Analytical and clinical validation of a dried blood spot assay for the determination of paclitaxel using high-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Paclitaxel (PCT) is a chemotherapeutic drug widely used for the treatment of several types of tumors, and its use is associated with severe adverse events, mainly neurologic and hematopoietic toxicities. The relation between systemic exposure and clinical response to PCT was previously described, making paclitaxel a potential candidate for therapeutic drug monitoring (TDM). The use of dried blood spot (DBS) sampling could allow complex sampling schedules required for TDM of PCT. The aim of this study was to develop and validate an LC-MS/MS assay for the quantification of PCT in DBS. METHODS: PCT was extracted from one 8 mm DBS punch with a mixture of methanol and acetonitrile, followed by chromatographic separation in a Kinetex C18 (50 × 4.6 mm, 2.6 µm) column. Detection was performed in a 5500-QTRAP® mass spectrometer, with a run time of 2.3 min. RESULTS: The assay was linear in the range of 2.5 to 400 ng mL-1. Precision (CV%) and accuracy at the concentration levels of 7.5, 40 and 150 ng mL-1 were 1.69-4.9% and 106.25 to 109.92%, respectively. PCT was stable for 21 days at 25 and 45 °C. The method was applied to DBS samples obtained from 34 patients under PCT chemotherapy. The use of a simple correction factor, derived from the correlation between PCT concentrations in plasma and DBS in this set of patients, allowed unbiased estimation of PCT plasma concentrations from DBS measurements, with similar clinical decisions using either plasma or DBS measurements. CONCLUSIONS: DBS testing of PCT concentrations represents a promising alternative for the dissemination of PCT dose individualization.

Evolutionary history of Podarcis tiliguerta on Corsica and Sardinia.

BACKGROUND: Podarcis tiliguerta is a wall lizard endemic to the Mediterranean islands of Corsica and Sardinia. Previous findings of high mtDNA and morphological diversity have led to the suggestion that it may represent a species complex. Here, we analysed mitochondrial and nuclear markers (mtDNA, 3110 bp; 6 nDNA loci, 3961 bp) in P. tiliguerta sampled from thirty-two localities across Corsica and Sardinia. RESULTS: We find much greater intraspecific genetic divergence than between sister species of other Mediterranean island Podarcis, i.e., between P. lilfordi and P. pityusensis. We detected three mtDNA clusters in Corsica (North, South-East and South-West) and either two or three in Sardinia (North vs. South) depending on the clustering method. Only one or two nDNA groups were identified within each main island (again, depending on the method). A Bayesian time-calibrated multispecies coalescent tree was obtained from mtDNA and provided statistical support for a Miocene origin of the species (13.87 Ma, 95% HPD: 18.30-10.77 Ma). The posterior mean divergence time for the Corsican and Sardinian lineages was 12.75 Ma ago (95% HPD: 16.94-9.04 Ma). CONCLUSION: The results support the evolutionary distinctiveness of Corsican and Sardinian populations and also indicate a lack of post-divergence migration despite periods of contact being possible. Further to this, species delimitation analyses of Corsican and Sardinian lineages provided statistical support for their recognition as distinct (sister) taxa. Our results provide new insights into the biogeography of the Mediterranean biodiversity hotspot, and contribute important findings relevant to the systematics and evolution of this speciose lizard genus.

Multilocus genetic diversity and historical biogeography of the endemic wall lizard from Ibiza and Formentera, Podarcis pityusensis (Squamata: Lacertidae).

Two monophyletic sister species of wall lizards inhabit the two main groups of Balearic Islands: Podarcis lilfordi from islets and small islands around Mallorca and Menorca and Podarcis pityusensis from Ibiza, Formentera and associated islets. Genetic diversity within the endangered P. lilfordi has been well characterized, but P. pityusensis has not been studied in depth. Here, 2430 bp of mtDNA and 15 microsatellite loci were analysed from P. pityusensis populations from across its natural range. Two main genetic groupings were identified, although geographical structuring differed slightly between the mtDNA and the nuclear loci. In general, individuals from islets/islands adjacent to the main island of Ibiza were genetically distinct from those from Formentera and the associated Freus islands for both mtDNA and the nuclear loci. However, most individuals from the island of Ibiza were grouped with neighbouring islets/islands for nuclear loci, but with Formentera and Freus islands for the mitochondrial locus. A time-calibrated Bayesian tree was constructed for the principal mitochondrial lineages within the Balearics, using the multispecies coalescent model, and provided statistical support for divergence of the two main P. pityusensis lineages 0.111-0.295 Ma. This suggests a mid-late Pleistocene intraspecific divergence, compared with an early Pleistocene divergence in P. lilfordi, and postdates some major increases in sea level between 0.4 and 0.6 Ma, which may have flooded Formentera. The program IMa2 provided a posterior divergence time of 0.089-0.221 Ma, which was similar to the multispecies coalescent tree estimate. More significantly, it indicated low but asymmetric effective gene copy migration rates, with higher migration from Formentera to Ibiza populations. Our findings suggest that much of the present-day diversity may have originated from a late Pleistocene colonization of one island group from the other, followed by allopatric divergence of these populations. Subsequent gene flow between these insular groups seems likely to be explained by recent human introductions. Two evolutionary significant units can be defined for P. pityusensis but these units would need to exclude the populations that have been the subjects of recent admixture.

