UGT1A1 Gene Mutations in Pakistani Children Suffering from Inherited Nonhemolytic Unconjugated Hyperbilirubinemias


Corresponding author: Muhammad Naeem, Ph.D., Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan. Tel: +925190644122; Fax: +925190644110; E-mail:


Two inherited unconjugated hyperbilirubinemias, Crigler–Najjar syndrome and Gilbert syndrome, arise due to deficiency of UGT1A1 enzyme activity. Crigler–Najjar syndrome type 1 (CN1) lies at the extreme severe end of the spectrum of UGT1A1 activity characterized by complete absence, followed by the less severe Crigler–Najjar syndrome type 2 (CN2). Gilbert syndrome is the mild form having only partial loss of UGT1A1 activity. The present study aimed to identify molecular genetic defects underlying unconjugated hyperbilirubinemias in children from six consanguineous Pakistani families. The patients were clinically diagnosed by exclusion of other unconjugated hyperbilirubinemias. Differential diagnosis of CN1 and CN2 was made on the basis of patient's response to phenobarbitone. The promoter region, coding exons, and adjacent splice sites of the UGT1A1 gene were PCR amplified from genomic DNA of all patients and their families, and were sequenced. DNA sequence analysis identified five different homozygous mutations: two novel missense mutations p.Y230C (proband A) and p.D36N (proband B), a 4-bp insertion c.622–625dupCAGC/p.Q208QfsX50 (probands C and E), a nonsense mutation p.R341X (proband D), and a TA insertion A(TA)7TAA in the promoter region (proband F). The present study extends the spectrum of UGT1A1 gene mutations and may be helpful in the diagnosis of Crigler–Najjar syndrome and Gilbert syndrome.


Bilirubin is a toxic metabolite formed during the catabolism of heme. The hepatic bilirubin UDP-glucoronosyltransferase (UGT) 1A1 enzyme (B-UGT; EC plays an important role in the detoxification of toxic bilirubin by conjugating it with glucuronic acid in the endoplasmic reticulum (ER) of hepatocytes. The resulting hydrophilic bilirubin mono and di-conjugates are then excreted in the bile (Bosma et al., 1994; Burchell et al., 1997). Mutations that lead to low expression or decreased activity of bilirubin UGT1A1 enzyme result in poor glucuronidation of bilirubin and subsequent development of unconjugated hyperbilirubinemia in the form of Crigler–Najjar syndrome or Gilbert syndrome, which are inherited as autosomal recessive diseases.

Crigler–Najjar syndrome (CN, OMIM# 218800), first described by Crigler and Najjar in 1952, is characterized by very high serum levels of unconjugated (indirect) bilirubin (Crigler & Najjar, 1952). On the basis of clinical severity, the CN syndrome can be divided into two subtypes, CN type 1 and CN type 2 (Arias et al., 1968). The Crigler–Najjar syndrome type 1 (CN-1; OMIM # 218800) is associated with almost complete absence of UGT1A1 activity (Crigler & Najjar, 1952; Seppen et al., 1994) in the liver and serum unconjugated bilirubin levels of 340–850 μmol/l or higher and often leads to brain damage (kernicterus). In CN-1 patients, the serum bilirubin concentration is noncompliant to phenobarbital treatment. The only effective treatment for CN-1 patients is liver transplantation (Ozçay et al., 2009).

Crigler–Najjar syndrome type 2 (CN-2; MIM # 606785), first described by Arias (1962), is characterized by intermediate serum indirect bilirubin levels (85–340 μmol/l). The UGT1A1 enzyme activity is moderately reduced in CN-2 patients but it can be induced by phenobarbital administration. CN-2 patients have higher bilirubin monoglucuronic and diglucuronic acids in the bile than CN-1 patients (Arias et al., 1968; Sampietro & Iolascon, 1999; Huang et al., 2006). Gilbert syndrome (GS; OMIM#143500) is a milder form of unconjugated hyperbilirubinemia (17–85 μmol/l) occurring in the absence of bilirubinuria or other evidence of hemolysis or liver disease (Bartlett & Gourley, 2011).

In this study, we report causative UGT1A1 gene mutations in six Pakistani children born to consanguineous parents and suffering from nonhemolytic unconjugated hyperbilirubinemia.

