Compound Heterozygote of a Novel Missense Mutation (p.K402T) and a Double Missense Mutation (p.[G71R;Y486D]) in Type II Crigler‐Najjar Syndrome
Articles > Article · 2011
Authors: Yoshihiro Maruo, Funda ÇETİN, Yu Mimura, Yoriko Ota, Katsuyuki Matsui, Hiroko Takahashi, Asami Mori, Takashi Taga, Tomoyuki Takano, Hiroshi Sato, Yoshihiro Takeuchi
Journal of Pediatric Gastroenterology and Nutrition, International, WoS, 2011, vol 52, pp 362-365
DOI: 10.1097/mpg.0b013e3181fcafb8
Summary
Crigler-Najjar syndrome type II (CN-II; # Mendelian Inheritance in Man [MIM] 606785) is a moderate type of hereditary unconjugated hyperbilirubinemia caused by mutations of the bilirubin uridine diphosphate-glucuronosyltransferase gene UGT1A1(1). Diagnosis of this syndrome is important in distinguishing it from the severe phenotype of hereditary unconjugated hyperbilirubinemia, known as Crigler-Najjar syndrome type I (CN-I; MIM #218800). The serum bilirubin concentration of CN-I is more than 20 mg/dL (343 μmol/L) and can reach 30 to 50 mg/dL (513–855 μmol/L). Patients with CN-I are in danger of kernicterus and must receive daily phototherapy right from the neonatal period (2). They should undergo liver transplantation to avoid kernicterus. Hyperbilirubinemia in patients with CN-II is less severe than CN-I (1). The serum bilirubin concentration of CN-II is seldom harmful after the neonatal period. The serum bilirubin concentration of patients with CN-I or CN-II is often intermediate between the values specifying the 2 syndromes. Accurate diagnosis of the 2 syndromes is necessary for appropriate treatment. A phenobarbital-loading test is effective in differential diagnosis (3), but the results are not always clear, in which case genetic diagnosis is necessary. The cDNA of UGT1A1 was first cloned in 1991, and a large number of mutations have been found in patients with CN-I, CN-II, and Gilbert syndrome (GS; MIM #143500) (4,5). Mutations of UGT1A1 differ according to ethnic grouping. East Asians display many unique mutations; in particular, a major polymorphic mutation is c.211G>A: p.G71R in exon 1 of UGT1A1 (6). p.G71R reduces glucuronidation ability to less than 32% of wild type and is an important cause of Gilbert syndrome (7). One of the most important causes of CN-II in Asians is a double missense mutation, p.[G71R;Y486D] (8). This double mutation has hardly ever been detected in white and African populations. We encountered a Turkish patient with moderate to severe unconjugated hyperbilirubinemia who was a compound heterozygote for a novel missense mutation, p.K402T, and the double missense mutation p.[G71R;Y486D]. The patient was diagnosed as CN-II on the basis of an in-vitro expression study. PATIENT Our patient is a 3-month-old Turkish boy. He is the second child of the family, born to nonconsanguineous parents. Although his parents and elder brother displayed no apparent jaundice, he developed severe jaundice 3 days after birth. He was treated with daily phototherapy and oral phenobarbital administration for prolonged hyperbilirubinemia; phenobarbital slightly reduced his serum bilirubin concentration. His serum total and indirect bilirubin concentrations were, respectively, 343.7 μmol/L (20.1 mg/dL) and 340.2 μmol/L (19.9 mg/dL). He showed no signs of infection or of haemolytic anaemia, pyrolic stenosis, liver dysfunction, or any other metabolic disease, according to the differential diagnosis of indirect hyperbilirubinemias. Because his serum bilirubin concentration was high, it was appropriate to perform genetic diagnosis of UGT1A1 to distinguish CN-II from CN-I. His psychomotor development was consistent with his age. METHODS Gene Analysis of UGT1A1 Genomic DNA was isolated from the leukocytes of the patient and his parents, with their informed consent. Amplification of exons, specifically the promoter region and enhancer regions of UGT1A1, was carried out by the polymerase chain reaction of genomic DNA, as described elsewhere (9,10). The amplified DNA fragments were sequenced directly (11). Construction of the Expression Vectors and Expression of UGT1A1 in COS-7 Cells The methods used to construct the expression vector using pCR3.1 (Invitrogen, San Diego, CA) and the expression study have been described elsewhere (12). To introduce the mutations into the expression vector, we used the following primers (the mutation points are indicated by underlining): 5′-CATCAGAGACAGAGCATTTTA-3′ for the G to A transversion at nucleotide 211 (c.211G>A) for p.G71R, 5′-GACAATGCAACGCGATGGAG-3′ for the A to C transversion at nucleotide 1205 (c.1205A>C) for p.K402T, and 5′-CCTGGTACCAGGACCATTCCT-3′ for the T to G transversion at nucleotide 1456 (c.1456T>G) for p.Y486D by means of the site-directed mutagenesis method using Mutan Km (TaKaRa, Kyoto, Japan). Three models were generated: wild-type UGT1A1, p.