Variability of the mc1r gene in melanic and non-melanic Podarcis lilfordi and Podarcis pityusensis from the Balearic archipelago.

The association between polymorphism at the mc1r locus and colour variation was studied in two wall lizard species (Podarcis lilfordi and P. pityusensis) from the Balearic archipelago. Podarcis lilfordi comprises several deep mitochondrial lineages, the oldest of which originated in the Pliocene, while much shallower mitochondrial lineages are found in P. pityusensis. Here, we examined whether specific substitutions were associated with the melanic colouration found in islet populations of these species. Homologous nuclear sequences covering most of the mc1r gene were obtained from 73 individuals from melanic and non-melanic Podarcis from different populations (the entire gene was also sequenced in six selected individuals). MtDNA gene trees were also constructed and used as a framework to assess mc1r diversity. Mc1r showed greater polymorphism in P. lilfordi than in P. pityusensis. However, we observed no substitutions that were common to all melanic individuals across the two species. Only one significant association was detected in the mc1r partial sequence, but this was a synonymous A/G mutation with A alleles being more abundant in melanic populations. In addition, there were no associations between the main dominant phenotypes (green and brown, blue and yellow spots and ventral colour) and synonymous or non-synonymous substitutions in the mc1r gene. There was no statistical evidence of selection on mc1r. This study suggests no relationship between mc1r polymorphism and colour variation in Balearic Podarcis.

Balearic archipelago: three islands, three beta-thalassemia population patterns.

The mutation spectrum of 175 ß-thalassemia (ß-thal) carriers, identified in pilot carrier screening on 22,713 individuals from Balearic Islands (Spain), is reported. The ß(0) CD39 (C>T) mutation is the most frequent (61.1%), followed by ß(+) IVS-I-110 (G>A) (12.0%), ß(+) IVS-I-6 (T>C) and ß(0) IVS-1-1 (G>A) (3.4% both) and eight other rare mutations (2.9-0.6%); with a distinct prevalence and distribution between islands. Minorca shows the highest prevalence in Iberian populations, with a single mutation, CD39 (C>T), present in most ß-thal carriers. Ibiza is the only Western Mediterranean population where the most frequent ß-thal mutation is IVS-I-110 (G>A). These results can be explained by a combination of historical-demographic characteristics together with evolutionary forces such as founder effect, genetic drift and probably selection by malaria. Knowledge of the mutational spectrum in the Balearic Islands will enable to optimize mutation detection strategy for genetic diagnosis of ß-thal in these islands.

Allele frequencies of 15 STRs in the Calchaqui Valleys population (North-Western Argentina).

Allele frequencies for 15 short tandem repeat (STR) loci were obtained from a sample of 110 individuals from the Calchaqui Valleys population (North-Western Argentina). The combined power of exclusion and combined power of discriminating for the 15 tested STR loci were 0.999964 and 0.9999999999999998, respectively. Matching probability was 1 in 4.58 × 10(15). Therefore, it may be concluded that the set of 15 STRs included in the AmpF STR Identifiler kit, represents a powerful tool for forensic applications, paternity testing and population genetics studies in the Calchaqui Valleys population.

Mitochondrial DNA effects on fitness in Drosophila subobscura.

