Search

Background: Carriers of the G2019S mutation of LRRK2 provide a great opportunity to investigate the premotor stages of Parkinson's disease (PD). We have studied by serial clinical and dopamine transporter single photon emission computed tomography (DaT-SPECT) evaluations a cohort of asymptomatic carriers of the LRRK2-G2019S mutation in order to evaluate the usefulness of these tools as biomarkers. Here we report the results of the extended follow-up of this cohort at 8 years. Methods: Seventeen participants, of the 25 available from the 4-year evaluation, completed the 8-year assessment. UPDRS-III, UPSIT test and DaT-SPECT imaging (123I-ioflupane) were performed. We used repeated-measures linear mixed effects models to examine the changes in DaT binding over time. Results: Three carriers had converted to PD at 4 years. One additional carrier converted at 8 years. PD-converters had lower striatal DaT binding at baseline than non-converters. There was a significant decline of DaT binding over time, with a mean annual rate of 3.5%, with somewhat inter-individual and intra-individual variability and comparable between PD-converters and non-converters. No carrier with DAT binding ratio above an undefined threshold between 0.5 and 0.8 developed PD symptoms. The age-adjusted UPSIT score did not change significantly over time. Conclusions: The rate of conversion to PD at 8 years in this cohort aged ~58 years at baseline was 16%. The observed decline of DaT binding over time and its association with the phenotype render DaT-SPECT a potentially useful tool for monitoring the premotor stage of the disease, although at the individual level its ability to predict phenoconversion is limited.

Genotoxicological studies are emerging as fundamental for knowing the hazards to our genome, to our health. Drosophila melanogaster is one of the preferable organisms for toxicological research considering its metabolic similarities (viz. on dietary input, xenobiotic metabolizing system, antioxidant enzymes and DNA repair systems) to mammals. Accordingly, somatic mutation and recombination tests (SMARTs) of D. melanogaster are fast and low-cost in vivo assays that have shown solid results evaluating genotoxicity. The w/w + SMART uses the white (w) gene as a recessive marker to monitor the presence of mutant ommatidia (eye units), indicating the occurrence of point mutations, deletions, mitotic recombination or/and nondisjunction. Additionally, several studies used SMARTs to assess antigenotoxicity, with some using the w/w + SMART. We reviewed the state of the art of the w/w + SMART used for antigenotoxicity analysis, focusing on published results, aiming to contribute to the conception of a reliable protocol in antigenotoxicity. As such, genotoxic agents with known action mechanisms, as streptonigrin (oxidative stress inducer), were used as a genotoxic insult for proving the antigenotoxic effects of natural substances (e.g. seaweeds), demonstrating the presence of antimutagens in their composition. These antigenotoxicity studies are crucial for promoting preventive measures against environmental genotoxics that affect humans daily.

Objectives A recent study showed that Arab-Berbers GG homozygous at rs2421947(C/G) in the dynamin 3 gene (DNM3) had 12.5 years earlier age at onset of leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD) (L2PD). We explored whether this variant modulates the L2PD age at onset in Spain. Methods We genotyped rs2421947 in 329 participants (210 L2PD patients, 119 L2PD nonmanifesting p.G2019S carriers), and marker rs356219 (A/G) in the α-synuclein gene (SNCA). Results By Kaplan Meier and Cox regression analyses, we did not find an association of the DNM3 polymorphism with L2PD age at onset. However, we found an association of the SNCA marker with up to an 11 years difference in the L2PD median age at onset (58 years for GG carriers vs 69 years for AA). Conclusion Our results indicate that SNCA rs356219 but not dynamin 3 DNM3 rs2421947 modifies the penetrance of the mutation G2019S in the Spanish population by influencing the L2PD age at onset. These findings suggest that different genetic modifiers may influence the L2PD age at onset in different populations. © 2018 International Parkinson and Movement Disorder Society.

El tabaquismo es un problema de salud publica dado que el consumo es cada vez mayor y presenta edades de inicio mas tempranas; en relacion con los procesos biologicos del tabaquismo se han identificado varios genes cuyas variantes han sido asociadas con este fenomeno. El objetivo de este estudio fue evaluar la relacion entre los polimorfismos rs6801272 y rs680244 en los genes CYP2A6 y CHRNA5 respectivamente, con el consumo de tabaco. Para esto se realizo un estudio de casos (fumadores-exfumadores versus controles), controlando las variables edad y sexo, ademas de considerar la edad de inicio y el numero de cigarrillos por dia. El locus rs6801272 se encontro monomorfico en la poblacion de estudio, presentando el alelo T asociado con el metabolismo normal de la nicotina; aunque el locus rs680244 se encontro polimorfico, no fue posible establecer la relacion de esta variante genetica con el consumo de cigarrillos (OR 3,2) o la capacidad de abandono (OR 5,3); tampoco fue posible establecer una correlacion entre el genotipo y el numero de cigarrillos por dia (R 2 = 0,001) o el genotipo y la edad de inicio (R 2 =0,015). En conclusion, no se detecto una relacion entre las variantes geneticas estudiadas y el consumo, la capacidad de abandono, el numero de cigarrillos por dia y la edad de inicio en la poblacion de Itagui.

Objective:To assess the value of baseline clinical and imaging biomarkers in a cohort of asymptomatic LRRK2 G2019S carriers for predicting conversion to Parkinson disease (PD) at 4 years.Methods:Thirty-two asymptomatic carriers of LRRK2 G2019S mutation underwent baseline and 4-year evaluation including clinical examination (Unified Parkinson's Disease Rating Scale, part III, olfaction University of Pennsylvania Smell Identification Test [UPSIT]) and dopamine transporter (DaT) SPECT (123I-ioflupane). Visual and semiquantitative analysis of images was performed. The specific striatal binding ratio was calculated (striatal region of interest [ROI] − occipital ROI/occipital ROI).Results:Three carriers, asymptomatic at baseline, had converted to PD at 4-year evaluation. Twenty-three participants were fully evaluated. PD converters had lower striatal DaT binding at baseline than nonconverters (p = 0.002). A baseline scan with a ratio of bilateral striatal uptake below 1 predicted conversion to PD within the 4-year period with high sensitivity and specificity (area under the curve 1; p = 0.006). The slope of DaT binding decline between the 2 scans was similar in PD converters and nonconverters. Age-adjusted UPSIT score at baseline and at 4 years was similar in both groups.Conclusions:Semiquantitative DaT-SPECT could be used to predict early conversion to PD in asymptomatic carriers of the LRRK2 G2019S mutation. Rate of conversion to PD at 4 years in this cohort aged ∼64 years was 12%. The slope of DaT binding decline on DaT-SPECT imaging seems to be similar across different stages of the premotor period.