[G71R;Y486D], and p.K402T UGT1A1; as controls, nontransfected cells were used. Protein content was measured using a BCA protein assay kit (Pierce, Rockford, IL). Assay of UGT1A1 Activity and Western Blot Analysis The methods we used in the in vitro expression study and assay for normal and mutated UGT1A1 activity were the same as reported previously, with a minor modification (12). The bilirubin glucuronide obtained was isolated by thin-layer chromatography (TLC) on TLC plastic sheet 5748 (Merck, Darmstadt, Germany), which was scanned on an Instant Imager (Packard, Meriden, CT). The amounts of enzyme expressed were checked by Western blotting analysis, using anti-UGT1A1 antibody to adjust enzyme activity. We calculated kinetic parameters using the Prism 3.0 software (Graph Pad Software, Inc, San Diego, CA) to perform nonlinear regression on the Michaelis-Menten equation. Molecular weights of mutated proteins were determined by NIH imaging. RESULTS Gene Analysis The patient was heterozygous for 3 mutations. Substitution of adenine for guanine at nucleotide 211 changed the codon from glycine to arginine at position 71 of the corresponding protein (c.211G>A: p.G71R). Substitution of adenine for cytosine at nucleotide 1205 in exon 4 changed the codon from lysine to threonine at position 402 of the corresponding protein (c.1205A>C: p.K402T) (Fig. 1). This missense mutation is novel. Substitution of thymine for guanine at nucleotide 1456 in exon 5 changed the codon from tyrosine to aspartate at position 486 of the corresponding protein (c.1456T>G: p.Y486D). The notation for the mutations is based on the recommendation of the Human Genome Variation Society. We detected no further mutation in the phenobarbital responsive enhancer module (gtPBREM), TATA box, exon 2, exon 3, or exon 4. The patient's mother was heterozygous for c.1205A>C. His father was heterozygous for c.211G>A and c.1456T>G, showing that a mutant allele derived from the father is a double missense mutation, c.[211G>A;1456T>G]: p.[G71R;Y486D]. Genetic analysis of the parents indicated that the patient is compound heterozygous for p.K402T and a double missense mutation, p.[G71R; Y486D].FIGURE 1: UGT1A1 nucleotide sequences amplified from the genomic DNA of the patient. The novel missense mutation, a transversion of A to T (arrow) at position 1205 in UGT1A1 cDNA, changes codon 402 from lysine to threnonine (p.K402T).Expression and Identification of UGT1A1 In Western blotting analysis, a protein band (Fig. 2) was detected at 52 kDa in wild-type expression models, but not in the mock transfection. The molecular weight of the band was identical to that reported previously in wild-type UGT1A1 and p.[G71R;Y486D]. The band of p.K402T enzyme shifted to a higher molecular weight of 57 kDa, however, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2).FIGURE 2: Western blotting of wild-type p.[G71R;Y486D] and p.K402T UGT1A1. The 54-kDa UGT1A1 band is shown by an arrow. (A) Nontransfected control; (B) wild type; (C) p.[G71R;Y486D]; (D) the p.L131P mutant protein band is higher than that for wild-type protein (57 kDa).Assay of Enzyme Activity The apparent Vmax, Km, and efficiency (Vmax/Km) of the wild-type expression model were, respectively, 7.19 pmol/min/mg protein, 8.23 μmol/L, and 0.873 μmol/L/min/mg. The apparent Vmax, Km, and efficiency of the p.[G71R;Y486D] enzyme were 1.39 pmol/min/mg protein, 38.9 μmol/L, and 0.053 μmol/L/min/mg (Table 1), respectively. The apparent Vmax, Km, and efficiency of the p.K402T enzyme were 1.43 pmol/min/mg protein, 28.1 μmol/L, and 0.050 μmol/L/min/mg, respectively. Enzyme activities for bilirubin glucuronidation of p.