Do Gene Polymorphisms Play a Role in Newborn Hyperbilirubinemia?

Hyperbilirubinemia is a common finding in the neonatal period and can sometimes lead to serious consequences such as kernicterus. Most cases of neonatal hyperbilirubinemia (NH) are physiological, and approximately 13.4% of the cases are non-physiological [1, 2]. In approximately half of cases of pathological jaundice, there is no identifiable factor [3]. Studies have shown that African newborns have lower serum bilirubin levels and Asian babies develop higher values than their Caucasian counterparts [4]. In a study, the incidence of non-physiological significant hyperbilirubinemia in Turkish newborns was found to be between approximately 10.5% and 25.3% [5]. This may be due to differences in the genetic backgrounds of the populations, suggesting the existence of genetic risk factors for the development of NH [6].

Unconjugated bilirubin is rapidly and selectively taken up across the basolateral membrane of the hepatocyte as a carrier-mediated process involving the partially dissolved carrier organic anion-carrying polypeptide-1B1/B3 (SLCO1B1 and SLCO1B3 genes). SLCO1B1 and SLCO1B3 are sinusoidal transporters that facilitate hepatic uptake of a wide variety of endogenous substrates [1, 2]. In hepatocytes, glutathione S-transferases (GSTs) are involved in binding to nonsubstrate ligands, such as unconjugated bilirubin [3]. Unconjugated bilirubin is then conjugated with glucuronate through the enzyme activity of uridine diphosphate glucuronyl transferase (UGT1A1 gene) [1]. In particular, polymorphisms of four genes specifically related to bilirubin production and metabolism (UGT1A1, SLCO1B1, SLCO1B3, and ⁄ or GST) may interact with environmental contributors to produce significant hyperbilirubinemia [3, 7].

The aim of this study is to investigate whether polymorphisms in the UGT1A1, SLCO1B1, SLCO1B3 and GST genes are a contributing factor to idiopathic hyperbilirubinemia or prolonged jaundice with unexplained etiology in Turkish newborns.

Demographic characteristics of the three groups are shown in Table 1. There was no difference between the groups in terms of mean gestational age and birth weight. There was no difference between the groups in terms of feeding patterns, gender and mode of delivery. There is a significant difference between the groups in terms of bilirubin levels.

The genotype distributions on the basis of nucleotides in these three groups are shown in Table 2. There was no significant difference in genotype distribution between the case and control groups (P>0.05 in all comparisons). There were no statistically significant differences in the prevalence of the variant of UGT1A1 gene at nt 211, variants of SLCO1B1 gene at nt 388, 463, 521 and 1463 variants of SLCO1B3 gene at nt 334, 727+118, 1865+19721 variants of GSTP1 gene at nt 313 and nt 341, and allele frequency of these genes (except SLCO1B3 at nt 334) among the three groups (P>0.05 in all comparisons, Table 2). The G allele frequency of the variant of SLCO1B3 gene at nt 334 of the infants in the idiopathic hyperbilirubinemia group was 85%, which was significantly lower than that in the control group (96%, P=0.03; Table 2). There was no statistically significant difference in the frequency of the G allele of the SLCO1B3 variant at nt 334 between infants in the prolonged jaundice and control groups (P=0.16). No variant of the SLCO1B1 gene was found at nt 1463 (rs59502379).

Neonatal jaundice is a good example of a complex condition in which the usual clinical factors examined alone cannot reveal the real cause. Although a few studies have been conducted to define the mechanisms involved in NH, more attention has recently begun to be paid to genetic factors as a contributing cause [8]. This study aimed to elucidate the contribution of multiple genetic modifiers affecting bilirubin metabolism to the development of pathological hyperbilirubinemia in Turkish newborns. UGT is the key enzyme of bilirubin conjugation. 211G > A (Gly71Arg), one of the most common UGT1A1 gene polymorphisms in the coding region, may cause unconjugated hyperbilirubinemia by reducing enzymatic activity [9]. Recently, in a meta-analysis of 21 studies including 4738 newborns, it was reported that UGT1A1 Gly71Arg variation may increase the risk of NH in Asian and African children [10]. There are two studies investigating the relationship between hyperbilirubinemia and the G71R variant in Turkish newborns. Kilic et al. [11] showed the frequency of heterozygous G71R variants in pathological jaundice, prolonged jaundice and control groups as 8.7%, 20.8% and 4.3%, respectively; however, these differences were not statistically significant. Narter et al. [12] reported a frequency of 33.3% and 27.1% for heterozygous G71R variants and 7.7% and 5.7% for homozygous G71R variants in the hyperbilirubinemia and control groups, respectively, and no statistically significant difference was found between these frequencies. Our study showed that the frequency of heterozygous G71R variants in idiopathic hyperbilirubinemia, prolonged jaundice, and control groups was 3.8%, 22.2%, and 7.9%, respectively. But these differences were not statistically significant. The frequency of homozygous G71R variant in idiopathic hyperbilirubinemia was 3.8%. However, no homozygous G71R variant was found in the prolonged jaundice and control groups.