Many studies have been conducted to understand the role of spontaneous diseases in cetacean conservation, focusing in particular on the interaction between the pathogen and its host. These interactions may be influenced by pollutants, but conclusive evidence of the influences of pollution in the development of a disease is not usually evident during postmortem investigations of cetaceans. For these reasons, the pathogen/host interaction must be first understood before these effects can be determined. Among the most significant emerging infectious diseases (EIDs) of cetaceans, those caused by Morbillivirus, Herpesvirus, Brucella ceti, and Toxoplasma gondii, along with a number of additional pathogens, have gained importance in recent years. EIDs have been reported in several cetacean species throughout the world, being responsible for dramatic mortality outbreaks and affecting reproductive function and biology, with associated population declines. A brief overview of the epidemiology of the aforementioned agents both in the Canary Islands archipelago and in the Mediterranean Sea is provided here.

Cisplatin, one of the most extensively used metallodrugs in cancer treatment, presents the important drawback of patient resistance. This resistance is the consequence of different processes including those preventing the formation of DNA adducts and/or their quick removal. Thus, a tool for the accurate detection and quantitation of cisplatin-induced adducts might be valuable for predicting patient resistance. To prove the validity of such an assumption, highly sensitive plasma mass spectrometry (ICP-MS) strategies were applied to determine DNA adduct levels and intracellular Pt concentrations. These two metal-relative parameters were combined with an evaluation of biological responses in terms of genomic stability (with the Comet assay) and cell cycle progression (by flow cytometry) in four human cell lines of different origins and cisplatin sensitivities (A549, GM04312, A2780 and A2780cis), treated with low cisplatin doses (5, 10 and 20 μM for 3 hours). Cell viability and apoptosis were determined as resistance indicators. Univariate linear regression analyses indicated that quantitation of cisplatin-induced G-G intra-strand adducts, measured 1 h after treatment, was the best predictor for viability and apoptosis in all of the cell lines. Multivariate linear regression analyses revealed that the prediction improved when the intracellular Pt content or the Comet data were included in the analysis, for all sensitive cell lines and for the A2780 and A2780cis cell lines, respectively. Thus, a reliable cisplatin resistance predictive model, which combines the quantitation of adducts by HPLC-ICP-MS, and their repair, with the intracellular Pt content and induced genomic instability, might be essential to identify early therapy failure.

Background Low serum uric acid (UA) levels have been associated with increased Parkinson's disease (PD) risk and accelerated disease progression. We analyzed the effect of polymorphisms in 9 genes influencing serum UA concentration on the risk of PD. Methods We genotyped SLC2A9 rs734553, ABCG2 rs2231142, SLC17A1 rs1183201, SLC22A11 rs17300741, SLC22A12 rs505802, GCKR rs780094, PDZK1 rs12129861, LRRC16A+SCGN rs742132, and SLC16A9 rs12356193 in 1061 PD patients and 754 controls. For each subject we calculated a cumulative genetic risk score (GRS), defined as the total number of PD-risk alleles (range, 2–15) associated to lower serum UA levels. Serum UA levels were measured in a subgroup of 365 PD cases and 132 controls. Results Serum UA levels were significantly lower in men with PD than in controls. Subjects (both men and women) carrying more than 9 risk alleles (third GRS tertile) had a 1.5 higher risk of developing PD than subjects with less than 8 risk alleles (first GRS tertile). An inverse correlation was observed between higher GRS and lower serum UA concentration in both men and women. Conclusions Genetic variability influencing serum UA levels might modify susceptibility to PD. © 2013 International Parkinson and Movement Disorder Society

Objective: To ascertain in a cross-sectional study whether substantia nigra (SN) echogenicity, olfaction, and dopamine transporter (DaT)-SPECT are reliable premotor biomarkers in a cohort of asymptomatic carriers of the LRRK2 G2019S mutation (AsG2019S+). Methods: These biomarkers were evaluated in 49 AsG2019S+ patients, and we also studied olfaction and SN echogenicity in 29 patients with G2019S-associated Parkinson disease (PD-G2019S), 47 relatives who were noncarriers of the LRRK2 G2019S mutation (AsG2019S−), 50 patients with idiopathic Parkinson disease (iPD), and 50 community controls. Results: Eighty-five percent of unaffected mutation carriers (AsG2019S+) showed pathologic SN hyperechogenicity, with a similar proportion observed among both PD-G2019S and iPD cases, and 41% of AsG2019S− also showing increased SN echogenicity. The proportion of hyposmic individuals was not statistically different in patients with PD-G2019S (50%) and iPD (82%), but hyposmia was significantly less common in both AsG2019S+ (26%) and AsG2019S− (28%). In AsG2019S+ cases, reduced striatal uptake in DaT-SPECT was observed in 43.7%. Conclusions: Independently of age at examination, the most frequently altered premotor biomarker in LRRK2 G2019S-associated PD was SN hyperechogenicity, whereas abnormal DaT-SPECT predominated in older, unaffected mutation carriers.

