Introduction
Neonatal hyperbilirubinemia(NH) and resultant jaundice are commonly seen in 50–60% of newborns and, to a greater extent, in premature infants [
1,
2]. Although most cases are benign and do not result in severe consequences, some infants will develop hazardous levels of bilirubin that directly threaten brain damage and may result in neuro-developmental abnormalities, such as hearing loss, athetosis, and intellectual deficit [
2]. In pathological hyperbilirubinemia, increased production of bilirubin, deficiency in hepatic uptake, impaired conjugation of bilirubin, and/or increased enterohepatic circulation of bilirubin are observed [
3]. Severe hyperbilirubinemia can be caused by maternofetal blood group isoimmunization (especially ABO hemolysis), G-6-PD deficiency, and severe infection [
4]. Laboratory testing can predict this part of infants in advance and help guide active intervention to prevent exchange transfusion and reduce the risk of bilirubin encephalopathy. However, routine laboratory tests do not identify the etiology of severe hyperbilirubinemia in over 50% of infants [
5,
6].
The American Academy of Pediatrics recommendations in 2004 identified East Asian ancestry, particularly mainland China, as a substantial risk factor for severe hyperbilirubinemia [
7]. A positive family history can be a marker for shared genetic susceptibility; several studies [
8,
9] have identified a previous sibling with a history of neonatal jaundice as an essential risk factor for neonatal hyperbilirubinemia. If an earlier sibling had a serum bilirubin > 15 mg/dL (257 µmol/L), the risk in subsequent siblings increased to 12.5-fold greater than controls [
10].In addition, Ebbesen’s [
11] research on bilirubin levels in identical and fraternal twins showed that after controlling for the factors known to regulate neonatal hyperbilirubinemia, genetics still has an important influence on neonatal bilirubin levels. Thus, genetic factors play an important role in the development of neonatal hyperbilirubinemia, especially genetic polymorphism of key enzymes involved in bilirubin metabolism. A growing body of evidence suggested that genetic variants in uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), solute carrier organic anion transporter family members 1B1 and 1B3 (SLCO1B1, SLCO1B3), glucose-6-phosphate dehydrogenase deficiency (G-6-PD), heme oxygenase 1 (HMOX1), and biliverdin reductase A (BLVRA) are closely associated with the incidence of severe hyperbilirubinemia.
However, the combined action of multiple genes on the occurrence of severe neonatal hyperbilirubinemia remains unclear. This study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in genetic variants in bilirubin metabolism and the risk of severe hyperbilirubinemia.
Materials and methods
Patient characteristics
Between January 1, 2019, and June 30, 2020, 144 neonates admitted to three hospitals in southwest China with severe hyperbilirubinemia and meeting the following inclusion criteria were enrolled as the case group: (1) gestational age(GA) ≥ 35 weeks, within seven days of birth; (2) indirect bilirubin was the predominate component of serum bilirubin, accounting for more than 80% of the total bilirubin; (3) total bilirubin level met the exchange transfusion criteria in compliance with the guideline of management of hyperbilirubinemia endorsed by American Academy of Pediatrics in 2004 [
7]. Meanwhile, a control group of 50 neonates was recruited, with GA ≥ 35 weeks and an age exceeding 7 days, who was in the absence of hyperbilirubinemia after brith or no phototherapy history. Additionally, their transcutaneous bilirubin levels monitored at community-based health facility after birth and blood bilirubin levels at admission were below the criteria requiring phototherapy [
7]. Those with definite infection, multiple organ malformations, evident ABO or Rh incompatibility, and G-6-PD deficiency were excluded from this study. The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the Children ‘s Hospital of Chongqing Medical University. Written informed consent was obtained from the parents or guardians.
SNP selection
UGT1A1, SLCO1B1, SLCO1B3, HMOX1, and BLVRA genes were chosen for their crucial involvement in bilirubin metabolism. TagSNPs selected for the validation were based on the following criteria: (1) SNPs within 2 kb upstream and downstream of the gene area; (2) when multiple associated SNPs were in strong linkage disequilibrium (LD, r2 > 0.8), SNPs previously reported in the literature were prioritized. Twelve tagSNPs were chosen based on these parameters, including six from UGT1A1, two from SLCO1B1, two from SLCO1B3, one from BLVRA, and one from HMOX1(Table
1).
