Pearson et al.SLC22A1 polymorphisms and metformin response

METHODS

The Genetics of Diabetes Audit and Research Tayside Scotland (Go-DARTS) resource

Patients were identified from an ongoing study of the Go-DARTS recruited in Tayside between 1st October 1997 and 1 February 2007 (n=6500) who could be linked to theprescribing data held in the Health Informatics Centre. The DARTS and linked datasets include validated prescribing, biochemistry and phenotypic historical data from 1992 to present, and prospective longitudinal data is collected on each person with type 2 diabetes recruited into the genetic study.The study was approved by the Tayside Medical Ethics Committee and informed consent was obtained from all subjects.

For this study, patients were selected to have type 2 diabetes on the basis of an age of diagnosis after the age of 30, with no progression to insulin within 6 months of diagnosis. Patients were excluded who were diagnosed with diabetes after 90 years of age. Prescription data were available between January 1992 and January 2007.

Determination of drug adherence and dose

We used population-based drug dispensing records to calculate the % maximum possible adherence for each patient (1). The average dose was determined as the mean dose of the prescriptions encashed between the index prescription and the treatment HbA1c.

Determination of Body Mass Index (BMI) and creatinine clearance

The BMI was taken as the mean of the BMI measures recorded 2 years either side of the index date. The creatinine clearance was calculated using the Cockcroft-Gault equation (2) using the mean of the creatinine and weight measurements recorded two years either side of the index date. Age at treatment initiation and sex were used to derive the creatinine clearance and therefore were not used as separate covariates.

Genotyping

We genotyped R61C (rs12208357) and 420del (no rs number available) of SLC22A1 by Taqman-based allelic discrimination using “Assays by Design” from Applied Biosystems which contained the following primer and probe sequences:

420del Forward primer: GCGTGGGCCGCATCTA

420 del reverse primer: CAACTTACCAACTTACCAGGTGAGA

420del probe 1(ATG+): VIC-CTCGTCATGATTTTTA-NFQ

420del probe 2(ATG-): FAM-CCTCGTCATTTTTA-NFQ

R61C Forward primer: CTGACCACCACTGCCAGAG

R61C Reverse primer CTCCGCAGGGCTCCAG

R61C Probe 1: VIC-CACAGCGCTGGCTC-NFQ

R61C Probe 2: FAM-CACAGCACTGGCTC-NFQ.

Assays were performed under standard conditions for Taqman-based assays as described by manufacturer (Applied Biosystems). Assays were performed on 10ng genomic DNA in 384 well plates; cycled using a H2OBIT thermal cycler (Thermo Scientific, Surrey); fluorescence detection and genotype calling were performed on an ABI 7900FastHT sequence detection system (Applied Biosystems).

Genotyping success rate for each SNP was ~97% and duplicate genotyping concordance was >99%.

REFERENCES

1. Donnan PT, MacDonald TM, Morris AD: Adherence to prescribed oral hypoglycaemic medication in a population of patients with Type 2 diabetes: a retrospective cohort study. Diabet Med 19:279-284, 2002

2. Cockcroft DW, Gault MH: Prediction of creatinine clearance from serum creatinine. Nephron 16:31-41, 1976

Supplementary Table A1.Linear regression of average HbA1c reduction on OCT1 variants

Del420 / R61C
Coef / p-value / Coef / p-value
Genotype / 0.071 / 0.122 / 0.039 / 0.582
Baseline HbA1c / 0.651 / <0.0001 / 0.651 / <0.0001
Creatinine clearance / -0.316 / <0.0001 / -0.316 / 0.025
Average dose / -0.018 / 0.008 / -0.018 / 0.582
Adherence / 0.099 / <0.0001 / 0.099 / <0.0001
Group / -0.485 / <0.0001 / -0.488 / <0.0001

Supplementary Table A2. Unbalanced repeated measurement analysis of metforminmonotherapy HbA1C between 6 and 42 months.

Del420 / R61C
Coef / p-value / Coef / p-value
Genotype / -0.113 / 0.444 / -0.032 / 0.750
Time / 0.083 / <.0001 / 0.076 / <.0001
Genotype * Time / -0.007 / 0.816 / 0.018 / 0.376
Baseline HbA1c / 0.129 / <.0001 / 0.128 / <.0001
Average dose / -0.011 / <.0001 / -0.011 / <.0001

Supplementary Table A3. Cox regression model oftime tomonotherapy failure.

Del420 / Del420
HR / 95% CI / p-value / HR / 95% CI / p-value
Genotype / 0.98 / (0.82,1.18) / 0.85 / 1.08 / (0.83,1.43) / 0.56
Baseline / 1.27 / (1.19,1.35) / <0.0001 / 1.27 / (1.19,1.34) / <0.0001
Adherence / 0.94 / (0.88,1.00) / 0.03 / 0.94 / (0.88,1.00) / 0.03
Creatinine Clearance / 1.004 / (1.002,1.01) / 0.002 / 1.004 / (1.002,1.01) / 0.002

Supplementary Table A4. Cox regression model of time to the composite event of either reaching 2g/day metformin or monotherapy failure.

420Del / R61C
HR / 95% CI / p-value / HR / 95% CI / p-value
Genotype / 0.96 / (0.82,1.13) / 0.65 / 1.1 / (0.86,1.41) / 0.45
Baseline / 1.23 / (1.16,1.30) / <0.0001 / 1.23 / (1.16,1.30) / <0.0001
Adherence / 0.94 / (0.89,1.00) / 0.03 / 0.94 / (0.89,1.00) / 0.03
Creatinine Clearance / 1.005 / (1.003,1.01) / <0.0001 / 1.005 / (1.003,1.01) / <0.0001