Introduction

The UDP-glucuronosyltransferases (UGT) represent a superfamily of enzymes bound to the membrane of the endoplasmic reticulum.^{1, 2} The human UGT family contains more than a dozen members from three subfamilies, UGT1A, 2A and 2B and are mainly of hepatic origin, but high UGT expression levels have been reported also in extrahepatic tissues such as intestine and kidney.^{3, 4, 5, 6, 7, 8} UGTs catalyze the conjugation of exogenous and endogenous compounds with uridine diphosphoglucuronic acid. The resulting glucuronides are more soluble in water and facilitate biliary or renal excretion. UGTs play an important role in cellular defense not only by eliminating xenobiotics and endogenous toxins, but also by detoxifying human carcinogens, such as heterocyclic amines as well as heterocyclic and polycyclic hydrocarbons.⁹

The UGT1A family is located on a single locus on chromosome 2q37 and consists of at least nine functional proteins.¹⁰ Exons 2 to 5 are shared by all UGT1A proteins, whereas exon 1 is specific for each isoform.¹¹ One specific UGT1A protein, UGT1A7, is present in the esophagus, stomach, lung and pancreas and catalyzes the glucuronidation of simple and complex phenols, flavones, coumarins and benzo[a]pyrenes.^{9, 12} UGT1A7 is highly polymorphic and so far 11 different alleles have been identified. These polymorphisms may have functional consequences as they reduce enzyme activity, resulting in a lower capacity for detoxification of human carcinogens.^{13, 14, 15, 16} Codons 129, 131 and 208 are highly variable among mammals and polymorphisms result in a range of separate UGT1A7 alleles: N129-R131-W208 (UGT1A7*1), K129-K131-W208 (UGT1A7*2), K129-K131-R208 (UGT1A7*3) and N129-R131-R208 (UGT1A7*4) (Table 1). On balance, this may have pathological consequences as, for example, the UGT1A7*3 allele has been associated with colorectal cancer, hepatocellular carcinoma, and orolaryngeal cancer and in particular with pancreatic cancer and alcoholic chronic pancreatitis (CP).^{16, 17, 18, 19, 20}

Table 1 - UGT1A7 alleles.

Full table

We recently performed a case–control study and examined the relationship between the UGT1A7*3 allele and pancreatic disease in a population of 973 pancreatitis or pancreatic cancer patients.¹⁵ In contrast to an earlier study,¹⁶ we failed to confirm an enrichment of UGT1A7*3 in patients with pancreatic disorders. Apart from study size and the possibility of non-random distribution of the alleles, we considered the possibility that the differences may be owing to polymerase chain reaction (PCR) bias. PCR bias leads to unfair representation of the quantity of a template present in the reaction. This might be caused not only by differences in template lengths, random variations in template number (especially with very small initial numbers) and random variations in PCR efficiency in each cycle, but also by the presence of a polymorphism at the primer-annealing site.

We hypothesized that the detection of UGT1A7*3 may be hampered by PCR bias. To establish this, we used a range of different primer sets and analyzed both human DNA samples and mixtures of cloned DNA for UGT1A7*3 detection.

Results

Genotyping

The results of the genetic analysis of 37 CP patients, which had previously been analyzed,¹⁶ differed in 14 cases (38%) (Table 2). Our analysis demonstrated that 11 out of 12 individuals carrying UGT1A7*1/*3 had been classified previously as having UGT1A7*1/*4 alleles.¹⁶ Sequencing of exon 1 revealed a T>G transversion at position -57 which appears to be in complete linkage disequilibrium with UGT1A7*3 (K129-K131-R208). This base pair change is precisely located at the annealing site of the forward primer (at position-19 away from the 3'-end of the primer) used in the previous analysis (Figure 1). PCR amplification of UGT1A7*1/*3 heterozygotes at an annealing temperature of 52°C with forward primers that contained either a wild-type T (11F and 13F) or a mutant G (12F and 14F) at position -57 resulted in a reduced amplification of the heterozygous allele with one mismatch. Higher annealing temperatures (56 and 60°C) led to complete amplification of the complementary allele in heterozygotes (data not shown). This suggests that the use of a primer without the mutation at position -57 in the sequence leads to uneven amplification of UGT1A7*3 (Figure 2).

