The study was conducted between October 2023 and May 2025 at seven hospitals in northern China and included neonates born at 35 weeks’ gestation or later during their first 28 days after birth. Cases were defined using an hour-specific bilirubin nomogram, with a total serum bilirubin concentration above the 95th percentile for postnatal age. Their elevated bilirubin was predominantly unconjugated, and routine etiological evaluation had not identified a common clinical cause. The investigators excluded infants with hemolytic disease, infection, polycythemia, clinically significant inadequate intake or delayed meconium passage, as well as those with previous allogeneic blood transfusion, congenital malformations, a known family history of hereditary disease or a clinically suspected or confirmed genetic disorder. The results therefore concern a specifically defined group with unexplained hyperbilirubinemia rather than neonatal jaundice of every cause.

Control neonates were enrolled at birth from six of the participating hospitals during the same period. They had not been diagnosed with hyperbilirubinemia on the day of enrollment, had normal Apgar scores and no history of asphyxia, and were subject to the same general exclusions concerning transfusion, congenital malformations, family history and suspected genetic disorders. Importantly, controls were not required to remain free of hyperbilirubinemia throughout the neonatal period. A telephone follow-up was conducted only for controls with detected target variants, and those observations were analyzed descriptively without reclassifying control status. The primary comparison was thus between hospitalized cases and newborns without a diagnosis at enrollment, rather than between cases and a uniformly verified unaffected group.

DNA analysis targeted 12 predefined hotspot loci in five genes involved in bilirubin production, transport or metabolism: UGT1A1, G6PD, SLC25A13, SLC10A1 and ABCC2. The variants were screened simultaneously by multicolor melting curve analysis. Genotypes were assigned by comparing each sample’s melting curves with known wild-type, heterozygous and homozygous controls, and approximately 10% of samples were randomly selected for validation by Sanger sequencing. The genotyping results showed 100% concordance with sequencing, and all enrolled neonates had successful calls for the 12 loci, with no assay failures reported. Because the panel covered only selected hotspots rather than the complete genetic landscape, finding no targeted variant did not establish that an infant lacked other genetic factors. Detection also did not imply that an allele was pathogenic or likely pathogenic.

UGT1A1 variants were detected in 60.3% of cases and 40.8% of controls, yielding an odds ratio of 2.20, a 95% confidence interval of 1.45–3.33 and a P value below 0.001. The UGT1A1 association remained significant after the study’s multiple-testing correction, with a false-discovery-rate-adjusted P value of 0.00063. UGT1A1 encodes the enzyme that catalyzes conjugation of unconjugated bilirubin with glucuronic acid, forming water-soluble conjugated bilirubin. Within this gene, c.211G>A, also known as UGT1A1*6, was the most frequently observed targeted locus. At least one copy of the variant was present in 53.3% of cases, compared with 37.5% of controls; the odds ratio was 1.90, with a 95% confidence interval of 1.25–2.89 and a P value of 0.002. This carrier association also remained significant after false-discovery-rate correction.

Genotype-specific comparisons produced higher odds ratios among homozygotes, although the confidence intervals showed substantial uncertainty. The heterozygous UGT1A1 c.211G>A genotype occurred in 40.7% of cases and 32.5% of controls, giving an odds ratio of 1.67 and a 95% confidence interval of 1.08–2.60. The homozygous genotype occurred in 12.6% of cases and 5.0% of controls, with an odds ratio of 3.37 and a wider 95% confidence interval of 1.39–8.17. Both genotype-specific comparisons met the study’s false-discovery-rate-adjusted significance threshold, with adjusted P values of 0.0413 and 0.0193, respectively. The pattern is compatible with a possible dose-related influence at this locus, but the case-control design cannot establish that c.211G>A directly causes hyperbilirubinemia, exclude effects from other linked genetic factors or predict an individual infant’s outcome.

