A Ge.F.I. - ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method.

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists - International Society of Forensic Genetics (Ge.F.I. - ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of -1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

Engraftment capacity of mesenchymal cells following hematopoietic stem cell transplantation in patients receiving reduced-intensity conditioning regimen.

The engraftment ability of mesenchymal cells was investigated in 26 patients receiving allogeneic transplantation from HLA-identical siblings with reduced-intensity conditioning (RIC). The stem cell source was bone marrow (BM) in eight patients and G-CSF-mobilized peripheral blood hematopoietic cells in 18 cases. A total of 32 patients engrafted very quickly and the chimerism evaluation (both on myeloid and on lymphoid subsets) showed that they were full donor by day 60. At the time of the study they were in complete hematological remission and displayed a full donor hematopoiesis. Two patients showed early disease progression while one did not engraft. Forty-eight out-marrow samples harvested from the 26 patients generated a marrow stromal layer adequate for the chimerism evaluation. Monocyte-macrophage contamination of marrow stromal layers was always reduced below 2% by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. The chimerism evaluation was performed by PCR analysis of STRs microsatellites and the amelogenin locus, by using capillary electrophoresis (CE) and by FISH analysis in case of the sex mismatch. In eight patients, a partial donor origin of stromal cells was shown (7-86% cells of donor). The source of hematopoietic cells was BM in three patients and mobilized peripheral blood in the other five.

Characterization of an outbreak of tetM-containing Neisseria gonorrhoeae in Argentina.

Phenotypic and molecular characterization of an outbreak of 9 Neisseria gonorrhoeae (NG) isolates exhibiting high-level plasmid mediated resistance to penicillin and tetracycline (PP-TRNG) that took place in Tandil, Argentina between February and April 1995. Comparison with the patterns of the 3 PP-TRNG strains previously isolated were made. We determined the following markers for each strain: antimicrobial susceptibility, serogroup, auxotype, plasmid profile, presence of tetM determinant and restriction pattern of the tetM-containing plasmid. Antimicrobial tests values were: tetracycline disk diameter 12-14 mm, minimum inhibitory concentration (MIC) 32 micrograms/ml; penicillin disk diameter 6 mm, MIC 32 micrograms/ml and sensitive by both methods to spectinomycin, cefuroxime, ceftriaxone and ciprofloxacin. All isolates were of the same serogroup (WI). Ten of the strains, including the 9 from Tandil outbreak, were arginine-requiring, while the other 2 were methionine and arginine-requiring. All of them demonstrate the same plasmid profile (2.6, 3.2, 25.2 MDa). They were positive for the tetM determinant and the restriction analysis identified it is a Dutch-type plasmid. In spite of the temporal and geographical dispersion, PP-TRNG strains in Argentina seem to be highly homogeneous in terms of antimicrobial susceptibility, serogroup, plasmid profiles and even auxotype.

PIP: This study determines the phenotypic and molecular characterization of an outbreak of 9 Neisseria gonorrhea isolates exhibiting high-level plasmid-mediated resistance to penicillin and tetracycline (PP-TRNG) that took place in Tandil, Argentina. Subjects included patients attending Tandil's Sanatamarina Hospital. Individuals infected with sexually transmitted disease were examined using a standardized questionnaire. Results show that all isolates produced B-lactamase, were penicillin resistant (disk zone diameter 6 mm, MIC 32 mcg/ml) and high-level tetracycline resistant (disk zone diameter 12-14 mm, MIC 32 mcg/ml). Moreover, all strains were fully sensitive to cefuroxime, ceftriaxone, ciprofloxacin, and spectinomycin; while autotype and serogroup results showed that all the strains were arginine-requiring and of serogroup WI. In spite of the temporal and geographical dispersion, PP-TRNG strains in Argentina seem to be highly homogeneous in terms of antimicrobial susceptibility, serogroup, plasmid profiles, and even auxotype.

Polymerase chain reaction typing of D21S11 short tandem repeat polymorphism by capillary electrophoresis. Allele frequencies and sequencing data in a population sample from central Italy.

