Co-inheritance of G6PD deficiency and 211 G to a variation of UGT1A1 in neonates with hyperbilirubinemia in eastern Guangdong

Neonatal hyperbilirubinemia can manifest as jaundice of skin, mucosa, and sclera, due to the excessive bilirubin in the neonates and the limited ability to deal with bilirubin originated from the decomposition of hemoglobin in red blood cells [1]. Bilirubin should be expelled from the body under normal condition, otherwise, its accumulation can induce cytotoxicity. Moreover, owing to the high renewal rate and short life span of red blood cells in neonates, neonatal bilirubin production is much faster than that of adults. On the other hand, the excretion of bilirubin in neonates is much slower than in adults [2, 3]. Therefore, bilirubin-related nuclear jaundice, nervous system damage, and even death often occur in neonates [4].

G6PD deficiency, the most common enzyme defect in humans, is one of the main risk factors of neonatal hyperbilirubinemia [5]. It is estimated that the global prevalence of G6PD deficiency is 4.9%, affecting about 330 million people, mostly from Africa, the Mediterranean, Southeast Asia, and Latin America [6, 7]. The incidence in China is higher in the Southern areas, including Hainan, Guangxi, Guangdong, Fujian, and Taiwan. To date, around 217 types of G6PD mutations have been reported worldwide while at least 31 mutations have been identified in the Chinese population [8, 9].

Non-conjugated bilirubin is bound to glucuronic acid to form conjugated bilirubin in the liver by uridine diphosphate glucuronosyltransferase (UGT) encoded by UGT1A1, and then excreted in vivo through a series of complex processes [10]. UGT1A1 gene mutations causing structural or functional defects of the enzyme can reduce the activity of UGT, resulting in increased serum bilirubin level. Among more than 100 mutation types of UGT1A1, the G to A variation at the nucleotide 211 of the UGT1A1 (UGT1A1*6, c.211G > A, p.Arg71Gly, rs4148323) is the most frequent mutation in the Asian cohort, contributes to hyperbilirubinemia in Asian neonates [11].

The purpose of this study was to explore the relationship between neonatal hyperbilirubinemia and G6PD deficiency and to investigate the effect of G6PD deficiency and UGT1A1 variant on bilirubin level of icteric neonates in Chaozhou, eastern Guangdong Province.

G6PD deficiency is a common hereditary blood disease in southern China. It is prevalent in Chaozhou, eastern Guangdong province. The rate of G6PD deficiency in Chaozhou neonates was 2.68% according to our previous screening [16]. In this study, we mainly focused on the neonates with hyperbilirubinemia and found that the incidence of G6PD deficiency in this cohort was 8.39% (74/882), which was 3-fold higher than that of neonates without hyperbilirubinemia (2.05%, 12/585). Thus, G6PD deficiency was a risk factor for neonatal hyperbilirubinemia. Importantly, for the 74 hyperbilirubinemia neonates with G6PD deficiency, 67 cases were males (90.5%) while only 7 cases were females (9.5%). This was likely attributed to the G6PD gene location on the X chromosome and the resulting sex linkage. Female heterozygotes may be difficult to diagnose because of X-chromosome mosaicism leading to a partial deficiency that will not be detected reliably with screening tests [17].

In the neonates with hyperbilirubinemia, the bilirubin level of 74 cases of G6PD deficiency was 334.43 ± 79.27 μmol/L, which was markedly higher than that of neonates with normal G6PD (300.30 ± 68.62 μmol/L). Furthermore, neonatal hyperbilirubinemia combining with the c.1388G > A mutation resulted in earlier jaundice and higher bilirubin level. Therefore, we should be alert to the G6PD deficiency when the neonates suffered from hyperbilirubinemia during clinical practice. In Guangdong province of China, G6PD screening was a routine procedure for newborns at birth, if G6PD deficiency was identified in neonates, their parents would be informed and G6PD deficiency associated disorders such as neonatal jaundice was especially cautioned, G6PD deficiency neonates should receive transcutaneous bilirubin measurement regularly. In this study, bilirubin encephalopathy occurred in three jaundiced newborns with G6PD deficiency. The pathogenesis of significant neonatal hyperbilirubinemia was often multifactorial, which might encompass several environmental and genetic contributors [3]. Therefore, the genetic detection was not enough for preventing severe hyperbilirubinemia. For those neonates with jaundice contributors, routinely transcutaneous bilirubin measurement was recommended to avoid severe hyperbilirubinemia and bilirubin encephalopathy.

