Influence of SLCO1B3 haplotype-tag SNPs on docetaxel disposition in Chinese nasopharyngeal cancer patients

Abstract

What is already known about this subject: SLCO1B3 is an influx transporter located at the hepatocyte basolateral membrane and it is involved in the uptake of a broad range of drug substrates including docetaxel. The pharmacogenetics of SLCO1B3 is not well characterized and previous in vivo and in vitro studies reported conflicting results with regards to the functional effects of the limited number of SLCO1B3 polymorphisms that were studied. Docetaxel displays a wide interindividual variability in its pharmacokinetics and pharmacodynamics and an understanding of SLCO1B3 pharmacogenetics might provide clinical benefits in guiding docetaxel dosing.

What this study adds: The SLCO1B3 gene was comprehensively screened in the local healthy Asian populations (n= 168). A strong linkage disequilibrium pattern was detected across a total of 88 polymorphisms and 15 haplotype-tag SNPs (htSNPs) were identified. These htSNPs were profiled in a cohort of Chinese nasopharyngeal cancer (NPC) patients (n= 50). Genotypic-phenotypic analysis showed that a haplotypic construct comprising of four variants [IVS4+76G>A, 699G>A(Met233Ile), IVS12-5676A>G, and *347_*348insA] was the critical determinant of docetaxel disposition. This study suggests that the comprehensive screening and haplotypic linkage analysis of SLCO1B3 can better elucidate its pharmacogenetic effects on interpatient variability of docetaxel and other putative drug substrates. Further studies are warranted in cancer patients belonging to other ethnic groups. AIMS To completely screen the SLCO1B3 gene in three distinct healthy Asian populations (Chinese, Malay and Indian, n= 168) and investigate the influence of haplotype-tag SNPs (htSNPs) on docetaxel disposition in 50 nasopharyngeal carcinoma patients.

Methods: Genomic DNA of individuals was screened for SLCO1B3 polymorphisms by direct sequencing. htSNPs were derived based on the sequence clustering algorithm and profiled in the patients. Population based genetic association analysis was performed using Haplostats package implemented in R and PLINK.

Results: A strong linkage disequilibrium pattern was detected across a total of 88 polymorphisms and 15-htSNPs were identified. The SLCO1B3 haplotypic region comprising seven htSNPs was found to be significantly associated with docetaxel clearance (P= 0.003). Conditional haplotype analyses revealed that the haplotypic constructs comprising the IVS4+76G>A, 699G>A(Met233Ile), IVS12-5676A>G, and *347_*348insA polymorphisms were critical determinants of variability in docetaxel disposition [clearance and area under the plasma concentration-time curve (AUC(0,∞)): r(2) = 29% and 22%, respectively]. Patients harbouring the GAG*347insA haplotype were significantly associated with a 30% decrease in clearance and a 40% increase in AUC(0,∞) of docetaxel compared with patients harbouring the reference haplotype, GGA*347wt (P= 0.025 and 0.018, respectively). In contrast, a 50% higher clearance was observed in patients carrying the GAG*347wt haplotype compared with those with the reference haplotype (P= 0.002). The functional SLCO1B3 haplotypic constructs included the widely studied Met233Ile variant and *347_*348insA located in the putative miR-890 binding site in the 3'-untranslated region which may influence the transport characteristics of SLCO1B3.

Conclusions: This study highlights the importance of SLCO1B3 polymorphic variations in influencing docetaxel disposition in nasopharyngeal carcinoma patients.

Figure 1

Graphical representation of identified polymorphisms in the SLCO1B3 gene. The locations of the identified polymorphisms (arrows) are indicated in relation to the promoter, exon and intron structures of SLCO1B3 (UCSC accession number NM_019844). The distal 5′UTR contains a non-coding exon 1. Exonic SNPs are labelled relative to the ATG translation initiation site in exon 2, with the A numbered as 1. The first nucleotide 5′ to the ATG site is −1, and the first nucleotide 3′ to the translation stop codon is *1. Intronic SNPs are labelled with the respective intronic number, followed by a + (downstream) or – (upstream) sign and the position in the intron, calculated from the nearest exon. Polymorphisms highlighted in red are the htSNPs selected and profiled in the patient cohort. The domain sizes are not drawn to scale

Figure 2

Linkage disequilibrium plot of SLCO1B3 polymorphisms in healthy (A) Chinese, (B) Malay and (C) Indian populations. LD values are denoted as (|D'|× 100). LD analyses, haplotype analyses and LD block construction were carried out by Haploview software v.3.32 (Daly lab, Broad Institute, MA, USA). The standard colour scheme in Haploview was chosen for LD display. Block 1 represented 5′ upstream and proximal UTR SNPs in all populations. Blocks 2 and 3 in the Chinese and Malay populations consisted of SNPs located between intron 2 and exon 14. The region represented by haplotype block 2 was the largest and comprised of 42, 41 and 33 exonic/intronic polymorphisms in the Chinese, Malay and Indian populations, respectively. Block 4 in the Chinese and Malay populations and block 3 in the Indian population consisted of SNPs from the 3′ UTR and downstream regions. The inter-block linkage was strong between blocks 2 and 3 (|D'| = 0.82–0.97), but moderate between blocks 1 and block 2 (|D'| = 0.55–0.78)

Figure 2

Linkage disequilibrium plot of SLCO1B3 polymorphisms in healthy (A) Chinese, (B) Malay and (C) Indian populations. LD values are denoted as (|D'|× 100). LD analyses, haplotype analyses and LD block construction were carried out by Haploview software v.3.32 (Daly lab, Broad Institute, MA, USA). The standard colour scheme in Haploview was chosen for LD display. Block 1 represented 5′ upstream and proximal UTR SNPs in all populations. Blocks 2 and 3 in the Chinese and Malay populations consisted of SNPs located between intron 2 and exon 14. The region represented by haplotype block 2 was the largest and comprised of 42, 41 and 33 exonic/intronic polymorphisms in the Chinese, Malay and Indian populations, respectively. Block 4 in the Chinese and Malay populations and block 3 in the Indian population consisted of SNPs from the 3′ UTR and downstream regions. The inter-block linkage was strong between blocks 2 and 3 (|D'| = 0.82–0.97), but moderate between blocks 1 and block 2 (|D'| = 0.55–0.78)

Figure 3

SLCO1B3 haplotypes in healthy (A) Chinese, (B) Malay and (C) Indian populations. Haplotype analyses and LD block construction were carried out by Haploview software v.3.32 (Daly lab, Broad Institute, MA, USA).The shaded box represents the presence of variant. C, Chinese; M, Malays; I, Indians; B, Block; H, Haplotype. The haplotype name is designated with block and haplotype number as suffix, e.g. C_B1 represents haplotype block 1 identified in the Chinese subjects. ¹ATATTCACTTGGTATCTG deletion at location −28 to −11. ²Denotes refererence haplotype

Figure 4

Effects of GAG*347insA and GAG*347wt in comparison with the reference haplotype (GGA*347wt) on docetaxel (A) CL and (B) AUC(0,∞). Patients carrying the GAG*347insA haplotype had significantly lower CL and higher AUC(0,∞) compared with those with the reference haplotype. In contrast, patients harbouring the GAG*347wt haplotype had significantly higher CL compared with those with the reference haplotype. Each bar represents the mean ± SE of the docetaxel pharmacokinetic parameters. P values were obtained from the haplotype specific generalized linear model implemented in the R haplostats package. AUC(0, ∞), area under plasma concentration−time curve from time zero to infinity; CL, plasma clearance; NS, non-significant