Can the contamination of urban street sediment be used as an indicator for traffic density? A case-study in the city of Leuven, Belgium.

The concentration, in urban street sediment, of 13 chemical elements was measured in the city of Leuven, Belgium. A total of 77 locations were sampled in pedestrian zones, streets with limited traffic, streets with much traffic, and on the ring road that has very busy traffic. The data were compared to NO2 concentrations measured by the CurieuzeNeuzen-Vlaanderen project, a large-scale study with 20,000 sampling points all over Flanders (the northern part of Belgium). NO2 is a frequently used indicator for traffic pollution. In Leuven the highest enrichment (strongest pollution) was measured for Cu, Sb, Pb and Zn. These elements could be related to brake wear and tire wear, respectively. For Cu and Zn the concentrations in Leuven exceed those in most of the other cities investigated in the literature. Moderate enrichment was measured for Cd, Cr, Ni and S. The other elements (As, Co, Fe, Mn and V) showed concentrations close to the background value in unpolluted soil. The Integrated Pollution Index (IPI) for Leuven is 8.15, which, according to criteria proposed by the literature on street sediment, classifies Leuven, on average over its surface area, as a "very highly polluted" city. As expected, for Cu and Sb the highest contamination is found on the ring road and the busy traffic circulation loops. For Zn and Pb, on the other hand, the highest contamination occurs in the city center, in the pedestrian zone where no traffic is allowed except for buses and taxis. We hypothesize that this is a result of historic accumulation of these elements at the time traffic was still allowed in this zone. In Leuven the chemical composition of street sediment did not correlate to the NO2 concentrations. This study shows that measurements of current pollution by traffic are not sufficient to determine the health risk because much exposure to toxic substances may be caused by resuspension, by traffic or wind, of substances that have accumulated in the city over time, sometimes decades ago, when regulations were much less stringent then today.

Immunotoxicological and neurotoxicological profile of health effects following subacute exposure to geogenic dust from sand dunes at the Nellis Dunes Recreation Area, Las Vegas, NV.

Exposure to geogenic particulate matter (PM) comprised of mineral particles has been linked to human health effects. However, very little data exist on health effects associated with geogenic dust exposure in natural settings. Therefore, we characterized particulate matter size, metal chemistry, and health effects of dust collected from the Nellis Dunes Recreation Area (NDRA), a popular off-road vehicle area located near Las Vegas, NV. Adult female B6C3F1 mice were exposed to several concentrations of mineral dust collected from active and vegetated sand dunes in NDRA. Dust samples (median diameter: 4.4 µm) were suspended in phosphate-buffered saline and delivered at concentrations ranging from 0.01 to 100 mg dust/kg body weight by oropharyngeal aspiration. ICP-MS analyses of total dissolution of the dust resulted in aluminum (55,090 µg/g), vanadium (70 µg/g), chromium (33 µg/g), manganese (511 µg/g), iron (21,600 µg/g), cobalt (9.4 µg/g), copper (69 µg/g), zinc (79 µg/g), arsenic (62 µg/g), strontium (620 µg/g), cesium (13 µg/g), lead 25 µg/g) and uranium (4.7 µg/g). Arsenic was present only as As(V). Mice received four exposures, once/week over 28-days to mimic a month of weekend exposures. Descriptive and functional assays to assess immunotoxicity and neurotoxicity were performed 24 h after the final exposure. The primary observation was that 0.1 to 100 mg/kg of this sand dune derived dust dose-responsively reduced antigen-specific IgM antibody responses, suggesting that dust from this area of NDRA may present a potential health risk.

Health effects following subacute exposure to geogenic dusts from arsenic-rich sediment at the Nellis Dunes Recreation Area, Las Vegas, NV.

