Predicting Hearing Loss in Testicular Cancer Patients after Cisplatin-Based Chemotherapy.

Testicular cancer is predominantly curable, but the long-term side effects of chemotherapy have a severe impact on life quality. In this research study, we focus on hearing loss as a part of overall chemotherapy-induced ototoxicity. This is a unique approach where we combine clinical data from the acclaimed nationwide Danish Testicular Cancer (DaTeCa)-Late database. Clinical and genetic data on 433 patients were collected from hospital files in October 2014. Hearing loss was classified according to the FACT/GOG-Ntx-11 version 4 self-reported Ntx6. Machine learning models combining a genome-wide association study within a nested cross-validated logistic regression were applied to identify patients at high risk of hearing loss. The model comprising clinical and genetic data identified 67% of the patients with hearing loss; however, this was with a false discovery rate of 49%. For the non-affected patients, the model identified 66% of the patients with a false omission rate of 19%. An area under the receiver operating characteristic (ROC-AUC) curve of 0.73 (95% CI, 0.71-0.74) was obtained, and the model suggests genes SOD2 and MGST3 as important in improving prediction over the clinical-only model with a ROC-AUC of 0.66 (95% CI, 0.65-0.66). Such prediction models may be used to allow earlier detection and prevention of hearing loss. We suggest a possible biological mechanism for cisplatin-induced hearing loss development. On confirmation in larger studies, such models can help balance treatment in clinical practice.

Association Study between Polymorphisms in DNA Methylation-Related Genes and Testicular Germ Cell Tumor Risk.

BACKGROUND: Testicular germ cell tumors (TGCT), histologically classified as seminomas and nonseminomas, are believed to arise from primordial gonocytes, with the maturation process blocked when they are subjected to DNA methylation reprogramming. SNPs in DNA methylation machinery and folate-dependent one-carbon metabolism genes have been postulated to influence the proper establishment of DNA methylation. METHODS: In this pathway-focused investigation, we evaluated the association between 273 selected tag SNPs from 28 DNA methylation-related genes and TGCT risk. We carried out association analysis at individual SNP and gene-based level using summary statistics from the Genome Wide Association Study meta-analysis recently conducted by the international Testicular Cancer Consortium on 10,156 TGCT cases and 179,683 controls. RESULTS: In individual SNP analyses, seven SNPs, four mapping within MTHFR, were associated with TGCT risk after correction for multiple testing (q ≤ 0.05). Queries of public databases showed that three of these SNPs were associated with MTHFR changes in enzymatic activity (rs1801133) or expression level in testis tissue (rs12121543, rs1476413). Gene-based analyses revealed MTHFR (q = 8.4 × 10-4), methyl-CpG-binding protein 2 (MECP2; q = 2 × 10-3), and ZBTB4 (q = 0.03) as the top TGCT-associated genes. Stratifying by tumor histology, four MTHFR SNPs were associated with seminoma. In gene-based analysis MTHFR was associated with risk of seminoma (q = 2.8 × 10-4), but not with nonseminomatous tumors (q = 0.22). CONCLUSIONS: Genetic variants within MTHFR, potentially having an impact on the DNA methylation pattern, are associated with TGCT risk. IMPACT: This finding suggests that TGCT pathogenesis could be associated with the folate cycle status, and this relation could be partly due to hereditary factors.

Corrigendum: Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias.

[This corrects the article DOI: 10.3389/fmicb.2017.01934.].

Ocular Surface Microbiota in Contact Lens Users and Contact-Lens-Associated Bacterial Keratitis.

Our objectives were to investigate whether the conjunctival microbiota is altered by contact lens wear and/or bacterial keratitis and to explore the hypothesis that commensals of conjunctival microbiota contribute to bacterial keratitis. Swab samples from both eyes were collected separately from the inferior fornix of the conjunctiva of non-contact-lens users (nparticipants = 28) and contact lens users (nparticipants = 26) and from patients with contact-lens-associated bacterial keratitis (nparticipants = 9). DNA from conjunctival swab samples was analyzed with 16S rRNA gene amplicon sequencing. Pathogens from the corneal infiltrates were identified by cultivation. In total, we identified 19 phyla and 283 genera; the four most abundant genera were Pseudomonas, Enhydrobacter, Staphylococcus, and Cutibacterium. Several pathogens related to bacterial keratitis were identified in the conjunctival microbiota of the whole study population, and the same bacteria were identified by both methods in the conjunctiva and cornea for four patients with contact-lens-associated bacterial keratitis. The overall conjunctival microbiota profile was not altered by contact lens wear or bacterial keratitis; thus, it does not appear to contribute to the development of bacterial keratitis in contact lens users. However, in some individuals, conjunctival microbiota may harbor opportunistic pathogens causing contact-lens-associated bacterial keratitis.

