Bumetanide oral solution for the treatment of children and adolescents with autism spectrum disorder: Results from two randomized phase III studies.

The efficacy and safety of bumetanide oral solution for the treatment of autism spectrum disorder (ASD) in children and adolescents was evaluated in two international, multi-center, randomized, double-blind, placebo-controlled phase III trials; one enrolled patients aged 7-17 years (SIGN 1 trial) and the other enrolled younger patients aged 2-6 years (SIGN 2). In both studies, patients were randomized to receive bumetanide oral solution twice daily (BID) or placebo BID during a 6-month double-blind treatment period. The primary endpoint was change in Childhood Autism Rating Scale 2 (CARS2) total raw score from baseline to Week 26. Key secondary endpoints included changes in Social Responsiveness Scale-2, Clinical Global Impression Scale, and Vineland Adaptive Behavior Scale. Each study enrolled 211 patients (bumetanide, n = 107; placebo, n = 104). Both studies were terminated early due to absence of any significant difference between bumetanide and placebo in the overall studied populations. In both studies, CARS2 total raw score decreased from baseline to Week 26 in the bumetanide and placebo groups, with no statistically significant difference between groups. No differences were observed between treatment groups for any of the secondary efficacy endpoints in either study. In both studies, treatment-emergent adverse events that occurred more frequently with bumetanide than placebo included thirst, polyuria, hypokalemia, and dry mouth. These large phase III trials failed to demonstrate a benefit of bumetanide for the treatment of pediatric ASD compared with placebo. Consequently, the sponsor has discontinued the development of bumetanide for the treatment of this condition. Trial registration: https://clinicaltrials.gov: SIGN 1: NCT03715166; SIGN 2: NCT03715153.

Evaluating the forensic informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale.

The impact of phylogeographic information on mtDNA forensics has been limited to the quality control of published sequences and databases. In this work we use the information already available on Eurasian mtDNA phylogeography to guide the choice of coding-region SNPs for haplogroup H. This sub-typing is particularly important in forensics since, even when sequencing both HVRI and HVRII, the discriminating power is low in some Eurasian populations. We show that a small set (eight) of coding-region SNPs resolves a substantial proportion of the identical haplotypes, as defined by control-region variation alone. Moreover, this SNP set, while substantially increasing the discriminating efficiency in most Eurasian populations by roughly equal amounts, discloses population-specific profiles.

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.

The place of the Basques in the European Y-chromosome diversity landscape.

There is a trend to consider the gene pool of the Basques as a 'living fossil' of the earliest modern humans that colonized Europe. To investigate this assumption, we have typed 45 binary markers and five short tandem repeat loci of the Y chromosome in a set of 168 male Basques. Results on these combined haplotypes were analyzed in the context of matching data belonging to approximately 3000 individuals from over 20 European, Near East and North African populations, which were compiled from the literature. Our results place the low Y-chromosome diversity of Basques within the European diversity landscape. This low diversity seems to be the result of a lower effective population size maintained through generations. At least some lineages of Y chromosome in modern Basques originated and have been evolving since pre-Neolithic times. However, the strong genetic drift experienced by the Basques does not allow us to consider Basques either the only or the best representatives of the ancestral European gene pool. Contrary to previous suggestions, we do not observe any particular link between Basques and Celtic populations beyond that provided by the Paleolithic ancestry common to European populations, nor we find evidence supporting Basques as the focus of major population expansions.

Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial.

We report the results of the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) Collaborative Exercise 2002-2003 on mitochondrial DNA (mtDNA) analysis. Six different samples were submitted to the participating laboratories: four blood stains (M1-M2-M3-M4), one mixture blood sample (M5), and two hair shaft fragments (M6). Most of the labs reported consensus results for the blood stains, slightly improving the results of previous collaborative exercises. Although hair shaft analysis is still carried out by a small number of laboratories, this analysis yielded a high rate of success. On the contrary, the analysis of the mixture blood stain (M5) yielded a lower rate of success; in spite of this, the whole results on M5 typing demonstrated the suitability of mtDNA analysis in mixture samples. We have found that edition errors are among the most common mistakes reported by the different labs. In addition, we have detected contamination events as well as other minor problems, i.e. lack of standarization in nomenclature for punctual and length heteroplasmies, and indels. In the present edition of the GEP-ISFG exercise we have paid special attention to the visual phylogenetic inspection for detecting common sequencing errors.

A Spanish population study of 17 Y-chromosome STR loci.

Haplotype, allele frequencies and population data of 17 Y-chromosome STR loci DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460 (GATA A7.1), DYS461 (GATA A7.2), GATA A10, GATA C4 and GATA H4 were determined from a sample of 148 unrelated male individuals from Spain. A total of 144 haplotypes were identified by the 17 Y-STR markers, of which 141 were unique, two were found in two individuals and one was found in three individuals. The haplotype diversity (99.95%) and discrimination capacity (97.30%) were calculated. Comparisons were made with previously published haplotype data on other Iberian population samples and no significant differences were found.