Material and Methods

Human Subjects

In the present study, we recruited six unrelated children suffering from unconjugated hyperbilirubinemia. These children belonged to consanguineous families originating from the Punjab and Khyber Pakhtunkhwa provinces of Pakistan. Clinical information was obtained for each proband with unconjugated hyperbilirubinemia. The diagnoses for the possible causes of unconjugated hyperbilirubinemia were based on clinical and laboratory parameters. Five recruited patients (referred to as probands “A,” “B,” “C,” “D,” and “E”) were diagnosed with Crigler–Najjar syndrome, while one patient (referred to as proband “F”) was diagnosed with Gilbert syndrome.

The study approval was obtained from the Quaid-i-Azam University Institutional Review Board. Informed consent was obtained from the family members who participated in the study. In the case of minors, consent was obtained from the guardians. Blood samples were collected from each proband and their parents, where available, and genomic DNA was isolated following a standard protocol. The isolated DNA was quantified by measurement of optical density at 260 nm wavelength in a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific, Wilmington, DE, USA) and diluted to 100 ng/μl for amplification by polymerase chain reaction (PCR).

Mutation Analysis

The promoter region, coding exons and splice junctions of the UGT1A1 gene were amplified from genomic DNA through PCR. The PCR amplification was carried out in a total volume of 50 μl containing 25 μl of 2X GoTaq Green Master Mix (Promega Corporation, Madison, WI, USA), 100 ng genomic DNA and 50 pmol of each primer (Macrogen Inc., Seoul, Korea). PCR was carried out for 35 cycles with the following thermal cycling conditions: 95°C for 1′, 60°C for 1′, and 72°C for 1′ followed by final extension at 72°C for 5′ in a Corbett palm cycler (QIAGEN, Melbourne, Australia).

The PCR products were purified using GeneJET Gel Extraction Kit (Thermo Fisher Scientific), and were subjected to DNA sequencing reaction using the Big Dye Terminator Cycle Sequencing Kit version 3.1 (PE Applied Biosystems, Foster City, CA, USA). The sequencing products were purified by ethanol precipitation and were sequenced in an ABI 3730xl automated sequencer. The primers used were designed from the intronic sequences of the UGT1A1 gene and are available on request. Sequence variants were identified using BioEdit Sequence Alignment Editor version (Hall, 1999).


Probands “B,” “C,” “D,” and “E” had Crigler–Najjar syndrome type 1 as phototherapy and enzyme induction by phenobarbital treatment were inefficient in reducing the serum bilirubin levels that were raised up to 600 μmol/l. Proband A had Crigler–Najjar syndrome type 2 with serum bilirubin levels up to 240 μmol/l that were reduced (to 100 μmol/l or less) with phenobarbital treatment. Proband “F” with Gilbert syndrome had raised serum bilirubin level only up to 50 μmol/l.

In all patients and their parents, immunological screening assays for the presence of hepatitis viruses were negative. The biochemical parameters including alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, and glucose-6-phosphate dehydrogenase activity were normal. Analysis of hematologic parameters did not reveal any evidence of hemolysis, hypothyroidism, polycythemia, or infection.

The promoter region, exons, and splice junctions of the UGT1A1 gene were sequenced from the genomic DNA of all probands and their parents (except the parents of proband “E,” who did not provide consent for the molecular analysis). DNA sequence analysis revealed an A > G transition at nucleotide position c.689 (exon 1) resulting in the substitution of tyrosine (partially hydrophobic with aromatic side chain) to cysteine (neutral) at amino acid position 230 (p.Y230C) in proband “A” (Fig. 1A); a G > A transition at nucleotide position 106 (exon 1), changing aspartic acid (negatively charged) into asparagine (neutral) at amino acid position 36 (p.D36N) in proband “B” (Fig. 1B); a 4-bp duplication in exon 1 (c.622–625dupCAGC) in probands “C” and “E” predicting a premature stop codon at amino acid position 258 (p.Q208QfsX50) (Fig. 1C); a C > T transition at nucleotide position c.1021 (exon 3) replacing arginine amino acid into a premature stop codon at amino acid position 341 (p.R341X) in proband “D” (Fig. 1D). These mutations were identified in the homozygous state in the patients while their parents were heterozygous carriers for the corresponding mutations. To ensure that these mutations were not neutral polymorphisms in the Pakistani population, 100 ethnically matched healthy control individuals were screened for the mutation by PCR followed by direct sequencing. None of the coding mutations described here was identified in the control samples. In proband “F,” no mutation was identified in the coding sequence of the UGT1A1 gene. However, a homozygous TA insertion polymorphism was identified in the TATAA element of the promoter sequence of the UGT1A1 gene (Fig. 1E).

