Etiology analysis for full-term newborns with severe hyperbilirubinemia in eastern Guangdong

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Methods Full-term newborns with severe hyperbilirubinemia in one hospital from January 2012 to December 2021 were retrospectively analyzed. The etiology was determined according to the laboratory results and clinical manifestations. Results Among 1602 full-term newborns with hyperbilirubinemia in Chaozhou area, 32.20% (580/1602) was severe hyperbilirubinemia, including 213 newborns with TSB levels reaching the recommended exchange transfusion levels and 52 cases diagnosed with prolonged jaundice. Among the causes of severe hyperbilirubinemia, neonatal hemolysis accounted for 15.17%, infection accounted for 10.17%, G6PD deficiency accounted for 9.13%, and the coexistence of multiple etiologies accounted for 6.55%, unknown etiology accounted for 50.00%. ABO hemolysis and G6PD deficiency were the most common causes in the 20 cases with bilirubin encephalopathy. 94 severe hyperbilirubinemia newborns were tested for UGT1A1*6 variant (rs4148323, c.211G > A, p.Arg71Gly,), 9 cases were 211 G to A homozygous variant, 37 cases were 211 G to A heterozygous variant, and 48 cases were wild genotypes. Conclusion The main cause for severe hyperbilirubinemia and bilirubin encephalopathy were the hemolytic disease of the newborn, G6PD deficiency and infection. UGT1A1 gene variant was also a high risk factor for neonatal hyperbilirubinemia. Targeted prevention and treatment according to the etiology may reduce the occurrence of bilirubin encephalopathy and kernicterus. Severe hyperbilirubinemia full-term newborns etiology Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) Glucose-6-phosphate dehydrogenase (G6PD) deficiency Figures Figure 1 0 Background Neonatal hyperbilirubinemia is one of the common diseases of newborns, which is mainly caused by the imbalance between the production and elimination of bilirubin in the body [ 1 ] . It can be manifested as yellow staining of skin mucosa and sclera and usually resolves within 1 week after birth, and most of neonates with jaundice have a good prognosis. However, if the total bilirubin level exceeds a certain threshold, it can lead to bilirubin encephalopathy, resulting in sequelaes such as hearing loss, motor and intellectual development disorders, and even death, which will bring a heavy burden to the society and family. The pathogenic factors of neonatal hyperbilirubinemia are complex. Different cases of hyperbilirubinemia may have a single or mixed etiology. The etiological composition and related genetic variants of hyperbilirubinemia in different regions may be different, resulting in different degrees of hyperbilirubinemia in clinic. The American Academy of Pediatrics published the percentile curve of hourly total serum bilirubin (TSB) of newborns in venous blood, which defined bilirubin more than 95% percentile as hyperbilirubinemia and should be intervened [ 2 ] . According to the level of bilirubin and clinical symptoms, hyperbilirubinemia can be divided into significant hyperbilirubinaemia (TSB ≥ 205µmol/L), severe hyperbilirubinaemia (TSB ≥ 342µmol/L), extreme hyperbilirubinaemia (TSB ≥ 428µmol/L), hazardous or critical hyperbilirubinaemia (TSB ≥ 513µmol/L), acute bilirubin encephalopathy, and kernicterus [ 3 , 4 ] . Early detection of high-risk factors for severe hyperbilirubinemia and prompt treatment are the key to the prevention of bilirubin encephalopathy. This study analyzed the clinical data of full-term newborns with severe hyperbilirubinemia retrospectively, to explore the etiology and clinical characteristics of severe hyperbilirubinemia in full-term newborns in Chaozhou, eastern Guangdong province, so as to provide basis for clinical prevention and early treatment of hyperbilirubinemia. 1 Materials and Methods 1.1 Subjects All of full-term newborns with hyperbilirubinemia (gestational age > 37 weeks) admitted to neonatal intensive care unit (NICU) of Chaozhou Central Hospital from January 2012 to December 2021 were selected. The age of admission was 1–28 days. Demographic and clinical data were collected from the medical records of the studied participants. TSB ≥ 342µmol/L was used as the diagnostic standard for severe hyperbilirubinemia. This study was initially approved by the Ethics Committee of Chaozhou Central Hospital in 2011 (No. 2011021), and then the second ethical approval was obtained in 2015 (No.2015001). As the patients’ data were analyzed anonymously, and the blood samples in this study were used after the clinical diagnosis, a waiver of written consent was approved by the Ethics Committee of Chaozhou Central Hospital. Our group had the administrative permissions to access the data according to associated regulation by national health commission of P. R. China. 1.2 Methods General information including sex, gestational age, birth weight, admission weight, mode of delivery, feeding pattern, admission age, and treatment were collected. Diagnostic information included hemolytic disease of the newborn (ABO hemolysis and Rh hemolysis), glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PD enzyme activity ≤ 1300U/L, Co-Heath Beijing Laboratories Co., Ltd. [ 5 ] ), infection (including sepsis, pneumonia, urinary tract infection, etc.), extravascular hemorrhage (including intracranial hematoma, scalp hematoma and other bleeding), polycythemia (venous red blood cells > 6×10 12 /L, hemoglobin > 220g/L, and hematocrit > 65%), congenital hypothyroidism (diagnosed by neonatal screening), insufficient breastfeeding (breastfeeding newborns with 10% birth weight loss after birth), breast milk jaundice, prolonged jaundice (onset in neonatal period, jaundice lasted for more than 2 weeks, bilirubin level > 150µmol/L), bilirubin encephalopathy (hyperbilirubinemia and typical clinical symptoms of nervous system), blood exchange indications (blood exchange reference standard recommended by American Academy of Pediatrics in 2004) [ 2 ] . The whole blood samples of EDTA anticoagulants in newborns with severe hyperbilirubinemia were prospectively collected after clinical diagnosis and stored at − 40°C in a biobank. The whole blood DNA extraction kit (Chaozhou Hybribio Co., Ltd.) was used to extract DNA according to the instructions of the kit, and then the DNA quantity and purity were tested (NanoDrop One, Thermo Fisher Scientific Co., Ltd.). MJ Mini Personal Thermal Cycler (Bio-Rad Company) was used for PCR amplification of G6PD , and then, G6PD gene variant was detected by reverse dot blot method (Chaozhou Hybribio Co., Ltd.) for 13 common G6PD mutation types c.95A > G (G6PD Gaohe), c.392G > T (G6PD Qing Yan), c.487G > A (G6PD Mahidol), c.493A > G (G6PD Taipei), c.592G > T (G6PD Coimbra), c.871G > A (G6PD Viangchan), c.1004C > T (G6PD Fushan), c.1024C > T (G6PD Chinese-5), c.1360C > T (G6PD Union), c.1376G > T (G6PD Canton), c.1387C > T (G6PD Keelung), c.1388G > A (G6PD Kaiping), c.1381G > A (G6PD Yunan) and one polymorphism c.1311C > T[5] . UGT1A1 gene was amplified and sequenced (PE Biosystems, CT, USA) to detect c.211G > A variant (UGT1A1*6, rs4148323), which was specifically described in our previous study [ 5 ] . 94 severe hyperbilirubinemia newborns were tested for UGT1A1*6 variant (rs4148323). The control 65 newborns for UGT1A1*6 test were the full-termed newborns without abnormality or illness, and with a TSB not requiring phototherapy, their clinical characteristics were described previously [ 6 ] . 1.3 Clinical data collection and statistical analysis SPSS 20.0 was used for data analysis. The continuous variables in the data group were expressed as mean ± standard deviation. Count data was described by frequency and percentage. The Pearson chi-square test was used, and P < 0.05 was considered as statistically significant. 