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SLCO1B1 Sequencing Study Suggests PGx Genotyping May Not ID Enough Rare Variants


By Molika Ashford

Rare variants, identified through deep exon sequencing of the SLCO1B1 gene, significantly contribute to variation in response to the drug methotrexate, a study by researchers at St. Jude Children's Research Hospital has shown.

The discovery of these rare variants' influence on methotrexate clearance suggests that future pharmacogenomics studies may require analysis beyond genotyping for common variants to avoid passing over subjects with less-common mutations associated with treatment response.

To provide personalized treatment for the greatest number of patients, sequencing may be required to deeply interrogate specific genomic regions to find influential rare SNPs, according to researchers led by Mary Relling, chair of the department of pharmaceutical sciences at St. Jude.

"As genetic testing moves from research to clinical implementation, it is important to define the relative contribution of common versus rare genetic variants in influencing phenotypic variation … If rare variants must be interrogated, clinical genetic testing will likely need to become 100 percent sequence-based to be adequate," the researchers wrote in a paper describing the study that was published online last week in Genome Research.

Methotrexate is an antimetabolite drug used to treat autoimmune disease and cancers, and is used widely to treat newly diagnosed acute lymphoblastic leukemia. Variation in clearance of the drug can influence its clinical effectiveness and toxicity. Relling's group has been studying the genomic underpinnings of methotrexate variation to better understand instances of poor response in children with ALL.

Previously, through a genome-wide association study conducted as part of the group's Pharmacogenomics of Anticancer Agents Research in Children project, Relling and colleagues found a number of common polymorphisms in the SLCO1B1gene associated with varying levels of methotrexate clearance. That effort, led by Relling, is part of the Pharmacogenomics Research Network's Clinical Pharmacogenetics Implementation Consortium and aims to define the pharmacogenomics of childhood acute lymphoblastic leukemia.

In their subsequent exon sequencing study, Relling and colleagues discovered several rare variants and quantified their relative contribution to variation in methotrexate clearance. She told PGx Reporter that this latest analysis is novel since there have been relatively few studies to date comparing the quantitative importance of rare versus common variants for a pharmacogenomic trait.

After studying close to 700 children, the researchers found that approximately 20 percent in their study population had low clearance of methotrexate on the basis of common variants, while 2.1 percent had low clearance as a result of rare variants. "If we took a strategy of only genotyping for common variants, you would miss the low-clearance phenotype in 2 percent of the population. Or, the other way to look at it is … about 18 percent of the variation in [methotrexate] clearance that is attributable to variation in SLCO1B1 is [due to] rare variants," Relling said.

The patient population, children with leukemia who had all been studied for their methotrexate disposition in the context of a St. Jude clinical trial, had a "very clean pharmacogenetic phenotype," according to Relling, and presented a good opportunity to explore the relative influence of rare and common variants in a way the study authors claim had not been sufficiently explored before.

Using pharmacokinetic modeling in the original GWAS to measure how the patient's bodies cleared methotrexate, the group created established phenotypes for each patient. With these in hand, they performed Sanger sequencing of SLCO1B1 exons in a total of 699 children in the new study, 640 of whom had been part of the original GWAS, and another 59 who had not.

The group sequenced exons 10-15 in the SLCO1B1 gene in all 699 patients, and then used a strategy of sequencing only the patients with the highest and lowest levels of methotrexate clearance for exons 1-9

The group found 39 SNPs in exons 10-15 and 21 in exons 1-9, and genotyped patients for all of these SNPs along with other previously published, non-synonymous polymorphisms, the authors reported.

Of the full 93 SNPs genotyped, 15 were non-synonymous. Three of these were common, with a minor allele frequency greater than five percent. One had low frequency, while another 11 were considered rare variants, with a frequency less than one percent. Overall, 2.1 percent of the children had one rare allele associated with reduced methotrexate clearance, which the study authors characterized as a "small but non-negligible percentage of patients."

After further analysis, the group concluded that the range of variants in SLCO1B1 accounted for 10.7 percent of all variability in methotrexate clearance. Of this portion, rare damaging variants made up about 18 percent. Rare variants also had larger effect sizes than common ones, the authors reported.

"What this means in a bigger sense," Relling said, "is that I think that as we move into clinical genotyping, to really identify all of the important variants that might be present in a patient population … you would have to sequence the gene in order to find all the important variants."

For example, Relling and her colleagues found one variant in exon_3_602 that was only observed one time in 1,400 chromosomes. The variant "conferred low clearance in that patient and when we tested the variant in the in vitro functional system, it conferred low function for the transporter in cell lines," she said.

"We feel like if we sequenced another 700 patients, we would find another couple of rare variants and in another 700 another one or two." Although there are some genes where genotyping will be sufficient, in other areas of the genome without sequencing, one could miss unanticipated rare non-functional or hypo-functional variants.

In the clinical setting, Relling noted that the SLCO1B1 gene is now becoming a target for genotyping, though not in the context of methotrexate in children, but because of a similar association with statin drugs. Vanderbilt University, which is also a member of the CPIC, is planning to begin screening patients prescribed simvastatin, the generic form of the statin Zocor, for alterations in SLCO1B1 that confer increased risk for muscle damage side effects (PGx Reporter 10/19/2011).

However, Relling said that it might be necessary for programs like the one at Vanderbilt to eventually switch to sequencing in order to identify rare variants. "If it's true that statins are similarly affected as methotrexate [is] by these low-function rare variants then the same principle would hold true," she said.

Of course, the question of how sequencing might be implemented on a widespread clinical basis is still an open one, she said.

In the meantime, Relling's group is planning to do some additional functional studies on SLCO1B1 and methotrexate, but more importantly, they plan to extend their deeper sequencing approach to other pharmacogenomically promising drug-gene combinations.

In the study, the group wrote they consider the project a "proof-of-principle for follow-up of a gene hit resulting from a GWAS in pharmacogenomics," with findings that "support the hypothesis that a combination of common and rare variants is likely to be important for pharmacogenetic phenotypes."

"We have really prioritized a few genes as very important for pharmacogenomics, and we are going to push for the idea that we will have to sequence these genes if we want to comprehensively interrogate them to find inactivating variants," said Relling.

"We'll probably start with methods based on genotyping, but we'll have to be willing to expand to include sequencing of clinically relevant pharmacogenetic genes in the near future," she said.

The CPIC has posted on the Pharmacogenomics Knowledge Base website a list of the genes and drugs it considers most promising. "Those are our top priority genes for clinical implementation, so they'll also be top priority to applying sequencing methods to eventually," she said.

Have topics you'd like to see covered in Pharmacogenomics Reporter? Contact the editor at mashford [at] genomeweb [.] com.

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