Table 1
Genotyping data for 12 tagSNPs genotyped from the candidate genes UGT1A1, SLCO1B1, SLCO1B3, BLVRA, and HMOX1
UGT1A1(2) | rs4148323 | 234,669,144 | exonic | 0.192 | 0.138 | A |
| rs3771341 | 234,673,239 | intronic | 0.113 | 0.127 | A |
| rs34946978 | 234,676,872 | exonic | 0.024 | 0.011 | T |
| rs114982090 | 234,680,955 | exonic | 0.005 | 0.007 | T |
| rs35350960 | 234,669,619 | exonic | 0.007 | 0.014 | A |
| rs34993780 | 234,681,059 | exonic | 0.002 | 0.003 | G |
SLCO1B1(12) | rs4149056 | 21,331,549 | exonic | 0.127 | 0.123 | C |
| rs1564370 | 21,335,190 | intronic | 0.221 | 0.244 | G |
SLCO1B3(12) | rs2417940 | 21,017,875 | intronic | 0.173 | 0.195 | T |
| rs2117032 | 21,074,122 | 3’-flanking | 0.421 | 0.461 | C |
BLVRA(7) | rs699512 | 43,810,764 | exonic | 0.310 | 0.317 | G |
HMOX1(22) | rs2071747 | 35,777,185 | exonic | 0.055 | 0.054 | C |
DNA isolation
Venous blood samples (2 to 3 ml) were collected and transferred to EDTA-anticoagulated tubes(Suzhou Bidi Medical Devices Co., Ltd.; Suzhou, China). The genomic DNA was isolated from EDTA-anticoagulated blood samples using the QIAamp DNA Kit (Qiagen, Hilden, Germany) and stored at − 80℃ for the subsequent experiments.
SNP genotyping [12]
The genotyping of 12 SNP loci in one ligation reaction was done in this work using an enhanced multiplex ligation detection reaction method. The 12 SNP sites weres amplified using a multiplex of PCR procedures. The PCR reaction in 10 µl contained 1x Takara GC-I buffer, 3.0 mM MgCl2, 0.3 mM dNTP mix, 1 U HotStar Taq polymerase(Qiagen Inc.), 1 µl of primer mixture and 1 µl of genomic DNA.
The PCR program for all reactions was 95℃ for 2 min; 11 cycles x (94℃ 20 s, 65℃-0.5℃/cycle 40 s, 72℃ 1 min 30 s); 24 cycles x (94℃ 20 s, 59℃ 30 s, 72℃1 min 30 s); and 72℃ for 2 min; holding at 4℃. Exonuclease I and shrimp alkaline phosphatase, which were digested at 37 °C for 1 h and 75 °C for 15 min, were used to purify 10 µl of PCR products. The labeling oligo mixture, probe mixture, 2 µl of ligation buffer, 80 U of Taq DNA Ligase (NEB), and 5 µl of the purified PCR product mixture are all included in the ligation reaction, which is contained in 10 µl.
The ligation cycling program was 95℃ for 2 min; 38 cycles x (94℃ 1 min, 56℃ 4 min); holding at 4℃. A 0.5 µl of ligation product was loaded in an ABI3730XL, and the raw data were analyzed by GeneMapper 4.1.All of the primers, probes and labeling oligos were designed by and ordered from Genesky Biotechnologies Inc (Shanghai, China).
Statistical analysis
Statistical analysis was performed by SPSS (version 19.0; SPSS, Inc., Chicago, IL, USA). Quantitative data were expressed as the mean ± SD and qualitative data as a percentage. Allelic frequencies were calculated by the gene-counting method. An unpaired Student’s t-test was utilized to compare the two groups. The Hardy-Weinberg test was used to evaluate the hereditary equilibrium. Linkage disequilibrium (LD) analyses were conducted using HaploView version 4.2 (Broad Institute, Cambridge, MA, United States) in Han Chinese in Beijing (CHB) populations from the 1000 Genomes Project phase 3 genotype data [
13]. The chi-square test or Fisher’s precision probability test was used to assess the frequencies of alleles and genotypes in the two groups under three distinct genetic models: dominant, recessive, and additive. We used multivariable logistic regression adjusting for GA and age. The genotype relative risk was calculated using odds ratios (ORs) and 95% confidence intervals (CIs). Logistic regression analysis was used to calculate the significance of differences in genotype and allele frequencies and investigate the association of tested SNPs with hyperbilirubinemia risk.
