Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo

Abstract

To evaluate whether alterations in the multidrug-resistance (MDR)-1 gene correlate with intestinal MDR-1 expression and uptake of orally administered P-glycoprotein (PGP) substrates, we analyzed the MDR-1 sequence in 21 volunteers whose PGP expression and function in the duodenum had been determined by Western blots and quantitative immunohistology (n = 21) or by plasma concentrations after orally administered digoxin (n = 8 + 14). We observed a significant correlation of a polymorphism in exon 26 (C3435T) of MDR-1 with expression levels and function of MDR-1. Individuals homozygous for this polymorphism had significantly lower duodenal MDR-1 expression and the highest digoxin plasma levels. Homozygosity for this variant was observed in 24% of our sample population (n = 188). This polymorphism is expected to affect the absorption and tissue concentrations of numerous other substrates of MDR-1.

Figures

Figure 1

Polymorphisms in the human MDR-1 gene. The positions of PCR fragments that we analyzed in our sequencing screen and the locations of the identified polymorphisms (arrows) are indicated in relation to the exon–intron structure of the human MDR-1 gene and the corresponding domain composition of PGP. *, SNP just before the translation start codon. White arrows depict noncoding polymorphisms, two of which are linked (broken line). Black arrows show the protein polymorphisms in exons 2, 5, and 11.

Figure 2

Detection of MDR-1 polymorphisms. Examples for the identification of homozygous and heterozygous polymorphisms by sequence analysis (A) and by allele-specific PCR (B). Shown is the polymorphism in exon 2 that directly precedes the ATG start codon at cDNA pos.-1. MDR-1 sequence deviations can be detected directly in the DNA sequence profiles. A shows a wild-type sequence (Left) and (Right) the sequence of a fragment with a heterozygous polymorphism. (B) Allele-specific PCR (agarose gel of the PCR fragments) defines the MDR-1 genotype, i.e., the presence and/or absence of alleles. PCR was performed with human genomic DNA as with allele-specific primer combinations 5′-ggtttctcttcaggtcgg-G3′(wild type) or 5′-ggtttctcttcaggtcgg-A3′(mut), and 5′-ctcagccaacaaacttctgc-3′ (reverse) and annealing temperatures of 54°C for both reactions. The wild-type sample shows exclusively a fragment with the wild-type–primer combination; the heterozygous sample is positive for the wild-type primer combination as well as the primer combination that specifically detects the polymorphism.

Figure 3

Correlation of the exon 26 SNP with MDR-1 expression. The MDR-phenotype (expression and activity) of 21 volunteers and patients was determined by Western blot analyses. The box plot shows the distribution of MDR-1 expression clustered according to the MDR-1 genotype at the relevant exon 26 SNP. The genotype–phenotype correlation has a significance of P = 0.056 (n = 21).

Figure 4

MDR-1 expression and PGP in vivo activity after rifampin induction. MDR-1 genotype in exon 26 and distribution of rifampin-induced PGP protein expression (A) in the duodenum. (B) Distribution of plasma levels of digoxin after rifampin induction. The plasma levels of digoxin area under the curve, μg × h × L−1 (AUC) are inversely proportional to PGP activity in the duodenum (9). The correlation of MDR genotype and PGP activity has a significance of P = 0.053 (n = 8).

Figure 5

Correlation of MDR-1 genotype and digoxin uptake in vivo. The MDR-1 genotype in exon 26 was analyzed in 14 healthy volunteers who participated in a clinical study that addresses plasma levels of digoxin during steady-state conditions (35). A statistically significant difference (P = 0.006; Mann–Whitney U two-sample test) was found in the comparison of maximum concentrations (Cmax) of digoxin between two groups of seven healthy volunteers harboring either T/T or C/C genotype. The mean difference of 38% in Cmax may reflect the importance of genotype on the absorption of digoxin after oral application. A 0.25 mg dose was applied on steady-state of digoxin.