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Technical Briefs |
1
Servizio di Genetica Medica, Instituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo and Università di Trieste, 34137 Trieste, Italy;
2
Dipartimento di Scienze Mediche, Università degli Studi del Piemonte Orientale "Amedeo Avogadro", Novara 28100, Italy;
3
Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Università di Trieste, 34137 Trieste, Italy;
a address correspondence to this author at: Servizio di Genetica, IRCCS Burlo Garofolo, Via dell’Istria, 65/1, 34137 Trieste, Italy
Gilbert syndrome (GS) is an inherited form of chronic mild unconjugated hyperbilirubinemia (1)(2)(3), although many patients do not have a clear family history (4). Hepatic glucuronidation of bilirubin is catalyzed by isoenzyme 1A1 of UDP-glucuronosyl transferase (UGT1A1). The majority of GS subjects were found to be homozygous for an extra TA in the TATA-box in the promoter region of UGT1A1 (5)(6)(7). Transcription of the (TA)7 allele is reduced by at least 70% compared with the wild-type (TA)6 allele. Because bilirubin UGT1A1 is the only enzyme with substantial bilirubin glucuronidating activity in humans (8), the presence of this extra TA in both alleles can explain the impaired conjugation of bilirubin found in Caucasoid GS patients (6).
A previous study of a large population found that the prevalence of the "abnormal" bilirubin UGT1A1 allele was 35–40% (9), leading to an expected frequency of homozygotes of ~16%; however, only 5% had increased serum concentrations of unconjugated bilirubin. Thus, a reduced expression of bilirubin UGT1A1, attributable to the (TA)7 abnormality in the promoter region, appears to be necessary, but not sufficient, for GS to be manifested clinically.
To date, the TA polymorphism has been detected by PCR amplification of the TATA-box element and high resolution polyacrylamide gel electrophoresis (9) or by direct sequencing (6). Recently, a new technique for sensitive, relatively inexpensive and automated high-throughput screening of mutations, denaturing HPLC (DHPLC), was introduced (10)(11)(12)(13). In this report, we evaluate the feasibility of applying DHPLC for the detection of TATA-box variants in the promoter region of the UGT1A1 gene in subjects with GS.
The UGT1A1 promoter was analyzed by both DHPLC and direct sequencing in 20 unrelated GS patients (16 males and 4 females; age range, 16–40 years) and 20 healthy controls with bilirubin concentrations within the reference interval (16 males and 4 females; age range, 13–35 years). The diagnosis of GS was based on the standard criteria of mild chronic, unconjugated hyperbilirubinemia in the presence of normal liver function and the absence of overt signs of hyperhemolysis (erythrocyte and reticulocyte counts, erythrocyte osmotic fragility, and immunoelectrophoretic patterns of erythrocyte hemoglobin were all normal) (14). None of the subjects had a history of hepatic or hematological disorders, excessive alcohol intake, or chronic use of medications or narcotics, and none received any drug during the 2 weeks before investigation.
In each subject, after an overnight fast, plasma concentrations of total and direct-reacting bilirubin were determined by a diazo method at least three times within 6 months before the study; indirect bilirubin was calculated as total minus direct bilirubin. Control subjects were pair-matched for age and sex. After informed consent, each patient and control was analyzed by both sequencing and DHPLC by investigators "blinded" to the plasma bilirubin concentrations.
DNA was isolated from EDTA-collected peripheral whole blood, using standard laboratory techniques (15).
The PCR reactions were performed in a 50-µL final volume containing 8 pmol of each primer, as described (6), and 1 U of AmpliTaq GOLD DNA polymerase (PE Applied Biosystems) in a thermal cycler 2400 (PE Applied Biosystems). After 35 cycles, PCR products were detected in a 2% agarose gel.
Heteroduplex molecules originate when a variation at the heterozygous state is present in a DNA fragment after denaturing and reannealing of the PCR product. DHPLC is based on the differential retention of homoduplex and heteroduplex molecules under the condition of partial heat denaturation. At low temperatures (50 °C), the two molecular types usually are coeluted. At increasing temperatures, the DNA starts to melt selectively in the region of mismatch of the heteroduplexes. Under these conditions, heteroduplex molecules are eluted ahead of homoduplexes, producing an additional peak.
To allow heteroduplex formation, PCR products were denatured for 3 min at 95 °C, followed by a gradual reannealing as temperature was decreased from 95 °C to 65 °C over 30 min in the thermal cycler. The reannealed duplexes were detected by scanning on an automated HPLC (Transgenomics). The stationary phase consisted of 2-µm nonporous alkylated poly(styrene-divinylbenzene) particles packed into a 50- x 4.6-mm (i.d.) column.
A 3- to 5-µL aliquot of each PCR product was injected onto the column and eluted at a flow rate of 0.9 mL/min with a mobile phase consisting of a mixture of buffers A and B; buffer A was 0.1 mol/L triethylammonium acetate (pH 7) and buffer B was 250 mL/L acetonitrile in 0.1 mol/L triethylammonium acetate (pH 7). The optimal temperature for HPLC was determined experimentally as 56 °C, and the PCR product separation was achieved through the following gradient: buffer B was increased from 57% to 62% over 30 s and then from 62% to 70% over 4 min. The eluted DNA fragments were detected at 260 nm.
DNA sequencing was performed on an automated ABI Prism 310 Genetic Analyzer (PE Applied Biosystems) with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) using forward and reverse primers (16).