To analyze the presence of phosphocalcic metabolism disorders in patients with osteopenia-osteoporosis without nephrolithiasis with respect to a control group.A cross-sectional study was conducted in patients with osteopenia-osteoporosis without nephrolithiasis (n = 67) in lumbar spine or femur and in a control group (n = 61) with no lithiasis or bone disorders. Blood bone markers, phosphocalcic metabolism, fasting urine, 24-h urine lithogenic risk factors, and densitometry were recorded in both groups. SPSS 20.0 was used for statistical analysis.In comparison with the controls, significantly higher blood calcium (9.27 ± 0.36 vs. 9.57 ± 0.38, p = 0.0001), intact parathormone (45.6 ± 14.9 vs. 53.8 ± 18.9, p = 0.008), and alkaline phosphatase (61.9 ± 20.9 vs. 70.74 ± 18.9, p = 0.014) levels were found in patients with osteopenia-osteoporosis. In the 24-h urine test, citrate (1010.7 ± 647.8 vs. 617.6 ± 315.8, p = 0.0001) and oxalate (28.21 ± 17.65 vs. 22.11 ± 16.49, p = 0.045) levels were significantly lower in osteopenia-osteoporosis patients than in controls, with no significant difference in calcium (187.3 ± 106.9 vs. 207.06 ± 98.12, p = 0.27) or uric acid (540.7 ± 186.2 vs. 511.9 ± 167.06, p = 0.35) levels. Patients with osteopenia-osteoporosis had significantly higher levels of lithogenic risk factors associated with bone remodeling, including significantly increased β-crosslaps and osteocalcin values and higher β-crosslaps/osteocalcin ratios.Patients with osteopenia-osteoporosis without nephrolithiasis showed phosphocalcic metabolism disorders as well as lower urinary citrate and higher β-crosslaps/osteocalcin and fasting calcium/creatinine ratios, which would increase the risk of nephrolithiasis. Hence, prospective studies are warranted to evaluate the long-term risks.

Patients with Parkinson's disease (PD) carrying the G2019S mutation of the LRRK2 gene provide an opportunity of studying in a homogeneous setting the molecular pathways involved in the pathogenesis of common idiopathic forms of PD. However, whether common mechanisms are involved in both conditions in not known. Here, we compared genome-wide gene expression (RNA sequencing) in peripheral blood between PD patients carrying the G2019S mutation of the LRRK2 gene and idiopathic PD cases, to deepen in the understanding of this topic. In addition, we compared the blood transcriptome between 2 cohorts of carriers of the G2019S mutation (symptomatic and asymptomatic) and 2 cohorts of noncarriers (symptomatic and asymptomatic) for detecting transcriptomic changes attributable to the presence of the G2019S mutation. We searched for gene enrichment in Reactome or Kyoto Encyclopedia of Genes and Genomes pathways. We found that despite some overlap, peripheral blood transcriptome differs widely between idiopathic and LRRK2 G2019S-associated PD, with only 4 deregulated pathways shared by both conditions (complement and coagulation cascades, cell adhesion molecules, hematopoietic cell lineage, and extracellular matrix organization). Changes in the blood transcriptome observed in asymptomatic carriers of the mutation included 6 genes known to be associated with PD in genome-wide association studies and also pathways related with immunity. Our findings emphasize the notion that PD is likely a pathogenically heterogeneous condition and suggest the existence of specific mechanisms involved in LRRK2-associated PD., This work was supported by the grant PI11/00228 from Instituto de Salud Carlos III.

Background Common variants in the LRRK2 gene influence risk of Parkinson’s disease (PD) in Asians, but whether the same is true in European-derived populations is less clear.

Objective Low serum uric acid (UA) levels have been associated with a worse cognitive function later in life and also with a higher risk and faster progression of Parkinson's disease (PD). Here we studied whether serum UA levels and the genetic variants related to its variability are associated with the presence of dementia in a cohort of patients with PD. Methods The study included 343 PD patients, which were examined for the presence of dementia according to the MDS Task Force criteria (level 1). The predominant PD phenotype and UPDRS-III and Hoehn-Yahr scales were recorded. Serum UA levels were determined in each participant. Genotyping of SLC2A9 rs734553, ABCG2 rs2231142, SLC17A1 rs1183201, SLC22A11 rs17300741, SLC22A12 rs505802, GCKR rs780094, PDZK1 rs12129861, LRRC16A + SCGN rs742132, and SLC16A9 rs12356193 was carried out by Taqman analysis. For each subject we calculated a cumulative genetic risk score (GRS), defined as the total number of risk alleles (range 2–15) associated to lower serum UA levels. Results 72 out of 343 PD patients (21%) presented dementia. Serum UA levels were not different between PD patients with or without dementia. No significant association was detected between any single SNP and the risk of PD-dementia. When we analyzed the combined effect of the eight SNPs using the cumulative GRS no significant association between the number of risk alleles and the risk of PD was observed. Conclusions Our data suggest that serum UA levels have not a significant impact on the risk of dementia in PD.

The aim of this study was to characterize the HIV-1 intersubtype recombinant forms generated during the follow-up of a dual natural infection with subtypes B and G. Near full-length sequences from plasma and peripheral blood mononuclear cell (PBMC) compartments were analyzed and the biological characteristics of their derived primary isolates studied. Different mutations were detected in V1, V2, and V3 sequences from primary isolates but not in sequences from plasma RNA or PBMC DNA. The HIV-1 near full-length sequence from the first collected plasma was of subtype G and the presence of subpopulations of subtypes B and G was observed with subtype-specific primers for protease and reverse transcriptase segments. Subsequent sequences from plasma, PBMCs, and primary isolates were obtained during a follow-up of 6 years; all of them were BG recombinants and showed identical intersubtype breakpoints between subtypes B and G in pol and nef. The env sequence from all primary isolates harbored a unique insert of subtype B. Specific primers for the V3 loop identified fluctuating subtype B and/or subtype G sequences either from plasma RNA or PBMC DNA.

