Main

Bilirubin is mainly produced in its unconjugated form by the turnover of erythrocytes. It may be transported by the organic anion transporter 2 (OATP 2) (1), and it is then conjugated with glucuronic acid through reaction with UDP-glucuronosyltransferase 1A1 (UGT1A1) in the liver before being excreted into the bile (2). The bilirubin level in neonates is much higher than in adults because the life span of the erythrocytes is relatively short and the capacity for bilirubin elimination is lower than in adults (3). The peak serum levels of unconjugated bilirubin in full-term Asian and American-Indian neonates are double those in white and black populations (4). The incidence of severe hyperbilirubinemia and kernicterus is also higher among newborn Asian infants (3). These findings suggest that genetic factors may be involved in the development of severe neonatal hyperbilirubinemia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common genetic defect, affecting >400 million individuals worldwide (5). Furthermore, G6PD deficiency was the main risk factor for development of severe hyperbilirubinemia in Taiwanese neonates in the past (6). The situation has improved, however, since neonatal G6PD screening tests and health education were instituted at the Cathay General Hospital (CGH) in Taipei and nationwide in Taiwan in 1981 (7) and 1987 (6), respectively. Recently, another genetic defect, a variation in the promoter area or within the coding region of the UGT1A1 gene, was associated with neonatal hyperbilirubinemia in whites and in Japanese and Taiwanese, respectively (8–12). It has been determined that the A(TA)7TAA promoter variant or homozygous G to A variation at nucleotide 211 in the UGT1A1 gene is an additive risk factor for neonatal hyperbilirubinemia in G6PD-deficient white (13–15) and Taiwanese neonates (16).

It was demonstrated recently that OATP 2 is responsible for the transportation of organic anions into hepatocytes (1). This mechanism may also be involved in the transportation of unconjugated bilirubin (1). Although variations at nucleotides 388, 463, 521, and 1463 in the OATP 2 gene have been reported (17,18), the relationship between these variants and hyperbilirubinemia has not been investigated.

Along with other authors, we have reported that at least 15 different types of single-point mutation are responsible for G6PD deficiency in Taiwanese (7,16,19–21). We found that most of the G6PD-deficient neonates who had suffered from hyperbilirubinemia carried the mutation at nucleotide 1376 (7). We also showed that the occurrence of the A(TA)7TAA allele was relatively rare (14.3 versus 40%) and that the variation rate within the coding region of the UGT1A1 gene was much higher (29.3 versus 0.1%) in Taiwanese compared with whites (22). Our previous research indicated that the variation at nucleotide 211 of the UGT1A1 gene was highly related to neonatal hyperbilirubinemia (12,16). As severe hyperbilirubinemia still occurs in Taiwanese neonates, it is necessary to understand the real cause(s) of this disease. In this study, therefore, all known risk factors for neonatal hyperbilirubinemia were investigated. To the best of our knowledge, this is the first such investigation of these three genes (G6PD, UGT1A1, and OATP 2) and the first using the OATP 2 gene for the case-control study of neonatal hyperbilirubinemia.

METHODS

Case subjects and control subjects.

Severe neonatal hyperbilirubinemia was diagnosed when a newborn infant had a peak bilirubin level ≥342 μM (20.0 mg/dL) in serum within 10 d of birth. From August 2001 through May 2003 at the CGH, 73 neonates (38 boys and 35 girls) received a diagnosis of this disease. Of the 73 neonates, a male neonate who had hepatitis was excluded. The remaining 72 neonates, with unconjugated bilirubin/total bilirubin ≥80%, were enrolled. Prolonged hyperbilirubinemia was defined when bilirubin was ≥150 μM (8.8 mg/dL) at day 14. All 72 neonates with hyperbilirubinemia were monitored for their bilirubin values at 1 mo of age. The 100 control subjects (50 boys and 50 girls) were selected from neonates who were born at the CGH and had peak serum bilirubin levels <256.5 μM (15.0 mg/dL) within the first 10 d of life. All parents of case and control neonates gave consent for their infants to participate in this study, which was approved by the research board of the CGH, Taipei, Taiwan.

Investigation of risk factors.