Geogenic dust from arid environments is a possible inhalation hazard for humans, especially when using off-road vehicles that generate significant dust. This study focused on immunotoxicological and neurotoxicological effects following subacute exposure to geogenic dust generated from sediments in the Nellis Dunes Recreation Area near Las Vegas, Nevada that are particularly high in arsenic; the naturally-occurring arsenic concentrations in these surficial sediments ranged from 4.8 to 346µg/g. Dust samples from sediments used in this study had a median diameter of 4.5µm and also were a complex mixture of naturally-occurring metals, including aluminum, vanadium, chromium, manganese, iron, cobalt, copper, zinc, strontium, cesium, lead, uranium, and arsenic. Adult female B6C3F1 mice exposed via oropharyngeal aspiration to 0.01 to 100mg dust/kg body weight, four times, a week apart, for 28days, were evaluated 24h after the last exposure. Peripheral eosinophils were increased at all concentrations, serum creatinine was dose responsively increased beginning at 1.0mg/kg/day, and blood urea nitrogen was decreased at 10 and 100mg/kg/day. Antigen-specific IgM responses and natural killer cell activity were dose-responsively suppressed at 0.1mg/kg/day and above. Splenic CD4+CD25+ T cells were decreased at 0.01, 0.1, 10, and 100mg/kg/day. Antibodies against MBP, NF-68, and GFAP were selectively reduced. A no observed adverse effect level of 0.01mg/kg/day and a lowest observed adverse effect level of 0.1mg/kg/day were determined from IgM responses and natural killer cell activity, indicating that exposure to this dust, under conditions similar to our design, could affect these responses.

Unraveling the genetic landscape of autosomal recessive Charcot-Marie-Tooth neuropathies using a homozygosity mapping approach.

Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes (ganglioside-induced differentiation protein 1­GDAP1, SH3 domain and tetratricopeptide repeats-containing protein 2­SH3TC2, histidine-triad nucleotide binding protein 1­HINT1) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 22% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.3% patients. Apart from a newly described founder mutation in GDAP1, the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.

The skin microbiome of caspase-14-deficient mice shows mild dysbiosis.

Caspase-14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase-14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2-pyrrolidone-5-carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase-14(-/-) mice show enhanced antibacterial response compared to wild-type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild-type and caspase-14(-/-) mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase-14 ablation leads to an increase in bacterial richness and diversity during steady-state conditions. Although both wild-type and caspase-14(-/-) skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase-14(-/-) mice. Altogether, our data demonstrated that caspase-14 deficiency causes the imbalance of the skin-resident bacterial communities.

Mutations in SEPT9 cause hereditary neuralgic amyotrophy.

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy affecting the brachial plexus. HNA is triggered by environmental factors such as infection or parturition. We report three mutations in the gene septin 9 (SEPT9) in six families with HNA linked to chromosome 17q25. HNA is the first monogenetic disease caused by mutations in a gene of the septin family. Septins are implicated in formation of the cytoskeleton, cell division and tumorigenesis.

Microplastic appraisal of soil, water, ditch sediment and airborne dust: The case of agricultural systems.

Although microplastic pollution jeopardizes both terrestrial and aquatic ecosystems, the movement of plastic particles through terrestrial environments is still poorly understood. Agricultural soils exposed to different managements are important sites of storage and dispersal of microplastics. This study aimed to identify the abundance, distribution, and type of microplastics present in agricultural soils, water, airborne dust, and ditch sediments. Soil health was also assessed using soil macroinvertebrate abundance and diversity. Sixteen fields were evaluated, 6 of which had been exposed to more than 5 years of compost application, 5 were exposed to at least 5 years of plastic mulch use, and 5 were not exposed to any specific management (controls) within the last 5 years. We also evaluated the spread of microplastics from the farms into nearby water bodies and airborne dust. We found 11 types of microplastics in soil, among which Light Density Polyethylene (LDPE) and Light Density Polyethylene covered with pro-oxidant additives (PAC) were the most abundant. The highest concentrations of plastics were found in soils exposed to plastic mulch management (128.7 ± 320 MPs.g-1 soil and 224.84 ± 488 MPs.g-1 soil, respectively) and the particles measured from 50 to 150 µm. Nine types of microplastics were found in water, with the highest concentrations observed in systems exposed to compost. Farms applying compost had higher LDPE and PAC concentrations in ditch sediments as compared to control and mulch systems; a significant correlation between soil polypropylene (PP) microplastics with ditch sediment microplastics (r2 0.7 p < 0.05) was found. LDPE, PAC, PE (Polyethylene), and PP were the most abundant microplastics in airborne dust. Soil invertebrates were scarce in the systems using plastic mulch. A cocktail of microplastics was found in all assessed matrices.