The HIV-1 subtype A epidemic affecting injecting drug users (IDU) in former Soviet Union (FSU) countries started dramatically in Odessa, southern Ukraine, in 1995, and is caused by a variant of monophyletic origin, often designated IDU-A. We phylogenetically analyzed one near full-length genome and two partial sequences of three HIV-1 subtype A viruses collected in St. Petersburg, Russia, heterosexually transmitted in 1992-1994. The sequences branched basally to the IDU-A clade, together with eight viruses from Odessa collected in 1993, all presumably acquired heterosexually, and two viruses from the Democratic Republic of Congo. Of all other FSU sequences in databases, only those from three recently collected viruses, one from Ukraine and two from northwestern Russia, at least one of them acquired heterosexually, branched basally to the IDU-A cluster. The results indicate that the FSU IDU-A variant derives from a strain that initially propagated heterosexually in Ukraine and originated in central Africa.

Human immunodeficiency virus type 1 (HIV-1) BF intersubtype recombinant viruses are common in Argentina and Uruguay, where CRF12_BF and related recombinants are frequently found, and, in a lower proportion, in Brazil. Full-length genome sequences have been characterized in several of these recombinant viruses. Here, we analyze six newly derived near full-length genome sequences of BF recombinant viruses, three from Chile, one from Venezuela and two from Spain. Five of them had known epidemiological links to Argentina. Genomes were amplified by PCR from plasma RNA or from peripheral blood mononuclear cells’ DNA. Mosaic structures and phylogenetic relationships were analyzed by bootscanning, neighbour-joining phylogenetic trees and by examination of subtype signature nucleotides. One virus from Spain had a mosaic structure fully coincident with CRF12_BF. The others had unique mosaic structures, except the viruses from two Chilean sisters infected vertically from the same mother, who showed identical recombination patterns. Each of the unique recombinants had one to six breakpoints coincident with CRF12_BF and three also had two or three breakpoints coincident with a previously characterized unique recombinant from Argentina (A025) related to CRF12_BF. A phylogenetic tree of concatenated subtype F segments supported the relationship of five recombinants with CRF12_BF. In trees of partial subtype F and B segments, four recombinants clustered with A025. The examination of CRF12_BF signature amino acids and nucleotides supported the common ancestry of all the analyzed viruses. Based on these results, a model of generation of HIV-1 BF recombinants of Argentinean ancestry by successive rounds of recombination along diverse lineages deriving from a common BF recombinant ancestor related to CRF12_BF is proposed.

In spite of differences between female and male germ cells, and although both of them contribute to the gene pool of future generations, most germ cell mutagenicity studies in higher eukaryotes have been carried out on males. To study the response of female germ cells to mutagen/carcinogen exposure, the mutagenicity of two model chemicals like diethyl sulfate (DES) and hexamethylphosphoramide (HMPA), and the monofunctional methylating chemotherapeutic drug streptozotocin (STZ), has been analysed on repair efficient females of Drosophila melanogaster. Results previously obtained with N-ethyl-N-nitrosourea (ENU), another model chemical, have also been included in the analysis. The activity of bypass tolerance mechanism (BTM; represented by the mus308 locus) and nucleotide excision repair (NER) on the removal of oxygen and nitrogen ethylations was studied by determining DES mutagenicity in NER deficient females, comparing it with existing results for ENU, and by analysing both chemicals on BTM deficient females. Results indicate that (1) all chemicals are mutagenic on repair efficient females; (2) a measure of mutagenic activity ranked from the lowest DES to STZ, HMPA, and ENU as the highest. This order correlates with the repair of the respectively induced DNA damages, and with the mutagenic and carcinogenic potency of these compounds, considering the toxicity of cross-linking agents; (3) NER efficiently repairs nitrogen ethylation damage and seems to contribute to the processing of oxygen damage in female germ cells; and (4) BTM is involved on the processing of oxygen ethylation damage, whereas the results on nitrogen ethylation are not clear. Finally, these results indicate that differences between male and female germ cells affect the response to chemical exposure, and therefore demonstrate the necessity of analysing also female cells in germinal mutagenicity studies. In addition, these studies can provide important mechanistic information about germ cell chemical mutagenesis, and even when the analysis of oogonia is not possible, since all female germ cells are pre-meiotic, studies of oocytes could be a model for pre-meiotic cells.

We report the near full-length sequence characterization of a HIV-1 DF intersubtype recombinant virus from Spain, X492, directly amplified from peripheral blood mononuclear cells' DNA. This isolate shares an identical mosaic structure and exhibits consistent phylogenetic clustering along the genome with VI961, a previously characterized DF recombinant virus. By contrast, VI1310, which may represent the same recombinant form as VI961 (CRF05_DF), is only partially homologous to VI961 and X492. Of three additional DF recombinant viruses previously characterized in gag-pol, only one, VI1267, clusters uniformly with VI961 and X492; the other two branch separately in a segment of pol. These results allow us to define an HIV-1 circulating recombinant form (CRF05_DF), characterized in near full-length genomes of two isolates (VI961 and X492) and in partial gag-pol sequences of a third virus (VI1267). Three other reported DF recombinant viruses, including the fully sequenced VI1310, exhibit incomplete homology to VI961 and X492.

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster.

We recently reported the finding of phylogenetically related HIV-1 BG intersubtype recombinant and G subtype nonrecombinant viruses circulating among injecting drug users in the region of Galicia in northwestern Spain. Here, we report the characterization of near full-length genome sequences of nine of these viruses (seven BG recombinant and two of nonrecombinant G subtype), obtained from epidemiologically unlinked individuals. Bootscan analysis reveals that six recombinant viruses share an identical mosaic structure, with two intersubtype breakpoints delimiting a B subtype segment comprising most of Env gp120 and the external portion of Env gp41, with the remaining portions of the genome being of subtype G, thus mimicking a pseudotype virion structure. The seventh BG recombinant virus exhibits breakpoints in env coincident with the other BG viruses but contains additional B subtype segments in gag and pol. In phylogenetic trees of complete genomes and of the B subtype segment of env, all seven BG viruses group in a monophyletic cluster. G subtype portions of the BG viruses group uniformly with the newly derived nonrecombinant G subtype viruses of Galicia in bootscan analysis, which points to the locally circulating G subtype strain as parental of the recombinants. These results allow us to define a new HIV-1 circulating recombinant form (CRF14_BG), the first reported to originate in Western Europe.

