Opioid Dependence Clinical Trial
Official title:
Influence of KCNH2 Polymorphisms on the QTc Interval in Kelantanese Malays Patients Receiving Methadone Maintenance Therapy (MMT) in Malaysia
Methadone maintenance therapy (MMT) is one of the modalities to prevent HIV transmission among injected drug users, particularly in opioid-dependent users. However, methadone-associated cardiotoxicity is one of the fatal adverse events that limit the widespread usage in certain groups of opioid-dependent patients. This is a cross-sectional study aimed to investigate the association between 4 KCNH2 SNPs (1539C>T, in exon 6 of KCNH2 gene; 1956T>C, in exon 8 of KCNH2 gene), 2350C>T (in exon 9 of KCNH2 gene), 2690A>C (Exon 11 of KCNH2 gene)) and prolongation of QTc interval in opioid-dependent Kelantanese Malays who are the recipients of Methadone Maintenance Therapy. The investigators hypothesized that subjects with minor alleles of those 4 SNPs will have longer QTc intervals than those with major alleles, adjusting for the effects of other confounding factors such as age and gender of the subjects, plasma methadone trough levels, hypokalemia, hypocalcemia and hypomagnesemia. The investigators also aimed to provide a model that will reliably predict the magnitude QTc based on the SNPs data and other covariates mentioned above. This will greatly assist in identifying methadone recipients who are at risk of developing prolonged QTc or the more fatal torsade de pointes.
Introduction
Methadone maintenance therapy (MMT) is one of the modalities to prevent HIV transmission
among injected drug users, particularly in opioid-dependent users. However,
methadone-associated cardiotoxicity is one of the fatal adverse events that limit the
widespread usage in certain groups of opioid-dependent patients.
Study hypotheses / aims
The investigators hypothesized that subjects with minor alleles of those 4 SNPs would have
longer QTc intervals than those with major alleles, adjusting for the effects of other
confounding factors such as age and gender of the subjects, plasma methadone trough levels,
hypokalemia, hypocalcemia and hypomagnesemia. The investigators also aimed to provide a model
that will reliably predict the magnitude QTc based on the SNPs data and other covariates
mentioned above. This will greatly assist in identifying methadone recipients who are at risk
of developing prolonged QTc or the more fatal torsade de pointes.
Study Design and Sample Size Calculation
This is a cross-sectional study aimed to investigate the association between 4 KCNH2 SNPs
(1539C>T, in exon 6 of KCNH2 gene; 1956T>C, in exon 8 of KCNH2 gene), 2350C>T (in exon 9 of
KCNH2 gene), 2690A>C (Exon 11 of KCNH2 gene)) and prolongation of QTc interval in
opioid-dependent Kelantanese Malays who are the recipients of Methadone Maintenance Therapy .
The sample size was calculated using single-proportion formula and the information required
was based on a similar prior study conducted among Singaporean Malay. It was concluded that
the sample size required is 105 patients. Since eligible patients were lacking, the
convenience (non-probability) sampling method was used.
During the initial visit, relevant clinico-demographic details such as age, gender, history
of drug addiction and psychiatric illnesses, drug dependency patterns, other drug usage and
treatment-related issues for each patient were gathered. Subsequently a validated Malay
version of Subjective Opioid Withdrawal Scale (SOWS) questionnaire was administered to assess
any opioid withdrawal symptoms experienced by study participants.
Five (5) mls of blood was then withdrawn for each subject for the ascertainment of relevant
biochemical profile (serum potassium, magnesium, calcium), plasma methadone trough levels,
and KCNH2 SNPs genotyping. Drug screening for substances such as MDMA, benzodiazepines
methamphetamine, cannabis, marijuana were also carried out using urine dipstick test at urine
collection point. The colour and temperature of the urine were also recorded.
QT measurement was obtained using calibrated Welch Allyn CP 50™ ECG (Electrocardiograph)
(Welch Allyn Australia Pty Ltd., New South Wales, Australia) machine, printed at a paper
speed of 25mm/s and voltage of 10mm/mV. QTc measurement was then manually calculated using
Fredericia's formula to correct for heart rate (R-R interval). All trained personnel who were
responsible for obtaining QTc measurement from each patient were blinded to other information
on serum biochemical profiles and methadone trough levels.
KCNH2 Genotyping
The DNA was extracted according to procedures modified from Bethesda Research Laboratories
based on the revisions made to the Brinboam and Doly method. The quantity and quality of the
extracted DNA were determined using NanoDrop ND-1000 Spectrophotometer (NanoDrop
Technologies, Inc. Wilmington, USA) with measurements done at 260 and 280 nm. The integrity
of the extracted DNA was determined using 2% agarose gel electrophoresis performed at 70
Volts for 90 minutes.
The DNA in all samples were amplified for all 4KCNH2 SNPs were performed using 2-step nested
allele-specific multiplex polymerase chain reaction (PCR). The primers (both forward and
reverse) were designed according to the published sequence for KCNH2 (NC_000007.13). To
improve primer specificity, mismatch at its 3´ ends that were specific to either the variant
sequence or wild-type DNA sequence at the specified locus was made to the primers. Besides,
the primers were also designed and manipulated to differentiate between the different single
nucleotide changes/alleles during PCR amplification. To verify primers specificity, the BLAST
program at NCBI (http://www.ncbi.nlm.nih.gov/ blast) was used.
In the first multiplex PCR, exon 6,8,9 and 11 were amplified under the following condition:
pre-denaturation at 95°C for 1 minute followed by 25-cycle of denaturation at 95°C for 15
seconds, annealing for 65°C for 15 seconds, and extension at 72°C for 10 seconds. Upon full
25-cycle completion, final extension phase lasted for 72°C for 7 minutes. The PCR products
(amplicons) were then resolved using 2% agarose gel electrophoresis at 130 Volts for 90
minutes.
The PCR products of the first multiplex PCR were then used as templates for second PCR which
targeted the 4 respective SNPs region for amplification. This was performed under the
following condition:pre-denaturation at 95°C for 1 minute followed by 25-cycle of
denaturation at 95°C for 15 seconds, annealing for 69°C for 30 seconds, and extension at 72°C
for 4 seconds. Upon full 25-cycle completion, final extension phase lasted for 72°C for 7
minutes. The PCR products (amplicons) were then resolved using 2% agarose gel electrophoresis
at 130 Volts for 90 minutes.
The first products of the amplified regions in the Exons 6, 8, 9, and Exon11 were
subsequently submitted for direct DNA sequencing. QIAquick PCR purification kit (Qiagen, USA)
was used for purification of the PCR products. DNA sequencing was carried out by applying
3130XL genetic analyzer DNA sequencer (ABI, USA). The results were compared with the
published sequences for KCNH2 in the NCBI, accession number (NC_000007.13)
Statistical Analysis
The select equally likely or more extreme samples (SELOME) version of the Fisher's exact test
was employed to examine whether the distribution of the SNP alleles and genotype follow the
Hardy-Weinberg Equilibrium (HWE) assumption. Those SNPs that significantly deviated from the
HWE assumption were dropped from further analysis.
To examine the associations between the 4SNPs and QTc interval (measured as continuous
variable) and build a statistical model that can reliably predict QTc intervals, simple and
multiple linear regression methods were used. Age and gender of the patients, plasma
methadone trough levels, serum potassium, calcium and magnesium were treated as confounding
factors whose effects on QTc were adjusted for.
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