Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.     

Evaluation of genetic markers for the analysis of THC levels of Cannabis sativa samples using principal component analysis – A preliminary study

Cannabis sativa is a worldwide commercial plant used for medicinal purposes, food and fiber production, and also as a recreational drug. Therefore, the identification and differentiation between legal and illegal C. sativa is of great importance for forensic investigations. In this study, principal component analysis (PCA), an exploratory data analysis technique, was tested to correlate the specific genotype with the concentration of tetrahydrocannabinol (THC) in the samples. C. sativa samples were obtained from legal growers in Piedmont, Italy, and from illegal drug seizures in the Turin region. DNA was extracted, quantified, amplified with a 13-loci multiplex STR and finally analyzed with an automated sequencer. The results showed a trend in the analyzed samples as they differed by their THC content and allele profiles. PCA yielded two clusters of samples that differed by specific allele profiles and THC concentrations. Further validation studies are needed, but this study could provide a new approach to forensic investigation and be a valuable aid to law enforcement in significant marijuana seizures or in tracing illicit drug trafficking routes.     

European validation of a Cannabis sativa 13-locus STR multiplex kit for genetic identification: A preliminary study

Cannabis sativa L. is a plant cultivated worldwide as a source of fiber, medicine and intoxicant. Traditionally, is divided into two main types: fiber type (hemp) and drug type (marijuana). Marijuana differs from hemp by the presence of a high quantity of the psychoactive drug, Δ9-tetrahydrocannabinol. The development of a validated method using short tandem repeats (STRs) could serve as an intelligence tool to link cases by means of genetic individualization or association of cannabis samples. For this purpose, a 13-locus STR multiplex method was developed, optimized, and validated by the Department of Forensic Science at Sam Houston State University (SHSU) according to relevant ISFG and SWGDAM guidelines. The European community considers C. sativa plants illegals, even though its consumption is accepted in precise and limited places (coffee shops or cannabis clubs in Netherlands and Spain). However, there are different gaps in the legislation of some European countries. For instance, in Italy, “weed” possession is decriminalized. Although trafficking and sale are prohibited, possession of small quantities of marijuana is considered only a civil offense. In order to proceed with the kit evaluation and inter-laboratory comparison, SHSU DNA laboratory sent blind cannabis DNA samples of known genotypes. Blind DNA samples were analyzed in different laboratories with different sequencers and analysis conditions. In this article, the goals were: a) to demonstrate that 13-locus STR kit for C. sativa is robust enough and reproducible, in all forensic laboratories, and b) to show the applicability of the STR system in association with Cannabis sativa cases for intelligence purposes to link multiple cases by means of genetic individualization or association of cannabis samples.     

Distribution of Chemical Phenotypes (Chemotypes) in European Agricultural Hemp (Cannabis sativa L.) Cultivars

In Europe, more than 50 approved cultivars of fiber hemp (Cannabis sativa L.) are in agricultural production. Their content of psychoactive tetrahydrocannabinol (THC) is legally restricted to <0.2% (%w/w in the dry, mature inflorescences). Cannabis strains with much higher THC contents are also grown, illegally or under license for drug production. Differentiation between these two groups relies on biochemical quantification of cannabinoid contents in mature floral material. For nonflowering material or tissue devoid of cannabinoids, the genetic prediction of the chemical phenotype (chemotype) provides a suitable method of distinction. Three discrete chemotypes, depending on the ratio of THC and the noneuphoric cannabidiol (CBD), can be distinguished: a “THC-predominant” type, a “CBD-predominant” type, and an intermediate chemotype. We present a systematic genetic prediction of chemotypes of 62 agricultural hemp cultivars grown in Europe. The survey reveals the presence of up to 35% B_T allele-carrying individuals (representing either a THC-predominant or an intermediate chemotype) in some cultivars—which is unexpected considering the legal THC limit of 0.2% THC. The fact that 100% of the seized drug-type seeds in this study revealed at least one B_T allele, reflects that plant breeding efforts have resulted in a fixation of the B_T allele in recreational Cannabis. To guarantee a sincere forensic application based on a genetic chemotype prediction, we recommend not to classify material of unknown origin if the samples size is below nine genetically independent individuals.     

A PCR marker Linked to a THCA synthase Polymorphism is a Reliable Tool to Discriminate Potentially THC‐Rich Plants of Cannabis sativa L.

