Vol. 46, No. 1, September 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 21-26
N E O N A T A L H Y P E R B I L I R U B I N E M I A AND M U T A T I O N OF THE BILIRUBIN URIDINE DIPHOSPHATE-GLUCURONOSYLTRANSFERASE GENE: A C O M M O N MISSENSE MUTATION AMONG JAPANESE, K O R E A N S AND C H I N E S E
Kazuhiro Akabal, Toshiyuki Kimural, Ayako Sasakil, Saori Tanabel, Tohru Ikegamil, Motoya Hashimot02, Hitoshi Umeda3, Hiroshi Yoshida4, Kazuo UmetsuS, Hitoshi Chiba6, Isao Yuasa7 and Kiyoshi Hayasakal* I Departments of Pediatrics and 5Forensic Medicine, Yamagata University School of Medicine, Yamagata 990-2331; 2Department of Neonatal Intensive Care Unit, Saiseikai Yamagata Saisei Hospital, Yamagata; 3Department of Pediatrics, Yamagata Prefectural Nihonkai Hospital, Sakata; 4Department of Pediatrics, Tsuruoka Municipal Shounai Hospital, Tsuruoka; 6Department of Laboratory Medicine, Hokkaido University School of Medicine, Sapporo; 7Department of Legal Medicine, Faculty of Medicine, Tottori University, Yonago, Japan ReceivedApril 20, 1998 Received after revision,May 15, 1998 SUMMARY. We analyzed the bilirubin uridine diphosphate-glucuronosyltransferase (B-UGT) gene in 42 Japanese newborns with hyperbilirubinemia and determined that 21 infants were heterozygous while 3 was homozygous for Gly71Arg. Allele frequency of GlyT1Arg was 0.32 in newborns with hyperbilirubinemia, which was significantly higher than 0.13 in healthy Japanese controls. This mutant allele is also prevalent among Korean and Chinese healthy controls with a frequency of 0.23 in both populations. However, this mutation was not detected in 50 healthy German controls. These data suggest that the high frequency of the GlyT1Arg mutation of the BUGT gene is associated with high incidence of neonatal hyperbilirubinemia in Japanese, Korean and Chinese populations. KEY WORDS: bilirubin uridine diphosphate-glucuronosyltransferase; hyperbilirubinemia INTRODUCTION
Hyperbilirubinemia in newborn infants has been recognized as a common physiologic phenomenon. Several physiologic conditions of the newborn contribute to the development of hyperbilirubinemia: cessation of bilirubin clearance by placental transport; increased production of bilirubin probably due to short life span of circulating erythrocytes, increased heme degradation of the fetal hematopoietic tissues and increased turnover of cytochromes; low uptake of bilirubin in liver; low activity of hepatic bilirubin uridine diphosphate-glucuronosyltransferase (B-UGT); and persistent enterohepatic circulation of biliruhin (1, 2). It is well known that there is a significant difference in the severity of physiologic jaundice among different ethnic populations. Mean maximal serum concentrations of unconjugated bilirubin in full-term Japanese, Korean, Chinese and American Indian newborns are approximately double those found in the white and black
*Corresponding author, Fax number: 81-?_3-628-5332, E-mail:
[email protected] 1039-9712/98/130021-06505.00/0 21
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populations (2). The incidence of kernicterus (bilirubin toxicity) is also significantly higher among Asian newborns.
Increased bilirubin production in Japanese newborn was speculated by
measuring the blood carboxyhemoglobin level (3). Lee et al. (4) reported that both increased production and decreased elimination of bilirubin contribute to physiologic jaundice in Korean neonates. Glucose-6-phosphate dehydrogenase deficiency is a common disease causing hemolytic anemia, especially in persons of Mediterranean, tropical African, and Asian descent.
Newborns
with glucose-6-phosphate dehydrogenase deficiency may develop a severe hemolytic crisis leading to a significant hyperbilirubinemia by exposure to drugs. The production of bilirubin estimated from the blood carboxyhemoglobin level is increased in the neonates with glucose-6-phosphate dehydrogenase deficiency, however, hyperbilirubinemia is detected in only limited cases and is not related to the severity of hemolysis (5, 6). Recently, Kaplan et al. (7) reported that a two-base-pair addition (TA) in the TATAA element of the B-UGT gene increases the incidence of neonatal hyperbilirubinemia in glucose-6-phosphate dehydrogenase deficiency. These data suggest that the deficiency of B-UGT activity is the central rate-limiting step in the development of neonatal hyperbilirubinemia. We analyzed the B-UGT gene in 42 Japanese newborns with hyperbilirubinemia and detected that approximately 60% of the infants had a missense mutation, which is prevalent among Japanese, Korean and Chinese populations. M A T E R I A L S and M E T H O D S
