Genetic susceptibility to adverse drug effects - Epidemiology ...

ABCB1 (MDR1) gene polymorphisms are associated with neuropsychiatric adverse effects of mefloquine
between baseline and follow-up. Subjects were followed from the time of prescription until the
follow-up visit at 3-4 weeks (i.e. after 3 doses of mefloquine), but always before departure to
the travel destination. All subjects received mefloquine in a dosage of 250 mg once a week.
After consent was obtained to perform neuropsychiatric testing, report adverse events and
provide blood, all personal identification was deleted from the data. Therefore, it was not possible
to obtain further consent for this specific analysis, which was not foreseen at the time
of sampling. The medical ethical committee gave permission for the genetic reanalysis of de
de-identified data from the previously approved study.
Genotyping. Genomic DNA was isolated from whole EDTA blood by use of a total nucleic
acid extraction kit (Roche, Basel, Switzerland) on a MagnaPure LC (Roche) according to
the manufacturer’s recommendations. Polymerase chain reaction (PCR)-restriction fragment
length polymorphism (RFLP) assays were performed by use of 5 ng of genomic DNA in a PCR
volume of 25 µl containing 1x buffer (10 mmol/l Tris-HCl, pH 8.3; 1.5 mmol/l MgCl 2
; 50 mmol/l
KCl, and 0.001% [wt/vol] gelatin [Perkin-Elmer, Wellesley, Mass]), 0.2 mol/l each of the deoxynucleotide
triphosphates (Roche), 1.25 U of AmpliTaq Gold (Perkin-Elmer), and 40 pmol of
each forward and reverse primer. For the C1236T polymorphism, forward primer 5’-CCT GAC
TCA CCA CAC CAA TG-3’ and reverse primer 5’-TAT CCT GTG TCT GTG AAT TGC C-3’ were
used. PCR conditions were 7 min at 94 0 C, followed by 35 cycles of 1 min at 94 0 C, 1 min at
55 0 C, and 1 min at 72 0 C, and a final incubation of 7 min at 72 0 C. The 370 bp PCR product was
digested with HaeIII. For the G2677T polymorphism, forward primer 5’-TAG TTT GAC TCA
CCT TCC CGG-3’ and reverse primer 5’-GGC TAT AGG TTC CAG GCT TG-3’ were used (the
underlined and in bold nucleotide is a mismatch with the MDR1 sequence, creating a restriction
site in the PCR product). PCR conditions were 7 min at 94 0 C, followed by 35 cycles of 1
min at 94 0 C, 1 min at 57 0 C, 1 min at 72 0 C; and a final incubation of 7 min at 72 0 C. The 218 bp
PCR product was digested with BanI (New England Biolabs). For the C3435T polymorphism
we used primers and PCR conditions and digestion as described previously. 12 All PCR digestion
products were analyzed on agarose/Trisborate-EDTA gels with ethidium bromide.
Outcome assessment. The primary outcome was neuropsychiatric adverse effects using
a broad definition that included type I complaints such as dizziness, nausea and sleeping
problems. 1, 3 Subjects were asked to report the occurrence of adverse effects on the diary
sheet used in previous studies. 2, 4 The diary sheet contained one question on adverse effects,
with an open-ended follow-up question: ‘Did you experience any adverse event(s) while using
mefloquine? If yes what kind of adverse event(s)?’ The answer to the question was only
filled out if the subject had symptoms. Not returning the diary sheet or questionnaires was an
exclusion criterion. Two independent physician-assessors classified reported adverse events
as being neuropsychiatric or not. All classification was performed with blinding to genotype.
We recorded neuropsychiatric side effects as present (‘yes’) or absent (‘no’) regardless of the
number or severity of reported events.
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