P < 0.05 was considered to be statistically significant.
Discussion
Hyperbilirubinemia, presenting as jaundice, is a ubiquitous and frequently benign condition in newborn babies, albeit a leading cause of hospitalization in the first week of life [
14]. Although the exact etiology of hyperbilirubinemia remains unclear [
15], the role of genetic factors in the pathogenesis of hyperbilirubinemia has received significant attention from neonatologists [
16]. The current study aims to determine the roles of several genetic bilirubin metabolism modifiers in developing severe hyperbilirubinemia in Chinese Han newborns. Our research indicated striking relationships between the vulnerability to severe hyperbilirubinemia and the gene polymorphisms for UGT1A1 and SLCO1B3.
UGT1A1 is a member of the UGT1 family of microsomal membranes and plays an essential role in converting the toxic form of bilirubin to its nontoxic form [
17]. The present study demonstrated that rs4148323 in the UGT1A1 gene is independently associated with total bilirubin levels. The frequency of the A allele in rs4148323 was associated with the incidence of severe hyperbilirubinemia. A prior study has reported that the A allele in rs4148323 is common in the East Asian population, with allele frequencies of 19.2% in Han Chinese and 19% in Korean populations [
18], and 16.2% in the Japanese population as well [
19], whereas it is monomorphic in the European populations (
http://www.ncbi.nlm.nih.gov/snp), indicating that rs4148323 might be specifically associated with serum bilirubin levels in Asians. In the control group, the A allele frequency was 13%, similar to another study in China [
20], and is much higher than in the severe hyperbilirubinemia group which had a frequency of 30.2%, similar to that observed in Japanese studies [
21].
SLCO1B3, located at 12p12, is highly expressed in the basolateral hepatocyte membrane. It is an organic anion transporter gene coding an organic anion transporter polypeptide (OATP/1B3) that mediates the extrusion of bilirubin [
22]. Genetic variants of SLCO1B3 contributed to idiopathic mild unconjugated hyperbilirubinemia [
23]. The variant of rs2417940, located in intron 7 of SLCO1B3, was significantly associated with total serum bilirubin levels [
18]. Our study demonstrated that the genotype CC and the frequency of allele C in the SLCO1B3-rs2417940 significantly correlated to the incidence of hyperbilirubinemia (81.3% vs. 58.0%,
P = 0.002, OR 2.989 and 90.6% vs. 78.0%,
P = 0.001, OR 2.726, respectively). Dai et al. [
24] reported that smoking and rs2417940 polymorphism in SLCO1B3 on total bilirubin levels had a significant interaction, and rs2417940 had a much stronger effect on serum bilirubin levels in nonsmokers than in smokers. The frequency of allele T in the control group with mild or no hyperbilirubinemia was 22.0%, similar to another report in China (18.3%) [
25].
We acknowledge that this study had several limitations. In this article, only twelve loci of genes that may affect bilirubin metabolism were assessed, including UGT1A1, SLCO1B1, SLCO1B3, HMOX1, and BLVRA. It is uncertain whether the polymorphism of other SNPs in the above genes or other genes would affect the serum bilirubin level. However, to our best knowledge, the current analysis was cost-effective, containing all relevant variations in these genes described in the Asian population. Another obvious limitation is that we could not examine the effect of medications on the metabolism of bilirubin levels or the activity of the target genes. However, medications are rarely prescribed for most neonates with hyperbilirubinemia in clinical settings.
Conclusion
This study demonstrated the influence of genetic polymorphisms of several hyperbilirubinemia-related genes, illuminating the complicated nature of this condition. However, severe neonatal hyperbilirubinemia is a multifactorial issue. Future studies focusing on the interactions of the bilirubin metabolism gene, other genes, and nongenetic factors will provide a more holistic insight into the pathogenesis of neonatal hyperbilirubinemia.
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