Figure 1.

DNA sequence analysis of the UGT1A1 gene. Sequencing chromatograms of: (A) exon 1 from proband A (i), carrier (ii), and control (iii). (B) Exon 1 from proband B (iv), carrier (v), and control (vi). (C) Exon 1 from proband C/E (vii), and control (viii). (D) Exon 3 from proband D (ix), carrier (x), and control (xi). (E) Promoter region from proband F (xii). The arrows indicate the positions of the mutations and the short bar above the sequence indicates a TA insertion in the promoter sequence.


The UDP-glucoronosyltransferase 1 gene family (UGT1) is a unique gene complex that spans 218 kb on chromosome 2q37 and encodes 13 UGT1 isoforms (Gong et al., 2001; Li et al., 2007). In each of these isoforms, a unique exon 1 (with a tissue specific upstream promoter which encodes the amino-terminal half of UGT1) is linked to four common exons to form overlapping transcripts through independent transcription initiation. The amino-terminal half of UGT1 isoforms includes an aglycone binding domain. The four common exons encode the carboxy terminal halves of UGT1 isoforms which specify interaction with the common sugar donor substrate, UDP-glucuronic acid. UGT1A1 is a transmembrane protein of 533 amino acids with approximately equal-sized amino-terminal and carboxy-terminal halves. It is expressed mainly in the liver and is located in the ER of hepatocytes and extra hepatic tissues (Ritter et al., 1992; Kurkela et al., 2003). The majority of the enzyme (including the aglycone and glucuronic acid binding sites) is located within the lumen of the ER cisternae, except for a membrane spanning segment of 17 amino acids and a 26 amino acid cytoplasmic tail (Kadakol et al., 2000; Kurkela et al., 2003).

There are 130 different mutations previously described in the UGT1A1 gene: 91 (70%) single nucleotide substitutions (77 missense and 14 nonsense), 21 (16.1%) deletions, 10 (7.6%) insertions, and 8 (6.1%) mutations that affect the promoter and introns (Canu et al., 2013). The severity of the functional deficiency of UGT1A1 is determined by the nature of the genetic lesion. CN-1 results from a number of nonsense (or frameshift) mutations that cause premature truncation or missense mutations that cause substitution of critical residues of the UGT1A1 protein. CN-2 is due to homozygous or compound heterozygous missense mutations and more rarely it is due to a combination of heterozygous nonsense/frameshift and missense mutations (compound heterozygous) or to an interaction between missense mutations and a TA insertion polymorphism in the TATAA promoter element of the UGT1A1 gene as described by Sampietro and Iolascon (1999) and Kadakol et al. (2001).

We have investigated molecular genetic defects in the UGT1A1 gene underlying unconjugated hyperbilirubinemia in unrelated children born to consanguineous parents in six Pakistani families. DNA sequence analysis of the UGT1A1 gene in the probands of these families led to the identification of five pathogenic sequence variants (Table 1). Three of these identified variants (AT(TA)7TAA, p.R341X, p.Q208QfsX50) have been previously reported (Moghrabi et al., 1993; Bosma et al., 1995; Kohli et al., 2010), while the two missense mutations (p.D36N, p.Y230C) are novel. Multiple sequence alignment of the UGT1A1 protein sequences from different species revealed that the amino acids aspartic acid (p.D36) and tyrosine (p.Y230) are evolutionarily highly conserved (Fig. 2). SIFT analysis predicted that the p.D36N and p.Y230C substitutions will affect the protein with a probability score of 0.00 and 0.01, respectively. The threshold score for intolerance of a substitution is 0.05; substitutions with probability score <0.05 are predicted to be deleterious.