Blood samples were collected from 100 individuals living in Central Italy and analysed for STR locus D21S11 by capillary electrophoresis on an ABI Prism 310 genetic analyzer. For fragment sizing, PCR amplification products, obtained using a 6-FAM 5'-labeled reverse primer and an unlabeled forward primer, were run with an internal size standard labeled with TAMRA dye and typed using the local reciprocal method. An allele ladder consisting of a mix of sequenced amplified products was also prepared. An Italian population database was established. No deviation from Hardy-Weinberg equilibrium was observed. The result of statistical analysis were highly informative (PD = 0.94; mean exclusion change = 0.66). DNA sequencing was performed on a set of representative alleles by Taq cycle sequencing using dye terminator labeling chemistry. A new structural variant was found.

Y-chromosome haplotypes in Italy: the GEFI collaborative database.

A sample of 1176 males from 10 Italian regions have been typed for DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, and DYS385. Individual haplotype data are available on line. A low degree of variation is present among regions. Use of this database is specifically recommended for forensic applications in Italy.

Polymorphism and sequence variations of the HumCD4 pentameric microsatellite in an Italian population sample.

A sample of 265 subjects from central Italy was analyzed at the HumCD4 locus by polymerase-chain-reaction (PCR). Phenotypes were identified by comparison with a sequenced ladder, after high-resolution horizontal polyacrylamide gel electrophoresis (PAGE) followed by silver staining. A set of representative alleles was sequenced by Taq-cycle-sequencing with dye terminator labeling and capillary gel electrophoresis strategies. Eight common alleles--5, 6, 7, 8, 9, 10, 11, 12--and a rare larger 14, never before described in Caucasians, were found. Allele and genotype frequencies were similar to those described in former studies on Caucasians, with a prevalency of alleles number 5, 6, 10. Sequence analysis showed that the polymorphism is due to a pentameric TTTTC basic motif, tandemly repeated, and that from allele number 10 onwards the fourth repeat presents a T to C translation (CTTTC). Instead, allele number 9 may exist in two forms, because 75% of alleles examined in this study presented the CTTTC motif at the fourth position, while the remaining 25% had the basic repeat structure.

Polymorphism of the mitochondrial DNA control region in Italians.

Mitochondrial DNA sequences of the hypervariable regions HVI and HVII were analysed in 83 Caucasians living in central Italy to expand the database for forensic identification purposes, and 75 different haplotypes resulting from 62 polymorphic positions in HVI and 44 in HVII were observed. The most frequent haplotype (263G, 309.1C, 315.1C) was shared by 7 individuals, 2 haplotypes were shared by 2 individuals, and 72 were unique. The genetic diversity was found to be 0.99 and the random match probability 1.9%. A condition of sequence heteroplasmy was found in only one case at nt 16311, whereas a length heteroplasmy was found in the homopolymeric stretch of cytosines 303-315. Our results indicate that in direct sequencing beyond the poly-cytosine stretch, the overlap is due to length heteroplasmy, whereas the blurred signal occurs when the stretch is composed of more than 10 cytosines.

PCR typing of the locus D17S30 (YNZ22 VNTR) in an Italian population sample.

A sample of 202 subjects living in 2 Italian provinces (Ancona and Parma) was tested for YNZ22 polymorphism by the polymerase chain reaction (PCR). After amplification, the phenotypes were separated by agarose gel electrophoresis, stained with ethidium bromide and identified by comparison with a molecular weight marker. No heterogeneity was found between the 2 populations. Alleles, pooled in 4 groups to calculate the Hardy-Weinberg (H-W) equilibrium, showed good accordance between observed and expected values. The power of discrimination (PD) was 0.95 and the chance of exclusion was 0.69. The allele comparison with previous studies on Caucasians showed no significant difference.

PCR analysis of the short tandem repeat (STR) system HUMVWA31. Allele and genotype frequencies in an Italian population sample.

A population study in a sample of 211 unrelated individuals from 2 cities in North and Central Italy was carried out to investigate the short tandem repeat (STR) system HUMVWA31. Separation of PCR- amplified DNA fragments was performed by high-resolution horizontal denaturing polyacrylamide gel electrophoresis (PAGE) followed by silver staining. The 7 common alleles were found, together with a new smaller allele. Distribution of the observed genotypes did not deviate from Hardy-Weinberg (H-W) equilibrium. The power of discrimination for this locus was 0.93 and the chance of exclusion was 0.61. Good agreement was found between the allele frequencies in 2 Italian population samples and previous studies on Caucasians. The results of this study suggest that this STR system may be a useful tool in forensic investigations.

