Francisco Corte-Real

Insights into the western Bantu dispersal: mtDNA lineage analysis in Angola

Africa is the homeland of humankind and it is known to harbour the highest levels of human genetic diversity. However, many continental regions, especially in the sub-Saharan side, still remain largely uncharacterized (i.e. southwest and central Africa). Here, we examine the mitochondrial DNA (mtDNA) variation in a sample from Angola. The two mtDNA hypervariable segments as well as the 9-bp tandem repeat on the COII/tRNAlys intergenic region have allowed us to allocate mtDNAs to common African haplogroups. Angola lies in the southern end of the putative western branch of the Bantu expansion, where it met the local Khoisan populations. Angolan mtDNA lineages show basically a Bantu substrate with no traces of Khoisan lineages. Roughly, more than half of the southwestern mtDNA pool can be assigned to west Africa, ~25% to central Africa and a significant 16% to east Africa, which points to the western gene pool having contributed most to the mtDNA lineages in Angola. We have also detected signals of extensive gene flow from southeast Africa. Our results suggest that eastern and western Bantu expansion routes were not independent from each other, and were connected south of the rainforest and along the southern African savannah. In agreement with historical documentation, the analysis also showed that the Angola mtDNA genetic pool shows affinities with the African lineages from Brazil, the main American destination of the slaves from Angola, although not all lineages in Brazil can be accounted for by the Angolan mtDNA pool.

Human settlement history between Sunda and Sahul: a focus on East Timor (Timor-Leste) and the Pleistocenic mtDNA diversity

BackgroundDistinct, partly competing, “waves” have been proposed to explain human migration in(to) today’s Island Southeast Asia and Australia based on genetic (and other) evidence. The paucity of high quality and high resolution data has impeded insights so far. In this study, one of the first in a forensic environment, we used the Ion Torrent Personal Genome Machine (PGM) for generating complete mitogenome sequences via stand-alone massively parallel sequencing and describe a standard data validation practice.ResultsIn this first representative investigation on the mitochondrial DNA (mtDNA) variation of East Timor (Timor-Leste) population including >300 individuals, we put special emphasis on the reconstruction of the initial settlement, in particular on the previously poorly resolved haplogroup P1, an indigenous lineage of the Southwest Pacific region. Our results suggest a colonization of southern Sahul (Australia) >37 kya, limited subsequent exchange, and a parallel incubation of initial settlers in northern Sahul (New Guinea) followed by westward migrations <28 kya.ConclusionsThe temporal proximity and possible coincidence of these latter dispersals, which encompassed autochthonous haplogroups, with the postulated “later” events of (South) East Asian origin pinpoints a highly dynamic migratory phase.

UPLC-MS/MS determination in blood of a mixed-drug fatal intoxication: a case report.

Trends in forensic toxicology show the introduction of rapid analytical methods for the simultaneous quantitative analysis of drugs. The authors present a fatal case involving a 32-year-old male, found dead in bed by his mother, with several blue, white and orange pills next to the body. Empty tablets were found in the trash bin and a suicide note was on the desk. He was diagnosed with bipolar disorder and had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide. A rapid method was developed to determine 55 different medicines and 32 benzodiazepines in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in positive and negative ion mode. Chromatographic analysis was preceded by an optimized solid-phase extraction procedure using Oasis(®) HLB (3 cc, 60 mg) extraction columns. The extracted analytes were separated by UPLC (Waters) with a reversed-phase Acquity UPLC(®) HSS T3 (2.1×100 mm id, 1.8 μm) column with acetonitrile and 0.1% formic acid in water as mobile phase, at 0.5 mL/min flow rate and a chromatographic run-time of 8 min. Analytes detection was achieved with a triple-quadrupole mass spectrometer in positive and negative electrospray ionization mode with multiple reaction monitoring (MRM). Two MRM transitions were monitored for each target-compound and one for each deuterated internal standards. Toxicological results showed high blood concentrations of antipsychotics (haloperidol, olanzapine and quetiapine), antidepressants (fluoxetine and paroxetine) and anxiolytics (bromazepam and lorazepam). Risperidone and other benzodiazepines were also present in therapeutic concentrations. Neither alcohol nor illicit drugs were present in the analyzed samples. The UPLC-MS-MS method showed to be appropriate for screening, identification and quantitation of antipsychotics, antidepressants, anxiolytics and antiepileptic drugs in blood after intake of therapeutic as well as toxic doses. The autopsy and toxicological results led the pathologist to rule that death was due to a mixed-drug intoxication. The manner of death was determined to be suicide.