A cohort investigation was subsequently conducted using the hospital records for every case of severe neonatal hyperbilirubinemia and each control subject. The known risk factors for neonatal hyperbilirubinemia [breast feeding (exclusive), ABO incompatibility (simply maternal–infant incompatibility in ABO system), premature birth (<38 wk), infection (by both clinical criteria and culture), cephalohematoma, asphyxia (Apgar score ≤6), G6PD deficiency, and carriage of the variant UGT1A1 gene] were investigated. Data for the first seven items were obtained from the chart as the records for these were complete and because neonatal screening of G6PD activity has been performed at the CGH since 1981 using umbilical-cord blood samples (7). For determination of UGT1A1 gene status, total genomic DNA was isolated from the umbilical-cord blood cells using a blood DNA isolation kit (Maxim Biotech Inc., San Francisco, CA). The PCR–restriction fragment length polymorphism (RFLP) method was applied to detect the known variant sites in the UGT1A1 gene in Taiwanese: promoter area, nucleotides 211, 686, 1091, and 1456 (12,16,22). The natural or mutagenic primers, restriction enzymes, and digested restriction fragment sizes of the five known variants are listed in Table 1. The PCR mixture (20 μL) consisted of 200 ng of DNA, 20 ng of each primer, 1.25 mM of each dNTP (3.2 μL), buffer solution (100 mM of Tris-HCl, 500 mM of KCl, 15 mM of MgCl2, and 0.01% gelatin), and 0.4 U of thermostable DNA polymerase (Dynazyme, Finnzymes Inc., Espoo, Finland). The PCR amplification was performed in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) for 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, primer extension for 1 min at 72°C, and a final extension for 10 min at 72°C. The PCR product was digested with the appropriate restriction enzyme and analyzed on a 3% agarose gel (NuSieve 3:1, Cambrex Bio Science Inc., Rockland, ME). For confirmation of results performed with the PCR-RFLP method, sequence of UGT1A1 gene in 20 randomly selected DNA samples were determined for exons 1 to 4, coding region of exon 5, their flanking intronic regions, and the promoter area with the technique described previously (22). The four known variants (nucleotides 388, 463, 521, and 1463) of the OATP 2 gene were detected using the PCR-RFLP method as described above except for the primers, restriction enzymes, and digested restriction-fragment sizes listed in Table 2. The same PCR-RFLP technique was used for identification of the G6PD mutation for the G6PD-deficient neonates using the 15 natural or mutagenic primers described previously (16). Figure 1 presents PCR-RFLP example results for the G6PD gene mutation and variants of the UGT1A1 and the OATP 2 genes.

Table 1 Natural or mutagenesis primers, restriction enzymes, and UGT1A1 gene variations
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Table 2 Natural or mutagenesis primers, restriction enzymes, and OATP2 gene variations
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Figure 1
figure 1

Example results using the PCR-RFLP method to determine G6PD gene mutation type and variants of the UGT1A1 and the OATP 2 genes (M, DNA marker). The 20 bp in the 1376 T-G6PD gene, 18 bp in the 211 G-UGT1A1 gene, 23 bp in the 388 G-OATP2 gene, and 20 bp in the 521 C-OATP2 gene are too small to be seen.

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Statistical analysis.

We analyzed the collected data using the statistical software package (Release 7.0) of Stata (Stata Co., College Station, TX). Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for severe hyperbilirubinemia of various risk factors by using univariate and multivariate logistic regression models. P < 0.05 or a 95% CI for OR ≥1.0 was defined as statistically significant.

RESULTS

Cohort investigation from chart records.

Table 3 presents the distributions for risk factors for neonatal hyperbilirubinemia. Of the seven risk factors obtained from chart records, the most prevalent was breast feeding, followed by ABO incompatibility, premature birth, infection, cephalohematoma, asphyxia, and G6PD deficiency, in descending order. Only three boys in the hyperbilirubinemic group and one boy of the control group were G6PD deficient.

Table 3 ORs and 95% CIs for severe neonatal hyperbilirubinemia associated with suspected factors
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Determination of genes.

