Our results demonstrated wide inter-individual variability of trough IM plasma levels in Asian patients with CML, confirming previous reports on Caucasian 
, Korean 
, Japanese 
, Chinese 
and other ethnic cohorts 
. In addition, we not only observed that the mean IM trough concentration in our cohort of Asian patients with CML was significantly higher than that previously reported in studies on Caucasians 
, 31 of 34 patients (94%) who received standard IM doses of 400 mg in fact had an IM trough concentration above the recommended threshold of >1000 ng/mL. This result is consistent with studies on the Chinese 
, Korean 
and Jordanian 
populations. These differences in IM levels may be attributed to several factors including poor medication compliance 
, intake of other drugs 
, gender as well as physical variables 
. In our study however, there was no evidence that any of the above factors could have accounted for the large inter-patient and inter-ethnic variability observed. Instead, the data indicated that the underlying reason may invoke intrinsic differences in cellular influx (e.g.
hOCT1) and efflux (e.g.
ABCB1) transporters 
. hOCT1 is known to mediate the active transport of IM into primary CML cells 
, and is likely to be a major determinant of hepatocyte uptake for systemic clearance 
. Low hOCT1 or high ABCB1 activity/expression in leukemic cells may result in reduced influx or increased efflux of drugs respectively, thereby reducing intracellular levels whilst increasing plasma levels. Simultaneously, the same effect on hepatocytes may impair drug uptake into the liver, leading to elevated plasma levels. Whilst it may be plausible that since some patients with CML achieve supra-therapeutic IM levels on standard 400 mg doses and may be considered for de-escalated regimens without aggravating their outcome 
, such observations may also reflect compromised cellular transport mechanisms resulting in high plasma levels and detrimentally-low intracellular levels.
In this exploratory study, we screened the entire SLC22A1
gene for polymorphic variation in three healthy Asian ethnic populations and further examined tag-SNPs in Asian CML patients. Our approach revealed a sub-haplotypic region encompassing 1 exonic SNP [1222A>G (rs628031)] surrounded by 2 intronic SNPs [IVS6-878C>A (rs3798168) and IVS7+850C>T] that is significantly associated with IM pharmacokinetics. The SLC22A1
gene was found to be highly polymorphic and displayed significant interethnic variations amongst healthy individuals of Chinese, Malay, and Indian ethnicity (Table S1
, see footnotes). For example, the allelic frequency of the 5′-upstream polymorphism [−1795G>A (rs6935207)], was significantly higher amongst Chinese (0.57) as compared to Malays (0.39), Indians (0.30) and Caucasians (0.21) 
. Interestingly, the majority of previously studied SLC22A1
coding SNPs in Caucasians 
were absent in the Asian ethnic groups examined, and only 1222A>G (rs628031) was found in common amongst Caucasian, Japanese 
and the current Asian cohort. Similarly, polymorphisms 181C>T (rs12208357) and 1260_1262delGAT (rs72552763) which have been linked to reduced transport activity of hOCT1 substrates in European-American groups 
, were not observed in Asians.
Amongst our cohort of Asian patients with CML, we were able to identify two groups based on unique haplotype profiles - one composed of those harboring 0 or 1 copy (Slow
) and the other carrying 2 copies of AGT or CGC haplotypes (Shigh
). Patients harboring Shigh
had 50% higher IM trough level and 33.4% lower clearance than patients with Slow
. The haplotype CGC was represented by the presence of the wild type allele of intronic polymorphisms IVS6-878C>A (rs3798168) and IVS7+850C>T, as well as the variant allele of the non-synonymous SNP 1222A>G (rs628031). On the other hand, the haplotype AGT was represented by the presence of the variant allele of all three polymorphisms. To the best of our knowledge, the intronic polymorphisms IVS6-878C>A (rs3798168) and IVS7+850C>T were not studied with phenotypic data previously. Although IM is a substrate of hOCT1 
, it has been shown in previous single marker studies that 1222A>G (rs628031) does not alter the function of the hOCT1 protein 
and does not affect response to IM therapy in CML patients 
. Our positive results from haplotype analysis is unsurprising, given that such an approach is known to be more robust than single marker analysis for identifying genomic regions enriched for phenotype-relevant casual variants 
. In the present study, a moderate to strong linkage pattern was observed amidst SLC22A1
variants in different ethnic groups. Particularly, strong linkage (|D′| >0.90) was detected amongst these three key variants in the Chinese population, which formed 84.2% of our patient cohort. Hence, the linkage effect of the region may have been the confounding factor in previous studies. The sub-haplotypic region is composed of two intronic and one coding SNP which suggests that the influence of haplotypes on IM pharmacokinetics could be mediated through interactions involving post-transcriptional modification. Taken together, out results highlight the importance of haplotype over single marker analysis to explain the variability in IM disposition and warrant further investigations in other ethnic groups.
Interestingly, we found that ABCB1
haplotypes may exert an indirect modulatory effect upon IM pharmacokinetics, depending on the specific SLC22A1
haplotype, with a trend towards high IM trough levels being observed from Slow
(). Previous studies focusing only on genotype rather than haplotype profiles obtained heterogeneous results, with Gurney et al.
reporting a lower IM clearance in individuals with the ABCB1
1236CC, 2677GG or 3435CC genotypes 
, Yamakawa et al.
demonstrating higher clearance in Japanese patients with the 3435CC genotype 
, and others reporting a lack of any association 
. In agreement with our finding though, Dulucq et al.
showed that both ABCB1
haplotype TTT as well as genotype 1236TT were correlated with high IM trough levels 
. These inconsistencies may perhaps be explained by a functional dependency of ABCB1
. In support of this thinking, it was noted in our study that patients with the SLC22A1
tended to possess the ABCB1
, whilst a previous microarray analysis on a panel of leukemic cell lines revealed that SLC22A1
was inversely related with gene expression of ABCB1
. This implies that specific ABCB1
haplotype profiles may affect IM levels by affecting their gene expression.
In conclusion, the present exploratory study comprehensively screened for SLC22A1 genetic variations and investigated its linkage and haplotype pattern in three distinct Asian populations, followed by its association with IM pharmacokinetics in CML patients. It is acknowledged that there are limitations in the current study and the results should be interpreted with care. Nonetheless, our report is the first to suggest that genetic polymorphisms and specific haplotypes in SLC22A1 and ABCB1 may contribute towards inter-individual and inter-ethnic variations in IM disposition in CML patients. The validation, as well as generalizability of results awaits future investigations in larger cohorts, including in other ethnic groups. More importantly, the functional and biological characterization of the haplotype variants ought to be further investigated.