Genetically Inferred Telomere Length and Testicular Germ Cell Tumor Risk.

BACKGROUND: Studies evaluating the association between peripheral blood leukocyte telomere length (LTL) and testicular germ cell tumor (TGCT) risk have produced conflicting results. METHODS: Using available genotype data from the Testicular Cancer Consortium (TECAC), polygenic risk score and Mendelian randomization analyses of genetic variants previously associated with LTL were used to assess potential etiologic associations between telomere length and TGCT risk. RESULTS: Genetically inferred telomere length was not associated with TGCT risk among 2,049 cases and 6,921 controls with individual-level genotype data (OR, 1.02; 95% confidence interval, 0.97-1.07). Mendelian randomization analyses using summary statistic data further indicated no evidence for an association between telomere length and TGCT risk among all available TECAC participants (3,558 cases and 13,971 controls). CONCLUSIONS: Our analyses in the largest molecular genetic testicular cancer study to date provide no evidence for an association between genetically inferred peripheral blood LTL and TGCT risk. IMPACT: The lack of evidence for an overall association indicates that peripheral blood LTL is likely not a strong biomarker for TGCT risk.

Acetaminophen metabolism revisited using non-targeted analyses: Implications for human biomonitoring.

The analgesic paracetamol/acetaminophen (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders in humans. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is critical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key issue of potential underestimation of APAP use using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. The question we address is whether investigating additional metabolites than direct phase II conjugates could improve the monitoring of APAP. Using non-targeted analyses based on high-resolution mass spectrometry, we identified, in a controlled longitudinal exposure study with male volunteers, overlooked APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, these conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods therefore provides an option to decrease potential underestimation of APAP use. Moreover, our data challenge the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of a non-persistent chemical such as APAP.

Ocular surface microbiota in patients with aqueous tear-deficient dry eye.

PURPOSE: An altered ocular surface microbiota may contribute to the pathophysiology of dry eye disease. The aim of the study was to explore potential differences in microbiota diversity and composition in aqueous tear-deficient dry eye (with and without ocular graft-versus-host disease) compared with controls. METHODS: Swab samples from the inferior fornix of the conjunctiva were obtained from patients with aqueous tear-deficient dry eye with and without ocular graft-versus-host disease (n = 18, n = 21, respectively) and controls (n = 28). Isolated bacterial DNA from swabs were analyzed with 16S rRNA gene amplicon sequencing. RESULTS: Decreased microbiota diversity was observed in patients with aqueous tear-deficient dry eye (p ≤ 0.003) who also showed a difference in microbiota composition compared with controls (p = 0.001). Although several genera were less abundant in aqueous tear-deficient dry eye, a minimal core ocular surface microbiota comprising five genera was shared by >75% of the study participants: Enhydrobacter, Brevibacterium, Staphylococcus, Streptococcus and Cutibacterium. Pseudomonas was identified as a bacterial biomarker for controls and Bacilli for patients with aqueous tear-deficient dry eye. CONCLUSIONS: Ocular surface microbiota in patients with aqueous tear-deficient dry eye was characterized by an aberrant microbiota composition in comparison to controls, with decreased diversity and reduced relative abundances of several genera. Additionally, a few genera were present in most of the study population, indicating that a minimal core ocular surface microbiota may exist.

Systems genetics analysis identifies calcium-signaling defects as novel cause of congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) occurs in almost 1% of newborn children and is considered a multifactorial disorder. CHD may segregate in families due to significant contribution of genetic factors in the disease etiology. The aim of the study was to identify pathophysiological mechanisms in families segregating CHD. METHODS: We used whole exome sequencing to identify rare genetic variants in ninety consenting participants from 32 Danish families with recurrent CHD. We applied a systems biology approach to identify developmental mechanisms influenced by accumulation of rare variants. We used an independent cohort of 714 CHD cases and 4922 controls for replication and performed functional investigations using zebrafish as in vivo model. RESULTS: We identified 1785 genes, in which rare alleles were shared between affected individuals within a family. These genes were enriched for known cardiac developmental genes, and 218 of these genes were mutated in more than one family. Our analysis revealed a functional cluster, enriched for proteins with a known participation in calcium signaling. Replication in an independent cohort confirmed increased mutation burden of calcium-signaling genes in CHD patients. Functional investigation of zebrafish orthologues of ITPR1, PLCB2, and ADCY2 verified a role in cardiac development and suggests a combinatorial effect of inactivation of these genes. CONCLUSIONS: The study identifies abnormal calcium signaling as a novel pathophysiological mechanism in human CHD and confirms the complex genetic architecture underlying CHD.