The mean (± SD) total serum bilirubin concentrations of GS subjects was 15.5 ± 4.0 mg/L, significantly (P <0.001) higher than in controls (6.6 ± 2.4 mg/L). In both controls and GS subjects, >80% of bilirubin was indirect reacting.
PCR products of a promoter region (from nucleotide -227 to nucleotide
132) containing the TATA-box element, were analyzed by DHPLC. The
presence of two well-resolved peaks in Fig. 1
A reveals the heterozygous condition (TA)6/(TA)7, as confirmed
by direct sequencing. The presence of one peak is characteristic of the
homozygous condition (Fig. 1
, B and C). To distinguish (TA)6 from (TA)7
homozygotes, each sample showing a single peak was mixed with
(TA)6/(TA)6 control DNA under conditions allowing heteroduplex
formation. The homozygous condition for (TA)7 was revealed by a double
peak (Fig. 1D
), whereas no change in the chromatogram showing a single
peak was detected for (TA)6 homozygotes. DHPLC results showed a 100%
match with those obtained by direct sequencing.
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The genotype frequencies in both Gilbert patients and controls are
reported in Table 1
.This distribution in healthy controls was significantly different from
that expected by Hardy-Weinberg law (
2 = 4.3;
P <0.05), mainly because of a higher than expected number
of homozygotes. The (TA)7 allele was significantly more frequent in GS
subjects than in controls (0.9 vs 0.25; 2 =
34.5; P <0.00001).
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Several useful techniques for detecting mutations have evolved in recent years. The most widely used simple single-step analytical method, single-strand conformational polymorphism, has a low sensitivity, whereas the more sensitive methods (i.e., direct sequencing and denaturing gradient gel electrophoresis) are often expensive and time-consuming (11).
As illustrated in our study, DHPLC technology for detection of mutants is a powerful and sensitive tool useful for the rapid, efficient screening of large numbers of samples. We found 100% concordance with direct sequencing, and the reagent costs of a DHPLC analysis (~$3 US per sample, PCR included) are lower than those of direct sequencing ($15 US). The labor requirements and operator time are also reduced, further increasing the cost-effectiveness. After temperature optimization, automated DHPLC analysis requires only a few seconds to load the autosampler and ~5 min to run each sample. The interpretation of the final chromatograms is also rapid. If a single peak is detected, however, a second run must be performed, with the PCR product mixed with a known homozygous control, to differentiate the two homozygous genotypes.
In the limited numbers of subjects in our study, we confirmed that the (TA)7 variation was significantly more frequent in GS subjects than in controls. The absence of Hardy-Weinberg equilibrium in controls may result from the small sample of individuals studied or from selection bias.
In conclusion, DHPLC can be chosen as a large-scale screening method for detection of the (TA)7 mutation in the promoter region of the UGT1A1 gene. It may be helpful in determining whether impaired glucuronidation contributes to clinical hyperbilirubinemia in subjects with abnormalities in bilirubin metabolism, as in subjects with heterozygous ß-thalassemia (17), glucose-6-phosphate dehydrogenase deficiency (18), neonatal icterus associated with glucose-6-phosphate dehydrogenase deficiency (19), or hereditary spherocytosis (20).
Acknowledgments
This work was supported in part by a grant from the Italian Ministry of Research Ministero dell’Università per la Ricerca Scientifica e Tecnologia (to C.T.) and grants from the Italian Ministry of Health (Grants RC 19/99 and RF 98.67 to A.A.). D. Pirulli is the recipient of a long-term fellowship from the University of Trieste. D. Puzzer is a recipient of a fellowship from Children’s Hospital Burlo Garofolo of Trieste. The financial support of Fondo Studi Fegato-ONLUS, Trieste, is also acknowledged. We thank Drs. J.D. Ostrow and J. Mihelcic for constructive discussions during the preparation of this manuscript.
Footnotes
fax 39-040-3785210, e-mail amoroso{at}burlo.trieste.it
References
The following articles in journals at HighWire Press have cited this article:
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  U. Ehmer, T. O. Lankisch, T. J. Erichsen, S. Kalthoff, N. Freiberg, M. Wehmeier, M. P. Manns, and C. P. Strassburg Rapid Allelic Discrimination by TaqMan PCR for the Detection of the Gilbert's Syndrome Marker UGT1A1*28 J. Mol. Diagn., November 1, 2008; 10(6): 549 - 552. [Abstract] [Full Text] [PDF]
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 J. R. Harraway and P. M. George Use of Fully Denaturing HPLC for UGT1A1 Genotyping in Gilbert Syndrome Clin. Chem., November 1, 2005; 51(11): 2183 - 2185. [Full Text] [PDF]
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E. Costa, E. Vieira, and R. dos Santos The Polymorphism c.-3279T>G in the Phenobarbital-Responsive Enhancer Module of the Bilirubin UDP-Glucuronosyltransferase Gene Is Associated with Gilbert Syndrome Clin. Chem., November 1, 2005; 51(11): 2204 - 2206. [Full Text] [PDF]
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N. von Ahsen, M. Oellerich, and E. Schutz DNA Base Bulge vs Unmatched End Formation in Probe-based Diagnostic Insertion/Deletion Genotyping: Genotyping the UGT1A1 (TA)n Polymorphism by Real-Time Fluorescence PCR Clin. Chem., December 1, 2000; 46(12): 1939 - 1945. [Abstract] [Full Text] [PDF]
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