Aline Hajj1–3 , Katell Peoc’h1,2, *, Kamilia Ksouda2,4 , Florence Vorspan2,4 , Jacques Callebert1,2 , Xavier Decle`ves2 , Jean-Pierre Lepine2,4 , Jean-Michel Scherrmann2 , Lydia Rabbaa-Khabbaz3 and Jean-Louis Laplanche1,2
1Service de Biochimie et Biologie Mole´culaire, Hoˆpital
Lariboisie`re, Paris, France, and Paris-Cite´ Descartes University (UFR des Sciences Pharmaceutiques et Biologiques), Paris, France
2INSERM U705, CNRS UMR8206:
Neuropsychopharmacologie des Addictions: vulne´rabilite´ et variabilite´ expe´rimentale et clinique, Faculte´ de Pharmacie, Universite´ Paris Descartes, Universite´ Paris Diderot, Paris, France
3Laboratoire de Pharmacologie Clinique et
Pharmacocine´tique, Faculte´ de Pharmacie, Universite´ Saint Joseph, Beyrouth, Liban
4Service de Psychiatrie, Hoˆpital Fernand Widal, Paris,
France
Keywords: addiction; buccal cells; DNA; single nucleotide
polymorphism.
Genomic studies are needed to identify gene polymorphisms (SNPs, single nucleotide polymorphisms) associated with drug abuse and dependence that can explain inter-individual variability in response to substitution drugs (1). However, blood is sometimes difficult to obtain from injection drug users, partly due to the vascular damage caused by injections. An alternative way to obtain DNA would be to collect buccal cells using swabs, a technical means that has been success- fully used in numerous clinical settings and epidemiological studies (2–4). However, buccal cell collection could be plagued by difficulties in poly-substance abusers due to oral dryness (xerostomia), poor oral health and sometimes the presence of lesions in the buccal mucosa due to malnutrition and HIV infection (5, 6). Moreover, xerostomia is a known side-effect of methadone treatment in opioid dependency (7). This pilot
*Corresponding author: Katell Peoc’h, Service de Biochimie et Biologie Mole´culaire, Hoˆpital Lariboisie`re, 2 rue A. Pare´, 75475 Paris Cedex 10, France
study was carried out to test the suitability of buccal cell collection for providing DNA in opiate addict patients treated with methadone before launching a large-scale genotyping study.
Buccal cells from 10 healthy volunteers and 20 heroin addict patients reluctant to undergo venipuncture were col- lected using the ‘‘Indicating’’ FTA cards (Whatman, Maid- stone, UK) according to the manufacturer’s instructions. The study was approved by the local Ethics Committee and written informed consent was obtained from all participants. Briefly, buccal cells were collected using a non-invasive sterile foam tipped applicator and then applied onto cards. Physicians declared that the collection technique was simple although they confirmed some difficulties due to the oral dry- ness in patients. Most of the patients were compliant and pre- ferred buccal cell collection than providing blood samples.
The cards were air-dried for 2 h and then conserved in a bag with desiccant at room temperature. DNA was used either directly as dry punches (3 mm diameter circle discs) as recommended by the manufacturer or after elution using the following protocol. Briefly, 3 mm diameter circle discs were punched from the FTA indicating card and rinsed twice
by pulse-vortexing in 500mL of distilled sterile water. Single
rinsed discs were transferred into 30mL of sterile water then
heated to 958C for 30 min. This protocol allowed us to eval- uate the DNA quantity present on the punches. DNA con- centration was determined using a Nanodrop spectrophoto-
meter (NanoDrop, Wilmington, NC, USA). DNA was
obtained in all addicts with yields ranging from 204 to 648 ng/punch (median: 336 ng/punch) vs. 123–636 ng/punch (median: 345 ng/punch) in controls (psNS). As the mean
estimated DNA quantity obtained per card was 14mg (cor-
responding to 40 punches per card), it appears that around 1000 genotyping reactions could be conducted per single DNA sample. No significant decrease in DNA quality was observed after at least 2 years at room temperature.
Dry punches or eluted DNA from buccal cells were used for genotyping two SNPs in the Opioid mu receptor gene
(OPRM1; c.118A)G, rs1799971) and the P-glycoprotein
gene (ABCB1; c.3435C)T, rs1045642) using Real-Time
PCR assays (Taqman, Applied Biosystems and Lightcycler, Roche) or denaturating high performance liquid chromato- graphy (dHPLC). Direct sequencing was also tested
(c.3435C)T ofABCB1). Positive controls for the two SNPs
were obtained by direct sequencing of blood DNA. DNA samples from 100% of the FTA punches were suc- cessfully genotyped at all loci using Real-Time PCR assays. No discrepancy was observed between blood DNA from
2 Hajj et al.: How to obtain DNA from injection drug users?
Article in press - uncorrected proof
Figure 1 A typical allelic discrimination plot for Taqman ABCB1 c.3435C)T genotyping assay on step one plus, displaying three
clusters corresponding to the three genotypes and, near the origin, the no template controls (NTC).
The samples with a dashed circle correspond to the amplified genomic blood DNA controls whereas the other samples to DNA from patients’ FTA cards.
dry punches or eluted DNA were tested using dHPLC and sequencing. dHPLC exhibited a 100% rate of genotyping success. In contrast to previous results (2), direct sequencing
ofABCB1 was successful. One hundred percent concordance
was attained for all successful genotyping of DNA samples between conventional PCR and Real-Time PCR.
We conclude that buccal cell collection is an efficient alternative to venipuncture to obtain DNA from addicted patients. It is well tolerated by patients often affected with altered oral mucosa and could be used to provide DNA with good yield and suitability in large-scale studies using differ- ent molecular genotyping techniques. In addition, it resolves the problem of low participation rates of the patients who are often forced to get to the collection site to give blood samples.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that
there are no conflicts of interest regarding the publication of this article.
Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.
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Q1: Please confirm running title
The American Journal on Addictions, 19: 458–459, 2010
CopyrightCAmerican Academy of Addiction Psychiatry ISSN: 1055-0496 print / 1521-0391 online
DOI: 10.1111/j.1521-0391.2010.00062.x