The commonest known cause of Parkinson's disease (PD) is the G2019S mutation of the LRRK2 gene, but this mutation is not sufficient for causing PD, and many carriers of the mutation never develop PD symptoms during life. Differences at the expression level of certain genes, resulting from either genetic variations or environmental interactions, might be one of the mechanisms underlying differential risks for developing both idiopathic and genetic PD. To identify the genes involved in PD pathogenesis, we compared genome-wide gene expression (RNA-seq) in peripheral blood of 20 PD patients carrying the G2019S mutation of the LRRK2 gene, 20 asymptomatic carriers of the mutation, 20 subjects with idiopathic PD, 20 controls and 7 PD patients before and after initiating dopaminergic therapy. We identified 13 common genes (. ADARB2, CEACAM6, CNTNAP2, COL19A1, DEF4, DRAXIN, FCER2, HBG1, NCAPG2, PVRL2, SLC2A14, SNCA, and TCL1B) showing significant differential expression between G2019S-associated PD and asymptomatic carriers and also between idiopathic PD and controls but not between untreated and treated patients. Some of these genes are functionally involved in the processes known to be involved in PD pathogenesis, such as Akt signaling, glucose metabolism, or immunity. We consider that these genes merit further attention in future studies as potential candidate genes involved in both idiopathic and LRRK2-G2019S-associated forms of PD., This work was supported by the grant PI11/00228 form Instituto de Salud Carlos III. Bioinformatic analyses were performed on the Altamira Supercomputer at the University of Cantabria.

Genotoxicity and DNA repair , Genotoxicity and DNA repair , کتابخانه مرکزی دانشگاه علوم پزشکی تهران

Molecular mutation spectra induced by N-ethyl-N-nitrosourea have been obtained in several organisms and test systems, frequently showing different results. In Drosophila melanogaster this spectrum has been analyzed in post-meiotic stages, resulting in good agreement between the adduct spectrum and mutational events, the majority being GC→AT transitions (61%). However, when collecting data about in vivo ENU-induced mutations in mouse germ cell stages mostly damage at A:T sites (89%) was observed. In this work we analyze the molecular spectrum induced with ENU in pre-meiotic repair-active male germ cells of D.melanogaster, using the specific locus test (SLT) with the vermilion locus as target. Results show that the most mutagenic sites in spermatogonial stem cells of Drosophila are A:T pairs (85%), with AT→TA transversions (50%) and AT

The white-ivory somatic assay of Drosophila melanogaster was developed to detect genotoxic agents which induce loss of a tandem duplication. Although the mechanism of this loss is not known, some suggestions point to intrachromosomal recombination as the main reversion mechanism. Since the few papers published to date on this assay present controversial methodologies, prior to a larger study of chemicals with different mechanisms of action, we have carried out an analysis to optimize some conditions of this assay. For this purpose, we have used three different strains and four well characterized mutagenic chemicals: N-ethyl-N-nitrosourea (ENU), methyl methane-sulfonate (MMS), ethyl methanesulfonate (EMS) and hexamethyl phosphoramide (HMPA). The results obtained allow us to conclude that: (i) the best strain for this assay is C(1)DX,y,f Dp(1:1:1)w i ,y 2 , although the use of strain FM6,l(1)66a / Dp(1:1:1:1)wi,y2;st / st could be considered for some mechanistical studies; (ii) developmental reasons make it necessary to use as estimate of reversion frequency the proportion of eyes showing at least one spot; (iii) reversion frequency cannot be used as estimate of mutation efficiency, neither can spot size evaluate time of spot induction; (iv) the four chemicals clearly induce loss of the wi duplication; according to their activities they rank ENU HMPA , MMS ≈ EMS .

Objective To establish cutoff points for markers of bone remodeling that allow for screening of patients at risk for serious lithogenic activity. Materials and Methods We conducted a cross-sectional study with 182 patients (aged between 25 and 60 years) divided into 3 groups: group 1, 56 patients without lithiasis; group 2, 67 patients with light calcium lithiasis; and group 3, 59 patients with severe calcium lithiasis. The criteria for inclusion in and exclusion from the study were established, and light and severe lithogenic activity were defined. Metabolic variables in blood and urine, along with bone densitometry, were studied for the groups. Statistical analysis of the results and preparation of receiver operating characteristic curves to establish optimal cutoff points were performed. Results The patients in group 3 showed the greatest bone mineral density loss and the highest values for markers of bone remodeling, together with increased 24-hour calciuria. Using the receiver operating characteristic curves developed and based on statistical significance ( P = .0001), the following cutoff points for severe lithogenic activity, with a sensitivity between 75% and 85%, were established: β-crosslaps >0.331 ng/mL; osteocalcin >13.2 ng/mL; β-crosslaps/osteocalcin >0.024; 24-hour calciuria >306.6 mg; and fasting urine calcium/creatinine >0.105. Conclusion Patients with calcium lithiasis and elevated values for osteocalcin, β-crosslaps, β-crosslaps/osteocalcin, 24-hour calciuria, and fasting urine calcium/creatinine may present a high risk of severe lithogenic activity.

Recent genome-wide association studies of Parkinson's disease have nominated 3 new susceptibility loci (PARK16-18) and confirmed 2 known risk genes (MAPT and SNCA) in populations of European ancestry. We sought to replicate these findings. We genotyped single-nucleotide polymorphisms in each of these genes/loci in 1445 Parkinson's disease patients and 1161 controls from northern Spain. Logistic regression was used to test for association between genotype and Parkinson's disease under an additive model, adjusting for sex, age, and site. We also performed analyses stratified by age at onset. Single-nucleotide polymorphisms in MAPT (rs1800547; P = 3.1 × 10(-4) ) and SNCA (rs356219; P = 5.5 × 10(-4) ) were significantly associated with Parkinson's disease. However, none of the markers in PARK16-18 associated with Parkinson's disease in the overall sample, or in any age stratum, with P values ranging from .09 to .88. Although our data further validate MAPT and SNCA as Parkinson's disease susceptibility genes, we failed to replicate PARK16, PARK17, and PARK18. Potential reasons for the discordance between our study and previous genome-wide association studies include effects of population structure, power, and population-specific environmental interactions. Our findings suggest that additional studies of PARK16-18 are necessary to establish the role of these loci in modifying risk for Parkinson's disease in European-derived populations. © 2011 Movement Disorder Society.