No evidence for GNAS copy number variants in patients with features of Albright's hereditary osteodystrophy and abnormal platelet Gs activity.

Albright's hereditary osteodystrophy (AHO) is characterized by short stature, round face, calcifications, obesity, brachydactyly and intellectual disability. AHO without hormone resistance is called pseudopseudohypoparathyroidism (PPHP), a rare clinical condition difficult to diagnose with highly variable features. PPHP is caused by paternally inherited loss-of-function mutations in the GNAS. Patients with 2q37 microdeletions or HDAC4 mutations are also defined as having an AHO-like phenotype with normal stimulatory G (Gs) function. We have studied 256 patients with AHO features but no other diagnosis. Their platelet Gs activity was determined via the aggregation-inhibition test showing Gs hypo- or hyperfuncton in 24% and 15% of the patients, respectively. Before initiating with detailed (epi)genetic GNAS studies, we here wanted to excluded copy number variants (CNVs) in GNAS as cause of AHO with a novel large-scale screening technique. Multiplex amplicon quantification (MAQ) for CNVs screening was developed for the 20q13.3 region including GNAS and potential long-range imprinting control elements such as STX16. This is the first large-scale GNAS CNV study in patients with common AHO features but no CNVs were detected. In conclusion, CNVs in the GNAS region are not likely to cause an AHO-like phenotype with or without abnormal platelet Gs activity. Future studies will be undertaken to find out whether these AHO patients with abnormal Gs function are characterized by GNAS coding or methylation defects.

Guanosine triphosphate cyclohydrolase 1 promoter deletion causes dopa-responsive dystonia.

BACKGROUND: Autosomal dominant dopa-responsive dystonia (AD-DRD) is caused by a biochemical defect primarily resulting from guanosine triphosphate cyclohydrolase 1 gene (GCH1) mutations. Few families have been reported without mutations in GCH1. METHODS: Genome-wide linkage analysis and positional cloning to identify the genetic defect in a Belgian AD-DRD family was carried out. RESULTS AND CONCLUSION: In this study, we report on the identification and characterization of a novel 24-kb deletion spanning exon 1 and the 5' regulatory region of GCH1 causing a wide spectrum of motor and nonmotor symptoms in a large Belgian AD-DRD family. This large-scale deletion of regulatory sequences leads to decreased GCH1 activity in all carriers, most probably resulting from allelic loss of transcription. We mapped the breakpoints of this deletion to the nucleotide level, allowing the development of a straightforward polymerase chain reaction assay for fast, efficient detection of this large deletion, which will prove valuable for preimplantation genetic diagnosis.

Rare copy number variants in neuropsychiatric disorders: Specific phenotype or not?

From a number of genome-wide association studies it was shown that de novo and/or rare copy number variants (CNVs) are found at an increased frequency in neuropsychiatric diseases. In this study we examined the prevalence of CNVs in six genomic regions (1q21.1, 2p16.3, 3q29, 15q11.2, 15q13.3, and 16p11.2) previously implicated in neuropsychiatric diseases. Hereto, a cohort of four neuropsychiatric disorders (schizophrenia, bipolar disorder, major depressive disorder, and intellectual disability) and control individuals from three different populations was used in combination with Multilpex Amplicon Quantifiaction (MAQ) assays, capable of high resolution (kb range) and custom-tailored CNV detection. Our results confirm the etiological candidacy of the six selected CNV regions for neuropsychiatric diseases. It is possible that CNVs in these regions can result in disturbed brain development and in this way lead to an increased susceptibility for different neuropsychiatric disorders, dependent on additional genetic and environmental factors. Our results also suggest that the neurodevelopmental component is larger in the etiology of schizophrenia and intellectual disability than in mood disorders. Finally, our data suggest that deletions are in general more pathogenic than duplications. Given the high frequency of the examined CNVs (1-2%) in patients of different neuropsychiatric disorders, screening of large cohorts with an affordable and feasible method like the MAQ assays used in this study is likely to result in important progress in unraveling the genetic factors leading to an increased susceptibility for several psychiatric disorders.