Variants in the other four tested genes were less common and did not differ significantly between cases and controls. SLC10A1 variants were detected in 3.9% of cases and 5.0% of controls, with an odds ratio of 0.77 and a 95% confidence interval of 0.29–2.00. Seven cases carried G6PD variants, while no controls did, but the difference was not statistically significant. One case carried the tested SLC25A13 deletion, and no ABCC2 variant was detected in either group. The cohort was characterized predominantly by unconjugated hyperbilirubinemia, whereas SLC25A13 deficiency-associated neonatal intrahepatic cholestasis and ABCC2-associated Dubin-Johnson syndrome typically involve intrahepatic cholestasis dominated by conjugated bilirubin. That biochemical pattern helps explain why variants in these pathways were uncommon in the selected cohort, but the study cannot determine their broader prevalence among northern Chinese newborns.

Several infants carried more than one targeted variant, but the multi-site detection rate did not differ significantly between groups: 8.2% of cases and 4.2% of controls had variants at multiple sites. All 25 neonates with multiple variants within one gene had combinations involving UGT1A1. The most frequent case-group combination was UGT1A1 c.211G>A with c.1091C>T, detected in 14 cases. Other combinations included c.211G>A with SLC10A1 c.800C>T, UGT1A1 c.1456T>G or G6PD c.1388G>A. Fourteen neonates carried variants in more than one gene. Three male cases with hemizygous G6PD c.1388G>A also carried heterozygous UGT1A1 c.211G>A, while seven cases carried both UGT1A1 c.211G>A and SLC10A1 c.800C>T. These findings document coexistence among variants affecting glucuronidation, hepatic uptake and red blood cell enzyme function, but they do not establish additive effects, biological interactions or a combined clinical risk.

Exploratory follow-up added evidence that some initially classified controls later developed hyperbilirubinemia. Among the 54 controls with detected target variants, 32, or 59.3%, subsequently developed the condition. This included 21 of 39 with a single heterozygous UGT1A1 c.211G>A variant and four of six with the homozygous variant. Other observed outcomes included two of five with heterozygous UGT1A1 c.1091C>T, one of two with heterozygous UGT1A1 c.1456T>G, none of one with heterozygous UGT1A1 c.686C>A and two of six with heterozygous SLC10A1 c.800C>T. Three of the five controls with multi-site variants also developed hyperbilirubinemia. These follow-up data were descriptive, were available only for variant-positive controls and were not used to make inferential comparisons, so they require cautious interpretation.

Sensitivity analyses restricted the sample to the six hospitals that enrolled both cases and controls. Multivariable logistic regression models adjusted for sex and recruitment center yielded effect estimates similar to those from the primary analyses for overall target-variant detection, UGT1A1 variants and carriage of UGT1A1 c.211G>A. Nevertheless, the study has important limitations. Detailed quantitative clinical phenotypes were not collected consistently across centers, limiting genotype–phenotype analysis, while the absence of family samples and haplotype analysis prevented the investigators from determining the phase of multiple UGT1A1 variants or evaluating possible compound heterozygous effects. The relatively small control group, targeted assay, uneven follow-up and possible selection bias also limit interpretation. Population-structure effects and residual confounding cannot be completely excluded, and extrapolation to other populations should be cautious. Overall, the study provides a regional description of selected bilirubin-metabolism variants among hospitalized northern Chinese neonates with unexplained hyperbilirubinemia, with the clearest statistical association at UGT1A1 c.211G>A rather than evidence that variants across all five genes are equally implicated.

Subject of Research: Targeted genetic variants associated with unexplained neonatal hyperbilirubinemia in northern Chinese neonates

Article Title: Bilirubin metabolism-related genetic variants in neonatal hyperbilirubinemia: a multicenter case-control study in northern China

Article References: Sheng, S., Li, Y., Ma, L., Chang, H., Guan, Y., Lei, Y., Tan, C., Arigonggaowa, Wen, H., Kou, C., & Wang, Y. (2026). Bilirubin metabolism-related genetic variants in neonatal hyperbilirubinemia: a multicenter case-control study in northern China. Frontiers in Pediatrics, 14, Article 1945311. https://doi.org/10.3389/fped.2026.1945311

Image Credits: AI Generated

DOI: 10.3389/fped.2026.1945311

Keywords: neonatal hyperbilirubinemia, UGT1A1, genetic variants, bilirubin metabolism, northern China, newborn jaundice, case-control study, G6PD, SLC10A1, multicenter research, Bilirubin, metabolism-related