2 Results 2.1 General clinical characteristics A total of 1602 newborns with hyperbilirubinemia (915 males and 687 females) were collected in this study, 580 newborns (346 males and 234 females) were severe hyperbilirubinemia (32.20%, 580/1602). Birth weight was 2050–5500 g, average 3211.6 ± 444.53g; gestational age was 37– 42 weeks, with an average of 39.07 ± 1.10 weeks; the age of the highest bilirubin value was 1– 28 d, with an average of 8 ± 5 d; 315 cases were vaginal delivery, 265 cases were cesarean section; 174 cases were breastfeeding, 165 cases were formula feeding, 195 cases were mixed feeding and 46 cases were unknown. The peak value of TSB was 342.05–817.8 µmol/L, with an average of 398.07 ± 55.09 µmol/L. Among them, 213 cases met the standard of blood exchange, 52 cases were prolonged jaundice (22 cases were breast feeding), and 20 cases were complicated with bilirubin encephalopathy (Table 1 ). Table 1 Demographic and clinical characteristics of severe hyperbilirubinemia Basic data Cases number (%) Sex Male 346 (59.66%) Female 234 (40.34%) Delivery pattern Caesarean section 265 (45.69%) Vaginal delivery 315 (54.31%) Feeding pattern Breast feeding 174 (29.90%) Mixed feeding 195 (33.62%) Formula feeding 165 (28.45%) Unknown 46 (7.93%) Proportion of exchange transfusion level 213 (36.72%) Prolonged jaundice 52 (8.97%) Bilirubin encephalopathy 20 (3.45%) Total 580 (100%) 2.2 Etiology composition Among the cases of severe hyperbilirubinemia with clear etiology, the most common was neonatal hemolysis (15.17%), followed by infection (10.17%), G6PD deficiency (9.13%), combined factors (6.55%) and breast milk jaundice (3.79% ), the etiology for the remaining 50% (290/580) cases were not clear. Among combined factors, neonatal hemolysis combined with infection (36.84%) was the most common, followed by G6PD deficiency combined with infection (26.32%) and infection combined with vascular bleeding (13.16%). In all cases of neonatal hemolysis, one case of Rh hemolysis was identified, the other 106 cases were ABO hemolysis (Fig. 1 ). 2.3 G6PD genotype and UGT1A1*6 variant Among 580 cases, 68 cases were G6PD deficient by enzyme activity test, and 43 newborns were tested for 14 common G6PD gene mutations in Chinese. Gene mutations were found in 40 cases, including 1 case c.95A > G (G6PD Gaohe), 1 case c.1024C > T (G6PD Chinese-5), 14 cases c.1376G > T (G6PD Canton), 23 cases c.1388G > A (G6PD Kaiping), and 1 case of composite mutation c.1311C > T combined with c.871G > A (G6PD Viangchan). No mutation was detected in the remaining 3 cases. A total of 94 neonates with severe hyperbilirubinemia were randomly selected to detect UGT1A1*6 variant (UGT1A1*6, c.211G > A , p.Arg71Gly, rs4148323). 48 cases were wild type, 37 cases were heterozygous variants, and 9 cases of homozygous variants were detected, with gene variant frequency of 48.94% (46/94). Among that 94 cases, a total of 18 cases of G6PD deficiency combined with UGT1A1 variants were found, including 2 cases of homozygous variants of UGT1A1*6 with c.1376G > T , 2 cases of homozygous variants of UGT1A1*6 with c.1388G > A , 4 cases of heterozygous variants of UGT1A1*6 with c.1376G > T , 10 cases of heterozygous variants of UGT1A1*6 with c.1388G > A (Table 2 ). Compared with our previous study on the UGT1A1 c. 211G > A variant in 65 full-term newborns with normal bilirubin [ 6 ] (Table 3 ). The proportion of c.211G > A variant in newborns with severe hyperbilirubinemia was higher than that in full-term neonates with normal bilirubin (P < 0.001). ABO hemolysis occurred in 34 of 94 cases, and 10 cases (10.64%, 10/94) were ABO hemolysis combined with UGT1A1*6 variant. Table 2 UGT1A1*6 variant combination with G6PD deficiency in 94 cases of severe neonatal hyperbilirubinemia. G6PD deficiency G6PD normal Total P G211A variation Wild type 16 32 48 0.787 c.211 hetereozygote 14 23 37 c.211 homozygote 4 5 9 Total 34 60 94 Table 3 The comparison of UGT1A1*6 variant between neonates with severe hyperbilirubinemia and the control group. Case Control* p G211A variation Wild type 48 51 < 0.001 c.211 hetereozygote 37 13 c.211 homozygote 9 1 Total 94 65 *Full-term newborns without major abnormality or illness, and with a TSB not requiring phototherapy according to the updated clinical guidelines by the China Neonatal Association. [ 6 ] 2.4 Bilirubin encephalopathy Among 580 cases of severe hyperbilirubinemia, 20 cases were bilirubin encephalopathy, including 11 cases of male and 9 cases of female, and TSB bilirubin level was 359–817.8 µmol/L, with an average of 480.21 ± 64.19 µmol/ L. The main causes of bilirubin encephalopathy were ABO hemolysis (6 cases), followed by infection (sepsis in 1 case, pneumonia in 4 cases), G6PD deficiency (4 cases), unknown causes (3 cases), ABO hemolysis combined with infection in 1 case, and ABO hemolysis combined with G6PD deficiency and infection in 1 case. 3 Discussion Bilirubin is the catabolic product of heme metabolism. Human bilirubin is mainly derived from the degradation of aging red blood cells, while the remaining derives from the ineffective erythropoiesis and the breakdown of other hemoproteins such as cytochromes, myoglobin, and catalase. Heme is degraded by heme oxygenase, resulting in the release of iron and the formation of carbon monoxide and biliverdin [ 7 ] . Biliverdin is converted to unconjugated bilirubin (UCB) by the action of biliverdin reductase. The UCB is lipophilic and tightly binds to albumin, which is transported to the liver. UCB dissociates from albumin and binds to proteins of the glutathione-S-transferases family in liver hepatocytes. UCB combines with glucuronic acid by the enzyme uridine diphospho-glucuronate glucuronosyltransferase (UGT1A1) to form conjugated bilirubin [ 8 ] . Conjugation increases the solubility of bilirubin in plasma and thereby make it easy to be eliminated from the body. After secreted by hepatocytes and discharged into the intestinal cavity, conjugated bilirubin can be hydrolyzed and reduced by intestinal flora to produce bilirubin, most of which are discharged through feces, while a small amount of bilirubin is reabsorbed into the circulation by intestinal mucosal cells [ 9 ] . When bilirubin is excessive, bilirubin can be excessively accumulated in the body, resulting in hyperbilirubinemia. Neonatal hyperbilirubinemia is one of the common diseases in neonatal period. In order to avoid the toxic effect of free bilirubin, which lead to cell damage in the central nervous system and cause acute bilirubin encephalopathy and nuclear jaundice, a relatively reasonable diagnosis and treatment guideline for neonatal hyperbilirubinemia has been developed. In recent years, there have been many studies on the etiology and related risk factors of neonatal hyperbilirubinemia in China and abroad [ 10 – 12 ] . However, the etiology of hyperbilirubinemia is easily affected by environment and other relative factors. Therefore, the analysis of the etiology of neonates with severe hyperbilirubinemia in eastern Guangdong is of great significance to reduce the incidence of severe hyperbilirubinemia and its serious complications in the region. Our study found that 32.20% of the 1602 hospitalized newborns with hyperbilirubinemia were severe hyperbilirubinemia. Hemolytic disease of the newborn (HDN) was the most common cause of severe hyperbilirubinemia in full-term newborns. ABO blood group incompatibility is one of the most common causes of HDN which often occurs in infants of blood type A or B with mothers of blood type O. The blood type antibody in mother combines with the corresponding antigen on the surface of fetal red blood cells during pregnancy. Macrophages and natural killer cells in newborn destroy sensitized red blood cells, resulting in hemolysis, jaundice and anemia after birth [ 13 ] . At the same time, 68 cases of G6PD deficiency was identified (11.72%, 68/580) in this study, including single G6PD deficiency (53 cases) and G6PD deficiency combined with other causes (15 cases). The incidence of G6PD