Identification of a CACNA2D4 deletion in late onset bipolar disorder patients and implications for the involvement of voltage-dependent calcium channels in psychiatric disorders.

The GWAS-based association of CACNA1C with bipolar disorder (BPD) is one of the strongest genetic findings to date. CACNA1C belongs to the family of CACN genes encoding voltage-dependent calcium channels (VDCCs). VDCCs are involved in brain circuits and cognitive processes implicated in BPD and schizophrenia (SZ). Recently, it was shown that rare copy number variations (CNVs) are found at an increased frequency in SZ and to a lesser extent also in BPD, suggesting the involvement of CNVs in the causation of these diseases. We hypothesize that CNVs in CACN genes can influence the susceptibility to BPD, SZ, and/or schizoaffective disorder (SZA). A search for CNVs in eight CACN genes in a patient-control sample of European decent was performed. A total of 709 BP patients, 645 SZ patients, 189 SZA patients, and 1,470 control individuals were screened using the Multiplex Amplicon Quantification (MAQ) method. We found a rare, partial deletion of 35.7 kb in CACNA2D4 in two unrelated late onset bipolar I patients and in one control individual. All three deletions shared the same breakpoints removing exons 17-26 of CACNA2D4, comprising part of the CACHE domain. Based on the data we cannot claim causality to BPD of the identified CACNA2D4 deletion but nevertheless this deletion can be important in unraveling the underlying processes leading to psychiatric diseases in general and BPD in particular.

Collection of human and environmental data on pesticide use in Europe and Argentina: Field study protocol for the SPRINT project.

Current farm systems rely on the use of Plant Protection Products (PPP) to secure high productivity and control threats to the quality of the crops. However, PPP use may have considerable impacts on human health and the environment. A study protocol is presented aiming to determine the occurrence and levels of PPP residues in plants (crops), animals (livestock), humans and other non-target species (ecosystem representatives) for exposure modelling and impact assessment. To achieve this, we designed a cross-sectional study to compare conventional and organic farm systems across Europe. Environmental and biological samples were/are being/will be collected during the 2021 growing season, at 10 case study sites in Europe covering a range of climate zones and crops. An additional study site in Argentina will inform the impact of PPP use on growing soybean which is an important European protein-source in animal feed. We will study the impact of PPP mixtures using an integrated risk assessment methodology. The fate of PPP in environmental media (soil, water and air) and in the homes of farmers will be monitored. This will be complemented by biomonitoring to estimate PPP uptake by humans and farm animals (cow, goat, sheep and chicken), and by collection of samples from non-target species (earthworms, fish, aquatic and terrestrial macroinvertebrates, bats, and farm cats). We will use data on PPP residues in environmental and biological matrices to estimate exposures by modelling. These exposure estimates together with health and toxicity data will be used to predict the impact of PPP use on environment, plant, animal and human health. The outcome of this study will then be integrated with socio-economic information leading to an overall assessment used to identify transition pathways towards more sustainable plant protection and inform decision makers, practitioners and other stakeholders regarding farming practices and land use policy.

Multiplex Amplicon Quantification (MAQ), a fast and efficient method for the simultaneous detection of copy number alterations in neuroblastoma.