The predominate variant in the UGT1A1 gene was the G to A substitution at nucleotide 211 (heterozygous and homozygous), followed by the heterozygous variation in the promoter area [A(TA)6TAA/A(TA)7TAA, 6/7], compound heterozygous variations (e.g. the heterozygous C to A substitution at nucleotide 686 plus 6/7), and the heterozygous C to T substitution at nucleotide 1091. No carriage of the T to G substitution at nucleotide 1456 was detected (Table 3). The status of UGT1A1 gene in the 20 randomly selected DNA samples were 10 for the heterozygous variation and two for the homozygous variation at nucleotide 211, six for the wild type, and two for the compound heterozygous variations (nucleotide 686 plus 6/7). All results were matched with those determined by the sequencing method. The most common variant of the OATP 2 gene was the G to A substitution at nucleotide 388 (heterozygous and homozygous), followed by the T to C substitution at nucleotide 521 (heterozygous and homozygous) and compound heterozygous variations of these two nucleotides. No carriage of the C to A substitution at nucleotide 463 or the G to C substitution at nucleotide 1463 was detected (Table 3). Two of the three G6PD-deficient neonates in the hyperbilirubinemic group and the G6PD-deficient subjects in the control group carried the G to T substitution at nucleotide 1376, whereas the remaining G6PD-deficient subjects in the hyperbilirubinemic group bore the G to A substitution at nucleotide 1388 (data not shown).

OR for development of severe hyperbilirubinemia.

For detailed analysis, the subjects with ABO incompatibility were divided by blood type into type A neonates who were born to type O mothers and type B neonates who were born to type O mothers. The subjects who carried the variant UGT1A1 gene were divided into nucleotides 211 and 1091, compound heterozygous variations, and 6/7; and the subjects who carried the variant OATP 2 gene were divided into nucleotides 388 and 521 and compound heterozygous variations. The results (OR and 95% CI) of the analysis for the relationship between development of severe hyperbilirubinemia and suspected factors are presented in Table 3. The statistically significant risk factors identified with the univariate logistic regression models were the breast feeding, the variant UGT1A1 gene at nucleotide 211, and the variant OATP 2 gene at nucleotide 388 (p < 0.001, p < 0.001, and p = 0.043, respectively). Statistical significance was not demonstrated, however, for the ORs for the other subgroups. The results of the multivariate logistic regression models adjusted for the three significant factors are presented in Table 4 and confirm the statistical significance of these factors (p < 0.001, p = 0.002, and p = 0.010, respectively). When the OR in the neonates who were fed with formula milk and carried wild types in the UGT1A1 gene and the OATP 2 gene was defined as 1.00, the ORs were 2.6 and 10.4 times higher in neonates who carried two and three risk factors than in those who carried one risk factor, as that presented in Table 5.

Table 4 Adjusted ORs and 95% CIs for severe neonatal hyperbilirubinemia associated with independent factors
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Table 5 Relationship between the number of risk factors and the OR of severe hyperbilirubinemia
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Neonates with prolonged hyperbilirubinemia.

All 72 neonates with hyperbilirubinemia received phototherapy with the serum bilirubin level except for two subjects, who rapidly declined below 342 μM. Blood exchange treatment was given to these two neonates (500 mL of packed red blood cells plus 500 mL of fresh frozen plasma and 1000 mL of packed red blood cells plus 250 mL of fresh-frozen plasma) to correct their serum bilirubin levels. However, 21 of the neonates, including one of the two who received blood exchange, subsequently experienced prolonged hyperbilirubinemia. The risk factor status for these neonates is presented in Table 6. Thirteen of the 21 newborns carried the variation at nucleotide 211 of the UGT1A1 gene (nine heterozygous and four homozygous). Of these 13 subjects, nine were breast fed and five carried the variant OATP 2 gene at nucleotide 388 (two were breast fed and the other three were not). All serum bilirubin values in the 72 neonates were normal [<25.65 μM (1.5 mg/dL)] at 1 mo of age.