deficiency was relatively high, which might be related to the geographical location of Chaozhou. G6PD deficiency was mainly prevalent in southern China. The prevalence of G6PD deficiency in Chaozhou was 3.36% [ 14 ] . The prevalence of G6PD deficiency in severe hyperbilirubinemia group was higher than that of the general population. G6PD deficiency causes increased susceptibility of erythrocytes to H 2 O 2 and other reactive oxygen species that can lead to hemolytic anemia, and neonatal hyperbilirubinemia resulting in neonatal kernicterus.. G6PD deficiency is an important reason for severe hyperbilirubinemia in full-term newborns. Of the cases with G6PD deficiency, 11 cases were combined with infection. Infection was the main cause of acute hemolysis in G6PD deficiency [ 15 ] . We performed gene analysis in 43 cases of G6PD deficiency and found that c.1388G > A and c.1376G > T were the most common variants in severe hyperbilirubinemia neonates. The bilirubin level in severe hyperbilirubinemia caused by hemolysis is high, and it occurs early and develops rapidly. In addition, neonatal blood-brain barrier is not fully developed, bilirubin encephalopathy and even death are prone to occur. Infection was another cause of severe hyperbilirubinemia (12.74%). Hyperbilirubinemia caused by infection may be due to the imperfect immune function of newborns and lack of mature defense ability, and the neonate is easy to be infected by viruses and bacteria. The destruction of the erythrocyte membrane by the pathogen themselves or secreted toxins, results in hemolysis and increased bilirubin production. At the same time, infection can cause liver dysfunction, inhibit the activity of UGT1A1, and reduce the ability of liver to deal with bilirubin. Hyperbilirubinemia may be the only manifestation of infection [ 16 ] . Because of the low positive rate of blood culture, lack of diagnostic basis for infection, the bilirubin may increase and progress to severe hyperbilirubinemia. Breast milk jaundice is a clinical diagnosis made after excluding the known causes of hyperbilirubinemia. At present, there is still a lack of reliable laboratory testing methods to get this diagnosis. Breast milk jaundice can be divided into early breastfeeding jaundice and late breast milk jaundice [ 17 ] . In our study, 9 cases (1.55%) of early breastfeeding jaundice and 22 cases (3.79%) of late breast milk jaundice were identified. Early breast feeding deficiency leads to insufficient calorie intake, reduced intestinal peristalsis, and increased bilirubin enterohepatic circulation, resulting in early hyperbilirubinemia, which is manifested by a weight loss of 10% within one week after birth. Late breast milk jaundice occurs relatively late, and the bilirubin generally peaks on about the 7th to14th days of life. The jaundice can last for 2–3 weeks or even 2–3 months, which may be caused by many factors [ 18 ] . Maruo [ 19 ] and Sato [ 20 ] reported that the variant of UGT1A1 c.211G > A was an important cause of breast milk jaundice in newborns, and the serum bilirubin level of breast milk jaundice newborns with this variant was significantly higher than that of breast milk jaundice newborns without this variant. This indicated that the presence of UGT1A1 c.211G > A variant could promote breast milk jaundice. The product encoded by UGT1A1 gene is bilirubin uridine diphosphate glucuronyltransferase. UGT1A1 variant make the activity of this enzyme reduced, so that bilirubin cannot combine with glucuronide to form conjugated bilirubin. The unconjugated bilirubin accumulates in the body, and resulting in different degrees of hyperbilirubinemia [ 21,22] . The UGT1A1 c.211G > A was a risk factor for neonatal severe hyperbilirubinemia in eastern Guangdong, which might occur with other causes simultaneously. Among the 94 cases with UGT1A1 variants data, we found that 18 cases were UGT1A1 gene variant combined with G6PD deficiency, 10 cases were UGT1A1 gene variant combined with ABO hemolysis. Our previous study found that in the newborns with hyperbilirubinemia which due to G6PD deficiency, bilirubin level showed an increasing trend with the accumulation of UGT1A1 c.211G > A variant [ 5 ] ; Yang, et al [ 23 ] reported that the risk of neonatal severe hyperbilirubinemia increased significantly when ABO hemolytic neonates were combined with UGT1A1 gene mutation (especially 211A > G homozygous mutation). For severe hyperbilirubinemia neonates with unknown etiology, due to limited conditions, we did not perform gene detection for all cases, resulting in some missing causes. Pathological jaundice, especially severe jaundice, is often the result of multiple influencing factors, while genetic factors are often not to be tested and neglected. Detection of bilirubin-related genes such as G6PD and UGT1A1 in neonates can be used as an auxiliary test for clinicians to assess the risk of neonatal severe hyperbilirubinemia, which will benefit for early prevention, etiological exploration and genetic counseling. 4 Conclusion The main causes of severe hyperbilirubinemia in Chaozhou are neonatal hemolysis and infection. Genetic factors, such as UGT1A1 variant and G6PD deficiency were also the important risk factors for neonatal severe hyperbilirubinemia. It is very important to clarify the etiology and to treat neonatal severe hyperbilirubinemia timely, which can reduce the occurrence of acute bilirubin encephalopathy and kernicterus. Abbreviations G6PD Glucose-6-phosphate dehydrogenase deficiency UGT1A1 Uridinediphosphateglucuronosyltransferase 1A1 TSB total serum bilirubin PCR polymerase chain reaction UCB unconjugated bilirubin HDN Hemolytic disease of the newborn Declarations Ethics approval and consent to participate Ethical approval All procedures performed in studies involving human participants followed the ethical standards of the study hospital and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical approval was granted by the Institutional Review Boards at Chaozhou Central Hospital Affiliated to Southern Medical University, and a waiver of written consent was approved by the Ethics Committee of Chaozhou Central Hospital. Consent for publication: Not Applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no conflict of interest. Funding This study was supported by the Natural Science Foundation of Guangdong Province (No: 2016A030307035), Special Research Plan 2019 of Chaozhou (2020xg01), and High Level Development Plan of People’s Hospital of Yangjiang (No: G2020007). The funder had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Author contributions LYY conceptualized and designed the study, coordinated and supervised data collection, and reviewed and revised the manuscript. JXX analyzed the data, drafted the initial manuscript, and revised the manuscript. FL collected the data, did the molecular analysis, and carried out the initial analysis. YHW and YBM participated in the sample and data collection and YHW performed the molecular analysis. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 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Underestimation of the contribution of 211 G to A variation in UGT1A1 to neonatal hyperbilirubinemia in China. J Pediatr. 2022; S0022-3476(22)00284–0. Yang H, Lin F, Chen ZK, Zhang L, Xu JX, Wu YH, Gu JY, Ma YB, Li JD, Yang LY. UGT1A1 mutation association with increased bilirubin levels and severity of unconjugated hyperbilirubinemia in ABO incompatible newborns of China. BMC Pediatr. 2021;21(1):259. