Sequencing CYP 2 D 6 for the detection of poor-metabolizers in post-mortem blood samples with tramadol

Tramadol concentrations and analgesic effect are dependent on the CYP2D6 enzymatic activity. It is well known that some genetic polymorphisms are responsible for the variability in the expression of this enzyme and in the individual drug response. The detection of allelic variants described as non-functional can be useful to explain some circumstances of death in the study of post-mortem cases with tramadol. A Sanger sequencing methodology was developed for the detection of genetic variants that cause absent or reduced CYP2D6 activity, such as *3, *4, *6, *8, *10 and *12 alleles. This methodology, as well as the GC/MS method for the detection and quantification of tramadol and its main metabolites in blood samples was fully validated in accordance with international guidelines. Both methodologies were successfully applied to 100 post-mortem blood samples and the relation between toxicological and genetic results evaluated. Tramadol metabolism, expressed as its metabolites concentration ratio (N-desmethyltramadol/Odesmethyltramadol), has been shown to be correlated with the poormetabolizer phenotype based on genetic characterization. It was also demonstrated the importance of enzyme inhibitors identification in toxicological analysis. According to our knowledge, this is the first study where a CYP2D6 sequencing methodology is validated and applied to post-mortem samples, in Portugal. The developed methodology allows the data collection of post-mortem cases, which is of primordial importance to enhance the application of these genetic tools to forensic toxicology and pathology. Lisbon, 30 September of 2015

We further state that this paper reports original work and is not under consideration for publication elsewhere.

Introduction 31
Tramadol is a centrally acting opioid analgesic commonly prescribed for treatment of 32 postoperative, dental, cancer, neuropathic and acute musculoskeletal pain control , with 33 high clinical efficacy, low incidence of adverse effects and low abuse potential. 34 Tramadol is administrated in a racemic mixture and undergoes extensive phase I and II 35 metabolization to 23 metabolites, mostly excreted in the urine. The main metabolites 36 resulting from the phase I metabolization are O-desmethyltramadol (ODT), catalyzed by 37 CYP2D6 enzyme, and N-desmethyltramadol (NDT), catalyzed by CYP3A4 and 38 CYP2B6 enzymes. Tramadol acts as a norepinephrine and serotonergic re-uptake 39 inhibitor, possesses low affinity for µ opioid receptors and no affinity for δ or κ opioid 40 receptors. The main opioid analgesic effect is attributed to ODT because it has 41 approximately 300 times more affinity to -opioid receptors than the parent compound 42 High blood concentrations of tramadol, due to accumulation or to increasing dosage, 82 can lead to adverse reactions, not directly related with the opioid depression of the 83 central nervous system, but specially with the inhibition of serotonin and 84     Table 3 . Five calibration curves were measured over a period of 15 days, using 236 seven levels of spiked blood samples in the working range (between 50 and 237 1000ng/mL) and three independent controls were prepared each day with the 238 concentrations of 150, 500 and 850ng/mL. The calibration model was chosen as 239 explained by Almeida et al [28] using as criteria the correlation coefficient higher than 240 0.99 and the best calibrators' accuracy (obtained by back calculating their 241 concentrations). The method was linear over the working range using a weighting factor 242 of 1/x2. Repeatability (within-day precision) was determined by analyzing six spiked 243 samples at the low, medium and high concentration levels simultaneously. The accuracy 244 and the precision were determined by the calculation of BIAS and the coefficient of 245 variation (% CV), using the concentration obtained for the triplicates of controls (see 246 Table 3). The limit of detection (LOD) was determined by the analysis of blood samples 247 spiked with decreasing amounts of the analytes, being the lowest concentration that 248 fulfilled the identification criteria, with the signal/noise of all the peaks above 3, in the 249 replicates (Table 3). The limit of quantitation (LOQ) was validated by analyzing six 250 replicates of spiked samples with a concentration of 50ng/mL (the first point of the 251 calibration curve) and verifying the coefficient of variation (<10%). Dilution of the 252 sample was tested for 1:2 and 1:5 using 10 real samples, covering a concentration range 253 between 50ng/mL to 5000ng/mL. The main results of the method validation are 254 summarized in Table 3Error The genetic and toxicological results were graphically and statistically compared, using 261 SPSS 17.0 software. The distribution of the results was tested for normality with 262 Kolmogorov-Smirnova test and the hypothesis was rejected. Non-parametric tests were 263 then used and the statistical differences between the medians of the genotype groups 264 were calculated using the Mann-Whitney test with 95% of confidence interval. 265 266