Table 6 Risk factor status for the 21 neonates with prolonged hyperbilirubinemia
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DISCUSSION

Neonatal hyperbilirubinemia is common in Asian populations, including Taiwanese. For example, our recent studies indicate that the incidence of neonatal hyperbilirubinemia was 7.8% (18 of 232) and 15.6% (33 of 212) in G6PD-normal and G6PD-deficienct Taiwanese neonates, respectively (16), with 14.6% (18 of 123) of the neonates with hyperbilirubinemia in one study having peak bilirubin levels ≥342 μM (12). These findings have enabled consecutive investigation of the cause of neonatal hyperbilirubinemia. Our previous research has focused on the contribution of G6PD deficiency and carriage of the variant UGT1A1 gene to the development of neonatal hyperbilirubinemia, to the exclusion of other possible risk factors (12,16). As the relative importance of these factors was not considered in the earlier studies, all eight of the known risk factors as well as an additional suspicious analog were investigated in the present study. No gender difference was demonstrated for any of the nine risk factors except for G6PD deficiency (X linkage), however. Moreover, subjects with G6PD-deficiency were rare (three boys in the affected group and one control). As the male/female ratio was ˜ 1:1 for both the case and control groups, the data obtained from the male and female neonates was pooled for analysis. Of the nine investigated factors, breast feeding was the most frequent, confirming the findings of our previous research into neonatal hyperbilirubinemia (12). It is known that, in breast milk, there are higher circulating levels of lipoprotein lipase activity, characterized by increased β-glucuronidase activity and containing 3α, 20β-pregnanediol (23). Furthermore, the increased release of fatty acids from triglycerides, catalyzed by the lipoprotein lipase, may interfere with the hepatic uptake and conjugation of bilirubin (23). The 3α, 20β-pregnanediol may also inhibit bilirubin conjugation, whereas the β-glucuronidase induces increased bilirubin resorption from the intestine. Thus, severe neonatal hyperbilirubinemia caused by breast feeding may be explained, in part, at least, by these mechanisms.

In this study, the second frequent risk factor was variation in the UGT1A1 gene. There are 1599 nucleotides in the UGT1A1 gene, which is located at chromosome 2q37 (2). The frequency of the A(TA)7TAA allele in the promoter area of the UGT1A1 gene is lower for Taiwanese (14.3%) (22), Chinese (16.2%) (24), Malaysians (18.8%) (24), and Japanese (10.0–16.8%) (10,25,26) in comparison with whites (35.7–41.5%) (27–30) and Indians (35.1%) (24). By contrast, the predominant variation of this gene is a G to A substitution at nucleotide 211 in Japanese (10,11,31) and Taiwanese (12,16,22); however, this expression has not been observed in whites (27). Although homozygous A(TA)7TAA and variant UGT1A1 gene at nucleotide 211 are reportedly associated with prolonged neonatal hyperbilirubinemia in breast-fed white and Japanese neonates, respectively (9,32), and homozygous A(TA)7TAA is a determining factor for neonatal hyperbilirubinemia ABO incompatibility (33), the relative predominance of multiple genetic (more than three) and nongenetic factors in neonatal hyperbilirubinemia has not been investigated. This study is the pioneer concerning that situation.

The serum bilirubin values in all of the neonates with hyperbilirubinemia were normal subsequently, indicating that no neonate experienced Criger-Najjar syndrome. Therefore, the determination of unexpected variants in the UGT1A1 gene is unnecessary. Moreover, the results of the UGT1A1 gene in the randomly selected DNA samples were identical between the PCR-RFLP and the sequencing methods. These data indicate that the results of the UGT1A1 gene, determined by the PCR-RFLP method, are reliable. The results of our study reveal that the prevalence (20%; 20 of 100) of variant UGT1A1 gene at nucleotide 211 in the control group is similar to that (21.4%; 62 of 290) in healthy Taiwanese adults (22). However, the OR for severe hyperbilirubinemia of this variation in the case group of the present study was statistically significant, indicating that neonates who carry variant 211 in the UGT1A1 gene are at high risk for experiencing severe hyperbilirubinemia. The UGT1A1 enzyme activities of the 211 G to A variation in the heterozygous and homozygous state were decreased to 60.2 and 32.2% of normal in an in vitro expression study (34). These decreased enzyme activities are thought to cause delayed elimination of bilirubin (2) and ultimately occurrence of hyperbilirubinemia. Moreover, most subjects (61.9%; 13 of 21) who experienced prolonged hyperbilirubinemia in our study carried the 211 variant, including the four of the seven homozygous variants. The high percentage (57.1%; four of seven) of prolonged hyperbilirubinemia in neonates who carry the homozygous 211 variant confirms the results of our previous study (12). The risk factors for the two neonates who received blood exchange treatment were carriage of the heterozygous 211 variant in the UGT1A1 gene plus breast feeding and carriage of the homozygous 211 variant in the UGT1A1 gene and the homozygous 388 variant in the OATP 2 gene. Analysis of these data shows that the variation at nucleotide 211 is probably the most important variant of the UGT1A1 gene in Taiwanese.