SLCO1B1/B3, the gene encoding the hepatic solute transporter organic anion transporter 1B1, a putative bilirubin transporter, may also be associated with increased susceptibility to NH by limiting bilirubin uptake [6, 13]. In our study, SLCO1B1 A388G in both the heterozygous and homozygous variants was found 72.2%, 75% and 53.7% in the idiopathic hyperbilirubinemia, prolonged jaundice and control groups, respectively. Other studies of SCLO1B1 in different countries showed similar findings to our study in terms of the incidence of polymorphism. In studies conducted on newborns with hyperbilirubinemia, the SLCO1B1 A388G variant was found at a rate of 77.7% in India and 87.7% in Malaysia [14, 15]. All these studies show that more than half of the studied population has the A388G variant. Our study showed that the SLCO1B1 A388G variant was not a significant risk factor for idiopathic and prolonged hyperbilirubinemia. There is tentative evidence from different studies in different populations regarding the association between SLCO1B1 A388G and high bilirubin levels in newborns. In a study conducted on Taiwanese newborns in 2004 by Huang et al. [16], it was reported that A388G was seen more frequently in babies with hyperbilirubinemia. Liu et al. [17] found that the A388G variant in Chinese newborns was associated with hyperbilirubinemia in the Guangdong population, but not in the Yunnan population. In our country, Büyükkale et al. [18] reported that polymorphic forms of 388 nucleotides of the SLCO1B1 gene were risk factors for idiopathic hyperbilirubinemia. Other studies conducted in Malaysian, Brazilian, American or Thai populations also failed to prove the association of the A388G variant with NH [15, 19,20,21].

In our study, there was no statistically significant difference in NH risk between SLCO1B1 T521C allele carriers (T/C+C/C) and T/T allele carriers; the same result was observed in the SLCO1B1 T521C variant when the T allele was compared with the C allele. A meta-analysis including five case-control studies (637 subjects with hyperbilirubinemia and 918 control subjects) from three countries examined the association between the SLCO1B1 T521C variant and NH. This meta-analysis reported that the SLCO1B1 T521C variant conferred protection for NH in the Chinese population but not in the Malaysian, Taiwanese, Brazilian, or American populations; when the T allele was compared with the C allele, the same situation was observed in the SLCO1B1 T521C variant [17].

Analysis of the SLCO1B1 C463A variant in our study showed a crossover variant in 14 of 61 (23%) neonates with idiopathic hyperbilirubinemia (13 heterozygous and one homozygous) and in 8 of 28 (28.6%) neonates with prolonged jaundice (7 heterozygous and one homozygous), and 7 of 39 (24.75%) control subjects (all heterozygous). The SLCO1B1 C463A variant was not a significant risk factor for idiopathic and prolonged hyperbilirubinemia in the present study. A meta-analysis of three case-control studies involving 286 cases of hyperbilirubinemia and 456 controls from three countries examined the association between the SLCO1B1 C463A variant and NH [17]. In studies conducted on Taiwanese and Thai newborns, carriage of the C to A substitution at nucleotide 463 was not detected [16, 21]; however, the study conducted by Watchko et al. [20] including American subjects showed that in those 31 of 153 newborns (20.26%) with hyperbilirubinemia (one homozygous and 30 heterozygous) and 74 of 299 control subjects (24.75%) (nine homozygous and 65 heterozygous). In that study (20), no statistically significant difference was found in the risk of NH between allele carriers (C/A+A/A) and (C/C) in the SLCO1B1 C463A variant; the same situation was observed when the A allele in the SLCO1B1 C463A variant was compared with the C allele; the same situation was observed when the A allele was compared with the C allele in the SLCO1B1 C463A variant.

There was no mutant allele in SLCO1B1 at Nt 1463 G>C. However, according to our research, no study investigating this variant has been found in the literature.

Similar to SLCO1B1 polymorphisms in this study, SLCO1B3 polymorphisms did not show statistical differences in genotype distribution. It was found in the present study that only the G allele at nt 344 of SLCO1B3 may be a protective factor for idiopathic NH. However, it does not protect against prolonged jaundice. No study investigating the relationship between SLCO1B3 nt 334 variant and NH has been found in the literature. However, there are very few studies on the C727+118G and C1865+19721T polymorphisms of this gene. Alencastro de Azevedo et al. [19] reported that the allelic and genotypic frequencies of SLCO1B3 gene C727+118G and C1865+19721T variants did not differ between idiopathic hyperbilirubinemia and control groups. They stated that only the T allele at nt 1865+19721 could be protective against NH in those without ABO incompatibility.

During the bilirubin conjugation process in hepatocytes, bilirubin binds to GST, also known as ligandin [22]. GST binds both bilirubin and bilirubin conjugates and reduces hepatocyte reflux into plasma [23]. The present study found that there is no statistically significant difference in the genotype and allele frequencies of GSTP1 A313G and C341T among the idiopathic hyperbilirubinemia, prolonged jaundice, and control groups. Muslu et al. [24], from Türkiye, reported that the frequencies of GSTM1 and GSTT1 were similar in newborns with hyperbilirubinemia and control groups.

In this study, no significant difference was found in terms of peak total bilirubin levels and onset time of hyperbilirubinemia in newborns in the idiopathic hyperbilirubinemia group with different genotypes.

The limitation of the present study is that the need for phototherapy was taken as a criterion, which does not have a strong discriminatory feature in determining genetic variability differences. Another limitation of our study is the small number of cases.

The role of UGT1A1, SLCO and GST polymorphisms in neonatal jaundice is still controversial and deserves further attention. Although they do not have the power to modulate neonatal jaundice, other genes that play a role in bilirubin metabolism (heme oxygenase and biliverdin reductase) other than the genes mentioned above can be investigated.