Low circulating levels of sex hormone-binding globulin (SHBG) are a strong predictor of the risk of type 2 diabetes. Androgenetic alopecia (AGA) has been related to an increase in cardiovascular risk, but the mechanism of this association has not been elucidated. AGA can be associated with low levels of SHBG and insulin resistance, which could be related to hyperglycemia and type 2 diabetes.The objective of this study was to evaluate SHBG and blood glucose levels in men and women with early-onset AGA and control subjects to determine whether low levels of SHBG are associated with hyperglycemia.This case-control study included 240 patients consecutively admitted to the outpatient clinic (Dermatology Department of San Cecilio University Hospital, Granada, Spain), 120 with early-onset AGA (60 men and 60 women) and 120 control subjects (60 men and 60 women) with skin diseases other than alopecia.Of patients with AGA, 39.1% presented with hyperglycemia (110 mg/dL) versus 12.5% of controls (P0.0001). AGA patients with hyperglycemia or diabetes presented lower significant levels of SHBG than alopecic patients without hyperglycemia or type 2 diabetes, respectively. Patients with AGA and hyperglycemia presented significantly lower levels of SHBG than controls with hyperglycemia (22.3 vs 39.4 nmol/L for AGA patients and controls, respectively, P = .004). No significant differences in SHBG levels were noticed between patients and controls without hyperglycemia. Binary logistic regression showed a strong association between lower SHBG levels and glucose levels greater than 110 mg/dL in patients with AGA even after additional adjustment for sex, abdominal obesity, and free testosterone (odds ratio = 3.35; 95% confidence interval = 1.9-5.7; P.001).The study of a wider sample of AGA patients would confirm these findings and would permit analysis of the pathogenic mechanisms underlying the increase in cardiovascular risk in patients with AGA.An association between early-onset AGA, hyperglycemia/diabetes, and low levels of SHBG was observed in the current study. Low levels of SHBG could be a marker of insulin resistance and hyperglycemia/diabetes in patients with AGA.

TheD. melanogaster mus308gene, highly conserved among higher eukaryotes, is implicated in the repair of cross-links and of O-ethylpyrimidine DNA damage, working in a DNA damage tolerance mechanism. However, despite its relevance, its possible role on the processing of different DNA ethylation damages is not clear. To obtain data on mutation frequency and on mutation spectra inmus308deficient (mus308-) conditions, the ethylating agent diethyl sulfate (DES) was analysed in postmeiotic male germ cells. These data were compared with those corresponding tomus308efficient conditions. Our results indicate that Mus308 is necessary for the processing of oxygen and N-ethylation damage, for the survival of fertilized eggs depending on the level of induced DNA damage, and for an influence of the DNA damage neighbouring sequence. These results support the role ofmus308in a tolerance mechanism linked to a translesion synthesis pathway and also to the alternative end-joinig system.

BACKGROUND: In Ghana programs to expand antiretroviral access are being implemented. In this context the dynamic genetic evolution of HIV-1 requires continuous surveillance particularly when diverse genetic forms co-circulate. METHODS: Phylogenetic and antiretroviral resistance analyses of HIV-1 partial pol sequences from plasma RNA samples from 207 Ghanaian individuals were performed. RESULTS: 66% of infections were CRF02_AG whereas 25% were unique recombinant forms (URFs). All 52 URFs were characterized by bootscanning. CRF02_AG was parental strain in 87% of URFs forming recombinants with genetic forms circulating in minor proportions: CRF06_cpx sub-subtype A3 CRF09_cpx and subtypes G and D. Two triple recombinants (CRF02_AG/A3/CRF06_cpx and CRF02_AG/A3/CRF09_cpx) were identified. Antiretroviral resistance analyses revealed that six individuals five of which were antiretroviral drug-experienced harbored mutations conferring high level of resistance to reverse transcriptase inhibitors. No major resistance mutations were identified in the protease although insertions of one and three amino acids were detected. CONCLUSIONS: The high frequency of URFs detected probably reflects a significant incidence of coinfections or superinfections with diverse viral strains which increases the genetic complexity of the HIV-1 epidemic in West Africa. Monitoring of HIV-1 drug resistance might provide data on the implications of intersubtype recombination in response to antiretrovirals.

Highly diverse HIV-1 genetic forms are circulating in Cuba, including subtypes B and G and two recombinant forms of African origin (CRF18_cpx and CRF19_cpx). Here we phylogenetically analyze pol sequences from a large collection of recent samples from Cuba, corresponding to 425 individuals from all Cuban provinces, which represents approximately 12% of prevalent infections in the country. RNA from plasma was used to amplify a pol segment by reverse transcription-polymerase chain reaction; phylogenetic analyses were performed with neighbour-joining trees and bootscanning. The distribution of genetic forms was subtype B, 41.2%; CRF19_cpx, 18.4%; BG recombinants, 11.6%; CRF18_cpx, 7.1%; subtype C, 6.1%; subtype G, 3.8%; B/CRF18 recombinants, 2.6%; subtype H, 2.1%; B/CRF19 recombinants, 1.7%; and others, 5.4%. Seventy-five (17.6%) viruses were recombinant between genetic forms circulating in Cuba. In logistic regression analyses, adjusting by gender and region, subtype B was more prevalent (OR 5.0, 95% CI 2.0-12.3) and subtype G less prevalent (OR 0.1, 95% CI 0.0-0.5) among men who have sex with men (MSM) than among heterosexuals. Within the main genetic forms of Cuba there were phylogenetic subclusters, several of which correlated with risk exposure or region. BG recombinants formed three phylogenetically related subclusters, corresponding to three different mosaic structures; most of these recombinants were from MSM from Havana City, among whom they have expanded recently, reaching 31% HIV-1 infections diagnosed in 2003. This study confirms the high HIV-1 diversity and frequent recombination in Cuba and reveals the recent expansion of diverse related BG recombinant forms in this country.

