2006 GEP-ISFG collaborative exercise on mtDNA: reflections about interpretation, artefacts, and DNA mixtures.

We report the results of the seventh edition of the GEP-ISFG mitochondrial DNA (mtDNA) collaborative exercise. The samples submitted to the participant laboratories were blood stains from a maternity case and simulated forensic samples, including a case of mixture. The success rate for the blood stains was moderate ( approximately 77%); even though four inexperienced laboratories concentrated about one-third of the total errors. A similar success was obtained for the analysis of mixed samples (78.8% for a hair-saliva mixture and 69.2% for a saliva-saliva mixture). Two laboratories also dissected the haplotypes contributing to the saliva-saliva mixture. Most of the errors were due to reading problems and misinterpretation of electropherograms, demonstrating once more that the lack of a solid devised experimental approach is the main cause of error in mtDNA testing.

Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group.

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.

Mutation rates at Y chromosome specific microsatellites.

A collaborative work was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) to estimate Y-STR mutation rates. Seventeen Y chromosome STR loci (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460, DYS461, DYS635 [GATA C4], GATA H4, and GATA A10) were analyzed in a sample of 3,026 father/son pairs. Among 27,029 allele transfers, 54 mutations were observed, with an overall mutation rate across the 17 loci of 1.998 x 10(-3) (95% CI, 1.501 x 10(-3) to 2.606 x 10(-3)). With just one exception, all of the mutations were single-step, and they were observed only once per gametogenesis. Repeat gains were more frequent than losses, longer alleles were found to be more mutable, and the mutation rate seemed to increase with the father's age. Hum Mutat 26(6), 520-528, 2005. (c) 2005 Wiley-Liss, Inc.

A STR mutation in a heteropaternal twin case.

A heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the first report on a STR mutation in a double paternity case where both biological fathers were indisputably identified. The STR systems enable the resolution of these complex genetic relationships even in a case where a mutation in one STR locus was encountered.

Sequence variation of new alleles at the short tandem repeat D19S253 locus.

This paper reports the sequences of two new alleles identified in a population database study on the short tandem repeat D19S253 locus. A Portuguese Caucasian population and a Portuguese African population were studied. Forty-four selected alleles were sequenced and 11 different alleles were found. All the sequenced alleles shown to possess a simple tetranucleotide GATA repeat region structure. The two new alleles, alleles 6 and 16, follow the simple repeat pattern. During paternity investigation casework, 1028 meiosis were analyzed and five isolated genetic incompatibilities detected. In one case, a non-detectable allele with the used set of primers could be the explanation. In the other four cases, single-step mutations could be considered. The mutation rate obtained for this locus was 3.89 x 10(-3).

Portuguese population and paternity investigation studies with a multiplex PCR--the AmpFlSTR Profiler Plus.

A Portuguese Caucasian population of 146 unrelated individuals was studied. DNA samples were amplified by multiplex PCR for D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 using the AmpFlSTR Profiler Plus PCR Amplification Kit (Perkin-Elmer). All loci met Hardy-Weinberg expectations. Forensic statistical parameters were according to those obtained by other authors. Statistical differences were observed concerning three loci when comparing the Portuguese Caucasian population and an Italian Caucasian population, although these differences mainly concern the less frequent alleles. Eighty-three paternity investigation cases were analysed. Exclusions in between three and nine loci were observed in all the 23 exclusion cases obtained. Most of the non-exclusion cases had probability of paternity > 99.9%. Two cases with an isolated genetic incompatibility between the alleged father and the child were detected, which may indicate probable mutation cases. These results demonstrate that the AmpFlSTR Profiler Plus is a suitable multiplex for paternity investigation in the Portuguese population.

South Portuguese population data on the loci HLA-DQA1, LDLR, GYPA, HBGG, D7S8 and Gc.

Five South Portuguese Caucasian subpopulations were analyzed for the HLA-DQA1, LDLR, GYPA, HBGG, D7S8 and Gc loci. Genotype distributions for these loci did not deviate from Hardy-Weinberg expectations. The allele and genotype frequencies found have been compared with previously published data from North and Central Portugal. A total of 11 out of 138 chi-square comparisons of allele frequencies between different Portuguese populations showed a certain degree of divergence. Alentejo, Algarve, Madeira Island and Azores Islands populations might be considered as different groups in a database. For forensic casework, a composite South Portuguese Caucasian population database was obtained for estimating multiple locus profile frequencies using the six PCR-based loci studied.

Alpha-1-antitrypsin and transferrin null alleles in the Portuguese population.

In a Portuguese family, a null allele was found in the Pi system. An apparent 'exclusion' of the mother was found to be due to the presence of null alleles in mother and child. A transferrin (Tf) null allele was found in a case of disputed paternity. The mother and putative father were heterozygous for Tf null alleles and the child was homozygous (TfQ0) and presented hypotransferrinemia.

Hyperamylasemia with an abnormal isoamylase distribution in patients with liver diseases.

Isoenzymes of amylase were studied in sera from 95 patients with various liver diseases and 82 controls. Twenty-two patients and two controls had hyperamylasemia. Four different zymograms were obtained by isoelectric focusing--normal, pancreatic, salivary/pancreatic, and double pancreatic. The prevalence of these zymograms was similar in controls and in patients with normal values of serum amylase, normal being the prevalent zymogram. Patients with hyperamylasemia had as predominant the salivary/pancreatic zymogram, corresponding to isoenzymes of similar isoelectric point common to both salivary and pancreatic secretions. The inhibitory assay revealed a predominant increase of pancreatic type isoamylase and showed a poor correlation with isoelectric focusing. Our findings indicate that hyperamylasemia occurs frequently in liver diseases and in most cases is associated with an abnormal electrophoretic pattern.