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1794289","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":116348470,"identity":"ba7b43db-a46b-45fe-895b-2a0c5c32c591","order_by":0,"name":"Jia-Xin Xu","email":"","orcid":"","institution":"Chaozhou Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia-Xin","middleName":"","lastName":"Xu","suffix":""},{"id":116348471,"identity":"04de89d5-a352-46df-a29c-853dd0bb7424","order_by":1,"name":"Fen Lin","email":"","orcid":"","institution":"Chaozhou Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fen","middleName":"","lastName":"Lin","suffix":""},{"id":116348472,"identity":"8bcd2b2e-fe30-483a-beb0-a5411605defe","order_by":2,"name":"Yong-Hao Wu","email":"","orcid":"","institution":"Chaozhou Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong-Hao","middleName":"","lastName":"Wu","suffix":""},{"id":116348473,"identity":"fb337ab1-c901-41d6-bafa-b82e108f8b72","order_by":3,"name":"Yu-Bin Ma","email":"","orcid":"","institution":"Chaozhou Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Bin","middleName":"","lastName":"Ma","suffix":""},{"id":116348475,"identity":"e71fc453-5726-4ad8-8892-b1baf872452e","order_by":4,"name":"Li-Ye Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYLCCB2CS+QBDAtFaICrZEkjWwmNAnGqD42cPv0iosEnsZ+/5/OHhDjsG/vZu/JYZnMlLs0g4k5Y4s+fsNonEM8kMEmfObsCv5UCOmUFi2+HEDTdytzEktjEzGEjkEtBy/g1Qy7//ifvvv3n8IbGtnggtN3KMHyQ2HEjcIMHDIAG0jrAWyRtvzBgSjiUbzziTZgbUcpyHoF/4zucYf/hQYyfb33748cefbdVy/O29+LUoHGBgkwDSjg1QAR68ykFAvoGB+QOQtieochSMglEwCkYuAAATGVCf39NV1wAAAABJRU5ErkJggg==","orcid":"","institution":"People’s Hospital of Yangjiang Affiliated to Guangdong Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Li-Ye","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2022-06-25 10:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1794289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1794289/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23141387,"identity":"d04067bc-206e-479a-8c08-c200fe38b8b1","added_by":"auto","created_at":"2022-06-27 19:22:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46946,"visible":true,"origin":"","legend":"\u003cp\u003eThe etiology of severe hyperbilirubinemia.\u003c/p\u003e\u003cp\u003eA .Unknown\u003c/p\u003e\u003cp\u003eB.Hemolytic disease of newborn\u003c/p\u003e\u003cp\u003eC.Infection\u003c/p\u003e\u003cp\u003eD.G6PD deficiency\u003c/p\u003e\u003cp\u003eE.Combined factors\u003c/p\u003e\u003cp\u003eF.Breast milk jaundice\u003c/p\u003e\u003cp\u003eG.Extravascular hemorrhage\u003c/p\u003e\u003cp\u003eH.Inadequate feeding\u003c/p\u003e\u003cp\u003eI.Congenital hypothyroidism \u003c/p\u003e\u003cp\u003eJ.Polycythemia\u003c/p\u003e\u003cp\u003eE1. Hemolytic disease of newborn + Infection\u003c/p\u003e\u003cp\u003eE2. G6PD deficiency + Infection\u003c/p\u003e\u003cp\u003eE3. Infection + Extravascular hemorrhage\u003c/p\u003e\u003cp\u003eE4. Extravascular hemorrhage + Inadequate feeding\u003c/p\u003e\u003cp\u003eE5. Hemolytic disease of newborn + Extravascular hemorrhage\u003c/p\u003e\u003cp\u003eE6. Hemolytic disease of newborn + G6PD deficiency \u003c/p\u003e\u003cp\u003eE7. G6PD deficiency + Extravascular hemorrhage\u003c/p\u003e\u003cp\u003eE8. G6PD deficiency + Inadequate feeding\u003c/p\u003e\u003cp\u003eE9. Inadequate feeding + Congenital hypothyroidism \u003c/p\u003e\u003cp\u003eE10.Hemolytic disease of newborn + G6PD deficiency + Infection\u003c/p\u003e\u003cp\u003eE11. G6PD deficiency + Infection+ Polycythemia\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1794289/v1/97b7e4b4563f548446139753.jpg"},{"id":25532553,"identity":"e11b2f24-97ab-4c6b-88a4-0bbccc9c5f18","added_by":"auto","created_at":"2022-08-23 07:29:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":281440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1794289/v1/198cae1c-b126-4a87-acdf-97d2cc678f41.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Etiology analysis for full-term newborns with severe hyperbilirubinemia in eastern Guangdong","fulltext":[{"header":"0 Background","content":"\u003cp\u003eNeonatal hyperbilirubinemia is one of the common diseases of newborns, which is mainly caused by the imbalance between the production and elimination of bilirubin in the body\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. It can be manifested as yellow staining of skin mucosa and sclera and usually resolves within 1 week after birth, and most of neonates with jaundice have a good prognosis. However, if the total bilirubin level exceeds a certain threshold, it can lead to bilirubin encephalopathy, resulting in sequelaes such as hearing loss, motor and intellectual development disorders, and even death, which will bring a heavy burden to the society and family.\u003c/p\u003e \u003cp\u003eThe pathogenic factors of neonatal hyperbilirubinemia are complex. Different cases of hyperbilirubinemia may have a single or mixed etiology. The etiological composition and related genetic variants of hyperbilirubinemia in different regions may be different, resulting in different degrees of hyperbilirubinemia in clinic. The American Academy of Pediatrics published the percentile curve of hourly total serum bilirubin (TSB) of newborns in venous blood, which defined bilirubin more than 95% percentile as hyperbilirubinemia and should be intervened\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. According to the level of bilirubin and clinical symptoms, hyperbilirubinemia can be divided into significant hyperbilirubinaemia (TSB\u0026thinsp;\u0026ge;\u0026thinsp;205\u0026micro;mol/L), severe hyperbilirubinaemia (TSB\u0026thinsp;\u0026ge;\u0026thinsp;342\u0026micro;mol/L), extreme hyperbilirubinaemia (TSB\u0026thinsp;\u0026ge;\u0026thinsp;428\u0026micro;mol/L), hazardous or critical hyperbilirubinaemia (TSB\u0026thinsp;\u0026ge;\u0026thinsp;513\u0026micro;mol/L), acute bilirubin encephalopathy, and kernicterus\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEarly detection of high-risk factors for severe hyperbilirubinemia and prompt treatment are the key to the prevention of bilirubin encephalopathy. This study analyzed the clinical data of full-term newborns with severe hyperbilirubinemia retrospectively, to explore the etiology and clinical characteristics of severe hyperbilirubinemia in full-term newborns in Chaozhou, eastern Guangdong province, so as to provide basis for clinical prevention and early treatment of hyperbilirubinemia.\u003c/p\u003e "},{"header":"1 Materials and Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003cp\u003e1.1 Subjects\u003c/p\u003e \u003cp\u003eAll of full-term newborns with hyperbilirubinemia (gestational age\u0026thinsp;\u0026gt;\u0026thinsp;37 weeks) admitted to neonatal intensive care unit (NICU) of Chaozhou Central Hospital from January 2012 to December 2021 were selected. The age of admission was 1\u0026ndash;28 days. Demographic and clinical data were collected from the medical records of the studied participants. TSB\u0026thinsp;\u0026ge;\u0026thinsp;342\u0026micro;mol/L was used as the diagnostic standard for severe hyperbilirubinemia.\u003c/p\u003e \u003cp\u003eThis study was initially approved by the Ethics Committee of Chaozhou Central Hospital in 2011 (No. 2011021), and then the second ethical approval was obtained in 2015 (No.2015001). As the patients\u0026rsquo; data were analyzed anonymously, and the blood samples in this study were used after the clinical diagnosis, a waiver of written consent was approved by the Ethics Committee of Chaozhou Central Hospital. Our group had the administrative permissions to access the data according to associated regulation by national health commission of P. R. China.\u003c/p\u003e \u003cp\u003e1.2 Methods\u003c/p\u003e \u003cp\u003eGeneral information including sex, gestational age, birth weight, admission weight, mode of delivery, feeding pattern, admission age, and treatment were collected. Diagnostic information included hemolytic disease of the newborn (ABO hemolysis and Rh hemolysis), glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PD enzyme activity\u0026thinsp;\u0026le;\u0026thinsp;1300U/L, Co-Heath Beijing Laboratories Co., Ltd.