BACKGROUND: Cancer genomes display characteristic patterns of chromosomal imbalances, often with diagnostic and prognostic relevance. Therefore assays for genome-wide copy number screening and simultaneous detection of copy number alterations in specific chromosomal regions are of increasing importance in the diagnostic work-up of tumors. RESULTS: We tested the performance of Multiplex Amplicon Quantification, a newly developed low-cost, closed-tube and high-throughput PCR-based technique for detection of copy number alterations in regions with prognostic relevance for neuroblastoma. Comparison with array CGH and the established Multiplex Ligation-dependent Probe Amplification method on 52 neuroblastoma tumors showed that Multiplex Amplicon Quantification can reliably detect the important genomic aberrations. CONCLUSION: Multiplex Amplicon Quantification is a low-cost and high-throughput PCR-based technique that can reliably detect copy number alterations in regions with prognostic relevance for neuroblastoma.

Strong evidence that GNB1L is associated with schizophrenia.

Evidence that a gene or genes on chromosome 22 is involved in susceptibility to schizophrenia comes from two sources: the increased incidence of schizophrenia in individuals with 22q11 deletion syndrome (22q11DS) and genetic linkage studies. In mice, hemizygous deletion of either Tbx1 or Gnb1l can cause deficits in pre-pulse inhibition, a sensory motor gating defect which is associated with schizophrenia. We tested the hypothesis that variation at this locus confers risk of schizophrenia and related disorders in a series of case-control association studies. First, we found evidence for a male-specific genotypic association (P = 0.00017) TBX1/GNB1L in 662 schizophrenia cases and 1416 controls from the UK. Moreover, we replicated this finding in two independent case-control samples (additional 746 cases and 1330 controls) (meta analysis P = 1.8 x 10(-5)) and also observed significant evidence for genotypic association in an independent sample of 480 schizophrenia parent-proband trios from Bulgaria with markers at this locus, which was again strongest in the male probands (P = 0.004). Genotyping the most significant SNPs in a sample of 83 subjects with 22q11DS with and without psychosis again revealed a significant allelic association with psychosis in males with 22q11DS (P = 0.01). Finally, using allele specific expression analysis, we have shown that the markers associated with psychosis are also correlated with alterations in GNB1L expression, raising the hypothesis that the risk to develop psychosis at this locus could be mediated in a dose sensitive manner via gene expression. However, other explanations are possible, and further analyses will be required to clarify the correct functional mechanism.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics.

Relative contribution of simple mutations vs. copy number variations in five Parkinson disease genes in the Belgian population.

The relative contribution of simple mutations and copy number variations (CNVs) in SNCA, PARK2, PINK1, PARK7, and LRRK2 to the genetic etiology of Parkinson disease (PD) is still unclear because most studies did not completely analyze each gene. In a large group of Belgian PD patients (N = 310) and control individuals (N = 270), we determined the mutation frequency of both simple mutations and CNVs in these five PD genes, using direct sequencing, multiplex amplicon quantification (MAQ), and real-time PCR assays. Overall, we identified 14 novel heterozygous variants, of which 11 were absent in control individuals. We observed eight PARK2 (multiple) exon multiplications in PD patients and one exon deletion in a control individual. Furthermore, we identified one SNCA whole-gene duplication. The PARK2 and LRRK2 mutation frequencies in Belgian PD patients were similar to those reported in other studies. However, at this stage the true pathogenic nature of some heterozygous mutations in recessive genes remains elusive. Furthermore, though mutations is SNCA, PINK1, and PARK7 are rare, our identification of a SNCA duplication confirmed that screening of these genes remains meaningful.

Paroxysmal exercise-induced dyskinesia and epilepsy is due to mutations in SLC2A1, encoding the glucose transporter GLUT1.