A Basque Country autochthonous population study of 11 Y-chromosome STR loci.

Haplotype, allele frequencies and population data of 11 Y-chromosome STR loci DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438 and DYS439 were determined from a sample of 168 unrelated autochthonous male individuals from the Basque Country. The eight surnames and birth places of the grandparents of all analyzed individuals were of Basque origin. A total of 89 haplotypes were identified by the 11 Y-STR loci. The haplotype diversity (97.49%) and discrimination capacity (52.98%) were calculated. Comparisons were made with previously published haplotype data on other Iberian population samples and significant differences were found.

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies.

We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed.

Results of the 1999-2000 collaborative exercise and proficiency testing program on mitochondrial DNA of the GEP-ISFG: an inter-laboratory study of the observed variability in the heteroplasmy level of hair from the same donor.

The Spanish and Portuguese working group (GEP) of international society for forensic genetics (ISFG) 1999-2000 collaborative exercise on mitochondrial DNA (mtDNA) included the analysis of four bloodstain samples and one hair shaft sample by 19 participating laboratories from Spain, Portugal and several Latin-American countries. A wide range of sequence results at position 16,093 of the HV1 (from T or C homoplasmy to different levels of heteroplasmy) were submitted by the different participating laboratories from the hair shaft sample during the first phase of this exercise. During the discussion of these results in the Annual GEP-ISFG 2000 Conference a second phase of this exercise was established with two main objectives: (i) to evaluate the incidence of the HV1 sequence heteroplasmy detected in Phase I across different sample types from the same donor including blood, saliva, and hair shafts, (ii) to perform a technical review of the electropherograms to evaluate the relative levels of heteroplasmies obtained by the different laboratories and also to examine the source of possible errors detected in Phase I. Anonymous review of the raw sequence data permitted the detection of three transcription errors and three errors due to methodological problems. Highly variable levels of heteroplasmy were found in the hair shaft and more stability in blood and saliva. Three laboratories found variable levels of heteroplasmy at position 16,093 across adjacent fragments from the same hair shaft. Two laboratories also described more than one heteroplasmic position from a single hair. The relevance of these findings for the interpretation of mtDNA data in the forensic context is also discussed.

The 2000-2001 GEP-ISFG Collaborative Exercise on mtDNA: assessing the cause of unsuccessful mtDNA PCR amplification of hair shaft samples.

We report the results of Spanish and Portuguese working group (GEP) of International Society of Forensic Genetics (ISFG) Collaborative Exercise 2001-2002 on mitochondrial DNA (mtDNA) analysis. 64 laboratories from Spain, Portugal and several Latin-American countries participated in this quality control exercise. Five samples were sent to the participating laboratories, four blood stains (M1-M4) and a sample (M5) consisting of two hair shaft fragments. M4 was non-human (Felis catus) in origin; therefore, the capacity of the labs to identify the biological source of this sample was an integral part of the exercise. Some labs detected the non-human origin of M4 by carrying out immuno-diffussion techniques using antihuman serum, whereas others identified the specific animal origin by testing the sample against a set of animal antibodies or by means of the analysis of mtDNA regions (Cyt-b, 12S, and 16S genes). The results of the other three human blood stains (M1-M3) improved in relation to the last Collaborative Exercises but those related to hairs yielded a low rate of success which clearly contrasts with previous results. As a consequence of this, some labs performed additional analysis showing that the origin of this low efficiency was not the presence of inhibitors, but the low quantity of DNA present in these specific hair samples and the degradation. As a general conclusion the results emphasize the need of external proficiency testing as part of the accreditation procedure for the labs performing mtDNA analysis in forensic casework.

Agomelatine in generalized anxiety disorder: an active comparator and placebo-controlled study.