Results and discussion 267
Post-mortem blood samples were studied searching for CYP2D6 genetic variants 268 responsible for the enzyme inactivation, and the results obtained were then compared 269 with the concentration of tramadol and its main metabolites: NDT and ODT. The sequencing methodology allowed the detection of 4 different alleles: CYP2D6*3 277 (2549delA); CYP2D6*4 (100C>T and 1846G>A); CYP2D6*6 (1707delT) and 278 CYP2D6*10 (100C>T). Sanger sequencing methodology can't detect the copy number 279 variation (CNV) of the gene, so it was not possible to identify the gene complete 280 deletion (allele *5). Nevertheless, in cases with allele *5 the PM phenotype assignment 281 is not necessarily compromised. In heterozigotes, this methodology will assign the 282 individual as if he was homozygote for the other allele: if it is null, the individual will 283 be designated as PM; if it's functional, he will not. On the other hand, in *5/*5 284 homozygotes, there will be no amplification product because there is no gene. So, when 285 the amplification fails, there may be two main explanations: the low quantity or the 286 degradation of the DNA in the sample, which can be evaluated using the degradation 287 index given by Quantifiler trio kit; or maybe the individual is homozygote for the 288 CYP2D6*5 allele, and this genotype should then be confirmed by a suitable method. 289

290
The allele with the higher prevalence was CYP2D6 *4, with a frequency of 19.6%. The 291 allele *10 was detected with a prevalence of 16,5%, which is higher than the expected 292 based on large European population studies and according to CYP2D6 Allelic Variation 293 Summary can decrease the enzymatic activity and change the substrate specificity of the enzyme 296 [13,33-35]. Only one allele *3 and one allele *6 have been detected. All the alleles that 297 hadn't any variant in the studied fragments were considered as wild type (WT). 298 The genotype distribution is presented in the Table 4. 299 300  In this study, six individuals were found to be poor-metabolizers (PM). All the available 366 information concerning these six cases is presented in the Table 6. 367 368

Conclusions 395
This study proved that Sanger sequencing methodology can be successfully applied to 396 the detection of genetic polymorphisms at CYP2D6 in post-mortem blood samples. The 397 method proved to be specific, accurate, with a good precision and limit of detection for 398 the null variants analyzed. 399 The results showed a good correlation between the PM genotype and the toxicology 400 results of the tramadol metabolic ratio NDT/ODT, appearing to be an alternative 401 parameter in the evaluation of the degree of metabolization of tramadol in post-mortem 402