\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e), infection (including sepsis, pneumonia, urinary tract infection, etc.), extravascular hemorrhage (including intracranial hematoma, scalp hematoma and other bleeding), polycythemia (venous red blood cells\u0026thinsp;\u0026gt;\u0026thinsp;6\u0026times;10\u003csup\u003e12\u003c/sup\u003e/L, hemoglobin\u0026thinsp;\u0026gt;\u0026thinsp;220g/L, and hematocrit\u0026thinsp;\u0026gt;\u0026thinsp;65%), congenital hypothyroidism (diagnosed by neonatal screening), insufficient breastfeeding (breastfeeding newborns with 10% birth weight loss after birth), breast milk jaundice, prolonged jaundice (onset in neonatal period, jaundice lasted for more than 2 weeks, bilirubin level\u0026thinsp;\u0026gt;\u0026thinsp;150\u0026micro;mol/L), bilirubin encephalopathy (hyperbilirubinemia and typical clinical symptoms of nervous system), blood exchange indications (blood exchange reference standard recommended by American Academy of Pediatrics in 2004)\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe whole blood samples of EDTA anticoagulants in newborns with severe hyperbilirubinemia were prospectively collected after clinical diagnosis and stored at \u0026minus;\u0026thinsp;40\u0026deg;C in a biobank. The whole blood DNA extraction kit (Chaozhou Hybribio Co., Ltd.) was used to extract DNA according to the instructions of the kit, and then the DNA quantity and purity were tested (NanoDrop One, Thermo Fisher Scientific Co., Ltd.). MJ Mini Personal Thermal Cycler (Bio-Rad Company) was used for PCR amplification of \u003cem\u003eG6PD\u003c/em\u003e, and then, \u003cem\u003eG6PD\u003c/em\u003e gene variant was detected by reverse dot blot method (Chaozhou Hybribio Co., Ltd.) for 13 common G6PD mutation types \u003cem\u003ec.95A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/em\u003e(G6PD Gaohe), \u003cem\u003ec.392G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e(G6PD Qing Yan), \u003cem\u003ec.487G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e(G6PD Mahidol), \u003cem\u003ec.493A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/em\u003e (G6PD Taipei), \u003cem\u003ec.592G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e(G6PD Coimbra), \u003cem\u003ec.871G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e (G6PD Viangchan), \u003cem\u003ec.1004C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e (G6PD Fushan), \u003cem\u003ec.1024C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e (G6PD Chinese-5), \u003cem\u003ec.1360C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e (G6PD Union), \u003cem\u003ec.1376G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e (G6PD Canton), \u003cem\u003ec.1387C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e (G6PD Keelung), \u003cem\u003ec.1388G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e (G6PD Kaiping), \u003cem\u003ec.1381G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e (G6PD Yunan) and one polymorphism \u003cem\u003ec.1311C\u0026thinsp;\u0026gt;\u0026thinsp;T[5]\u003c/em\u003e. UGT1A1 gene was amplified and sequenced (PE Biosystems, CT, USA) to detect \u003cem\u003ec.211G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e variant (UGT1A1*6, rs4148323), which was specifically described in our previous study \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e94 severe hyperbilirubinemia newborns were tested for UGT1A1*6 variant (rs4148323). The control 65 newborns for UGT1A1*6 test were the full-termed newborns without abnormality or illness, and with a TSB not requiring phototherapy, their clinical characteristics were described previously \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e1.3 Clinical data collection and statistical analysis\u003c/p\u003e \u003cp\u003eSPSS 20.0 was used for data analysis. The continuous variables in the data group were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Count data was described by frequency and percentage. The Pearson chi-square test was used, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"2 Results","content":"\u003cp\u003e2.1 General clinical characteristics\u003c/p\u003e \u003cp\u003eA total of 1602 newborns with hyperbilirubinemia (915 males and 687 females) were collected in this study, 580 newborns (346 males and 234 females) were severe hyperbilirubinemia (32.20%, 580/1602). Birth weight was 2050\u0026ndash;5500 g, average 3211.6\u0026thinsp;\u0026plusmn;\u0026thinsp;444.53g; gestational age was 37\u0026ndash; 42 weeks, with an average of 39.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 weeks; the age of the highest bilirubin value was 1\u0026ndash; 28 d, with an average of 8\u0026thinsp;\u0026plusmn;\u0026thinsp;5 d; 315 cases were vaginal delivery, 265 cases were cesarean section; 174 cases were breastfeeding, 165 cases were formula feeding, 195 cases were mixed feeding and 46 cases were unknown. The peak value of TSB was 342.05\u0026ndash;817.8 \u0026micro;mol/L, with an average of 398.07\u0026thinsp;\u0026plusmn;\u0026thinsp;55.09 \u0026micro;mol/L. Among them, 213 cases met the standard of blood exchange, 52 cases were prolonged jaundice (22 cases were breast feeding), and 20 cases were complicated with bilirubin encephalopathy (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical characteristics of severe hyperbilirubinemia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasic data\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCases number (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e346 (59.66%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e234 (40.34%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDelivery pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaesarean section\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e265 (45.69%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaginal delivery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e315 (54.31%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeeding pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBreast feeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e174 (29.90%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixed feeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e195 (33.62%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormula feeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e165 (28.45%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46 (7.93%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProportion of exchange transfusion level\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e213 (36.72%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProlonged jaundice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52 (8.97%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBilirubin encephalopathy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (3.45%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e580 (100%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e2.2 Etiology composition\u003c/p\u003e \u003cp\u003eAmong the cases of severe hyperbilirubinemia with clear etiology, the most common was neonatal hemolysis (15.17%), followed by infection (10.17%), G6PD deficiency (9.13%), combined factors (6.55%) and breast milk jaundice (3.79% ), the etiology for the remaining 50% (290/580) cases were not clear. Among combined factors, neonatal hemolysis combined with infection (36.84%) was the most common, followed by G6PD deficiency combined with infection (26.32%) and infection combined with vascular bleeding (13.16%). In all cases of neonatal hemolysis, one case of Rh hemolysis was identified, the other 106 cases were ABO hemolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e2.3 G6PD genotype and UGT1A1*6 variant\u003c/p\u003e \u003cp\u003eAmong 580 cases, 68 cases were G6PD deficient by enzyme activity test, and 43 newborns were tested for 14 common G6PD gene mutations in Chinese. Gene mutations were found in 40 cases, including 1 case \u003cem\u003ec.95A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/em\u003e (G6PD Gaohe), 1 case \u003cem\u003ec.1024C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e (G6PD Chinese-5), 14 cases \u003cem\u003ec.1376G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e (G6PD Canton), 23 cases \u003cem\u003ec.1388G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e (G6PD Kaiping), and 1 case of composite mutation \u003cem\u003ec.1311C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e combined with \u003cem\u003ec.871G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e (G6PD Viangchan). No mutation was detected in the remaining 3 cases.