Twenty-five exonic SNPs were revealed in the American population, 16 of which are newly-identified as compared to the NCBI database. In the Ashkenazi Jewish population, the SNP in position 3435T was significantly more common than in the American population (χ2 is 39.927 p<0.001). The estimated frequency of the most common haplotype, 1236T/2677T/3435T, is 23.6%.
Analysis of the locations of the SNPs demonstrates that all non-synonymous SNPs occur in coding regions predicted to be intracellular, and moreover, none of them appear at the ATP binding sites. Nonetheless, several non-synonymous SNP sites could potentially affect either the nearby ATP sites, or the function of the ABCB
1 protein as a whole. The intracellular loops of the ABCB
1 sequence are less conserved (20%) than the extracellular loops (40%) among various species, which is consistent with the higher probability of SNPs in the intracellular loops (http://genome.ucsc.edu
To date, the most commonly reported polymorphism linked to different responses of patients to various ABCB
1 substrates is located at exon 26, 3435C>T, and does not result in an amino acid change [30
]. The 1236C>T and 2677G>T/A/C polymorphisms have also been linked to several diseases such as pharmacoresistant epilepsy and Parkinsons disease [31
]. Studies on these polymorphisms have yielded contradictory results, possibly due to small sample sizes and the isolated nature of the study populations. Also, some studies did not include a control group for comparison with cancer patients. Several researchers performed an in-depth analysis of the polymorphisms along the ABCB
1 gene, while others researched specific SNPs without investigating the existence of other polymorphic locations. The contradictory results may indicate that SNPs of the ABCB
1 gene should be analyzed according to complete haplotypes instead of individually. Several different studies have found the 3435C>T polymorphism to be associated with a change in the expression of P-gp [30
], although in transfection studies of cells, no differences in either mRNA or protein levels are observed [47
Ostrovsky and colleagues found a significantly different frequency of the T allele at site 3435 of the ABCB
1 gene among Near Eastern Jews (from Iraq, Iran, and Buchara) when compared with other Jewish populations (Ashkenazi, Yemenite and North-African) [23
]. Our results for the homozygous polymorphism at this site (38.6%) are more comparable to those found in Near Eastern Jews (31%) than the Ashkenazi Jews (12%) in that study. A comparison between the allele and genotype frequencies of the Ashkenazi population and other ethnic groups from previous studies is summarized in . In each of the three SNP locations, the Ashkenazi population is comparable in its allele and genotype frequencies to that of Caucasian populations. According to studies submitted to the National Center of Bioinformatics, the incidence of the 3435T allele in the studied American population is similar to African American populations (24.2% and 16% respectively). Studies on the 3435C>T SNP show a correlation between allele frequency and risk of cancer development, as well as various responses to drug treatments. While no association has been observed between the TT genotype and the lung cancer phenotype [9
], homozygous and heterozygous carriers of the T allele have been linked to a greater risk of developing nonclear cell renal cell carcinoma than individuals carrying the C allele [49
]. Carriers of the TT genotype are more at risk of developing Acute Lymphoblastic Leukemia (ALL) than other individuals, whereas the CC genotype carriers exhibit a different prognosis [50
The American and Ashkenazi populations both expressed similar percentages of the 1236C>T and 2677G>T/A/C SNPs in the ABCB
1 gene. However, the percentage of individuals expressing the 3435C>T polymorphism in the Ashkenazi population was significantly higher when compared to the American population (χ2
is 39.927 p<0.001). The high prevalence of this SNP in the Ashkenazi population may reflect a founder effect that preceded the large expansion of the Ashkenazi Jews between the 16th
to the 19th
centuries. Later on, the polymorphism was fixed in this population due to its rapid expansion and not necessarily because of a selective advantage of carrying this SNP. Of note, the high prevalence of BRCA
1 185delAG and APC 11307K in Ashkenazi Jews was similarly explained by a founder effect occurring between 947 and 195 BC rather than by selective advantage [51
An increased risk for the development of colorectal cancer has also been found in carriers of the 3435T allele under the age of fifty. Carriers of the ABCB
1 3435TT genotype had a 2.7-fold greater risk of the development of colorectal cancer [32
]. This SNP may therefore be linked to a high prevalence of colon cancer in the Ashkenazi population. Similarly, refractory Crohn’s disease has also been found to be associated with the 2677G>T/A/C, 3435C>T, and 1236C>T SNPs [31
]. This could also be linked to a high prevalence of the disease in the Ashkenazi Jewish population, as noted by the high prevalence of the 3435T allele discovered in our Ashkenazi population. The mutation ΔF508 in Cystic fibrosis also has a high incidence in the Ashkenazi population [52
]. This mutation in another ATP transporter was associated with protection against cholera and typhoid fever [53
]. However, it has yet to be determined if the high prevalence of the 3435C>T SNP has a heterozygote advantage in these diseases.
In summary, this study has investigated differences in the number and location of single nucleotide polymorphisms along the ABCB1 gene between an ethnically diverse American population and an ethnically homogeneous Ashkenazi Jewish population. Using sequencing methods as well as PCR-RFLP, polymorphisms were located at three primary locations along the ABCB1 gene (1236C>T, 2677G>T/A/C, and 3435C>T). The PCR-RFLP method proved to be a very accurate and reliable method for identifying specific SNPs at a lower cost then genomic sequencing. Results of this analysis showed that the 3435C>T polymorphism plays a significant role in the determination of haplotypes of the ABCB1 gene. However, due to the prevalence of the 1236T/2677T/3435T haplotype in the Ashkenazi population in individuals that possess the 3435C>T SNP, it may be beneficial to also search for this particular haplotype.
The overall frequency of SNPs is consistent with current understanding of the prevalence of SNPs in the genome. Notably, a comparative analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous mouse and rat genes has shown that on average, non-synonymous exonic changes between these two species were 3-fold less common compared with intronic inter-species changes [28
]. Thus, we can conclude that the higher degree of conservation of the ABCB
1 gene may indeed reflect the function of the ABCB
1 protein as a poly-specific detoxifying transporter of foreign compounds and its need to adapt to new challenges.