We tested different fitness components on a series of conspecific mtDNA haplotypes, detected by RFLPs in Drosophila subobscura. Additionally, haplotype VIII, endemic to the Canary Islands, was tested upon its own native nuclear DNA background and upon that of the rest of mtDNAs tested herein. We found that both nuclear and mitochondrial DNA can have a significant effect upon their hosts' fitness, and that negative selection is one of the mechanisms that can intervene in this species' mtDNA haplotype pattern. We discuss the importance of this mechanism in relation to genetic drift, in the form of periodic population bottlenecks, and how the latter can enhance the former. We also detected a significant positive effect of haplotype VIII upon fitness that could explain in part the dominance of this endemic haplotype on some of the Canary Islands, and a mitochondrial heterosis involving this haplotype when on a foreign nuclear DNA background.

Clinic and genetic evaluation of variegate porphyria (VP) in a large family from the Balearic Islands.

Variegate porphyria (VP) (an autosomal dominant disease), is clinically characterized by skin photosensitivity and/or acute neurovisceral crises and biochemically by high levels of faecal protoporphyrin and coproporphyrin. It results from the partial deficiency of protoporphyrinogen oxidase (PPOX gene). Genetic heterogeneity has been reported in this gene, although no genotype-phenotype correlation has been evidenced. We have sequenced 27 members of a single large Majorcan family with several individuals that exhibit VP symptoms: two of the eight patients had only skin symptoms (25%), one patient had only acute visceral crises (12.5%), one patient had both manifestations (12.5%) and the rest were completely asymptomatic (50%). In eight individuals, a T>A transversion at the intron 6 consensus splicing site was found (IVS6+2T>A), but only four of them presented clinical symptoms. We have also detected four polymorphic positions, three non-coding and one non-synonymous coding: c.-414A>C; IVS2+121G>C; c.1188G>A and IVS12+34C>T. Although IVS12+34C>T change has been reported to cause VP, generalized linear model (GLM) analyses showed no significant association between these SNPs and phenotypic manifestations. Only three mtDNA haplogroups were detected in this family: H, K and U(5a1). Two of them are relatively common in Balearic Islands. Our family evidenced a positive correlation between the clinically overt VP and haplogroup H. Thus, it seems that, in this family, the haplogroup H could be involved in the expression of the disease. The GLM analyses evidenced an association between haplogroup H, mutation IVS6+2T>A and clinically overt variegate porphyria.

Genetic sub-structure in western Mediterranean populations revealed by 12 Y-chromosome STR loci.

Haplotype and allele frequencies of 12 Y-chromosome short tandem repeat (Y-STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385 a/b, DYS437, DYS438 and DYS439) included in the Powerplex(R) Y System were determined in seven western Mediterranean populations from Valencia, Majorca, Ibiza (eastern Spain), Sicily and three Calabrian provinces (southern Italy). Amongst the 554 males included in the study, 443 different haplotypes were observed of which 372 were only observed once. The other haplotypes were shared by two to seven men. The overall haplotype diversity was 0.9988 +/- 0.0002. These Y-STRs markers showed a low capacity of discrimination (56.3%) in the Ibiza population probably due to genetic drift. Comparisons between the populations studied and other neighbouring populations showed a clear genetic sub-structure in the western Mediterranean area.

Bayesian estimation of post-Messinian divergence times in Balearic Island lizards.

Phylogenetic relationships and timings of major cladogenesis events are investigated in the Balearic Island lizards Podarcislilfordi and P.pityusensis using 2675bp of mitochondrial and nuclear DNA sequences. Partitioned Bayesian and Maximum Parsimony analyses provided a well-resolved phylogeny with high node-support values. Bayesian MCMC estimation of node dates was investigated by comparing means of posterior distributions from different subsets of the sequence against the most robust analysis which used multiple partitions and allowed for rate heterogeneity among branches under a rate-drift model. Evolutionary rates were systematically underestimated and thus divergence times overestimated when sequences containing lower numbers of variable sites were used (based on ingroup node constraints). The following analyses allowed the best recovery of node times under the constant-rate (i.e., perfect clock) model: (i) all cytochrome b sequence (partitioned by codon position), (ii) cytochrome b (codon position 3 alone), (iii) NADH dehydrogenase (subunits 1 and 2; partitioned by codon position), (iv) cytochrome b and NADH dehydrogenase sequence together (six gene-codon partitions), (v) all unpartitioned sequence, (vi) a full multipartition analysis (nine partitions). Of these, only (iv) and (vi) performed well under the rate-drift model. These findings have significant implications for dating of recent divergence times in other taxa. The earliest P.lilfordi cladogenesis event (divergence of Menorcan populations), occurred before the end of the Pliocene, some 2.6Ma. Subsequent events led to a West Mallorcan lineage (2.0Ma ago), followed 1.2Ma ago by divergence of populations from the southern part of the Cabrera archipelago from a widely-distributed group from north Cabrera, northern and southern Mallorcan islets. Divergence within P.pityusensis is more recent with the main Ibiza and Formentera clades sharing a common ancestor at about 1.0Ma ago. Climatic and sea level changes are likely to have initiated cladogenesis, with lineages making secondary contact during periodic landbridge formation. This oscillating cross-archipelago pattern in which ancient divergence is followed by repeated contact resembles that seen between East-West refugia populations from mainland Europe.