Figure 1.

UGT1A7 -57T>G polymorphism. The first line shows the forward primer used in a previous report;¹⁶ the second line shows the genomic sequence of UGT1A7. The GC-rich clamp at the 5'-end of the primer that does not align with the UGT1A7 sequence is underlined. The position of the mutated nucleotide is indicated in bold.

Full figure and legend (8K)

Figure 2.

Constructs. Construct 1 represents the normal wild-type allele present in humans cloned into the pGEM-T vector with a wild-type thymidine at position -57. The star indicates polymorphisms and construct 2 represents the UGT1A7*3 allele in humans with a mutant guanine at position -57. Construct 4 contains the mutant allele but with a wild-type thymidine at position -57 and construct 3 contains wild-type allele with a mutant guanine at position -57.

Full figure and legend (19K)

Table 2 - UGT1A7 genotyping results obtained from 37 chronic pancreatitis patients analyzed with different primer combinations.

Full table

Constructs

Next, we examined how the sequence of the primer affects the efficacy to detect UGT1A7*1 and UGT1A7*3 constructs by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. We tested two forward primers, one with a wild-type T (13F) and another with mutant G at position -57 (14F). We found that both primers detected UGT1A7*1 and UGT1A7*3 well at both annealing temperatures, from a mixture of UGT1A7*1 and UGT1A7*3 (with or without the mutation at position -57) (Figure 3a, b, e and f).

Figure 3.

Melting curves with FRET probes for codons 129 and 131 of the constructs. The blue line represents detection with primer 13F, the red line represents detection with primer 14F. Panels (a) and (b) represent melting curves for PCRs with constructs 1 and 4 (both -57T) as templates at annealing temperatures of 52°C (a) and 57°C (b). Panels (c) and (d) depict melting curves of PCRs with constructs 4 (-57T) and 3 (-57G) as input at annealing temperatures of 52°C (c) and 57°C (d). Panels (e) and (f) show a melting curve for constructs 2 and 3 (both -57G) at annealing temperatures of 52°C (E) and 57°C (f), and lastly, panels (g) and (h) are representative examples of melting curves with constructs 1 (-57T) and 2 (-57G) at annealing temperatures of 52°C (g) and 57°C (h). Construct 1: -57t, N129, R131, W208; construct 2: -57g, K129, K131, R208; construct 3: -57 g, N129, R131, W208; construct 4: -57t, K129, K131, R208 (see also Figure 2).

Full figure and legend (354K)

If we use a mixture of UGT1A7*1 with G at position -57 (construct 3) together with UGT1A7*3 with a T at position -57 (construct 4) as a template for the PCR, both primers will amplify both constructs at the annealing temperature of 52°C in a balanced manner. However, we found that upon increasing the annealing temperature to 57°C, the primer with a wild-type T at position -57 (13F) merely amplified construct UGT1A7*3 with a T at position -57. (Figure 3c and d). In contrast, at 57°C the primer with the mutant G at position -57 (14F) preferably amplified UGT1A7*1 with G at position -57 (construct 3). Likewise, a mixture of UGT1A7*1 and UGT1A7*3 constructs (representative of a compound heterozygote UGT1A7*1/*3 carrier) yielded similar results. Both primers amplify UGT1A7*1 and UGT1A7*3 evenly at 52°C, but at 57°C, the primer with T at position -57 (13F) preferentially amplifies UGT1A7*1 whereas the primer with G at position -57 (14F) only amplifies UGT1A7*3 (Figure 3g and h).