\u003c/p\u003e \u003cp\u003eA total of 94 neonates with severe hyperbilirubinemia were randomly selected to detect UGT1A1*6 variant (UGT1A1*6, \u003cem\u003ec.211G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e, p.Arg71Gly, rs4148323). 48 cases were wild type, 37 cases were heterozygous variants, and 9 cases of homozygous variants were detected, with gene variant frequency of 48.94% (46/94). Among that 94 cases, a total of 18 cases of G6PD deficiency combined with UGT1A1 variants were found, including 2 cases of homozygous variants of UGT1A1*6 with \u003cem\u003ec.1376G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e, 2 cases of homozygous variants of UGT1A1*6 with \u003cem\u003ec.1388G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e, 4 cases of heterozygous variants of UGT1A1*6 with \u003cem\u003ec.1376G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e, 10 cases of heterozygous variants of UGT1A1*6 with \u003cem\u003ec.1388G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared with our previous study on the UGT1A1 c. 211G\u0026thinsp;\u0026gt;\u0026thinsp;A variant in 65 full-term newborns with normal bilirubin\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The proportion of \u003cem\u003ec.211G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e variant in newborns with severe hyperbilirubinemia was higher than that in full-term neonates with normal bilirubin (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). ABO hemolysis occurred in 34 of 94 cases, and 10 cases (10.64%, 10/94) were ABO hemolysis combined with UGT1A1*6 variant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUGT1A1*6 variant combination with G6PD deficiency in 94 cases of severe neonatal hyperbilirubinemia.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG6PD deficiency\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG6PD normal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eG211A variation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.787\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.211 hetereozygote\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.211 homozygote\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe comparison of UGT1A1*6 variant between neonates with severe hyperbilirubinemia and the control group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eG211A variation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.211 hetereozygote\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.211 homozygote\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Full-term newborns without major abnormality or illness, and with a TSB not requiring phototherapy according to the updated clinical guidelines by the China Neonatal Association. \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e2.4 Bilirubin encephalopathy\u003c/p\u003e \u003cp\u003eAmong 580 cases of severe hyperbilirubinemia, 20 cases were bilirubin encephalopathy, including 11 cases of male and 9 cases of female, and TSB bilirubin level was 359\u0026ndash;817.8 \u0026micro;mol/L, with an average of 480.21\u0026thinsp;\u0026plusmn;\u0026thinsp;64.19 \u0026micro;mol/ L. The main causes of bilirubin encephalopathy were ABO hemolysis (6 cases), followed by infection (sepsis in 1 case, pneumonia in 4 cases), G6PD deficiency (4 cases), unknown causes (3 cases), ABO hemolysis combined with infection in 1 case, and ABO hemolysis combined with G6PD deficiency and infection in 1 case.\u003c/p\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eBilirubin is the catabolic product of heme metabolism. Human bilirubin is mainly derived from the degradation of aging red blood cells, while the remaining derives from the ineffective erythropoiesis and the breakdown of other hemoproteins such as cytochromes, myoglobin, and catalase. Heme is degraded by heme oxygenase, resulting in the release of iron and the formation of carbon monoxide and biliverdin\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Biliverdin is converted to unconjugated bilirubin (UCB) by the action of biliverdin reductase. The UCB is lipophilic and tightly binds to albumin, which is transported to the liver. UCB dissociates from albumin and binds to proteins of the glutathione-S-transferases family in liver hepatocytes. UCB combines with glucuronic acid by the enzyme uridine diphospho-glucuronate glucuronosyltransferase (UGT1A1) to form conjugated bilirubin\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Conjugation increases the solubility of bilirubin in plasma and thereby make it easy to be eliminated from the body. After secreted by hepatocytes and discharged into the intestinal cavity, conjugated bilirubin can be hydrolyzed and reduced by intestinal flora to produce bilirubin, most of which are discharged through feces, while a small amount of bilirubin is reabsorbed into the circulation by intestinal mucosal cells\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhen bilirubin is excessive, bilirubin can be excessively accumulated in the body, resulting in hyperbilirubinemia. Neonatal hyperbilirubinemia is one of the common diseases in neonatal period. In order to avoid the toxic effect of free bilirubin, which lead to cell damage in the central nervous system and cause acute bilirubin encephalopathy and nuclear jaundice, a relatively reasonable diagnosis and treatment guideline for neonatal hyperbilirubinemia has been developed. In recent years, there have been many studies on the etiology and related risk factors of neonatal hyperbilirubinemia in China and abroad\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, the etiology of hyperbilirubinemia is easily affected by environment and other relative factors. Therefore, the analysis of the etiology of neonates with severe hyperbilirubinemia in eastern Guangdong is of great significance to reduce the incidence of severe hyperbilirubinemia and its serious complications in the region.\u003c/p\u003e \u003cp\u003eOur study found that 32.20% of the 1602 hospitalized newborns with hyperbilirubinemia were severe hyperbilirubinemia. Hemolytic disease of the newborn (HDN) was the most common cause of severe hyperbilirubinemia in full-term newborns. ABO blood group incompatibility is one of the most common causes of HDN which often occurs in infants of blood type A or B with mothers of blood type O. The blood type antibody in mother combines with the corresponding antigen on the surface of fetal red blood cells during pregnancy. Macrophages and natural killer cells in newborn destroy sensitized red blood cells, resulting in hemolysis, jaundice and anemia after birth \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt the same time, 68 cases of G6PD deficiency was identified (11.72%, 68/580) in this study, including single G6PD deficiency (53 cases) and G6PD deficiency combined with other causes (15 cases). The incidence of G6PD deficiency was relatively high, which might be related to the geographical location of Chaozhou. G6PD deficiency was mainly prevalent in southern China. The prevalence of G6PD deficiency in Chaozhou was 3.36%\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The prevalence of G6PD deficiency in severe hyperbilirubinemia group was higher than that of the general population. G6PD deficiency causes increased susceptibility of erythrocytes to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and other reactive oxygen species that can lead to hemolytic anemia, and neonatal hyperbilirubinemia resulting in neonatal kernicterus.. G6PD deficiency is an important reason for severe hyperbilirubinemia in full-term newborns. Of the cases with G6PD deficiency, 11 cases were combined with infection. Infection was the main cause of acute hemolysis in G6PD deficiency \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. We performed gene analysis in 43 cases of G6PD deficiency and found that \u003cem\u003ec.1388G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e and \u003cem\u003ec.1376G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/em\u003e were the most common variants in severe hyperbilirubinemia neonates. The bilirubin level in severe hyperbilirubinemia caused by hemolysis is high, and it occurs early and develops rapidly. In addition, neonatal blood-brain barrier is not fully developed, bilirubin encephalopathy and even death are prone to occur.