Lin et al. have shown that c.1376G > T, c.1388G > A, and c.1024C > T were the most common mutations of G6PD in Chaozhou, accounting for 75.6% of G6PD-deficient cases [18]. Similarly, we found that the majority of G6PD variants were c.1376G > T (34/74, 45.95%) and c.1388G > A (26/74, 35.14%), and there was only one case with c.1024C > T. The diagnostic criteria for severe hyperbilirubinemia was bilirubin level over 342 μmol/L [19]. In our study, the mean peak bilirubin of neonates with c.1388G > A was 367.88 ± 75.79 μmol/L, indicating that c.1388G > A lead to severe neonatal hyperbilirubinemia easily. This result was consistent with a previous study in Malaysian Chinese [20]. Theoretically, c.1388G > A causes substitution of the Arg⁴⁵³ of G6PD to a histidine, which is close to the NADP binding region and affects the integration and function of G6P and NADP [21]. Therefore, c.1388G > A often causes severe clinical symptoms. Due to the limited number of cases studied, the relationship between bilirubin level and neonatal hyperbilirubinemia could not be determined in other types of G6PD mutation.

Most patients with G6PD deficiency have only one mutation, while a few patients have multiple mutations. In our study, 1 case (1 female) showed compound mutation of c.1388G > A/c.95A > G, and the bilirubin in this neonate was 280.1 μmol/L. Whether the level of bilirubin is related to the types of multiple mutations remains to be further studied.

During bilirubin metabolism process, gene mutation can cause bilirubin conjugation blockage and abnormal accumulation in vivo, resulting in neonatal hyperbilirubinemia. Whether the cumulative mutations of G6PD and UGT1A1 are related to more severe neonatal hyperbilirubinemia requires further study. TATA mutation of UGT1A1 promoter coupled with G6PD deficiency was indicated as an important factor for neonatal hyperbilirubinemia in Nigeria [22]. Yang et al. showed that G6PD deficiency combined with UGT1A1c.211G > A homozygous mutation can increase the risk of severe hyperbilirubinemia [15]. In the current study of hyperbilirubinemia group with G6PD deficiency, we showed that 6 cases were UGT1A1 c.211G > A homozygous mutation, 27 cases were heterozygous, and 41 cases were wild. Although the jaundice tended to be more severe with the increase of mutations, the difference in bilirubin levels among them was not statistically significant. It may be due to the limited number of cases and requires larger enrollment of neonates in further study.

Unexpectedly, common G6PD mutations were not detected in 4 G6PD deficient neonates with hyperbilirubinemia (3 males, 1 female) and 3 G6PD deficient neonates without hyperbilirubinemia (3 males). This may be due to that the kit for detection of G6PD mutation can only detect 14 common mutations in the Chinese population, and there may be unusual mutations in these cases, which need to be verified by gene sequencing. Moreover, mutations of other genes, such as heme oxygenase-1 (HO-1), biliverdin reductase A (BLVRA), and solute carrier organic anion transporter family member 1B1 (SLCO1B1) could also affect the serum bilirubin [23].

G6PD deficiency has important clinical significance for neonatal hyperbilirubinemia. We proposed that the neonates with hyperbilirubinemia should be screened for G6PD activity actively and timely. This can help to analyze the etiology and characteristics of neonatal jaundice and to prevent serious kernicterus or even death with prompt and effective treatment. If possible, universal screening for G6PD deficiency at birth was recommended, percutaneous bilirubin in newborns with G6PD deficiency should be monitored regularly to avoid severe jaundice happening.