Neither absolute THC content nor morphology allows the unequivocal discrimination of fiber cultivars and drug strains of Cannabis sativa L. unequivocally. However, the CBD/THC ratio remains constant throughout the plant's life cycle, is independent of environmental factors, and considered to be controlled by a single locus (B) with two codominant alleles (B_T and B_D). The homozygous B_T/B_T genotype underlies the THC‐predominant phenotype, B_D/B_D is CBD predominant, and an intermediate phenotype is induced by the heterozygous state (B_T/B_D). Using PCR‐based markers in two segregating populations, we proved that the THCA synthase gene represents the postulated B locus and that specific sequence polymorphisms are absolutely linked either to the THC‐predominant or the THC‐intermediate chemotype. The absolute linkage provides an excellent reliability of the marker signal in forensic casework. For validation, the species‐specific marker system was applied to a large number of casework samples and fiber hemp cultivars.     

DYS448基因座分型缺失分析

目的调查分析DYS448基因座分型缺失,为法医学提供有意义的数据。方法收集中国汉族5487名无关男性个体血样,其中4479份样本用Y-filerTM试剂盒,1008份样本应用Yfiler PlusTM试剂盒进行复合扩增,所有样本应用AGCU Y-24试剂盒复核;统计DYS448基因座出现基因分型缺失的概率。结果在5487名无关个体的Y-STR数据中,观察到35个个体的35种单倍型中DYS448基因座分型出现缺失,其中2个样本在其它基因座位同时出现多谱带。结论 DYS448基因座分型缺失率为0.637%,在Y-STR数据库与父系鉴定应用中应予以关注。     

Validation of the AMPFℓSTR® MiniFilerTM PCR Amplification Kit for Use in Forensic Casework*

Abstract: The AmpF?STR^® MiniFiler^TM PCR Amplification Kit is designed to genotype degraded and/or inhibited DNA samples when the AmpF?STR^® Identifiler^TM PCR Amplification Kit is incapable of generating a complete genetic profile. Validation experiments, following the SWGDAM guidelines, were designed to evaluate the performance of MiniFiler. Data obtained demonstrated that MiniFiler, when used in conjunction with Identifiler, provided an increased ability to obtain genetic profiles from challenged samples. The optimum template range was found to be between 0.2 and 0.6 ng, with 0.3 ng yielding the best results. Full concordance was achieved between the MiniFiler kit and Identifiler kit except in a single case of a null allele at locus D21S11. Numerous instances of severe heterozygous peak imbalance (<50%) were observed in single source samples amplified within the optimum range of input DNA suggesting that caution be taken when attempting to deduce component genotypes in a mixture.     

Whole Plastome Sequences of Two Drug-Type Cannabis: Insights Into the Use of Plastid in Forensic Analyses

DNA is one of the fastest growing tools in forensic sciences, increasing reliability in forensic reports and judgments. The use of DNA has increased in different areas of the forensic sciences, such as investigation of plant species, where plastid DNA has been used to elucidate and generate evidence in cases of traceability of genetically modified and controlled plants. Even with several advances and the practice of using DNA in forensic investigations, there are just few studies related to the identification of genetic tools for the characterization of drug and nondrug-types of Cannabis. Herein, the whole plastomes of two drug-type Cannabis are presented and have their structures compared with other Cannabis plastomes deposited in the GenBank, focusing in the forensic use of plastome sequences. The plastomes of Cannabis sativa “Brazuka” and of the hybrid Cannabis AK Royal Automatic presented general structure that does not differs from the reported for other C. sativa cultivars. A phylogenomic analyses grouped C. sativa “Brazuka” with the nondrug C. sativa cultivars, while the hybrid Cannabis AK Royal Automatic placed isolated, basal to this group. This suggests that the analysis of plastomes is useful toward genetic identification of hybrids in relation to C. sativa.     

DNA Analysis of Natural Fiber Rope*

Abstract: When rope is found at a crime scene, the type of fiber is currently identified through its microscopic characteristics. However, these characteristics may not always unambiguously distinguish some types of rope from others. If rope samples contain cells from the plants of origin, then DNA analysis may prove to be a better way to identify the type of rope obtained from a crime scene. The objective of this project was to develop techniques of DNA analysis that can be used to differentiate between ropes made from Cannabis sativa L. (hemp), Agave sisalana Perrine (sisal), Musa textilis Née (abaca, “Manila hemp”), Linum usitatissimum L. (flax), and Corchorus olitorus L. (jute). The procedures included extracting the DNA from the rope, performing polymerase chain reaction (PCR) using the extracted DNA as a template, and analyzing the DNA products. A primer pair for PCR, chosen from within a chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase, was designed to be specific for plant DNA and complementary to the genes from all five plants. The resulting PCR fragments were approximately 771 base pairs long. The PCR fragments, distinguished through base sequence analysis or restriction enzyme analysis, could be used to identify the five different rope types. The procedure provides a useful addition to visual methods of comparing rope samples.     