Frequency of HLA DQA1 alleles in an Italian population.

A sample of 103 Italians was tested for HLA-DQA1 polymorphism using the polymerase chain reaction (PCR) and dot blot hybridization. Results were in Hardy-Weinberg equilibrium. The power of discrimination was 0.91 and the rate of exclusion 56.7. The frequencies of the DQA1*0201 and DQA1*0301 alleles were found to be significantly different from other Caucasian populations.

PCR analysis of the STR HUMF13A01 in a population sample of central Italy.

A population genetic study was carried out on an Italian sample of Central Italy (n = 159) using the short tandem repeat (STR) system HUMF13A01. After vertical electrophoresis in denaturing polyacrylamide gel, 11 alleles were found. No deviation from the Hardy-Weinberg equilibrium were observed. The power of discrimination (PD) was 0.91 and the chance of exclusion was 0.59. The allelic comparison with previous Italian population studies showed no significant difference.

Opiate-related death: morphometric study of neurons from the dorsal motor nucleus of the vagus nerve.

The aim of this work is to verify the presence of objectively observable morphological alterations caused by the direct action of opiates on brain structures controlling respiration in cases of drug-related deaths. A computer-assisted morphometric study was carried out on the Dorsal Motor Nucleus of the Vagus Nerve (DMNV) in seven subjects (four of whom died of drug overdose, and 3 controls). Serial sections 10-15 microns thick were stained according to Nissl. The sections were then examined under the optical microscope and 200 fields per subject were digitalized. The resulting images, regarding at least 700 neurons per subject were segmented using a semi-automatic procedure and analysed by the Optilab software. The data obtained were subjected to cluster analysis to identify the neuron classes in the DMNV. Each subject was characterized with a vector of percentages indicating the fraction of neurons falling in each of these classes. Discriminant analysis was then applied to verify to what extent the variables discriminated between the two populations. Results indicated that: the statistical methods adopted have a good power of discrimination; in subjects who died of opiate overdose, the neurons are distributed to a greater extent in classes composed of smaller neurons than in controls.

Allele typing of short tandem repeats by capillary electrophoresis.

Capillary electrophoresis with laser-induced fluorescence was applied to the analysis of six STRs and the amelogenin sex test with the purpose of verifying accuracy and precision of the sizing method with the GS500 internal standard. Sequenced dye-labeled, PCR-amplified alleles from amelogenin, HumVWA31, HumTH01, HumF13A01, HumFIBRA, D21S11 and HumCSF1PO loci were run several times on the same capillary and on multiple capillaries and the offset of computer-measured fragment sizes from the expected molecular weights was calculated and analysed. All loci except D21S11 showed a poor degree of accuracy. Precision results from run-to-run and day-to-day injections displayed a maximum standard deviation (SD) > 0.15 nt for HumVWA31, HumF13A01, D21S11 and HumFIBRA, although the maximum range of calculated sizes in multiple runs was lower than 1 basepair. No variation in precision was observed according to the quality of the DNA template. Allele typing by comparison with allelic ladders for each locus is recommended.

Ventricular enlargement in adults with profound mental retardation who demonstrate violent/destructive behaviors.

The authors determined brain ventricular size (ventricular brain ratio [VBR]) in 39 ambulatory adult mentally retarded individuals who did or did not require psychotropic medication for violent/destructive behavior. The mean VBR of mentally retarded patients who required psychotropic medication was significantly greater than the VBR of those who did not require such medication.

Italian population data on the loci LDLR, GYPA, HBGG, D7S8 and GC.

Allele and genotype frequencies for the five loci LDLR, GYPA, HBGG, D7S8 and GC were determined for 374 unrelated Italians using a multiplex PCR-amplification and typing commercial kit. The distribution of the genotype frequencies showed no deviations from Hardy-Weinberg expectations. The combined power of discrimination and chance of exclusion for all five loci were 0.999 and 0.702, respectively. A test for homogeneity was performed and no significant differences were observed among the Caucasian population samples.