Circulating recombinant forms (CRFs) represent a substantial proportion of HIV-1 isolates in the global pandemic. Characterization of HIV-1 genetic forms, including CRFs, may be relevant to studies on molecular epidemiology, recombination, superinfection, vaccine development, and antiretroviral therapy. This study analyzes near complete genomes of 4 epidemiologically unlinked viruses from Cuba, originally characterized as D/A intersubtype recombinants in pol and env segments. The genomes of 3 viruses exhibited virtually coincident mosaic structures, with multiple segments of subtypes A, D, and G and uniform phylogenetic clustering with each other along the genome. These results allow us to define a new CRF (CRF19_cpx). The 4th analyzed Cuban virus was recombinant between CRF19_cpx and CRF18_cpx (which also circulates in Cuba). CRF19_cpx exhibited homology to an AG intersubtype recombinant virus from Cameroon (CM53392) along approximately 5 kb and clustered with a subtype D virus from Gabon (G109) in gag. Four other viruses from central or west Africa were also phylogenetically related to CRF19_cpx in env fragments. These results allow us to define CRF19_cpx as a second novel CRF of African origin circulating in Cuba, to identify putative representative viruses of its parental strains, and to characterize a unique CRF18/CRF19 recombinant virus.

The oligonucleotide ligation assay is a genotypic assay for the detection of resistance-associated mutations to reverse transcriptase and protease inhibitors in human immunodeficiency virus type 1 subtype B. This assay has been modified and developed for non-B subtypes and recombinant strains and has been evaluated with sequencing, resulting in a more sensitive assay than sequencing for non-B subtypes.

There are few data on drug resistance-associated mutations in the former Soviet Union since, studies have usually been focused on the env or gag genes for subtype information. This study examines the prevalence and patterns of resistance-associated mutations to reverse transcriptase and protease inhibitors (RTI, PRI) in 278 HIV-1-infected treatment-naive subjects from countries of Eastern Europe, and defines characteristic polymorphisms of RT and PR sequences in HIV-1 subtype A viruses. Blood samples were collected between 1997 and 2004. Plasma RNA was used for PR-RT amplification by reverse transcription coupled with nested PCR and sequencing. Phylogenetic analysis was done with neighbor-joining trees and bootscanning. Analysis of drug resistance mutations, with Stanford University HIV Drug Resistance Database's algorithm, resulted in an overall prevalence of 12.9% resistance to RTI and 3.9% to PRI. The most frequent substitutions in the RT region were at positions 62 and 236. V77I substitution in PR was found in 47.8% of samples. Polymorphisms in subtype A sequences were identified. This is the first study reporting the prevalence and patterns of both PRI and RTI resistance-associated mutations in naive HIV-1 infected patients from the former Soviet Union. These data underline the importance of genotypic resistance testing of chronically HIV-1-infected patients before initiating treatment, in order to select the most suitable drug regimen. J. Med. Virol. 77:337–344, 2005. © 2005 Wiley-Liss, Inc.

Background: Analysis of partial pol and env sequences have indicated a high diversity of HIV-1 genetic forms in Cuba, including two potential novel circulating recombinant forms (CRF): U pol /H env and D pol /A env . Objectives: To determine whether U pol /H env recombinant viruses from Cuba, detected in 7% of samples, represent a novel HIV-1 CRF, and to identify non-Cuban viruses related to this recombinant form. Methods: Near full-length genome amplification was carried out by nested polymerase chain reaction in four overlapping DNA segments of two epidemiologically unlinked viruses in uncultured peripheral blood mononuclear cells. The sequences were analysed phylogenetically. Recombinant structures and phylogenetic relationships were analysed by bootscanning and by maximum likelihood. Searches for related viruses in databases were initially based on sequence homology and sharing of signature nucleotides. Results: Both Cuban viruses clustered uniformly in bootscans all along the genome with each other and with a virus from Cameroon, CM53379, indicating that all three represent the same recombinant form. Their genome comprised multiple segments clustering with subtypes A1, F, G, H and K, as well as segments failing to cluster with recognized subtypes. The newly defined CRF, designated CRF18―cpx, was phylogenetically related in partial segments to CRF13―cpx, CRF04―cpx and 36 additional viruses, most of them from Central Africa. One of the viruses from Cameroon, sequenced in the near full-length genome, was a CRF18―cpx/subtype G secondary recombinant. Conclusions: A novel HIV-1 complex circulating recombinant form (CRF18―cpx) has been identified that is circulating in Cuba and Central Africa.

The natural occurrence of primary resistance mutations in reverse transcriptase (RT) and protease (PR) genes of HIV-1 isolates from untreated patients has been reported and it may have important implications for the response to drug treatment. It is predictable that the same occurs in the HR1 region of gp41 sequence from patients who have never received T20 therapy, and in this regard it would be important to know not only the mutation frequencies at HR1 region but also the natural polymorphisms at resistance-associated positions present in the absence of this drug.The objectives of this study are to investigate the existence of natural resistance-associated mutations to T20 in HR1 gp41 region corresponding to different HIV-1 genetic forms from T20 naive patients and to determine their prevalence.Two hundred HIV-1 gp41 sequences were included: subtype B: 164 (81.3%); subtype A: 15 (8.2%); subtype G: 10 (4.6%); subtype F: 6 (3.5%); subtype C: 3 (1.8%); subtype K: 1 (0.6%); and subtype D: 1 (0.6%). We analyzed the resistance-associated mutations previously described: Q32H/R, G36D/S, I37V, V38A/M, Q39R/H, Q40H, N42T/D/Q/H, N43D/S/K/Q, L44M, L45M, R46M and V69I.Natural resistance mutations to T20 were found at a high frequency: 10.5%, corresponding to 9.1% in subtype B and 16.7% in non-B subtype samples. Polymorphisms were more frequent in non-B and recombinant forms than in subtype B (p0.001). Different substitutions were related to subtypes: N42S in subtypes A, B, G and C, but not in F, Q56R in subtype A from CRF02_AG, and L54M in subtype B from CRF14_BG.To our knowledge this is the first study describing natural-resistance to T20 among different HIV-1 subtypes, warranting a study of the biological significance of this mutations and their clinical relevance. The detection of differences between subtypes may have an influence on the rate and patterns of resistance in patients undergoing T20 treatment.

