MDR1 ABCB1 Gene ID 5243 as a primary target locus

Officially known as ATP-binding cassette, sub-family B member 1 (ABCB1), MDR1 (multidrug resistance protein 1) encodes a P-glycoprotein (GP170) that has been studied extensively for its critical role as a drug transporter. Up-regulation of MDR1 alters the kinetics of many therapeutic agents being used for treating patients with cancer (Rund et al., 1999) and HIV-1 infection (Fellay et al., 2002). In both disease systems, enhanced efflux of intra-cellular drugs have been associated with germline and somatic mutations at the MDR1 locus. Uneven distribution of certain MDR1 variants in human ethnic groups (Rund et al., 1999, Schaeffeler et al., 2001) may be further responsible for the racial disparity in response to certain therapy. In contrast, the influence of MDR1 promoter methylation on MDR1 function is not so clear.

Table 10.2 Summary of qPCR-based tests of three MDR1 genomic regions (nucleotide reference positions are based on GenBank sequence X58723).

Specifics

Promoter fragment 1 (MDR1-PCR 1)

Promoter fragment 2 (MDR1-PCR 2)

Intron 1 fragment (MDR1-PCR 3)

Forward primer positions

nt. 768 > 790

nt. 888 > 907

nt. 1316 > 1336

Reverse primer positions

nt. 888 < 907

nt. 1178 < 1193

nt. 1579 < 1598

Amplicon size

140 bp

308 bp

283 bp

GC content

43%

55%

66%

PCR annealing

60°C

60°C

60°C

temperature

Amplicon melting

80.5°C

88.5°C

90.0°C

temperature

CpG sites within

14

3

22

amplicon

EcoR 1 site (GAATTC)

0

0

0

BstU 1 site (CGCG)

1

0

6

qPCR standard curve a 1

log10Y = 7.75-0.29X

log10Y = 7.81-0.27X

log10Y = 7.60-0.26X

r2 for curve 1

0.992

0.994

0.994

qPCR standard curve a 2

log10Y = 7.85-0.30X

log10Y = 7.56-0.26X

log10Y = 7.89-0.28X

r2 for curve 2

0.998

0.990

0.998

a For all standard curves, Y is the input DNA copy number number determined automatically by the iCycler® (BioRad).

in each reaction, while X

is the threshold cycle (Ct)

Methylation at various CpG sites within a 1000-bp region (including MDR1 exon 1 to exon 2) appears to regulate MDR1 gene expression (Nakayama et al., 1998, Kusaba et al., 1999). Our ongoing studies of several HIV/AIDS cohorts deal with both genetic and epigenetic characteristics of the MDR1 gene, primarily because MDR1 is important to the metabolism of HIV-1 protease inhibitors (Kim et al., 1998). The work described here aims to define CpG methylation in the MDR1 promoter and intron 1 sequences.

Was this article helpful?

0 0

Post a comment