Detection of genotype recycling fraud in U.S. immigrants

Abstract: Relationship testing laboratories provide genetic evidence to support or refute claims of kinship between U.S. citizen petitioners and potential immigrant beneficiaries. One female beneficiary presented a male amelogenin type and alleles at 15 autosomal loci that were identical to an alleged brother’s. Laboratory records showed that her alleged father had petitioned to have 15 children emigrate from Ghana. The petitioner’s 15 paternity indices exceeded 10⁵, but the children shared only four short tandem repeat (STR) profiles, suggesting fraudulent reuse of genotypes in this alleged pedigree (AP). To determine the extent of this “genotype recycling,” I examined the laboratory’s 555 APs from Ghana and 532 control APs from Nigeria. Seventeen Ghanaian APs (3.1%) but no Nigerian APs showed genotype recycling. Of 90 tested people in the 17 APs, 56 shared identical STR profiles with others in their AP. Of these 56 people, 10 were petitioners with unexpectedly high parentage indices. Seven of 56 had amelogenin types that disagreed with their declared genders. Database searches for identical multilocus genotypes in allegedly different people would best detect this fraud.     

Short tandem repeat (STR) DNA markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations

Short tandem repeat (STR) markers are the DNA marker of choice in forensic analysis of human DNA. Here we extend the application of STR markers to Cannabis sativa and demonstrate their potential for forensic investigations. Ninety-three individual cannabis plants, representing drug and fibre accessions of widespread origin were profiled with five STR makers. A total of 79 alleles were detected across the five loci. All but four individuals from a single drug-type accession had a unique multilocus genotype. An analysis of molecular variance (AMOVA) revealed significant genetic variation among accessions, with an average of 25% genetic differentiation. By contrast, only 6% genetic difference was detected between drug and fibre crop accessions and it was not possible to unequivocally assign plants as either drug or fibre type. However, our results suggest that drug strains may typically possess lower genetic diversity than fibre strains, which may ultimately provide a means of genetic delineation. Our findings demonstrate the promise of cannabis STR markers to provide information on: (1) agronomic type, (2) the geographical origin of drug seizures, and (3) evidence of conspiracy in production of clonally propagated drug crops.     

Alcohol dependence and criminal behavior: preliminary results of an association study of environmental and genetic factors in an italian male population

The aim of this study is to propose an innovative approach evaluating the connection between alcohol use disorders and criminal behavior. The research, structured as a case–control study, was based on the analysis of environmental (social variables) and genetic factors (single nucleotide polymorphisms of glutamic acid decarboxylase) in a population (N = 173) of Italian alcohol‐dependent men. Group 1 (N = 47, convicted subjects) was compared with Group 2 (N = 126, no previous criminal conduct). Grade repetition, work problems, and drug problems were statistically associated with criminal behavior. Having daily family meals together and having children were inversely related to convictions. The genotype distribution of the two groups was similar. The association between environmental factors and antisocial behavior confirms previous findings in the literature. The lack of genetic association does not exclude the role of the gamma‐aminobutyric acid (GABA) system in determining antisocial behavior; further studies with larger samples are needed, together with investigation of other components of the GABA pathway.     

RAPD analysis distinguishes Cannabis sativa samples from different sources

Samples of the seeds and seedlings of Cannabis sativa, and its dried leaves and flowerheads (marijuana), could be reliably distinguished by RAPD-PCR (Random Amplified Polymorphic DNA using the Polymerase Chain Reaction). DNA was best extracted from fresh tissues using buffers and the detergent cetyltrimethylammonium bromide; poorly dried tissue or inviable seed yielded coloured samples of degraded DNA. DNA was isolated from 51 C. sativa and two Humulus lupulus (hops) samples. Of the C. sativa samples 43 were from Australia (ten from Canberra gardens, eight from a New South Wales crop and 25 from two Queensland crops) and eight were from Papua-New Guinea (P-NG). A total of 102 different bands were obtained using four 10-nucleotide primers with arbitrarily chosen sequences. Banding patterns were compared by calculating pairwise distances using various algorithms, and presented using the neighbour-joining tree and multidimensional scaling methods. These showed a clear difference between C. sativa and H. lupulus, and separated the samples of the latter into three distinct groups; one group comprised all the P-NG samples, another the Canberra samples, and the third, the three crop samples.     