BACKGROUND: Agomelatine was efficacious in reducing symptoms in a short-term placebo-controlled trial in generalized anxiety disorder (GAD) and in preventing relapse in a longer term placebo-controlled study. An additional short-term placebo-controlled study is required by regulatory agencies to confirm the efficacy of agomelatine in GAD. METHOD: This 12-week, placebo-controlled, double-blind, randomized, parallel group, international, multicenter study was designed to confirm the efficacy of agomelatine 25-50 mg/d in the treatment of patients with a primary DSM-IV-TR diagnosis of GAD. The primary outcome measure was the Hamilton Anxiety Rating Scale (HARS) total score. Assay sensitivity was evaluated by including an escitalopram (10-20 mg/d) group. SETTINGS: The study was undertaken in 45 clinical centers in Argentina, Czech Republic, Finland, South Korea, Poland, Russia, and Slovakia from April 2010 to July 2011. RESULTS: One hundred thirty-nine outpatients were included in the agomelatine group, 131 in the placebo group, and 142 in the escitalopram group. Agomelatine significantly reduced mean (SD) HARS total score (agomelatine-placebo difference: 4.71 [1.03], P <.0001) and had significant effects on secondary outcome measures, including psychic and somatic HARS subscales, response rate (estimate [standard error]) (agomelatine-placebo difference: 27.4% [5.9%], P< .0001), remission on the HARS (agomelatine-placebo difference: 16.8% [5.4%], P = .002), Clinical Global Impressions-Severity of Illness scale (CGI-S) (P < .001), functional impairment (P < .0001), and sleep quality (P < .001). Findings were confirmed in the subset of more severely ill patients (HARS total score ≥ 25 with or without CGI-S ≥ 5 at baseline). Agomelatine was well tolerated by patients, with no more adverse events than placebo. Escitalopram was similarly efficacious but was accompanied by a higher incidence of adverse events compared to placebo. CONCLUSIONS: In clinical practice, agomelatine has at least similar efficacy to that of escitalopram for the short-term treatment of GAD and is well tolerated. TRIAL REGISTRATION: Controlled-Trials.com identifier: ISRCTN03554974.

Agomelatine prevents relapse in generalized anxiety disorder: a 6-month randomized, double-blind, placebo-controlled discontinuation study.

OBJECTIVE: This study evaluated the efficacy and tolerability of agomelatine in the prevention of relapse in patients with generalized anxiety disorder (GAD). METHOD: Patients with GAD (Hamilton Anxiety Rating Scale [HARS] ≥ 22, with items 1 and 2 ≥ 2, item 1 + 2 ≥ 5; Montgomery-Asberg Depression Rating Scale [MADRS] ≤ 16; and < 20% decrease in HARS total score between screening and baseline) who responded to a 16-week course of agomelatine 25-50 mg/d treatment were randomly assigned to receive continuation treatment with agomelatine (n = 113) or placebo (n = 114) for 26 weeks. The main outcome measure was time to relapse during this maintenance period. The estimated risk of relapse was calculated using the Kaplan-Meier method, and groups were compared using a log-rank test stratified for country. The study was undertaken in 31 clinical centers in Canada, Denmark, Estonia, Finland, Hungary, and Sweden from November 2007 to September 2009. RESULTS: During the 6-month maintenance period, the proportion of patients that relapsed during the double-blind period in the agomelatine group (22 patients, 19.5%) was lower than in the placebo group (35 patients, 30.7%). The risk of relapse over time was significantly lower for patients who continued treatment than for those switched to placebo (P = .046, log-rank test stratified for country). Agomelatine was also superior to placebo in preventing relapse in the subset of more severe patients with baseline HARS total score ≥ 25 and CGI-S score ≥ 5. The tolerability of agomelatine was good throughout the study, and there were no differences in discontinuation symptoms after withdrawal of agomelatine in comparison to maintenance on agomelatine. CONCLUSIONS: The present study extends the positive findings of an earlier short-term study of agomelatine in GAD, demonstrating that agomelatine is effective and well-tolerated in the longer-term treatment of this chronic disorder. TRIAL REGISTRATION: www.isrctn.org identifier: ISRCTN38094599.

Forensic analysis of dog (Canis lupus familiaris) mitochondrial DNA sequences: an inter-laboratory study of the GEP-ISFG working group.

A voluntary collaborative exercise aiming at the mitochondrial analysis of canine biological samples was carried out in 2006-2008 by the Non-Human Forensic Genetics Commission of the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Genetics (ISFG). The participating laboratories were asked to sequence two dog samples (one bloodstain and one hair sample) for the mitochondrial D-loop region comprised between positions 15,372 and 16,083 using suggested primers and PCR conditions, and to compare their results against a reference sequence. Twenty-one participating laboratories reported a total of 67.5% concordant results, 15% non-concordant results, and 17.5% no results. The hair sample analysis presented more difficulty to the participants than the bloodstain analysis, with a high percentage (29%) failing to obtain a result. The high level of participation showed the interest of the community in the analysis of dog forensic samples but the results reveal that crucial methodological issues need to be addressed and further training is required in order to respond proficiently to the demands of forensic casework.

The specificity in the interaction between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 is mainly determined by the copper protein.

The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural differences when compared with the homologous complexes from other cyanobacteria and plants, with electrostatic and hydrophobic interactions being differently involved in each case. Here, five negatively charged residues of a recombinant form of cytochrome f from Nostoc have been replaced with either neutral or positively charged residues, and the effects of mutations on the kinetics of electron transfer to wild-type and mutant forms of plastocyanin have been measured by laser flash absorption spectroscopy. Cytochrome f mutants with some negative charges replaced with neutral residues exhibit an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than that of the wild-type species, whereas the mutants with negative charges replaced with positive residues exhibit a significantly lower reactivity. Taken together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. Moreover, cross reactions between mutants of both proteins reveal the presence of some short-range specific electrostatic interactions. Our findings also make evident the fact that in Nostoc the main contribution to the electrostatic nature of the complex is provided by the small domain of cytochrome f.

