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.

DNA damage promotes mistyping in the allele specific oligonucleotide probing analysis of forensic samples.

Five polymerase chain reaction (PCR) products which could not be reliably typed by allele-specific oligonucleotide (ASO) probing at the human leukocyte antigen (HLA) DQA1 locus were analyzed by polyacrylamide gel electrophoresis and direct sequencing. The first method revealed the preferential amplification of only one of the two alleles in two cases. Direct sequencing of PCR products allowed unambiguous genetic typing but a high number of artifacts was observed. Several of these artifacts occurred in the sequences recognized by the ASOs. This finding provides an explanation for the mistyping in the ASO probing procedure because Taq polymerase errors both created new genetic specificities and eliminated site-specific polymorphisms. Reversed-phase HPLC-MS of the five forensic templates showed a high degree of DNA damage. These data together indicate that the risk of mistyping when using the ASO probing procedure cannot be neglected in the forensic analysis of damaged DNA samples.

Highly informative Y-chromosomal haplotypes by the addition of three new STRs DYS437, DYS438 and DYS439.

The Y chromosome STRs DYS437, DYS438 and DYS439 were selected from publicly available genome databases and used to analyse an Italian population sample. A tetraplex PCR reaction including the highly informative DYS385 locus, was set up and used for the analysis of 131 male samples to determine allele frequencies and STR diversity values. The number of different haplotypes and the haplotype diversity value found from the analysis of the STRs included in the tetraplex reaction were very similar to those found from the analysis of the basic set of 7 Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392 and DYS393) previously carried out on the same population sample. By combining the allelic states of the 11 Y-chromosomal STRs we could construct highly informative haplotypes that allowed the discrimination of 93.8% (120 out of 128) of the samples tested. This approach represents a very powerful tool for individual identification and paternity testing in forensic medicine.

Fidelity of polymerase chain reaction-direct sequencing analysis of damaged forensic samples.

Polymerase chain reaction (PCR) direct sequence analysis was performed on aged forensic samples, six or thirteen years old. This method allowed unambiguous genetic typing, but PCR products from such samples showed several artifacts. Control samples generated sequence ambiguities at a frequency of 1 in 567 bases, but the aged samples had an error frequency about 30-fold higher. In order to study the molecular composition of these aged DNA samples, reversed-phase high performance liquid chromatography (HPLC) was performed. Reduced amounts of the four DNA bases were observed and anomalous peaks were found. These peaks were analyzed by ionization mass spectrometry and identified as molecular products of DNA oxidation. The frequency of sequencing artifacts was found to be proportional to the decay of the PCR templates. Although PCR fidelity is a relevant concern in the forensic analysis of damaged samples, our data indicate that the risk of mistyping is circumventable by sequencing both strands and by performing replicate amplifications from the same PCR template.

Y-chromosomal DNA haplotype differences in control and infertile Italian subpopulations.

Y-chromosomal DNA haplotypes were determined in 74 infertile and 216 control Italian males using eight biallelic markers. A significant difference in haplotype frequency was found, but could be explained by the geographical origins of the samples. The Y chromosome is thus a sensitive marker for population substructuring and may be useful for determining whether two population samples come from a single population, for example in association studies.

Mapping the human Y chromosome by fingerprinting cosmid clones.

We have used Y-specific cosmid clones in a random fingerprinting approach to build contigs on the human Y chromosome. Clones derived from two libraries have been analyzed. The construction of one library is described here, the second was the Y chromosome-specific library LLOYNCO3 "M" (Lawrence Livermore National Laboratory). To date, we have fingerprinted 4430 cosmids: 377 contigs have been constructed containing from 2 to 39 clones. Along with the singletons, we estimate that we have covered 72.5% of the euchomatic portion of the Y chromosome with fingerprinted clones. Sequence tagged sites are being used to anchor cosmids and contigs onto the YAC framework.

DNA fingerprint analysis for the detection of induced mutations in mammalian cells in culture.

A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence of phenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with bothHinfI andHae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion withHae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.

Classification of Italian isolates of Borrelia burgdorferi into three genomic groups.

In this study we investigated the genotypic characteristics of some locally isolated strains of B. burgdorferi by three different methodologies: restriction endonuclease analysis (REA), Southern blot hybridization with whole DNAs from Borrelia strains and Southern blot hybridization with rRNA 16 + 23S genes derived from E. coli. REA fingerprintings were evaluated by cluster analysis, according to the principles of numerical taxonomy. The genomas of the locally isolated strains were compared with borreliae originating from different countries of Europe, including Sweden and with the American reference strain B31. Among the European strains, some already described by Baranton (Baranton et al., 1992) as representatives of different genomic groups Borrelia sensu stricto and Borrelia garinii were used. By the different techniques the isolates were included in three genomic groups which could correspond to the three genospecies identified by Baranton, namely B. burgdorferi sensu stricto, B. garinii and B. group VS461: in fact two strains were included in a homogeneous group, probably corresponding to the VS461 genomic group, together with other European borreliae; one isolate was included in a group consisting of B31 and some other European strains already described as belonging to Borrelia burgdorferi in sensu stricto. Finally two isolates were ascribed to a third genomic group probably corresponding to the genospecies indicated as Borrelia garinii. These findings indicate that a small number of Borrelia strains isolated from a very restricted area can be genetically heterogeneous.

Loss of heterozygosity and K-ras gene mutations in gastric cancer.