Increasing Methamphetamine Detection in Cases of Early Childhood Fatalities

Age and organ maturity can influence drug toxicity in children; however, most clinical data and literature are based on drug concentrations in adults. Therefore, the interpretation of drugs detected in children is often difficult or not possible. Retrospective reviewing of pathology and toxicology information from postmortem cases may assist in future interpretations or identify drug trends. A search of the Forensic Science SA case files was undertaken over 15 years from January 2002 to December 2016 for all children (<13 years). Of the 412 pediatric coronial cases, toxicological information was available on 373. At least one drug was detected in 94 cases with paracetamol, ibuprofen, codeine and hospital-administrated lignocaine and morphine among the most commonly detected agents. Methamphetamine, one of the most commonly abused illicit drugs in Australia, was found in seven cases. In the methamphetamine cases, deaths were associated with shared sleeping in three, pneumonia in one, and stillbirth in one. Methamphetamine was considered potentially contributory to death in two cases. The causes of death in the remaining two cases were undetermined. As six of the seven positive cases occurred in the 2012–2016 (n = 106) timeframe, an increase has occurred over recent years in the number of infants and young children presenting to forensic autopsy in South Australia who have detectable concentrations of methamphetamine. If this is an indication of a more generalized increased childhood exposure in the community there may be significant long-term physical and psychological effects.     

A fatal doxepin poisoning associated with a defective CYP2D6 genotype

It has been suggested that the polymorphism of the CYP2D6 gene can contribute to occurrence of fatal adverse effects. We therefore investigated postmortem toxicology cases of fatal drug poisonings related to CYP2D6 substrates, with the manner of death denoted as accidental or undetermined. CYP2D6 genotypes were determined in 11 consecutive cases with samples available for DNA analysis. A case of fatal doxepin poisoning with an undetermined manner of death was found to coincide with a completely nonfunctional CYP2D6 genotype (*3/*4), indicating a total absence of CYP2D6 enzyme and suggesting a poor metabolizer phenotype. The doxepin concentration was 2.4 mg/L, the concentration of nordoxepin 2.9 mg/L, and the doxepin/nordoxepin ratio 0.83, the lowest found among the 35 nordoxepin-positive postmortem cases analyzed during the same year. No alcohols or other drugs were detected in the case. The CYP2C19 genotype was determined as that of an extensive metabolizer. The high N-desmethylmetabolite concentration is not consistent with acute intoxication. It is therefore probable that the defective genotype has contributed to the death, possibly involving repeated high dosage of doxepin. Our case strongly emphasizes that a pharmacogenetic analysis in postmortem forensic setting may reveal new insight to the cause or manner of death.     

国产Goldeneye~(TM) 20A试剂盒性能指标验证

目的测试国产GoldeneyeTM20A试剂盒技术性能指标,评估其法医学应用能力。方法从方法学验证、准确性、峰值均衡性、灵敏度、批次间试剂及稳定性测试、耐受性、不同检材的适应性与一致性、种属特异性、混合样本等9个方面对GoldeneyeTM20A试剂盒进行测试。结果阳性DNA样本分型正确,内标和等位基因分型标准物符合要求;等位基因间的均衡性≥83%,同一荧光标记基因座间的均衡性≥55%,不同标记物间的均衡性≥52%;0.125ng DNA阳性样本可检出全部STR基因座分型,不同批次间和反复冻融后试剂盒测试可以获得正确分型,对降解检材和混有抑制剂的样本等具有一定的耐受性,能对案件中多种检材进行分型且分型结果一致,具有一定的种属特异性和混合DNA样本检测能力。结论国产GoldeneyeTM 20A试剂盒可用于法庭科学实际检案与建库。     

Alleles responsible for ABO phenotype-genotype discrepancy and alleles in individuals with a weak expression of A or B antigens

ABO types obtained from evidentiary samples have been used effectively to obtain the initial information leading to the apprehension of culprits in Japanese criminal investigations. A simple ABO genotyping method using multiplex sequence-specific PCR and capillary electrophoresis was developed as a supplement to serological ABO typing. Limitations in predicting a phenotype based on genotype were evaluated using 1134 randomly selected Japanese peripheral blood samples. A concordance rate of 99.82% (1132/1134 samples) was found between genotypes and phenotypes defined as Groups A, B, AB, and O. Sequencing analysis revealed that one discrepant sample contained an O allele having a previously unreported point mutation at the primer binding site in exon 6, and another discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the O301 allele). Therefore, the existence of such alleles must be given some consideration when predicting phenotype based on genotype.     