Mitochondrial DNA sequence variation in Jewish populations.

Sequence analysis of HVRI and HVRII mitochondrial DNA was carried out on 107 Jewish samples from Ashkenazi, Oriental, North African, and Sephardic origins. Control region sequences were assigned to a haplogroup by means of the analysis of the RFLP motif -7025 AluI or by using sequence motifs. A total of 88 different haplotypes were observed with a lower incidence of unique haplotypes (68.2%) than in other populations. Four individuals with one position of sequence heteroplasmy at nucleotides 16093, 16134, 16169, and 235, respectively, were detected. The mean pairwise difference in the Jewish population was 9.7 nucleotides. The gene diversity was 0.996, and the random match probability was 1.3%. When the data were compared with the autosomal and Y-chromosome markers previously studied in these populations, sex-specific differences could be observed in the Jewish populations. This fact must be taken into account for choosing suitable databases to correctly weigh the value of the evidence of a mtDNA and/or Y profile match.

Prevalence of HFE C282Y and H63D in Jewish populations and clinical implications of H63D homozygosity.

The frequencies of C282Y and H63D mutations of the HFE gene vary between different populations. A previous study showed an unexpectedly high H63D frequency in Chuetas (a population of Jewish descent). The present study addressed the question of the distribution of these mutations in Jewish populations from different origins and studied the possible causes of the high H63D frequency in Chuetas. Moreover, to improve the understanding of the controversial relationship between H63D homozygosity and iron overload, a group of patients with altered iron metabolism were studied. The high frequency of H63D mutation in Chuetas is not due to a high prevalence of this mutation in Sephardic Jews. Jewish populations have low C282Y and moderate H63D frequencies, suggesting slight gene flow from their surrounding populations. In accordance with historical and demographic data, genetic drift is the most probable cause for the singular H63D frequency in Chuetas. Clinically, this study of H63D homozygotes supports the conclusion that this genotype must be taken into account, because it confers an increased risk of iron overload and therefore genetic susceptibility to developing hereditary hemochromatosis or to aggravating other diseases.

Differential maternal and paternal contributions to the genetic pool of Ibiza Island, Balearic Archipelago.

We report on a comparison of the genetic diversity between Ibiza and the population of the other Balearic islands and also between the archipelago with respect to circum-Mediterranean populations. For such a comparison, autosomal and Y-chromosome STRs, as well as mtDNA sequence data analyzed from the same individuals, were studied. Analysis of 14 autosomal STRs showed that Ibiza had significant differentiation with respect to other Balearic populations and also with respect to insular and continental populations from the Mediterranean area. Nevertheless, the results obtained from the analysis of eight Y-STRs showed a high level of genetic homogeneity for eight western Mediterranean populations. On the other hand, these populations did not show a compacted group when mtDNA diversity was analyzed, since they showed genetic differentiation among them. The analyses of haplotypes shared between populations indicated that mtDNA haplotypes have drifted to higher frequencies than the Y chromosome. This fact could be due to a shared recent history between Ibiza and other western Mediterranean populations, with numerous male displacements originated by wars and, especially, commercial relations. The results of mtDNA from the Ibiza population could be due to a maternal Carthaginian/Phoenician founder effect, together with genetic drift, in accordance with the historical and demographic data of the area.

Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: focus on the mtDNA profile of a mixed semen-saliva stain.

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.

Mitochondrial DNA HVRI variation in Balearic populations.

