The GHEP-EMPOP collaboration on mtDNA population data--A new resource for forensic casework.

Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org).

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.

Allele frequencies of six miniSTR loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) in a Spanish population.

Allele frequencies and forensic parameters for six miniSTR autosomal loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) were obtained from a sample of 264 unrelated individuals from Spain. No significant deviations from Hardy-Weinberg expectations were found. Due to the small PCR products (<125 bp), the use of these non-CODIS (NC) miniSTRs can increase the probability that a degraded sample can be typed. Additionally, these systems can be used in routine paternity analyses where more markers are needed to increase the power of exclusion or in complex paternity cases (e.g. involving closely related individuals).

Mitochondrial DNA haplotyping revealed the presence of mixed up benign and neoplastic tissue sections from two individuals on the same prostatic biopsy slide.

DNA typing was requested to investigate a presumptive cancer diagnosis error by confirming whether benign and cancerous prostatic tissue in the same presurgical haematoxylin and eosin stained slide belonged to the same person. After independent histological re-examination of the slide by a pathologist, manual slide dissection was used to guarantee independent and high recovery DNA isolation from each tissue section, avoiding carryover and background contamination. Nuclear DNA quantification performed by real time polymerase chain reaction (PCR) revealed the absence of human DNA for short tandem repeat (STR) typing. Mitochondrial DNA was only obtained by performing PCR of very short fragments ( approximately 100 bp), indicating high DNA degradation. Different low frequency hypervariable region I haplotypes were obtained from each tissue section (normal tissue section haplotype: 16224C, 16234T, 16311C, 16356C; cancer tissue section haplotype: 16256T, 16270T, 16293G). Only the normal tissue section haplotype matched that obtained from the patient's blood sample, indicating that the cancer tissue section originated from an unknown patient. These results supported the hypothesis of sample mix up during block processing or slide preparation by a carryover mechanism. Mitochondrial genetic typing is recommended to exclude the possibility of carryover artefacts when low DNA content and high degradation compromise conventional STR typing.

DNA typing from skeletal remains: evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples.

AIM: To evaluate the performance of three multiplex short tandem repeat (STR) systems (AmpflSTR Profiler, AmpflSTR Profiler Plus, and AmpflSTR COfiler), and a megaplex STR system (PowerPlex 16) on DNA extracted from the skeletal remains. By performing a microbial DNA challenge study, we also evaluated the influence of microbial DNA on human DNA typing. METHODS: A subset of 86 DNA extracts isolated from 8-50 years old bone and teeth samples, corresponding to 20 identification cases from mass graves in Croatia and Bosnia and Herzegovina, and to 4 paternity cases involving deceased parents in Spain, were analyzed by the above systems. RESULTS: Bone samples with no detectable human DNA (tested with Quantiblot), as well as teeth samples with detectable human DNA, were successfully amplified. Surprisingly, even in highly degraded samples, PowerPlex 16 offered very robust amplification for the both Penta E and Penta D markers. We observed a few non-specific extra peaks of 202 and 308 base pairs, which appeared to match 16S rRNA of the Pseudomonas halodenitrificans. CONCLUSION: AmpflSTR Profiler Kit, AmpflSTR Profiler Plus Kit, the AmpflSTR COfiler Kit, and the PowerPlex 16 system are very sensitive multiplex STR amplification systems, which can be successfully used to obtain a multilocus STR profile from old teeth and bone samples with minimal amounts (pg) of human DNA or even with no detectable human DNA.

Spanish population data on the four STR loci D8S1179, D16S539, D18S51 and D21S11.

Population data were generated for four tetrameric short tandem repeat loci systems (D8S1179, D16S539, D18S51 and D21S11) for a Spanish Caucasian population sample (n = 218-219 individuals) using PCR. All loci were highly polymorphic, met Hardy-Weinberg expectations and the results demonstrated the assumption of independence of the loci analysed. The allele frequency data can be used in identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Spanish population.

Characterization of the visual performance with soft daily wear disposable contact lenses.