[G71R;Y486D] and p.K402T were 6.1% and 5.8% of the wild-type model, respectively.TABLE 1: Kinetic parameters of wild-type and mutant UGT1A1DISCUSSION Bilirubin uridine diphosphate-glucuronosyltransferase (UGT1A1) is a critical enzyme for bilirubin glucuronidation (13), and mutations in the enzyme can induce unconjugated hyperbilirubinemias. GS and CN-II are mild and moderate forms of hyperbilirubinemia, but CN-I causes severe hyperbilirubinemia and sometimes results in death by kernicterus (2,3). Genetic analysis revealed that the patient was heterozygous for 3 UGT1A1 mutations: p.G71R in exon 1, p.K402T in exon 4, and p.Y486D in exon 5 (Fig. 1). Genetic analysis of the parents indicates that the patient is compound heterozygous for p.[G71R;Y486D] and p.K402T. Although p.[G71R;Y486D] is the most prevalent mutation in east Asian patients with CN-II, p.K402T is novel, and residual activity of the mutated enzyme is unknown. The patient experienced prolonged hyperbilirubinemia, with serum bilirubin concentration more than 20 mg/dL (342 μmol/L), and we undertook an in vitro expression study to find out whether p.K402T has residual enzyme activity. UGT1A1 with p.[G71R;Y486D] and UGT1A1 with p.K402T both exhibited reduced enzyme activity for bilirubin glucuronidation to 6.1% and 5.8% of wild type, respectively. These residual enzyme activities are compatible with CN-II, so that the patient was diagnosed as having CN-II. Western blotting analysis revealed that the apparent molecular weight of the p.K402T protein shifted to 57 kDa from 52 kDa for the wild type and p.[G71R;Y486D] protein (Fig. 2). This change suggests that p.K402T changes the protein structure radically and may alter the structure of its active site, resulting in reduced enzyme activity (5.8%). According to Western blotting analysis of a gene expression experiment we performed, p.L131P mutations of UGT1A1 with missense mutation induce loss of enzyme activity and change the mobility of the mutated enzyme (14). The mutated protein with p.L131P also shifted from 52 to 57 kDa. A predicted 3-dimensional model indicated that p.L131P loses β-sheet structure around codon 131. As with p.L131P, the structural change in UGT1A1 with p.K402T is capable of reducing glucuronidation activity to 5.8% of wild type. UGT1 has a unique gene structure. There are 8 functional exon 1s from A1 to A10, and exons 2 to 5 are used in common for all of the mRNAs expressed from the gene (15). Because p.Y486D is located on the common exon 5, it affects the glucuronidation of all of the isoforms (UGT1A1 to 1A10) expressed from UGT1. For example, UGT1A6 with p.Y486D reduced glucuronidation of 2-amino-5-nitro-4-trifluoromethylphenol (a major metabolite of flutamide) to less than 1% of wild type (16). Also, p.Y483D of UGT1A9, which corresponds to Y486 of UGT1A1, reduces glucuronidation for entacapone by half, but doubles it for scopoletine compared to wild type (17). It is known that p.Y483D of UGT1A9 also decreases or increases enzyme activity depending on the substrate (mycophenolic anid, naringenin, and 1-naphthol) (18). The mutation p.Y486D was first detected in a Japanese patient with Crigler-Najjar syndrome type II. The gene frequency in the Japanese population is 0.004 (19). Originally, p.Y486D was believed to be distributed only among east Asians. A carrier of p.Y486D has recently been reported in Germany, however (20). Our finding confirms that p.Y486D is not limited to Asia, but is distributed among populations in the Middle East and probably in Europe. The UGT1 isoforms constitute 1 of the major phase II drug-metabolising enzymes, and they have substrate specificities for a considerable number of medicines in general use. Consequently, p.Y486D is important when considering the side effects of drugs. If it is easy to check glucuronidation, then it is a good idea to examine the activity of UGT1s with p.Y486D to prevent severe side effects of newly developed medicines that are metabolised by UGT1s. An in vitro expression study of UGT1A1 is valuable for diagnosing patients as having CN-II by measurement of residual enzyme activity of the novel mutation, whenever clinical tests fail to distinguish between CN-I and CN-II. Acknowledgments We thank M. Suzaki and R. Okamoto of the Central Research Laboratory, Shiga University of Medical Science, for technical assistance.