Extraction and isolation of cannabinoids from marijuana seizures and characterization by 1H NMR allied to chemometric tools

Marijuana, a drug derived from the Cannabis sativa L. plant, is the world's most consumed illicit drug. In this paper, a total of 156 marijuana samples seized in the state of Espírito Santo (ES), Brazil were studied and analysed by proton nuclear magnetic resonance (¹H NMR) spectroscopy to identify the major cannabinoids present. A crude extract of all samples was purified using high performance liquid chromatography so that these compounds could serve as reference substances. Nine fractions were obtained and analysed by ¹H NMR and gas chromatography–mass spectrometry (GC–MS), with five presented cannabinoids. ?⁹-THC (Δ⁹-trans-tetrahydrocannabinol), ?⁹-THCA (?⁹-tetrahydrocannabinolic acid), ?⁸-THC (?⁸-tetrahydrocannabinol), 11-hydroxycannabinol, CBV (cannabivarin), and CBN (cannabinol) were found, and their chemical structures were confirmed by GC–MS. The latter compound was obtained with high purity (≈100%), while the others were obtained as less complex mixtures with purity higher than 75% (except for Δ⁸-THC). Principal component analysis (PCA) was used on the ¹H NMR spectra of the 156 samples, and it was found that the samples were grouped according to the months, differentiating into two groups (from July 2014 to January 2015 and from February 2015 to July 2015), where non-grouping was observed from four macro-regions of the ES state (North, Central, Metropolitan, and South). The chemical profile of the seized samples was correlated to the ¹H NMR spectrum of an isolated CBN sub-fraction, in which the group formed by samples seized in the year 2015 presented lower CBN content in the chemical composition. From the PCA score plot, two groups of samples were confirmed using the partial least squares discriminant analysis and orthogonal projections to latent structures classification methods.     

Evaluating probabilistic genotyping for low-pass DNA sequencing

Most genomic methods consider the sample genotype. Data are evaluated at some location, and if the signal strength is sufficient, a genotype call is made. Conversely, sites that lack sufficient signal are treated as missing data. Such methods for genotype calling are binary, and this dichotomy limits genomic analyses to relatively high-coverage (and high-cost) massively parallel sequencing (MPS) data. It follows that bioinformatic methods that rely on genotypes may not be ideal for trace DNA samples, such as those sometimes encountered in forensic investigations, but even when applicable such analyses can be expensive. However, there are some genomic analyses where having many uncertain genotypes (with measured uncertainty) assayed over the entirety of the genome may be more powerful than current multi-locus approaches that consider a limited number of well-characterized markers. Methods for such problems may rely on genotype likelihood, which expresses the likelihood of alternative genotype calls in addition to the most likely call. One application that can benefit from genotype likelihoods is kinship analysis. NgsRelate is a bioinformatic tool that infers pairwise relatedness using a probabilistic genotyping framework, which accommodates the uncertainty associated with genotype calls for low-pass MPS data. Here, NgsRelate was used to infer kinship coefficients from low-pass whole genome sequencing data from a known pedigree. Multiple samples in a titration series (ranging from 50 ng to 0.5 ng) on a single MPS S4 flow cell were assessed. A reproducible scientific bioinformatic workflow was developed to evaluate kinship coefficients considering up to 3rd degree relatives. NgsRelate was found to provide robust assessments of kinship. Further, the use of low-pass MPS data provides a more cost-effective way to conduct forensic investigations.     

Changes in methamphetamine impurity profiles induced by tert-butoxycarbonylation

Analysis of impurities in methamphetamine (MA) can be used to characterize MA seizures, investigate the relationship among MA seizures, and provide information on their synthetic routes. Recently, chemically derivatized MA, such as tert-butoxycarbonyl (t-Boc) MA, has been seized and attracted attention because routine forensic analysis methods may fail to correctly identify them. Chemical derivatization is a simple method for protection and deprotection of a compound, and protection of MA using t-Boc can be used to mask the MA. Although t-Boc derivatization might alter the impurity profile of MA, the actual changes in the impurity profile have not been investigated. In this study, changes in the MA impurity profile with tert-butoxycarbonylation were explored. MA and some typical impurities were derivatized using di-tert-butyl dicarbonate and water. Analysis of the impurities in five MA samples by gas chromatography showed that peaks both appeared and disappeared for the deprotected MA compared with the original MA. However, typical impurities important for characterizing MA seizures were conserved after derivatization and deprotection. Most of the new peaks were speculated to be contaminants introduced during derivatization and deprotection. A peak giving a mass spectrum similar to that of t-Boc MA was detected in the chromatograms of t-Boc MA and deprotected MA. Although the origin of this peak was not determined, it might be a marker for the MA involving tert-butoxycarbonylation. These results indicate that tert-butoxycarbonylation can alter the MA impurity profile; therefore, care is needed when interpreting results for derivatized MA.