The other novel finding of our research is that neonates who bear the variant 388 in the OATP 2 gene are also at high risk to develop severe hyperbilirubinemia. The locus of the OATP 2 gene is at chromosome 12p12 (35). As there are 2073 nucleotides and 14 exons in this gene (35), determination of the full sequence of the OATP 2 gene would be tedious. The results of the OATP 2 gene in this report are preliminary, however, because only variations at nucleotides 388, 463, 521, and 1463, determined for white and Japanese populations (17,18), were measured. We found that Taiwanese carry the variations only at nucleotides 388 and 521. Variation at nucleotide 521 (T to C) reduces the transportation function for estrone-3-sulfate to <50% of normal in an experiment using HeLa cells (17). In our study, however, the OR for severe hyperbilirubinemia in neonates who carry this variation was not significant. The results may indicate that the 174th amino acid (valine, encoded by nucleotides 520–522) of OATP 2 is the binding site for estrone-3-sulfate but not for bilirubin. In this study, the prevalence of variant 388 in the OATP 2 gene in the case and control groups was 37.5 and 22%, respectively, the former higher than the latter, and the OR for severe hyperbilirubinemia of this variation in the case group was statistically significant. Although it has been reported that OATP 2 is involved in the transportation of both conjugated and unconjugated bilirubins (1), the results of a more recent study showed that a role for OATP 2 in hepatocyte bilirubin transport is unlikely (36). However, the results of this study show that the change of amino acid (aspartic acid to asparagine, encoded by nucleotides 388–390) at codon 130 of OATP 2 may reduce the function in unconjugated bilirubin elimination, and the mechanism is worthy of further investigation.

In contrast to the significant risk factors of severe hyperbilirubinemia described above, statistical significance was not demonstrated for the relationships between risk factors such as ABO incompatibility (type A neonates who were born to type O mothers and type B neonates who were born to type O mothers), premature birth, infection, cephalohematoma, asphyxia, and G6PD deficiency and development of severe neonatal hyperbilirubinemia. As the subjects with these risk factors among both the case and control groups were few, the results seem not to exclude absolutely their relationships with severe hyperbilirubinemia. However, it is reasonable to conclude that these risk factors are less important in the development of severe hyperbilirubinemia in neonates. Although the incidence of hyperbilirubinemia in G6PD-deficient male Taiwanese neonates was double that for control subjects with normal G6PD activity (16), the results of this study demonstrated that the incidence of G6PD deficiency in the case group was low (4.2%; three of 72) and that the OR for severe hyperbilirubinemia caused by G6PD deficiency is not significantly different from that of the control group. At the CGH, we are aware of any G6PD deficiency within 3 d of birth, the administration of any oxidative chemical or drugs is avoided, and health education is provided to parents before the infant's discharge from the hospital (7,16). On the basis of this experience, we conclude that severe hyperbilirubinemia caused by G6PD deficiency, without associated polymorphisms in the UGT1A1 or the OATP2 gene, is preventable.

In conclusion, neonates who carry the 211 and 388 variants in the UGT1A1 and OATP 2 genes, respectively, as well as feed with breast milk are at high risk to develop severe hyperbilirubinemia. Our results also indicate that the OR is correlated directly with the cumulative number of these three risk factors as present in individual infants. Because our results show that the serum bilirubin concentration in most neonates with hyperbilirubinemia is typically reduced after phototherapy, it seems reasonable to suggest that breast feeding should not be discouraged solely on the basis of temporary severe hyperbilirubinemia that is responsive to phototherapy. Determination of the status of the UGT1A1 and OATP 2 genes is strongly recommended in any investigation of unconjugated hyperbilirubinemia in neonates, in both epidemiologic and consultative contexts.