In order to identify relevant genetic lesions in gastric carcinoma, we searched for tumor suppressor gene inactivation and K-ras gene mutations by analyzing tumor and control DNAs from 34 patients. These were from an epidemiologically defined area of Italy characterized by one of the world's highest incidences of stomach cancer. Allele losses were investigated by the Southern blotting procedure at 16 polymorphic loci on 11 different chromosomes. Our data demonstrate that chromosomal regions 5q, 11p, 17p and 18q are frequently deleted, and that 7q and 13q chromosome arms are also involved, although at a lower frequency. Loss of heterozygosity (LOH) at region 11p was not found during other surveys carried out on patients of different geographic origins. No specific combination of allelic losses could be recognized in the samples analyzed, the only exception being that tumors with 17p allelic loss also showed LOH on the 18q region. When matching frequent LOH events and the stage of progression of the tumors, we observed a trend of association between advanced stages and allelic losses on 17p and 18q chromosome arms. The analysis of K-ras, carried out by the polymerase chain reaction and denaturing gradient gel electrophoresis, demonstrated transforming mutations in only 3 out of 32 cases. Colorectal tumorigenesis proceeds by the accumulation of genetic alterations, including K-ras mutations and inactivation of tumor suppressor genes on the 5q, 17p and 18q regions. Our data indicate that, although gastric and colorectal neoplasias share common genetic alterations, they probably progress through different pathways.

Sex identification by polymerase chain reaction of alpha-satellite in aged tissue samples.

In vitro amplification of the alphoid repeated sequences clustered in the centromeric regions of the human X and Y chromosomes was carried out. A modification of the amplification conditions reported by Witt and Erickson gave clear amplification bands from 10 pg of genomic DNA template. Results were obtained from the following aged tissue samples: 1 microL three-year-old blood stain, one-year-old single hair roots, six-month-old saliva stains and twelve-year-old bone fragments. Good results were obtained even when the template was artificially fragmented into fragments of less than 1 kbp.

Variation of minisatellites in chemically induced mutagenesis and in gene amplification.

A mutation assay in cultured mammalian cells was developed based on direct analysis of minisatellite DNA. Chinese hamster cells (V79) were mutagenized with nitrosoguanidine and independent colonies were isolated and expanded. DNA fingerprints were then obtained after digestion with HinfI or HaeIII and hybridization with 33.15 and 33.6 probes (Jeffreys et al., 1985). 12 colonies from untreated cells were also analyzed. Digestion with HaeIII and hybridization with 33.15 probe detected the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic effect of chemical agents. We have also analyzed the DNA fingerprints of 17 independent Chinese hamster (CHO) cell lines carrying amplification of the CAD gene. The DNA fingerprint analysis showed a variation in minisatellite regions in 3 lines while no variation was observed in independent colonies from the CHO parental cell line. The results suggest that these sequences may be hot spots for recombination during gene amplification.

Mixed chimerism in thalassemic patients after bone marrow transplantation.

Seventy-four patients with homozygous beta-thalassemia who underwent allogeneic bone marrow transplantation (BMT) were analysed in order to evaluate the incidence and the significance of mixed chimerism (MC). Using a panel of four single locus specific minisatellite DNA probes, MC was found in 36.5%, 34.7% and 16.7% of the patients at 2, 6 and 12 months respectively after BMT. Moreover we found that different pretransplant conditioning regimens could be responsible for variations in the incidence of MC. The level of residual host cells found 2 months after BMT correlated with the occurrence of rejection.

DNA homology comparison between American and European Borrelia burgdorferi strains.

Seven strains of Borrelia burgdorferi isolated from ticks and from human beings in Europe and U.S.A. were analyzed for DNA restriction patterns with several enzymes and for DNA homology in Southern blot hybridizations. The restriction patterns showed a moderately high variability. In Southern blot hybridization, strain B31 (U.S.A.) DNA gave a strong signal with itself, strain Bsf (U.S.A.) and Alcaide (isolated in Italy but presumably contracted in Venezuela). Strain B45 (F.R.G.) hybridized to itself, strain BITS (Italy) and to strain D.A. (Italy). Strain Nancy (Italy) gave a signal only when hybridized to itself, although it was classified as Borrelia on the basis of the clinical manifestations, SDS-PAGE protein pattern and antigenic determinants. No hybridization differences were observed for strains isolated from different hosts in the same continental geographical area.

Sex determination and species exclusion in forensic samples with probe cY97.

A total of 120 human samples of blood, saliva and semen stains, hair roots, bone and skin fragments, obtained from 30 males and 16 females were analyzed in Southern blots with probe cY97. Only the male samples gave a specific band of 5.7 kb. In dot blot, under high stringency conditions, male DNA gave signals equivalent to a quantity of female DNA eight times higher. Probe cY97 did not react with 9 different vertebrate species but gave a signal for monkey DNA when used at low stringency. The advantage of using a probe specific for the centromeric region for sex determination and species exclusion is discussed.

Restriction endonuclease analysis of four Borrelia burgdorferi strains.

A restriction endonuclease analysis was performed on four strains of Borrelia burgdorferi: one isolated from man (SF), one from Ixodes dammini (B31) and two from I. ricinus (BITS in Italy and B45 in Germany). Digestion by Taq I and Hae III gave the best resolution of the DNA fragments. Three different restriction patterns were obtained: BITS and B45 showed only one band difference. These results correlate with the reactivity of the four strains with monoclonal antibodies.