In summary, upon testing two different primers using two UGT1A7 constructs with different nucleotides on position -57 as a template, we found that the primer with a matching sequence at that position prefers to amplify the analogous template. Spiking the PCR template with mouse genomic DNA did not affect the obtained results.

Discussion

To examine whether the differences in genotyping results from previous studies^{16, 17, 18} arose from PCR amplification bias, we analyzed 37 CP samples that had been part of a previous case–control study focused on the role of UGT1A7 polymorphisms in pancreatic disorders.¹⁶ In approximately 40% of these samples, we obtained different genotyping results (Table 2). Upon sequencing, we first identified a T>G transversion at position -57 of the UGT1A7 gene, corroborating data from a recent report.²¹ This polymorphism appears to be in complete linkage disequilibrium with UGT1A7*3 (K129-K131-R208) and was detected both in a heterozygous or homozygous state in all UGT1A7*3 heterozygotes or homozygotes, respectively, but not in individuals without a UGT1A7*3 allele. -57T>G is located within the annealing sequence of the forward primer used in previous studies,^{16, 17, 18, 22} and is located in 19 nucleotides upstream from the 3'-end of the chosen primer. In principle this should not affect PCR amplification, but the region downstream of -57 is extremely AT-rich, making the 3'-end of the chosen oligonucleotide primer unstable. Moreover, a GC-rich clamp, which does not align with the UGT1A7 sequence, was attached in previous studies at the 5'-end of this primer, which further impairs the affinity to its target (Figure 1).

We examined the possibility that -57T>G affects PCR amplification, and designed several primers either with a T or a G at position -57. Subsequent melting curve analysis showed that in -57T>G heterozygotes, primarily the -57T or -57G allele was amplified depending on the chosen primer.

As a next step, we used cloned DNA that allows normalization of a template input with a genomic reference sequence for the most accurate quantification of PCR bias. We prepared four constructs with different combinations of nucleotides at position –57, 129, 131 and 208 of UGT1A7. We found that the nucleotide at -57 was critical as in heterozygous samples; an unmatched primer pair led to unbalanced amplification with a clear preference for the template with the matching sequence. We found that reactions with -57 homozygous constructs led to successful amplifications regardless of the nature of the nucleotide at -57. This observation has immediate consequences as -57T>G appears to be in complete linkage disequilibrium with K129-K131-R208 (UGT1A7*3). As a result, this polymorphism reduces the amplification of the UGT1A7*3 allele in the presence of UGT1A7*1 or UGT1A7*2 allele (containing the K129 and K131 polymorphisms), and confounds the interpretation of the results.

We found that the frequency of the UGT1A7*3 allele in our healthy controls is 40%.¹⁵ This is in line with frequencies of 32, 36 and 37% in control samples reported by others,^{13, 14, 26} but is appreciably higher than the 16–21% detected by early reports from the group that studied the role of UGT1A7 in pancreatic diseases.^{16, 17, 18, 22} Our data suggest that the relatively low frequency of UGT1A7*3 alleles in these studies can be explained by this PCR bias.

On the other hand, PCR bias cannot explain the reported differences in the frequency of UGT1A7*3 alleles between controls and patients. One may speculate that the over-representation of the UGT1A7*3 allele in the patient group is due to the fact that samples from each group of subjects to be compared (e.g., cases and controls) have not been included in each batch analyzed as recommended as a quality measure in genetic association studies. As shown in Figure 3, the occurrence of uneven allelic amplification is dependent on the chosen primer-annealing temperature. Thus, a slight variation of annealing temperature (or of ion concentration of the PCR reaction mix) may drive the reaction to an even amplification of both alleles or to PCR bias with preferred amplification of the UGT1A7*1.

Although the present study explains the basic principle of the genotyping error reported by previous studies, it does not answer why the -57T>G alteration led to *1/*4 instead of *1/*1 genotypes in UGT1A7*1/*3 heterozygotes. If the upstream primer does not appropriately anneal to UGT1A7*3, one would expect an exclusive amplification of the second allele, UGT1A7*1, and that these individuals would be classified as UGT1A7*1/*1 homozygotes.