Paroxysmal exercise-induced dyskinesia (PED) can occur in isolation or in association with epilepsy, but the genetic causes and pathophysiological mechanisms are still poorly understood. We performed a clinical evaluation and genetic analysis in a five-generation family with co-occurrence of PED and epilepsy (n = 39), suggesting that this combination represents a clinical entity. Based on a whole genome linkage analysis we screened SLC2A1, encoding the glucose transporter of the blood-brain-barrier, GLUT1 and identified heterozygous missense and frameshift mutations segregating in this and three other nuclear families with a similar phenotype. PED was characterized by choreoathetosis, dystonia or both, affecting mainly the legs. Predominant epileptic seizure types were primary generalized. A median CSF/blood glucose ratio of 0.52 (normal >0.60) in the patients and a reduced glucose uptake by mutated transporters compared with the wild-type as determined in Xenopus oocytes confirmed a pathogenic role of these mutations. Functional imaging studies implicated alterations in glucose metabolism in the corticostriate pathways in the pathophysiology of PED and in the frontal lobe cortex in the pathophysiology of epileptic seizures. Three patients were successfully treated with a ketogenic diet. In conclusion, co-occurring PED and epilepsy can be due to autosomal dominant heterozygous SLC2A1 mutations, expanding the phenotypic spectrum associated with GLUT1 deficiency and providing a potential new treatment option for this clinical syndrome.

Dust dynamics in off-road vehicle trails: Measurements on 16 arid soil types, Nevada, USA.

Soil analyses and measurements with the Portable In Situ Wind Erosion Laboratory (PI-SWERL) were conducted on 16 soil types in an area heavily affected by off-road vehicle (ORV) driving. Measurements were performed in ORV trails as well as on undisturbed terrain to investigate how ORV driving affects the vulnerability of a soil to emit PM10 (particles<10microm), during the driving as well as during episodes of wind erosion. Particular attention is paid to how the creation of a new trail affects those properties of the topsoil that determine its capability to emit PM10. Also, recommendations are given for adequate management of ORV-designed areas. The type of surface (sand, silt, gravel, drainage) is a key factor with respect to dust emission in an ORV trail. Trails in sand, defined in this study as the grain size fraction 63-2000microm, show higher deflation thresholds (the critical wind condition at which wind erosion starts) than the surrounding undisturbed soil. Trails in silt (2-63microm) and in drainages, on the other hand, have lower deflation thresholds than undisturbed soil. The increase in PM10 emission resulting from the creation of a new ORV trail is much higher for surfaces with silt than for surfaces with sand. Also, the creation of a new trail in silt decreases the supply limitation in the top layer: the capacity of the reservoir of emission-available PM10 increases. For sand the situation is reversed: the supply limitation increases, and the capacity of the PM10 reservoir decreases. Finally, ORV trails are characterized by a progressive coarsening of the top layer with time, but the speed of coarsening is much lower in trails in silt than in trails in sand or in drainages. The results of this study suggest that, to minimize emissions of PM10, new ORV fields should preferably be designed on sandy terrain rather than in silt areas or in drainages.

Detailed analysis of the serotonin transporter gene (SLC6A4) shows no association with bipolar disorder in the Northern Swedish population.

Through active reuptake of serotonin into presynaptic neurons, the serotonin transporter (5-HTT) plays an important role in regulating serotonin concentrations in the brain, and it is the site of binding for tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs). Therefore it has been hypothesized that this transporter is involved in the etiology of bipolar (BP) disorder. Inconsistent association study results for the SLC6A4 gene encoding 5-HTT reported in literature emphasize the need for more systematic and detailed analyses of this candidate gene. We performed an extensive analysis of SLC6A4 on DNA of 254 BPI patients and 364 control individuals from a Northern Swedish isolated population. This analysis consisted of a HapMap LD-based association study including three widely investigated polymorphisms (5-HTTVNTR, 5-HTTLPR, and rs3813034), a copy-number variation (CNV) analysis and a mutation analysis of the complete coding sequence and the 3'-UTR of SLC6A4. No single marker showed statistically significant association with BPI, nor did any of the haplotypes. In the mutation analysis 13 novel variants were detected, including 2 amino acid substitutions M389V and I587L, but these are probably not implicated in risk for BP. No deletions or duplications were detected in the CNV analysis. We conclude that variation in the SLC6A4 gene or its regulatory regions does not contribute to the susceptibility for BP disorder in the Northern Swedish population.

TP53 Outperforms Other Androgen Receptor Biomarkers to Predict Abiraterone or Enzalutamide Outcome in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi).Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models. RESULTS: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18-3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001). CONCLUSIONS: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.