cases. 403
The presence of enzymatic inhibitors affects significantly the degree of metabolization, 404 which can be seen in the results obtained. By this reason, is very important to include 405 that compounds in the toxicology screening. 406 The detection of allelic variants described as non-functional can be useful to explain 407 some circumstances of death in the study of tramadol positive cases and the results 408  * Secondly, tramadol analgesia is mediated not only by the polymorphic mu opioid receptor but also through modulation of norepeniphrine ad serotonergic re-uptake.
A: I have reformulated the introduction as suggested.
* Thirdly, tramadol is not always given in controlled doses (as stated), but commonly prescribed in outpatient settings. Therefore, the assumption that higher than expected tramadol doses would arise from malpractice or neglect is short-sighted and should probably be removed from the manuscript althogether as it is outside the scope of this work.
A: Removed as suggested.
2. Please provide a reference to support the statement (page 3) that PM of tramadol have reduced analgesic effects with tramadol and reduced adverse effects with tramadol.
A: Done.
3. The postmortem concentrations should not, in general, be compared to those in clinical studies/therapeutic studies. Please discuss at length the various issues with postmortem findings (post-mortem redistribution, variances in drug collection sites (femoral blood, etc), death interval) as they pertain to the population in this study, and as they pertain to tramadol specifically.
A: We have added the information required.
4. The methods section should clearly state which concomitant medications were considered in this study as CYP2D6 substrates. Furthermore, not all CYP2D6 substrates are "inhibitors". Please 5. There are over 100 allelic variants of CYP2D6. In this study, the author's method only categorizes 4 of these variants. The authors then subscribe individuals to either poor, intermediate, or extensive metabolizers based on only 4 variants and make several assumptions on their tested population to rationalize this approach. As their testing approach is not the gold standard in pharmacogenetic testing, given its limited scope of testing, they need to compare their methodology with a platform that tests for the majority of CYp2D6 alleles in order to justify these assumptions. A false positive and false negative rate of attributing an individual to the poor metabolizer phenotype based on only 4 variants needs to be provided.
A: We have clarified in the manuscript the aims of our work.
The focus was to detect the PM. We have tried to explain better the exceptions. We changed also the assignment of the genotypes based in the variants searched to restrict it to the PM.
On the other hand, we described in more detail the validation of the method. It is not possible for us to compare our results with other methodology as suggested, but we had used reference materials to evaluate the accuracy of the method.
We would like to thank both reviewers for the deep appreciation of our manuscript and for all the suggestions. We tried to fulfill all the requirements and we will answer point-by point to the comments Reviewer #2: 1. The present manuscript describes a Sanger sequencing method for detection of some CYP2D6 polymorphisms causing a reduced or absent enzyme function, as well as a GC/MS method for the quantitation of tramadol, O-desmethyltramadol and N-desmethyltramadol. The methods were applied to 100 tramadol positive post-mortem samples. The purpose of doing that is however not clearly stated. Several papers on CYP2D6 sequencing methods and quantitative tramadol methods have been published previously. Furthermore, many of those are better described in terms of validation and performance and are using techniques able to detect also the CYP2D6 ultra-rapid metabolizers and quantifying the enantiomers of tramadol and its metabolites. If the authors clearly state the aim of identifying poor metabolizers and further describe and discuss the six cases being poor metabolizers the present manuscript could however add on to the current knowledge within this field.
A: We have clarified in the manuscript the aims of our work.