A recent study from the same researchers who reported the enrichment of UGT1A7*3 in pancreatic disorders reported a -57G allele frequency of 0.39 among 427 control subjects.²¹ Similar to our results, they found that -57G is in complete linkage disequilibrium with UGT1A7*3. As a corollary, it is more than likely that the 'real' UGT1A7*3 frequency in their control population is close to 0.39.

In most studies, UGT1A7 polymorphisms have been determined by sequencing PCR fragments of the gene. Although sequencing has been generally accepted as the 'gold standard' for mutation detection, a recent study demonstrated that in some instances PCR products that were heterozygous in the restriction fragment length polymorphism (RFLP) assay came back as homozygous on sequencing.²³ This effect was even more pronounced if primers were used that were not completely aligned (mismatch) with the template sequence. This leads us back to UGT1A7 because we observed that most other studies^{16, 17, 18, 22} used a 'mismatched' primer (because of the -57T>G polymorphism). This will cause uneven allele amplification during PCR and erroneous results in the subsequent sequencing reaction. The observed differences in UGT1A7 gene analysis of the 37 subjects can best be explained by a primer-dependent PCR bias for the various alleles. Accordingly, as most analytical methods such as sequencing as the gold-standard, RFLP, TaqMan or hybridization probes depend on a PCR product that is generated by the mismatched primer, all diagnostic methods will always lead to incorrect results. In this respect, our report represents an important methodological example of how PCR amplification bias results in genotyping errors. As additional clinically significant SNPs are being discovered, assessment of PCR bias will be increasingly important. The implications of this finding extend beyond analysis of UGT1A7 gene variants and may have an impact on other genetic association studies. Indeed, this is well illustrated by a study that initially indicated an association between a microsomal epoxide hydrolase gene polymorphism and Crohn's disease.²⁴ Subsequent testing demonstrated a genotyping error because of a silent polymorphism that interfered with proper annealing of the primer and led to misinterpretation of the RFLP.²⁵ The positive association completely disappeared on proper reanalysis. Thus, assessment of assay quality by using a second independent set of primers or sequencing of both primer regions to exclude genetic variants that affect primer annealing is strongly recommended.

Materials and methods

Genotyping

We investigated 37 CP patients with a N34S SPINK1 mutation (courtesy of Dr Volker Keim, Leipzig, Germany). These participants were part of an earlier study and had been genotyped for UGT1A7 by others.¹⁶ We genotyped UGT1A7 alleles by melting curve analysis with FRET probes in the LightCycler (Roche Diagnostics, Mannheim, Germany). The FRET probes for detection of the polymorphisms at codons 129 and 131 and the W208R mutation were designed and synthesized by TIB MOLBIOL, Berlin, Germany. For detection of the polymorphisms at codons 129 and 131 the sensor probe was 5'-LC 640-TTAAGTATTCTACTAATTTTTTGTCCTT-ph (LC: LightCycler Red attached to 5' terminus; ph: phosphate) and the anchor probe 5'-GGATC GAGAAACACTGCATCAAAACAACTCTCC-FL (FL: 5,6-carboxyfluorescein attached to 3'-O-ribose) were used. For identification of W208R the sequence of the sensor probe was 5'-TGATGTGGTTCCGTACTCTCTCCTT-FL and of the anchor probe was 5'-LC 705-AAAGTCATGGCGTCTGAG AACCCTAAG-ph. Both sensor probes were complementary to the mutant sequences (129K/131K and 208R, respectively). During melting curve analysis, the mutant allele forms a more stable duplex than the wild-type allele, resulting in an allele-specific melting curve (N129K/R131K: 58 vs 47°C; W208R: 65 vs 60.5°C).