We investigate the visual performance associated with adaptation to a daily wear soft contact lens on the human eye. For this purpose, we used four parameters, one of which was an objective parameter, while the rest were subjective parameters. The objective parameter was a single quality parameter, a Merit function (Mf) derived from the modulation transfer function (MTF) of the overall [eye + contact lens] system The subjective parameters were the visual acuity (VA), the contrast sensitivity function (CSF) and the standard adaptation criterion of Terry et al. (1993). The normality criterion for the MTF was determined by evaluating the fluctuations of the Mf over a day in five emmetropic observers. Fluctuations with no statistically significant differences in the merit function (p > 0.05) and their standard deviation (8%) defined our standard criterion. The CSF and the VA were similarly measured (for emmetropic observers). The results obtained with emmetropic observers allowed us to establish a standard criterion for the evaluation parameters we propose. When this criterion is applied to daily soft wear disposable contact lenses, their performance proves to be good, since both the objective (MTF) and the subjective parameters (CSF, VA, adaptation criterion) always lie within the range defined by our criterion.

Basque Country autochthonous population data on 7 short tandem repeat loci.

Blood samples from 202-208 unrelated Basque Country autochthonous individuals were amplified, typed and their allele frequencies were determined. Results demonstrate the assumption of independence within and between the loci analyzed. Therefore, a Basque population database can be used in identity testing to estimate the frequency of a multiple PCR-based locus DNA profile.

A Spanish population study of the STR loci HumLPL, D5S818, D7S820 and D13S317.

Allele and genotype frequencies for four tetrameric short tandem repeat loci were determined in a Spanish population sample (N=193-225) using PCR. All loci met Hardy-Weinberg expectations and the results demonstrated the assumption of independence of the loci analysed. The allele frequency data can be used in identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Spanish population.

A simple technique for measurement of corneal thickness.

Measurement of corneal thickness has potential benefit both in the fitting of contact lenses and in monitoring any pathology that could affect corneal thickness. Corneal thickness measurement is undertaken using an accessory to the biomicroscope, the optical pachometer, or by means of expensive apparatus such as the ultrasonic pachometer. There are other complex methods, such as laser Doppler interferometry, or ultrasonic rasters of the cornea. In this study, an easier and low cost method, based on the measurement of the optical section formed of the cornea by the biomicroscope illumination system, is proposed. The advantages of this new method are the simplicity of its experimental set-up which consists of a calibrated graticule in an eyepiece of the biomicroscope, the speed of the measurement which increases with practice and the low cost, due to the fact that the biomicroscope is standard equipment in the ophthalmic consulting room.

Detection of somatic mutations in the mitochondrial DNA control region of colorectal and gastric tumors by heteroduplex and single-strand conformation analysis.

Each entire hypervariable region of the mitochondrial DNA control region was screened for mutations from paired normal and tumor DNA corresponding to a group of 21 patients (13 colorectal and 8 gastric adenocarcinomas) using both heteroduplex analysis and single-strand conformation analysis. These two mutation scanning strategies allowed the identification of sequence alterations in 3/13 (23%) colorectal tumors and in 3/8 (37%) gastric tumors. Heteroduplex analysis showed the heteroplasmic state of the majority of these tumor mutations. Sequence analysis revealed two A:T/G:C transitions (nucleotide positions: 16241 and 16166) in hypervariable region 1 (HV1) and two C:G/T:A transitions (nucleotide positions: 76 and 312), one A:T/G:C transition (nucleotide position: 93), a 1-basepair C:G deletion (nucleotide position: 309), and a 2-base-pair CC:GG insertion (nucleotide position: 309) in the HV 2 region. A considerable proportion of these mutations was found in homopolymeric regions which are highly polymorphic among humans. Different mechanisms (clonal expansion, increased oxidative damage, and nuclear mutator mutations) were suggested to explain the increased mitochondrial DNA mutation rate observed in cancer.

A review of the collaborative exercises on DNA typing of the Spanish and Portuguese ISFH Working Group. International Society for Forensic Haemogenetics.

Since 1992 the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Haemogenetics (ISFH) has been organizing collaborative exercises on DNA profiling with the aim of making progress on standardization and discussing technical and statistical problems in DNA analysis. A total of four exercises (GEP-92 to GEP-95) have been carried out until now. A consequence of these exercises was the creation of a quality control programme in Spain and Portugal in 1995 which was carried out simultaneously with the GEP-95 exercise. The number of participating laboratories increased from 10 in the first exercise (GEP-92) to 19 in the last exercise (GEP-95). Despite this increasing number of participating laboratories, results remained satisfactory. In the last exercises, all the laboratories used PCR-based DNA polymorphisms with an increasing number of markers obtaining good results. SLPs were used by only 30% of laboratories in the last two exercises but the results indicated a good level of expertise in most of these laboratories. The reasons for these successful results are the common use of the EDNAP protocol for SLP analysis and commercially available kits or common sequenced allelic ladders for PCR-based DNA polymorphisms.