Paternal and maternal lineages in Guinea-Bissau population

The aim of the present work was to study the origin of paternal and maternal lineages in Guinea-Bissau population, inferred by phylogeographic analyses of mtDNA and Y chromosome defined haplogroups. To determine the male lineages present in Guinea-Bissau, 33 unrelated males were typed using a PCR-SNaPshot multiplex based method including 24 Y-SNPs, which characterize the main haplogroups in sub-Saharan Africa and Western Europe. In the same samples, 17 Y-STRs (included in the YFiler kit, Applied Biosystems) were additionally typed. The most frequent lineages observed were E1b1a (xE1b1a4,7)-M2 (68%) and E1a-M33 (15%). The European haplogroup R1b1-P25 was represented with a frequency of 12%. The two hypervariable mtDNA regions were sequenced in 79 unrelated individuals from Guinea-Bissau, and haplogroups were classified based on control region motifs using mtDNA manager. A high diversity of haplogroups was determined in our sample being the most frequent haplogroups characteristic of populations from sub-Saharan Africa, namely L2a1 (15%), L3d (13%), L2c (9%), L3e4 (9%), L0a1 (8%), L1b (6%) and L1c1 (6%). None of the typical European haplogroups (H, J and T) were found in the present sample of Guinea-Bissau. From our results, it is possible to confirm that Guinea-Bissau presents a typically West African profile, marked by a high frequency of the Y chromosome haplogroup E1b1a(xE1b1a4,7)-M2 and a high proportion of mtDNA lineages belonging to the sub-Saharan specific sub-clusters L1 to L3 (89%). A small European influx has been also detected, although restricted to the male lineages.

An UPLC–MS/MS method for the determination of valproic acid in blood of a fatal intoxication case

Valproic acid (VPA) has been used as an anticonvulsant for the treatment of epilepsy. The authors present a fatal case involving a 45-year-old female, found dead lying in bed with empty tablets of Diplexil(®) next to her. She was a chronic alcoholic and epileptic who had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide. A rapid method was developed and validated to determine VPA in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in negative ion mode. The method involved sample treatment with phosphoric acid followed by solid-phase extraction. Chromatographic separation was achieved using an Acquity UPLC(®) BEH (2.1 × 50 mm id, 1.7 μm) column and a mobile phase containing ammonium acetate and acetonitrile, at a 0.5 mL/min flow rate. Detection and quantification of VPA was achieved using multiple reaction monitoring (MRM). The MS/MS transitions used for monitoring were m/z 143.1-143.1 for valproic acid and m/z 296.1-205.0 for hydrochlorothiazide used as an internal standard (IS). The limit of quantification (LOQ) was 0.5 μg/mL and the method was linear in the concentration range of 0.5-100 μg/mL. The coefficients of variation obtained for accuracy and precision were less than 10% and the mean recovery was 95% for the three concentrations levels studied (5 μg/mL, 10 μg/mL and 50 μg/mL). Toxicological results showed high concentration of VPA (556 μg/mL) and therapeutic concentrations of tiapride, mirtazapine, oxazepam and nordiazepam. Blood sample analysis also revealed the presence of ethanol at a concentration of 1.34 g/L. A specific, selective and sensitive method for the determination of VPA in blood was developed and can be used in routine forensic investigation. Toxicological results led the pathologist to rule that death was due to an intoxication caused by the simultaneous ingestion of high VPA concentrations and alcohol, with a suicidal legal-medical etiology.

Spanish public awareness regarding DNA profile databases in forensic genetics: what type of DNA profiles should be included?

The importance of non-codifying DNA polymorphism for the administration of justice is now well known. In Spain, however, this type of test has given rise to questions in recent years: (a) Should consent be obtained before biological samples are taken from an individual for DNA analysis? (b) Does society perceive these techniques and methods of analysis as being reliable? (c) There appears to be lack of knowledge concerning the basic norms that regulate databases containing private or personal information and the protection that information of this type must be given. This opinion survey and the subsequent analysis of the results in ethical terms may serve to reveal the criteria and the degree of information that society has with regard to DNA databases. In the study, 73.20% (SE 1.12%) of the population surveyed was in favour of specific legislation for computer files in which DNA analysis results for forensic purposes are stored.

Biological Evidence Management for DNA Analysis in Cases of Sexual Assault

Biological evidence with forensic interest may be found in several cases of assault, being particularly relevant if sexually related. Sexual assault cases are characterized by low rates of disclosure, reporting, prosecution, and conviction. Biological evidence is sometimes the only way to prove the occurrence of sexual contact and to identify the perpetrator. The major focus of this review is to propose practical approaches and guidelines to help health, forensic, and law enforcement professionals to deal with biological evidence for DNA analysis. Attention should be devoted to avoiding contamination, degradation, and loss of biological evidence, as well as respecting specific measures to properly handle evidence (i.e., selection, collection, packing, sealing, labeling, storage, preservation, transport, and guarantee of the chain custody). Biological evidence must be carefully managed since the relevance of any finding in Forensic Genetics is determined, in the first instance, by the integrity and quantity of the samples submitted for analysis.

