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations. This corroborated other findings in most of the Asians (9,23). However, discrepant conclusions were reached for Caucasians. Indeed, (TA)7 insertion is the most common mutation causing Gilbert syndrome in Caucasians (6,7), and multiple studies have revealed its relationship with neonatal hyperbilirubinemia in Caucasians (14,24,25,26,27,28,29,30). Roy-Chowdhury et al. (24) reported that the (TA)7 allele may be associated with high TSB levels. Agrawal et al. (14) reported that (TA)n promoter variations are significantly associated with neonatal hyperbilirubinemia in North India. However, the majority of studies assessing Caucasians, including two recent ones, have shown that (TA)7 insertion alone does not necessarily cause neonatal hyperbilirubinemia (20,25,26,27), in agreement with Asian studies. But it increases the risk for hyperbilirubinemia when the neonates also have G6PD deficiency, are breastfed, or have concomitant ABO incompatibility (20,28,29,30).

Previous studies have suggested breastfeeding as a significant risk factor for hyperbilirubinemia (29,31). The possible explanation is that breast milk contains β-glucuronidase, which increases the entero-hepatic circulation and inhibits the hepatic excretion of bilirubin (32). Unlike the studies mentioned above, we did not find any contributory role of breastfeeding in neonatal hyperbilirubinemia, as breastfeeding was universally present in both the case and control groups, in agreement with a previous report (11).

In conclusion, our results showed that UGT1A1 G71R mutation is a strong risk factor for neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations. Meanwhile, (TA)7 insertion mutation is not associated with neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations. Therefore, a large sample investigation is warranted to confirm our findings.

Methods

Study Subjects

The case-control study method was applied. A total of 408 subjects were included, all full-term newborns who were born in The First Affiliated Hospital of Guangxi Medical University and People’s Hospital of Napo, between June 2012 and October 2013. They were 2–14 d old, with birth weights of 2.5–4.0 kg. The diagnostic standard of neonatal hyperbilirubinemia was done following the 2004 American Academy of Pediatrics guidelines (33). The case group included 218 neonates (118 of Heiyi Zhuang origin, 100 of Han origin) with unexplained hyperbilirubinemia (total serum bilirubin level above the 95th percentile of Bhutani nomogram (33). The neonates had no known risk factors for neonatal hyperbilirubinemia (such as congenital malformations, glucose-6- phosphate dehydrogenase(G6PD) deficiency, ABO incompatibility, small size for gestational age, asphyxia, cephalohematoma, infection, abnormal liver function, polycythemia, low blood sugar and the mother with pregnancy-induced hypertension, anemia, and/or diabetes during pregnancy). The control group comprised 190 neonates (110 of Heiyi Zhuang origin and 80 of Han origin) without hyperbilirubinemia (total serum bilirubin level below the 40th percentile of Bhutani nomogram (33).

The study was approved by the Institutional Review Board and ethics committee of The First Affiliated Hospital of Guangxi Medical University and People’s Hospital of Napo. Signed informed consent forms were obtained from the parents of all infants.

Genotyping

Peripheral blood samples were collected in tubes containing ethylene diamine tetraacetic acid (EDTA). Genomic DNA was extracted using the BloodGen Mini Kit (CWBIO, Beijing, China). The promoter TATA-box and exon 1 of the UGT1A1 gene were amplified by polymerase chain reaction (PCR), using previously described primers (34,35). The PCR mixture (50 µl) was consisted of 2 µl of DNA(≥100 ng/µl); 1 µl of each primer; 25 µl of 2× Taq Master Mix (CWBIO) containing 0.3 U Taq DNA Polymerase, 2× Taq PCR Buffer, 3 mmol/l MgCl2, and 400 µmol/l dNTP mix; 21 µl of deionized water. Fragment 1 was amplified under the following conditions: predenaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 60 s, annealing at 60 °C for 60 s, extension at 72 °C for 60 s; final extension at 72 °C for 5 min. Fragment 2 was amplified as follows: predenaturation at 94 °C for 2 min; 30 cycles of denaturation at 94 °C for 15 s, annealing at 62 °C for 30 s, extension at 68 °C for 30 s; final extension at 68 °C for 5 min. The PCR products were analyzed by 2% Agarose gel (DOC2000, Bio-Rad laboratories, Hercules, CA) electrophoresis.

Statistical Analysis

The SPSS software (revision 17.0, SPSS, Chicago, IL) was used for statistical analysis. Data were expressed as medians (5–95 percentiles) for continuous variables. T-test was used to compare continuous variables while chi-squared test (χ2) was utilized to compare categorical variables between the case and control groups. Logistic regression analysis was performed to identify the risk factors for neonatal hyperbilirubinemia in Heiyi Zhuang and Han ethnic groups. A P value of less than 0.05 or a 95% CI for OR ≥ 1.0 was considered statistically significant.

Statement of Financial Support

This study was supported by the National Natural Science Foundation of China (Grant 81060055).

Disclosures:

None of the authors have competing financial interests.