Contact lenses: do they really change the optical performance?

In this investigation the quality of two types of optical correction, hydrogel contact lenses and ophthalmic lenses, was compared by measuring the modulation transfer function (MTF) of the correcting lens plus visual optics system using an objective method. A merit function was defined in order to allow the direct comparison between the optical performance with the two corrections. A study on 10 subjects was undertaken, measuring the MTF of both corrections by using the double pass method. The results show that the optical quality of contact lenses was higher than that with ophthalmic lenses. The contrast sensitivity function (CSF) and visual acuity (VA) were determined as subjective parameters of visual performance.

Influence of the tear film on optical quality of the eye.

The optical quality of the tear film of the eye was tested by measuring the retinal image before and after the break-up time (BUT). An objective method was used based on the evaluation of the retinal image of a point test projected onto the fovea. The loss of an entire tear film would result in a decrease in the optical quality because of corneal irregularities and the formation of an irregular tear lens after the BUT. Our results confirm the expected loss both of non-contact lens wearers and contact lens wearers. Also, the fact that the optical deterioration found after the BUT is greater for contact lens wearers confirms that soft contact lens wear produces a disruption of the tears.

Rapid detection of sequence polymorphisms in the human mitochondrial DNA control region by polymerase chain reaction and single-strand conformation analysis in mutation detection enhancement gels.

The article describes a rapid approach for the detection of sequence polymorphisms in the mitochondrial (mt)DNA control region that involves enzymatic amplification of each entire mtDNA control region (HV1 and HV2) and the subsequent analysis of the PCR products by single-strand conformation analysis (SSCA) in mutation detection enhancement (MDE) gels, followed by silver stain detection. HV1 and HV2 SSC reference ladders were developed to standardize the classification of the different mtDNA types. Twenty-five mtDNA types were observed among the 45 Spanish individuals analyzed: 11 types were observed in the HV1 region as compared with 10 types in the HV2 region. This mutation scanning strategy could be a promising method of potential use not only in forensic genetics but also in population and evolutionary studies.

A HinfI polymorphism in the 5' flanking region of the human VNTR locus D1S80.

A restriction fragment length polymorphism (RFLP) characterized by the presence (HinfI+) or absence (HinfI-) of a HinfI site has been found in the 5' flanking region of the VNTR locus D1S80. RFLP-allele frequencies were determined from 82 unrelated individuals: HinfI+ = 0.49, HinfI- = 0.51. The RFLP/VNTR haplotype frequencies show an absolute association between the HinfI+ allele and the VNTR allele of 18 repeat units and an extreme association between the HinfI- allele and the VNTR allele of 24 repeat units. The remaining VNTR alleles associate more randomly with the 2 flanking HinfI alleles.

Spanish population data on 7 tetrameric short tandem repeat loci.

Allele and genotype frequencies for 7 tetrameric short tandem repeat loci were determined in a Spanish population sample (N = 186-244) using PCR and subsequent analysis of the PCR products by denaturing polyacrylamide gel electrophoresis followed by silver staining. The loci were HUMFES/FPS, HUMVWA, HUMTHO1, HUMF13B, HUMCSF1PO, HUMF13A1 and HUMTPOX and all loci met Hardy-Weinberg expectations. In addition, little evidence was found for association of alleles among the 7 loci. Thus the allele frequency data can be used in identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Spanish population.

Amplified fragment length polymorphism analysis of the VNTR locus D1S80 in central Spain.

The polymorphism of the D1S80 locus has been analyzed in a population sample of 203 unrelated individuals living in Madrid (central Spain) by PCR and subsequent semi-dry discontinuous polyacrylamide gel electrophoresis (Tris-chloride/Tris-glycine buffer system) followed by silver staining. The electrophoretic system described in this study offers high resolution in the separation of the different D1S80 alleles allowing the detection of microvariability around the allele T22 in the spanish population. Twenty different alleles containing 17-40 repeats of the basic 16 bp unit were distinguished. The alleles T18 and T24 were found to be relatively common in Spain, as in other populations, with frequencies of 0.224 and 0.372, respectively. No evidence of significant deviations from Hardy-Weinberg equilibrium was found in these preliminary population data.