\u003c/p\u003e \u003cp\u003eInfection was another cause of severe hyperbilirubinemia (12.74%). Hyperbilirubinemia caused by infection may be due to the imperfect immune function of newborns and lack of mature defense ability, and the neonate is easy to be infected by viruses and bacteria. The destruction of the erythrocyte membrane by the pathogen themselves or secreted toxins, results in hemolysis and increased bilirubin production. At the same time, infection can cause liver dysfunction, inhibit the activity of UGT1A1, and reduce the ability of liver to deal with bilirubin. Hyperbilirubinemia may be the only manifestation of infection\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Because of the low positive rate of blood culture, lack of diagnostic basis for infection, the bilirubin may increase and progress to severe hyperbilirubinemia.\u003c/p\u003e \u003cp\u003eBreast milk jaundice is a clinical diagnosis made after excluding the known causes of hyperbilirubinemia. At present, there is still a lack of reliable laboratory testing methods to get this diagnosis. Breast milk jaundice can be divided into early breastfeeding jaundice and late breast milk jaundice\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In our study, 9 cases (1.55%) of early breastfeeding jaundice and 22 cases (3.79%) of late breast milk jaundice were identified. Early breast feeding deficiency leads to insufficient calorie intake, reduced intestinal peristalsis, and increased bilirubin enterohepatic circulation, resulting in early hyperbilirubinemia, which is manifested by a weight loss of 10% within one week after birth. Late breast milk jaundice occurs relatively late, and the bilirubin generally peaks on about the 7th to14th days of life. The jaundice can last for 2\u0026ndash;3 weeks or even 2\u0026ndash;3 months, which may be caused by many factors \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMaruo\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e and Sato\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e reported that the variant of UGT1A1 \u003cem\u003ec.211G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e was an important cause of breast milk jaundice in newborns, and the serum bilirubin level of breast milk jaundice newborns with this variant was significantly higher than that of breast milk jaundice newborns without this variant. This indicated that the presence of UGT1A1 \u003cem\u003ec.211G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e variant could promote breast milk jaundice. The product encoded by UGT1A1 gene is bilirubin uridine diphosphate glucuronyltransferase. UGT1A1 variant make the activity of this enzyme reduced, so that bilirubin cannot combine with glucuronide to form conjugated bilirubin. The unconjugated bilirubin accumulates in the body, and resulting in different degrees of hyperbilirubinemia \u003csup\u003e[ 21,22]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe UGT1A1 \u003cem\u003ec.211G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e was a risk factor for neonatal severe hyperbilirubinemia in eastern Guangdong, which might occur with other causes simultaneously. Among the 94 cases with UGT1A1 variants data, we found that 18 cases were UGT1A1 gene variant combined with G6PD deficiency, 10 cases were UGT1A1 gene variant combined with ABO hemolysis. Our previous study found that in the newborns with hyperbilirubinemia which due to G6PD deficiency, bilirubin level showed an increasing trend with the accumulation of UGT1A1 \u003cem\u003ec.211G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/em\u003e variant\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e ; Yang, et al\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e reported that the risk of neonatal severe hyperbilirubinemia increased significantly when ABO hemolytic neonates were combined with UGT1A1 gene mutation (especially 211A\u0026thinsp;\u0026gt;\u0026thinsp;G homozygous mutation).\u003c/p\u003e \u003cp\u003eFor severe hyperbilirubinemia neonates with unknown etiology, due to limited conditions, we did not perform gene detection for all cases, resulting in some missing causes. Pathological jaundice, especially severe jaundice, is often the result of multiple influencing factors, while genetic factors are often not to be tested and neglected. Detection of bilirubin-related genes such as G6PD and UGT1A1 in neonates can be used as an auxiliary test for clinicians to assess the risk of neonatal severe hyperbilirubinemia, which will benefit for early prevention, etiological exploration and genetic counseling.\u003c/p\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eThe main causes of severe hyperbilirubinemia in Chaozhou are neonatal hemolysis and infection. Genetic factors, such as UGT1A1 variant and G6PD deficiency were also the important risk factors for neonatal severe hyperbilirubinemia. It is very important to clarify the etiology and to treat neonatal severe hyperbilirubinemia timely, which can reduce the occurrence of acute bilirubin encephalopathy and kernicterus.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eG6PD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose-6-phosphate dehydrogenase deficiency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUGT1A1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUridinediphosphateglucuronosyltransferase 1A1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etotal serum bilirubin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUCB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eunconjugated bilirubin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHemolytic disease of the newborn\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants followed the ethical standards of the study hospital and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical approval was granted by the Institutional Review Boards at Chaozhou Central Hospital Affiliated to Southern Medical University, and a waiver of written consent was approved by the Ethics Committee of Chaozhou Central Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication: \u003c/strong\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of Guangdong Province (No: 2016A030307035), Special Research Plan 2019 of Chaozhou (2020xg01), and High Level Development Plan of People\u0026rsquo;s Hospital of Yangjiang (No: G2020007). The funder had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLYY conceptualized and designed the study, coordinated and supervised data collection, and reviewed and revised the manuscript. JXX analyzed the data, drafted the initial manuscript, and revised the manuscript. FL collected the data, did the molecular analysis, and carried out the initial analysis. YHW and YBM participated in the sample and data collection and YHW performed the molecular analysis. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement: \u003c/strong\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKaplan M, Muraca M, Hammerman C, et al. Imbalance between production and conjugation of bilirubin: a fundamental concept in the mechanism of neonatal jaundice. Pediatrics. 