Insights into the "isolation" of the Basques: mtDNA lineages from the historical site of Aldaieta (6th-7th centuries AD).

We analyzed the hypervariable region I (HVR-I) sequence variability of the mitochondrial DNA (mtDNA) of individuals buried at Aldaieta (6th-7th centuries AD) in order to find out more about the biosocial implications of this cemetery. The results, fully authenticated by means of diverse criteria (analysis of duplicates, replication in an independent laboratory, quantification of target DNA, and sequencing and cloning of polymerase chain reaction products), suggest that Aldaieta largely consists of autochthonous individuals who shared common funereal customs with the late Ancient North Pyrenean cemeteries of Western Europe (the Reihengräberfelder), a cultural influence possibly accompanied by a certain genetic flow. Furthermore, the distribution of mtDNA lineages in the cemetery highlighted the existence of a significant number of family relationships, supporting the belief that it was a stable settlement and not a group that had haphazardly settled in the area. Finally, this paper stresses the importance of ancient DNA data for reconstructing the biological history of human populations, rendering it possible to verify certain hypotheses based solely on current population data. The presence at Aldaieta of an mtDNA lineage originating in Northwest Africa testifies to the existence of contact between the Iberian Peninsula and Northwest Africa prior to the Moorish occupation. Both this latter discovery and the high frequency of haplogroup J at the Aldaieta cemetery raise questions about the generally accepted belief that, since ancient times, the influence of other human groups has been very scarce in the Basque Country.

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies.

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.

Laser flash-induced kinetic analysis of cytochrome f oxidation by wild-type and mutant plastocyanin from the cyanobacterium Nostoc sp. PCC 7119.

Oxidation of the soluble, truncated form of cytochrome f by wild-type and mutant species of plastocyanin has been analyzed by laser flash absorption spectroscopy in the cyanobacterium Nostoc (formerly, Anabaena) sp. PCC 7119. At low ionic strengths, the apparent electron transfer rate constant of cytochrome f oxidation by wild-type plastocyanin is 1.34 x 10(4) s(-)(1), a value much larger than those determined for the same proteins from other organisms. Upon site-directed mutagenesis of specific residues at the plastocyanin interaction area, the rate constant decreases in all cases yet to varying extents. The only exception is the D54K variant, which exhibits a higher reactivity toward cytochrome f. In most cases, the reaction rate constant decreases monotonically with an increase in ionic strength. The observed changes in the reaction mechanism and rate constants are in agreement with the location of the mutated residues at the interface area, as well as with the peculiar orientation of the two partners within the Nostoc plastocyanin-cytochrome f transient complex, whose NMR structure has been determined recently. Furthermore, the experimental data herein reported match well the kinetic behavior exhibited by the same set of plastocyanin mutants when acting as donors of electrons to photosystem I [Molina-Heredia, F. P., et al. (2001) J. Biol. Chem. 276, 601-605], thus indicating that the copper protein uses the same surface areas-one hydrophobic and the other electrostatic-to interact with both cytochrome f and photosystem I.

High-resolution mtDNA evidence for the late-glacial resettlement of Europe from an Iberian refugium.

The advent of complete mitochondrial DNA (mtDNA) sequence data has ushered in a new phase of human evolutionary studies. Even quite limited volumes of complete mtDNA sequence data can now be used to identify the critical polymorphisms that define sub-clades within an mtDNA haplogroup, providing a springboard for large-scale high-resolution screening of human mtDNAs. This strategy has in the past been applied to mtDNA haplogroup V, which represents <5% of European mtDNAs. Here we adopted a similar approach to haplogroup H, by far the most common European haplogroup, which at lower resolution displayed a rather uninformative frequency distribution within Europe. Using polymorphism information derived from the growing complete mtDNA sequence database, we sequenced 1580 base pairs of targeted coding-region segments of the mtDNA genome in 649 individuals harboring mtDNA haplogroup H from populations throughout Europe, the Caucasus, and the Near East. The enhanced genealogical resolution clearly shows that sub-clades of haplogroup H have highly distinctive geographical distributions. The patterns of frequency and diversity suggest that haplogroup H entered Europe from the Near East approximately 20,000-25,000 years ago, around the time of the Last Glacial Maximum (LGM), and some sub-clades re-expanded from an Iberian refugium when the glaciers retreated approximately 15,000 years ago. This shows that a large fraction of the maternal ancestry of modern Europeans traces back to the expansion of hunter-gatherer populations at the end of the last Ice Age.