Qualitative and quantitative analysis of a group of volatile organic compounds in biological samples by HS-GC/FID: application in practical cases

A simple and sensitive procedure, using n-propanol as internal standard (IS), was developed and validated for the qualitative and quantitative analysis of a group of 11 volatile organic substances with different physicochemical properties (1-butanol, 2-propanol, acetaldehyde, ethyl acetate, acetone, acetonitrile, chloroform, diethyl ether, methanol, toluene and p-xylene) in whole blood, urine and vitreous humor. Samples were prepared by dilution with an aqueous solution of internal standard followed by Headspace Gas Chromatography with a Flame-ionization Detector (HS GC-FID) analysis. Chromatographic separation was performed using two capillary columns with different polarities (DB-ALC2: 30m×0.320mm×1.2μm and DB-ALC1: 30m×0.320mm×1.8μm), thus providing a change in the retention and elution order of volatiles. This dual column confirmation increases the specificity, since the risk of another substance co-eluting at the same time in both columns is very small. The method was linear from 5 to 1000mg/L for toluene and p-xylene, 50-1000mg/L for chloroform, and 50-2000mg/L for the remaining substances, with correlation coefficients of over 0.99 for all compounds. The limits of detection (LOD) ranged 1 to 10mg/L, while the limits of quantification (LOQ) ranged from 2 to 31mg/L. The intra-day precision (CV<6.4%), intermediate precision (CV<7.0%) and accuracy (relative error ±10%) of the method were in conformity with the criteria normally accepted in bioanalytical method validation. The method developed has been applied to forensic cases, with the advantages that it uses a small sample volume and does not require any extraction procedure as it makes use of a headspace injection technique.

Genetic study of 15 STRs loci of Identifiler system in Angola population.

Angola is located in the African continent, in the area of southern Africa and has a population of approximately 14 million inhabitants. The Angola population has origin from Occidental and Southern Bantu people that came from the great lakes region, creating the most ever known African migration of our days. Allele frequencies for the 15 STRs loci in the AmpFlSTR Identifiler kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, HUMTH01, D13S317, D16S539, D2S1338, D19S433, HUMVWA, TPOX, D18S51, D5S818, HUMFIBRA/FGA and including the segment of the X-Y homologous gene amelogenin) were studied for Angola population. The genotype frequency of the 15 STR loci showed no significant deviations from Hardy-Weinberg equilibrium expectations and great values for the combined power of discrimination and combined power of a priori exclusion validate the application of these markers in forensic genetics. Comparative analyses between Angola population data and other relevant population database from Africa, Europe and American are presented.

Validated UPLC-MS/MS assay for the determination of synthetic phosphodiesterase type-5 inhibitors in postmortem blood samples

The use of synthetic phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of erectile dysfunction: sildenafil citrate (Viagra(®)), tadalafil (Cialis(®)) and vardenafil hydrochloride (Levitra(®)) has increased dramatically over the past 2 years. These substances are prescription drugs and must be used under medical supervision. However, they can easily be obtained over the internet from illegal sites, being a potential for a threat to public health. The development of an electrospray ionisation (ESI) ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) procedure for the simultaneous identification and quantification of three PDE5 inhibitors in blood samples was desired. Samples were prepared using Oasis(®) HLB solid-phase cartridges (3 cc, 60 mg) and chromatographic separation was achieved on an Acquity UPLC(®) HSS T3 (100 × 2.1 mm i.d., 1.8 μm particles) column with a gradient mobile phase of 0.1% formic acid and acetonitrile at a 0.5 mL/min flow rate. Quantification was achieved by multiple reaction monitoring (MRM) of two transitions per compound: m/z 475.1 > 58 e m/z 475.1 > 311.1 for sildenafil; m/z 389.9 > 267.9 e m/z 389.9 > 134.8 for tadalafil and m/z 489 > 71.9 e m/z 489 > 150.9 for vardenafil. Zolpidem-d6 (m/z 314.5 > 235.3) was used as the internal standard. Calibration curves were linear over the concentration range of 5-1000 ng/mL, with a coefficient of determination better than 0.997. The lower limits of detection and quantification for these substances were ≤ 3 ng/mL and ≤ 8 ng/mL, respectively. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. A rapid, selective and sensitive UPLC-MS/MS method using solid-phase extraction was developed for the simultaneous determination and quantification of sildenafil, vardenafil and tadalafil in blood samples.