DNA isolation from Japanese newborns with hyperbilirubinemia and from healthy Japanese, Korean, Chinese and German controls. After informed consent was obtained, genomic DNA was isolated from the white blood cells from 42 Japanese full-term newborns with hyperbilirubinemia of unknown etiology. For this study, hyperbilirubinemia in a child weighing 2500 g or more at birth was defined as a total serum bilirubin concentration 6 mg/dl at day 0, 10 mg/dl at day 1, 12.0 mg/dl at day 2, 14.0 mg/dl at day 3, 15.0 mg/dl at day 4, 16.0 mg/dl at day 5, 17.0 mg/dl at day 6, and 18.0 mg/dl at day 7 or more. For a child weighing less than 2,500 g at birth weight, the definition was adjusted to lower levels of bilirubin than for a child weighing 2500 g or more at birth. Basic characters of the 42 neonates were as follows: gestational periods were 277.2 _+9.2 (mean _+SD) days, birth weight was 3091.8 _+508.5 (mean • SD) g and the male-tofemale ratio was 22:20. Genomic DNA was obtained from 101 healthy Japanese controls, who had no episodes of apparent hyperbilirubinemia. Most of the controls were born in Yamagata prefecture of Japan as were the newborns with hyperbilirubinemia. They included 53 men and 48 women, with ages ranging from 18 and 49 years old. Genomic DNA was also obtained from 50 healthy Korean, Chinese and German controls. PCR amplification and sequencing analysis of the B-UGT gene. Promoter and coding regions including exon-intron boundaries of the B-UGT gene were obtained as four fragments by PCR. Fragment 1 encompassing the 5' promoter region including the TATAA element was amplified using primers PF: 5'-GAGGTTCTGGAAGTACTTTG-3' and PR: 5'-TCCGTCTCT GATGTACAACG-3'. Fragment 2 encompassing exon 1 was amplified with IF: 5'-AAGTAG G A G A G G G C G A A C C - 3 ' and 1R: 5 ' - G T C C C A C T C C A A T A C A C A C - 3 ' . Fragment 3 encompassing exons 2-4 and fragment 4 encompassing exon 5 were amplified with primers E2F: 5'-GAAGTAAAGGAGAGGAAAAT-3' and FAR: 5'-TTTTATCATGAATGCCATGACC-3' and primers E5F:5'-AGCATAAAGAGAGGATTGTT-3' and E5R:5'-CACCAGAGGGGGCACGAT AC-3', respectively. PCR was performed for 35 cycles consisting of denaturation at 94~ for 1 min, annealing at 60"C for 1 rain, and extension at 72~ for 1 min. Primers PF, 1R, E2F and E5R were biotinylated at the 5' end, and single strand DNA was prepared for sequence determination
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using streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin, Dynal, Oslo, Norway). Sequences were determined by the dideoxy chain termination method using the following primers: SEQPRO: 5'-CAGCAGGCCCAGGACAAGTG-3' for promoter; SEQ1A: 5'-CACTTGTCCTGG GCCTGCTG-3', SEQ1B: 5'-CGTTGTACATCAGAGACGG A-3', SEQ1C" 5'-GCACAACAA GGAGCTCATGG-3', SEQID: 5'-GCTCATTGCCTTTTCA CAGAA-3' for exon l; SEQEX2: 5'-AGCGCTCCTGTGAAAATA-3' for exon 2; SEQEX3: 5'-TTAATTTGA CCCTGGTT-3' for exon 3; SEQEX4: 5'-TGAATGCCATGACCAAA-3' for exon 4; SEQEX5A: 5'-GAGGATTGTT CATACCA-3' and SEQEX5B: 5'-GATTGGTTTCCTCTTGG-3' for exon 5. Oligonucleotide primers used for PCR and sequencing were designed based on genomic information of the B-UGT gene (8, 9).
Gene frequency analysis of Gly71Arg in Japanese, Korean, Chinese and German and analysis of TA repeat promoter polymorphism in Japanese. Allele specific oligonucleotide hybridization (ASO) analysis was performed to detect the Gly71Arg mutation. The PCR products (using primers 1F and 1R) were prepared and an aliquot of the PCR products was denatured and blotted onto Hybond-N+ membranes. The membranes were prehybridized and hybridized with normal or mutant ASO probes that were 5'-end-labeled with[~,-32p]ATP by T4 polynucleotide kinase. The ASO probes were 5'-AGAGACGGAGCATT-3' and 5'-AGAGACAGAGCATT-3', for Gly71 (normal) and Arg71 (mutant), respectively. Following hybridization, the membranes were washed and autoradiographed overnight. For the analysis of TA repeat promoter polymorphism, fragment 1 encompassing the 5' promoter region including the TATAA element was amplified from the genomic DNA of 50 healthy Japanese controls and 42 Japanese newborns with hyperbilirubinemia and its sequence was determined. RESULTS
The coding region and the 5' promoter region including the TATAA element of the B-UGT gene were sequenced after amplification of genomic DNA from 42 Japanese newborns with hyperbilirubinemia. Direct sequencing revealed the presence of a G-to-A substitution of nucleotide 295, leading to a Gly71Arg substitution in 24 patients. Twenty one of them were heterozygous and 3 were homozygous for the mutation. The gene frequency of Gly71Arg was 0.32 in the patients and significantly higher than 0.13 in healthy Japanese controls. To investigate whether this mutation was specific to Japanese, the allele frequency was determined in Korean, Chinese and German populations by ASO analysis. As shown in Table 1, the allele frequency was 0.23 in Koreans and Chinese. However, this mutation was not detected among Germans. As for the TA repeat promoter polymorphism, none of 42 newborns with hyperbilirubinemia had (TA)7 allele (Table 2). The allele frequency of (TA)7 was 0.10 among Japanese healthy controls. These results indicate that the Gly71Arg mutation is a common mutation among Japanese, Koreans and Chinese and that the high frequency of the mutation seems to be associated with high incidence of neonatal hyperbilirubinemia in those ethnic populations. DISCUSSION Neonatal hyperbilirubinemia is a physiological phenomenon and severe cases have been treated by phototherapy or exchange transfusion. However, it is important clinically to identify predisposing factors to establish a definitive treatment. Recently, we studied several patients with
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Table 1. Gly71Arg allele frequency in various populations and Japanese newborns with hyperbilirubinemia
Ethnic background
No. tested
Japanese 101 Korean 50 Chinese 50 German 50 Japanese newborns 42 with hyperbilirubinemia
Gly71Arg Heterozygote I-Iomo~gote 23 19 17 0 21
2 2 3 0 3
Allele frequency 0.13 0.23 0.23 0 0.32"
*Significant difference (p