In addition, we used a set of 12 different primers to genotype the various UGT1A7 polymorphisms (Table 3). These primers were designed to flank the site of polymorphism of interest at exon 1 of UGT1A7 and are based on the published nucleotide sequence (Table 3; GenBank no. U39570).

Table 3 - Primers used for PCR amplification of UGT1A7.

Full table

Cloning of UGT1A7 constructs

To examine how the selection of a primer affects the detection of UGT1A7 codon 129 and 131 polymorphisms, we designed four different constructs. (Figure 2) We used DNA isolated from homozygous UGT1A7*1/*1 and UGT1A7*3/*3 carriers as a template for our constructs. These constructs were built in such a way that they contained four different allelic versions of the UGT1A7*1 or *3 allele with or without a mutation at position -57 of the UGT1A7 gene (Figure 2).

To clone constructs 1 (representing UGT1A7*1) and 2 (representing UGT1A7*3), the fragment that contained part of the UGT1A7 gene was amplified using PCR. The 50 l reaction mixture contained 200 ng of genomic DNA, 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% TRITON, 2 mM MgCl₂, 0.25 mM dNTPs, 10 pmol of primers 1F and 2R (Table 3), and 3.0 U Taq-DNA-polymerase. The PCR product was subjected to electrophoresis on a 1% agarose gel and we isolated the 808 bp fragments using the QIAEXII Gel Extraction Kit (Qiagen, Hilden, Germany). The fragments were subsequently cloned into a pGEM-T Vector (Promega, Madison, WI, USA).

Constructs 1 and 2 contain two NcoI restriction sites, one located in the multiple cloning site of the vector immediately before the UGT1A7 fragment, whereas a second site is at nucleotide 97 of the UGT1A7 cDNA sequence. To prepare constructs 3 and 4 (representatives for UGT1A7*1 and UGT1A7*3 but now both with a mutation at position -57), we digested constructs 1 and 2 with NcoI. Subsequent electrophoresis on a 1% agarose gel separated a 219 bp fragment. The gel-isolated fragment of construct 1 was then cloned in NcoI digested construct 2, creating a new construct 3 (UGT1A7*1 with a G at position -57). In the same way, we cloned the fragment of construct 2 in NcoI digested construct 1, thus creating construct 4 (UGT1A7*3 with a T at position -57). (Figure 2) All constructs were sequenced to verify the presence of the mutations.

To mimic PCR conditions of human genomic DNA, the 25 l reaction mixture contained 100 ng of mouse genomic DNA spiked with eight combinations of 10⁵ copies of 2 constructs representing two alleles, 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% TRITON, 2 mM MgCl₂, 0.25 mM dNTPs, 0.2 M of 14F or 15F, 0.2 M of primer 2R, 100 nM of sensor probe A, 100 nM of anchor probe A, and 1.5 U AmpliTaq Gold. The reaction mix was denatured at 95°C for 3 min followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 52 or 57°C for 30 s, elongation at 72°C for 60 s and a final extension step for 2 min at 72°C. The program for analytical melting was 95°C for 60 s, 35°C for 40 s and an increase to 75°C at a 1°C/10 s ramp rate and was analyzed using the i-Cycler (Biorad, Laboratories BV, Veenendaal, The Netherlands).

Notes

Duality of Interest

The authors declare no duality of interest.

References

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Acknowledgements

We thank Volker Keim (Leipzig, Germany) for providing DNA samples and genotyping data of N34S SPINK1 chronic pancreatitis patients. We also thank Mrs Christiane Jechorek (Magdeburg) and Claudia Güldner (Berlin) for their excellent technical assistance. Dr Joost PH Drenth is supported by an NWO-VIDI grant. This study was partly supported by the Deutsche Forschungsgemeinschaft (Wi 2036/1-1 and Wi 2036/2-1), by grants from the Dutch Foundation of Digestive Diseases (MLDS WS00-21) and Sonnenfeld-Stiftung, Berlin, Germany (Witt), and by IGA MZCR 00000064203/6112 to MM