We analyze the recombinant structures and phylogenetic relationships of nine near full-length genome sequences of HIV-1 BF intersubtype recombinant viruses from Brazil, eight of them newly derived. These were obtained by PCR amplification from peripheral blood mononuclear cells (PBMCs) DNA or PBMCs culture supernantant RNA. The recombinants exhibited unique mosaic structures, except two viruses with a single near coincident breakpoint. Comparison with CRF12_BF revealed only two coincident breakpoints in two recombinants. Phylogenetic analyses failed to support a common ancestry of Brazilian recombinants or their relationship to CRF12_BF, which widely circulates in Argentina. Intersubtype breakpoint distribution along the genome was uneven, with the highest mean frequency in the polymerase domain of reverse transcriptase, and the lowest in env. These results indicate that HIV-1 BF recombinants from Brazil have independent origins and are unrelated to CRF12_BF, and that intersubtype breakpoints are frequent in pol segments analyzed for drug resistance detection.

Although N-nitrosodiethylamine (NDEA) is a potent carcinogen in rodents and a probable human carcinogen, little attempts were made to characterize its mutation spectrum in higher eukaryotes. We have compared forward mutation frequencies at multiple (700) loci with the mutational spectrum induced at the vermilion gene of Drosophila, after exposure of post- and pre-meiotic male germ cells to NDEA. Among 30 vermilion mutants collected from post-meiotic stages were 12 G:C-->A:T transitions (40%), 8 A:T-->T:A transversions (27%), and 4 structural rearrangements (13%). The remainder were three A:T-->G:C transitions, two G:C-->C:G transversions and one G:C-->T:A transversion. The results show that although NDEA induces predominantly transitions (40% G:C-->A:T and 10% A:T-->G:C), the frequencies of transversions (37%, of which 27% of A:T-->T:A transversions) and especially of rearrangements (13%) are remarkably high. This mutation spectrum differs significantly from that produced by the direct-ethylating agent N-ethylnitrosourea (ENU), although the relative distribution of ethylated DNA adducts is similar for both carcinogens. These differences, in particular the occurrence of rearrangements, are most likely the result of the requirement of NDEA for bioactivation. Since all four rearrangements were collected from non-metabolizing spermatozoa (or late spermatids), it is hypothesized that they derived from acetaldehyde, a stable metabolite of NDEA. Due to its cytotoxicity, attempts to isolate vermilion mutants from NDEA-exposed pre-meiotic cells were largely unsuccessful, because only two mutants (one A:T-->G:C transition and one 1bp insertion) were collected from those stages. Our results show that NDEA is capable of generating carcinogenic lesions other than base pair substitutions.

This work was supported by grants from the Parkinson's disease Foundation; Department of Veterans Affairs (1I01BX000531), National Institutes of Health (P50 NS062684, R01 NS065070 and R25 TW009345), Fondo de Investigacion Sanitaria (FIS, PI11/00228, PFIS, FI 11/00259, IFIMAV, and WLA 04/11), Instituto de Salud Carlos III (PI11/00093 and PI08/0915), Spanish Ministry of Economy and Competitiveness, European Social Fund, and the Asociación Parkinson Asturias.

Seven carcinogenic compounds (urethane, ethionine, auramine O, safrole, amitrole, acetamide and thioacetamide) were tested using the white-ivory (Wi) assay of Drosophila melanogaster. These compounds were chosen because they were considered as Ames-test negative but produced positive results in the yeast DEL assay, which estimates the introduction of intrachromosomal recombination. Only one compound, urethane, produced positive results in the Wi assay, while the remaining were classified as negative. These results indicate that, in contrast with which has been postulated in yeast, these carcinogens do not induce any event associated to intrachromosomal recombination in D. melanogaster.

The hypersensitivity of the mus308 mutant of D. melanogaster to cross-linking agents has been suggested to be the consequence of a possible defect of this mutant in DNA cross-link repair. Moreover, the mus308 mutation has been proposed as an animal model for the study of Fanconi's anemia. In order to obtain more information about the function controlled by this locus, we have measured the mutability of the mus308 mutant to several mutagens with different modes of action using the sex-linked recessive lethal test. We show that this mutation confers hypermutability not only to the cross-linking agents tested, hexamethylphosphoramide and hexamethylmelamine, but to the point mutagen N-ethyl-N-nitrosourea as well, whereas the response to methyl methanesulfonate was normal. The results suggest that the mus308 locus is not defective in a repair pathway specific for cross-links but is rather involved in a step of a more general post-replication repair process responsible for the removal of non-excised adducts.

The DNA base sequence changes induced by diethyl sulfate (DES) were analyzed in postmeiotic male germ cells of Drosophila melanogaster. 31 transmissible vermilion mutants were recovered in F1 and F2 generations, with a frequency of 2.6 x 10(-4) for the F1, and of 1.8-13 x 10(-4) for the F2. The results show that DES induces both base pair substitutions (93%) and deletions (7%). In accord with its relatively high ability to alkylate oxygens in DNA, the most frequent type of sequence alteration among the basepair changes are GC-AT transitions, accounting for 73% of mutations, followed by transversions AT-TA (10%). DES also induced AT-GC transitions and AT-CG transversions. Both induced deletions were intralocus deletions, not occurring between basepair repeats. No influence of neighboring bases on the mutation position was found.