Table 1. Summary of UGT1A1 gene mutations identified in the current study
ProbandsDiagnosisMutationMutant protein(TA) PolymorphismReferences
  1. CN2, Crigler–Najjar syndrome type 2; CN1, Crigler–Najjar syndrome type 1; GS, Gilbert syndrome.

ACN 2c.689A > Gp.Y230CTA6/TA6Novel
BCN 1c.106 G > Ap.D36NTA6/TA6Novel
CCN 1c.622-625dupCAGCp.S208fsX258TA6/TA6(Kohli et al., 2010)
DCN 1c.1021C >Tp. R341XTA6/TA6(Moghrabi et al., 1993)
ECN 1c.622-625dupCAGCp.S208fsX258TA6/TA6(Kohli et al., 2010)
FGSA(TA)7TAATA7/TA7(Bosma et al., 1995)
Figure 2.

Partial amino acid sequence comparison of the human UGT1A1 protein with other orthologs. The missense mutations (p.D36N and p.Y230C) affect the evolutionarily conserved amino acid residues indicated by the arrows.

The mutations p.Y230C and p.D36N lie in the amino-terminal half (first 267 residues) of UGT1A1 that binds aglycone and imparts acceptor substrate specificity to the enzyme (Kadakol et al., 2000). In recent studies, the acceptor-binding site of the human UGT1A1 protein has been predicted as a deep kinked pocket adjacent to the donor-binding site. Bilirubin can fit into this pocket, which is larger in size than the bilirubin. D36 is one of the residues that lie in the predicted acceptor-binding pocket (Ritter et al., 1992; Bosma et al., 1995). Moreover, a membrane-attached region (between 140 and 240 residues; more specific location unknown) has been predicted to help highly lipophilic substances (bilirubin substrates) to reach the active site (Kadakol et al., 2000; Li et al., 2007). Therefore, the identified amino acid substitutions (p.Y230C and p.D36N) in the amino terminal region could alter the acceptor binding site, reducing the affinity of the enzyme for bilirubin substrate and/or interfering with the binding of bilirubin to human UGT1A1. These enzyme defects could decrease or abolish its bilirubin glucuronidating activity consistent with the unconjugated hyperbilirubinemia phenotypes. However, expression studies are required to confirm the effect of these novel mutations on the enzyme activity.

The identified mutations p.Q208QfsX50 and p.R341X predict truncated proteins of 258 and 340 amino acids, respectively, for all the UGT1 gene products. These truncated proteins might be expected to be nonfunctional and to be found in the cytoplasm (unlike normal proteins, which are anchored on the luminal surface of the ER) due to an absent or incomplete carboxy terminal half (which would include the ER anchorage domain). An immunoblotting study in a Pakistani patient previously reported to harbor the p.R341X mutation did not detect truncated UGTs, which might be due to nonsense mediated mRNA decay (Moghrabi et al., 1993).

In proband “F” who was diagnosed with Gilbert syndrome, a homozygous TA insertion was identified in the TATA element of the UGT1A1 promoter sequence (a polymorphism known as the UGT1A1*28 allele). Homozygosity for the TA insertion in the TATA box promoter element of the UGT1A1 gene is associated with Gilbert's syndrome (Bosma et al., 1995; Minucci et al., 2010). The normal TATA box promoter region of the UGT1A1 gene has the AT(TA)6TAA sequence. The presence of a longer TATAA element in the promoter region reduces the efficiency of transcription of the UGT1A1 gene leading to unconjugated hyperbilirubinemia (Bosma et al., 1995).


We present a spectrum of UGT1A1 gene mutations in Pakistani children suffering from nonhemolytic unconjugated hyperbilirubinemia. These data should help in the prenatal diagnosis and genetic counseling of the affected families. However, there is a need to define the mutation spectrum in a larger sample size from the Pakistani population for downstream application in molecular diagnostics.

Conflict of Interest

The authors declare that they have no conflict of interest.


We are grateful to the patients and their family members for participation in the study. The study was funded by a grant received from the Higher Education Commission (HEC) of Pakistan, and the University Research Fund (URF) of Quaid-i-Azam University, Islamabad, Pakistan. GS is supported by an HEC indigenous Ph.D. fellowship program.