The Balearic archipelago (Majorca, Minorca, and Ibiza islands and the Chuetas, a small and inbred community of descendants of Sephardic Jews) and Valencia were studied by means of the sequencing of a 404-bp segment of hypervariable region I (HVRI) mtDNA in 231 individuals. In total, 127 different haplotypes defined by 92 variable positions were identified. The incidence of unique haplotypes was very low, especially in Ibiza and the Chuetas. A remarkable observation in the Chueta community was the high frequency (23%) of preHV-1, a Middle Eastern lineage that is closely related, though not identical, to many others found at high frequencies in different Jewish populations. The presence of this haplogroup convincingly supported the Jewish origin of the Chueta community. The studied populations showed a reduced African contribution, and no individuals were detected with North African haplogroup U6, indicating a lack of maternal contribution from the Moslem settlement to these populations. Only Ibiza showed a lower diversity, indicating a possible genetic drift effect, also supported by the historical information known about this island. The variability in the sequence of mtDNA hypervariable region I correlated well with the existing information from the populations, with the exception of that of the Y-chromosome, which could indicate a differential contribution of the maternal and paternal lineages to the genetic pool of the Balearic Islands. The phylogenetic trees showed the intermediate position of the Chueta population between the Middle Eastern and Majorcan samples, confirming the Jewish origin of this population and their Spanish admixture.

Fitness and life-history traits of the two major mitochondrial DNA haplotypes of Drosophila subobscura.

Mitochondrial DNA restriction site analyses on natural populations of Drosophila subobscura have proved the existence of two common, coexisting haplotypes (I and II), as well as a set of less frequent ones derived from them. To explain this distribution, experiments to date point practically to all possible genetic mechanisms being involved in the changes of gene frequencies (cytonuclear coadaptation, direct natural selection on mtDNA and genetic drift). In an attempt to find differences that help to understand the dynamics of these haplotypes and to detect the effect of selection, we measured certain fitness components and life-history traits (egg-larva and larva-adult viabilities and developmental times, longevity, resistance to desiccation and optimal density) of the two main haplotypes I and II when maintained in laboratory population cages. As a general trend, haplotype II showed a higher net fitness than haplotype I, which explains the superiority of haplotype II over haplotype I in experimental populations but not their coexistence in nature, where additional factors must be considered.

Linkage disequilibria between mtDNA haplotypes and chromosomal arrangements in a natural population of Drosophila subobscura.

The association between mtDNA haplotypes and chromosomal arrangements in a natural population of Drosophila subobscura from Calvia (Balearic Islands, Spain) was studied in order to search for linkage disequilibria, in an attempt to explain the populational dynamics of the mtDNA haplotypes of this species in nature. The presence of Wolbachia was not detected. Two main haplotypes (I and II) were found, as well as a series of less common ones. The Tajima D-test seemed to indicate some kind of seasonal population bottleneck. An analysis of linkage disequilibrium and factorial analysis of correspondences detected an association between haplotype I and the J(ST) inversion and haplotype II and the J(1) inversion.

Genetic variability at 14 STR loci in the Puna population of north western Argentina.

Frequency data of the short tandem repeat (STR) loci D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, HUMTH01, D4S243, D18S535, HUMF13A1 and D12S391 were determined in the Puna population (Andean region from north western Argentina). In this study, 5 out of the 14 loci did not meet Hardy-Weinberg expectations. The excess of homozygotes observed in most of the markers could be due to a Wahlund effect and/or inbreeding. The frequencies were significantly different from those of other compared populations from Europe and America. Forensic parameters indicated that the 14 loci studied in the Puna region are highly discriminating, but the lack of Hardy-Weinberg equilibrium in some of the markers must be taken into account in the application of these results to paternity and forensic casework.

Distribution of HFE C282Y and H63D mutations in the Balearic Islands (NE Spain).

The HFE gene contains two main missense mutations: C282Y and H63D. Individuals with these mutations carry a risk of developing hereditary haemochromatosis (HH). The common form of this disease is due to homozygosity for the C282Y mutation. Population studies have shown the variation of the prevalence of these mutations in different countries and ethnic groups. The purposes of this current study were to determine the prevalence of the C282Y and H63D mutations in the Balearic Islands and the genotypic characterization of patients diagnosed with HH, as well as those with iron overload and liver diseases. A total of 1330 Balearic chromosomes were analyzed. The results showed that the populations of the Balearic Islands were not homogeneous. No C282Y carriers were observed in a group of descendants of Majorcan Jews (Chuetas) and the frequency was very low in Minorca (1.2%) in comparison with the other islands of Majorca (4.7%) and Ibiza (6.5%). The carrier frequency of the H63D mutation was similar in the three islands and very high (43.1%) in the descendants of Majorcan Jews. The study of patients was carried out in 129 individuals. The homozygous C282Y genotype was the principal one involved in hereditary haemochromatosis (90%), whereas the other HH patients were C282Y/H63D compound heterozygous and H63D homozygous.