Abstract:
* "A Sanger sequencing methodology was developed for the detection of genetic variants that cause absence of CYP2D6 activity, such as *3, *4, *6, *8, *10 and *12 alleles". As mentioned later in the manuscript *10 causes a decreased function of the enzyme, not a total absence. Consider to reformulate, for example "…genetic variants that cause absent or reduced CYP2D6 activity". * The allele frequency of the null allele *5 (which was not searched for) is higher than that of *3 and *6 (which was searched for) in a Caucasian population. Therefore consider to reformulate the following sentences on page 7 "All the alleles that hadn´t any variant in the studied fragments were considered as wild type. This assumption was considered acceptable because the prevalence of the other null alleles that weren´t searched for is very low in Caucasian population".
A: The sentence was removed and we have tried to explain better the exceptions, focusing in the identification of the PM, which is the main purpose of the study. * In section 3.2 it is written "Since this method is not able to detect CNV events, the ultrametabolizers were included in the group of EM. This assumption was considered acceptable once the final objective of the present work was to detect the PM". What was the purpose of constructing figure 3 if only PMs were important to find? Consider to reformulate.
A: We changed the assignment of the genotypes based in the variants searched to restrict it to the PM.
Thank you for your suggestions.
* A further description of the INIB group would be valuable. Were those cases PM, IM or EM?
What was the concentrations of the inhibitors? To be able to draw conclusions about the significance of the inhibitors a comparison between for example EMs with and without inhibitors seems more valuable than putting all cases with inhibitors in one group, regardless of genotype.
A: Since we had focused in the PM cases, we removed the EM and IM assignment. On the other hand, and unfortunately, the available information of the cases is scarce. We were not able to do this.
* Table 3  * Why does the authors conclude that the NDT/ODT ratio is a better measure of TMD to ODT metabolism than the TMD/ODT ratio? The correlation seemed better for the NDT/ODT ratio, yes, although other factors than the CYP2D6 genotype might affect this ratio. A discussion concerning the impact of CYP2B6 and CYP3A4 genotype for the formation of NDT would be meaningful. If the authors think that the correlation between the TMD/ODT ratio and CYP2D6 genotype was less than expected a discussion concerning reasons for this would be highly valuable. Could for example the inability of the sequencing method to detect allele *5 and multiple copies of the gene have had an impact on the results? Or could the classification into IMs and EMs have had an impact? The present classification is not wrong although according to other definitions EMs have two functional alleles. The present EM group includes individuals with both one, two and multiple copies of CYP2D6 alleles.
A: We have tried to explain it better, especially for post-mortem cases.
* The information in table 4 is interesting, as well as the measured concentrations in table 5, although the concentration unit must be given in table 5. This information could however be compiled in one table, while DNA-concentration, degradation index and polymorphisms are left out. The important thing is that all individuals are PMs and that information is given in the table text.
A: Done as suggested.
* The results of table 4 and 5 are only discussed with a few sentences on page 11, saying that in three cases the tramadol concentration was higher than the therapeutic range but ODT concentrations were acceptable. What does that mean, what are the conclusions drawn? Case 2 and 3 have "unknown" and "natural" stated as the cause of death, respectively, in spite of high tramadol concentrations. Was the tramadol concentrations not considered toxic (because of low ODT concentrations)? What were the circumstances of death? Any comments regarding the other cases? Please discuss the results in more detail.
* What substances were included in the toxicological screening? It could be interesting to describe case 3,4 and 5 (without other substances present) in more detail.
* It could perhaps be of interest to show the concentrations and CYP2D6 genotypes for the 10 intoxication cases mentioned in section 2.1.
A: Done as suggested, although we cannot discuss better the results because of the lack of information.
The study of the autopsy results of these cases together with the pathologists would be indeed very interesting but it was not possible yet.

Conclusions:
* "The Sanger sequencing method proved to be specific, accurate, with a good precision and limit of detection". As mentioned previously results for all validation parameters are not presented in the manuscript, for what reason it is difficult for the reader to be able to draw the same conclusion.
A: We have reformulated the manuscript and included the validation parameters.
* "The results of genotypes showed a good correlation with toxicology results of the tramadol metabolic ratio NDT/ODT, appearing to be a better parameter to know the degree of metabolization of TMD to ODT, than the usual metabolic ratio as TMD/ODT". See also the comments above in the "results and discussion" section. In my opinion this is not a correct conclusion.
A: We have reformulated the results and discussion and also the conclusions in the manuscript.
* "The presence of enzymatic inhibitors affects significantly the degree of metabolization" See also the comments above in the "results and discussion" section. It is known that some drugs acts as inhibitors of CYP2D6 and therefore affect the metabolism of tramadol. In my opinion it is however not satisfactory shown in this manuscript.
A: We have reformulated the conclusions in the manuscript.
* "The detection of allelic variants described as non-functional were useful to explain some circumstances of death in the study of tramadol positive cases and demonstrate the importance of this genetic tool to forensic toxicology and pathology". What circumstances were clarified in this study? A more profound discussion regarding the results is desirable.
A: As said above, it was not possible yet. Nevertheless, the cases number 1 and 3 can be good examples of the future applications of this approach.