2002;110(4):e47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmercian Academy of Pediatrics, Subcommittee on Hyperbilirubinemia,Management of Hyperbilirubinemia in the Newborn Infant 35 or More Weeks of Gestation.Pediatrics.2004;114(1):297\u0026ndash;316.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhutani VK, Vilms RJ, Hamerman-Johnson L. Universal bilirubin screening for severe neonatal hyperbilirubinemia. J Perinatol.2010;30 (suppl): S6\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlusanya BO, Ogunlesi TA, Kumar P, et al. Management of late-preterm and term infants with hyperbilirubinaemia in resource-constrained settings. BMC Pediatr. 2015; 15: 39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu JX, Lin F, Chen ZK, Luo ZY, Zhan XF, Wu JR, Ma YB, Li JD, Yang LY. Co-inheritance of G6PD deficiency and 211 G to a variation of UGT1A1 in neonates with hyperbilirubinemia in eastern Guangdong. BMC Pediatr. 2021;21(1):564.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Wang Q, Zheng L, Zheng XB, Lin M, Zhan XF, Yang LY. Clinical Significance of UGT1A1 Genetic Analysis in Chinese Neonates with Severe Hyperbilirubinemia. Pediatr Neonatol.2016;57(4):310\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSticova E, Jirsa M. New insights in bilirubin metabolism and their clinical implications. World J Gastroenterol.2013;19(38):6398\u0026ndash;6407.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh A, Koritala T, Jialal I. Unconjugated Hyperbilirubinemia. 2022 Apr 2. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan. PMID: 31747203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson NB, Calkins KL. Neonatal Indirect Hyperbilirubinemia. Neoreviews. 2020;21(11):e749-e760.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Yang C, Zhu W, Chen S, Zeng Y, Wang J, Zhao H, Chen Y, Lin F. Identification of Genetic Risk Factors for Neonatal Hyperbilirubinemia in Fujian Province, Southeastern China: A Case-Control Study. Biomed Res Int. 2018;2018:7803175.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMei H, Dong X, Wu B, Wang H, Lu Y, Hu L, Wang J, Cao Y, Zhang R, Cheng G, Wang L, Li Z, Yang L, Zhou W. Clinical and Genetic Etiologies of Neonatal Unconjugated Hyperbilirubinemia in the China Neonatal Genomes Project. J Pediatr. 2022;243:53\u0026ndash;60.e9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsuorah CDI, Ekwochi U, Asinobi IN. Clinical evaluation of severe neonatal Hyperbilirubinaemia in a resource-limited setting: a 4-year longitudinal study in south-East Nigeria. BMC Pediatr. 2018;18(1):202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChristensen RD, Baer VL, MacQueen BC, O'Brien EA, Ilstrup SJ. ABO hemolytic disease of the fetus and newborn: thirteen years of data after implementing a universal bilirubin screening and management program. J Perinatol. 2018;38(5):517\u0026ndash;525.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin F, Wu J, Yang H, Lin M, Yang L. [Molecular epidemiology of G6PD deficiency in Chaozhou area of eastern Guangdong Province]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2016;33(1):26\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarcke SJ, Rizzolo D, Harcke HT. G6PD deficiency: An update. JAAPA. 2019;32(11):21\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmar C, Hamza S, Bassem AM, Mariam R.Urinary tract infection and indirect hyperbilirubinemia in newborns.N Am J Med Sci.2011;3(12):544\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGartner LM. Breastfeeding and jaundice. J Perinatol. 2001; 21: S25-9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBratton S, Cantu RM, Stern M. Breast Milk Jaundice. 2021 Nov 16. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan\u0026ndash;. PMID: 30726019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaruo Y, Morioka Y, Fujito H, Nakahara S, Yanagi T, Matsui K, Mori A, Sato H, Tukey RH, Takeuchi Y. Bilirubin uridine diphosphate-glucuronosyltransferase variation is a genetic basis of breast milk jaundice. J Pediatr. 2014;165(1):36\u0026ndash;41.e1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato H, Uchida T, Toyota K, Kanno M, Hashimoto T, Watanabe M, Nakamura T, Tamiya G, Aoki K, Hayasaka K. Association of breast-fed neonatal hyperbilirubinemia with UGT1A1 polymorphisms: 211G \u0026gt; A (G71R) mutation becomes a risk factor under inadequate feeding. J Hum Genet. 2013;58(1):7\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarici SU, Saldir M. Genetic factors in neonatal hyperbilirubinemia and kernicterus. Turk J Pediatr. 2007;49(3):245\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang LY. Underestimation of the contribution of 211 G to A variation in UGT1A1 to neonatal hyperbilirubinemia in China. J Pediatr. 2022; S0022-3476(22)00284\u0026ndash;0.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Lin F, Chen ZK, Zhang L, Xu JX, Wu YH, Gu JY, Ma YB, Li JD, Yang LY. UGT1A1 mutation association with increased bilirubin levels and severity of unconjugated hyperbilirubinemia in ABO incompatible newborns of China. BMC Pediatr. 2021;21(1):259.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Severe hyperbilirubinemia, full-term newborns, etiology, Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), Glucose-6-phosphate dehydrogenase (G6PD) deficiency","lastPublishedDoi":"10.21203/rs.3.rs-1794289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1794289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo explore the etiological characteristics of severe hyperbilirubinemia in full-term newborns of eastern Guangdong.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFull-term newborns with severe hyperbilirubinemia in one hospital from January 2012 to December 2021 were retrospectively analyzed. The etiology was determined according to the laboratory results and clinical manifestations.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong 1602 full-term newborns with hyperbilirubinemia in Chaozhou area, 32.20% (580/1602) was severe hyperbilirubinemia, including 213 newborns with TSB levels reaching the recommended exchange transfusion levels and 52 cases diagnosed with prolonged jaundice. Among the causes of severe hyperbilirubinemia, neonatal hemolysis accounted for 15.17%, infection accounted for 10.17%, G6PD deficiency accounted for 9.13%, and the coexistence of multiple etiologies accounted for 6.55%, unknown etiology accounted for 50.00%. ABO hemolysis and G6PD deficiency were the most common causes in the 20 cases with bilirubin encephalopathy. 94 severe hyperbilirubinemia newborns were tested for UGT1A1*6 variant (rs4148323, c.211G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.Arg71Gly,), 9 cases were 211 G to A homozygous variant, 37 cases were 211 G to A heterozygous variant, and 48 cases were wild genotypes.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe main cause for severe hyperbilirubinemia and bilirubin encephalopathy were the hemolytic disease of the newborn, G6PD deficiency and infection. UGT1A1 gene variant was also a high risk factor for neonatal hyperbilirubinemia. Targeted prevention and treatment according to the etiology may reduce the occurrence of bilirubin encephalopathy and kernicterus.\u003c/p\u003e","manuscriptTitle":"Etiology analysis for full-term newborns with severe hyperbilirubinemia in eastern Guangdong","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-27 19:22:46","doi":"10.21203/rs.3.rs-1794289/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00d72bda-faca-4bd1-9eee-6170fe857132","owner":[],"postedDate":"June 27th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-23T07:29:22+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-27 19:22:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1794289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1794289","identity":"rs-1794289","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}