Prediction of Nephrotoxicity Associated With Cisplatin-Based Chemotherapy in Testicular Cancer Patients.

BACKGROUND: Cisplatin-based chemotherapy may induce nephrotoxicity. This study presents a random forest predictive model that identifies testicular cancer patients at risk of nephrotoxicity before treatment. METHODS: Clinical data and DNA from saliva samples were collected for 433 patients. These were genotyped on Illumina HumanOmniExpressExome-8 v1.2 (964 193 markers). Clinical and genomics-based random forest models generated a risk score for each individual to develop nephrotoxicity defined as a 20% drop in isotopic glomerular filtration rate during chemotherapy. The area under the receiver operating characteristic curve was the primary measure to evaluate models. Sensitivity, specificity, and positive and negative predictive values were used to discuss model clinical utility. RESULTS: Of 433 patients assessed in this study, 26.8% developed nephrotoxicity after bleomycin-etoposide-cisplatin treatment. Genomic markers found to be associated with nephrotoxicity were located at NAT1, NAT2, and the intergenic region of CNTN6 and CNTN4. These, in addition to previously associated markers located at ERCC1, ERCC2, and SLC22A2, were found to improve predictions in a clinical feature-trained random forest model. Using only clinical data for training the model, an area under the receiver operating characteristic curve of 0.635 (95% confidence interval [CI] = 0.629 to 0.640) was obtained. Retraining the classifier by adding genomics markers increased performance to 0.731 (95% CI = 0.726 to 0.736) and 0.692 (95% CI = 0.688 to 0.696) on the holdout set. CONCLUSIONS: A clinical and genomics-based machine learning algorithm improved the ability to identify patients at risk of nephrotoxicity compared with using clinical variables alone. Novel genetics associations with cisplatin-induced nephrotoxicity were found for NAT1, NAT2, CNTN6, and CNTN4 that require replication in larger studies before application to clinical practice.

Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial.

OBJECTIVE: To investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality. DESIGN: 60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed. RESULTS: 50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (p<0.0001). Compared with refined grain, whole grain did not significantly alter glucose homeostasis and did not induce major changes in the faecal microbiome. Also, breath hydrogen levels, plasma short-chain fatty acids, intestinal integrity and intestinal transit time were not affected. The whole grain diet did, however, compared with the refined grain diet, decrease body weight (p<0.0001), serum inflammatory markers, interleukin (IL)-6 (p=0.009) and C-reactive protein (p=0.003). The reduction in body weight was consistent with a reduction in energy intake, and IL-6 reduction was associated with the amount of whole grain consumed, in particular with intake of rye. CONCLUSION: Compared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation. TRIAL REGISTRATION NUMBER: NCT01731366; Results.

A low-gluten diet induces changes in the intestinal microbiome of healthy Danish adults.

Adherence to a low-gluten diet has become increasingly common in parts of the general population. However, the effects of reducing gluten-rich food items including wheat, barley and rye cereals in healthy adults are unclear. Here, we undertook a randomised, controlled, cross-over trial involving 60 middle-aged Danish adults without known disorders with two 8-week interventions comparing a low-gluten diet (2 g gluten per day) and a high-gluten diet (18 g gluten per day), separated by a washout period of at least six weeks with habitual diet (12 g gluten per day). We find that, in comparison with a high-gluten diet, a low-gluten diet induces moderate changes in the intestinal microbiome, reduces fasting and postprandial hydrogen exhalation, and leads to improvements in self-reported bloating. These observations suggest that most of the effects of a low-gluten diet in non-coeliac adults may be driven by qualitative changes in dietary fibres.

NT5C2 germline variants alter thiopurine metabolism and are associated with acquired NT5C2 relapse mutations in childhood acute lymphoblastic leukaemia.

The antileukaemic drug 6-mercaptopurine is converted into thioguanine nucleotides (TGN) and incorporated into DNA (DNA-TG), the active end metabolite. In a series of genome-wide association studies, we analysed time-weighted means (wm) of erythrocyte concentrations of TGN (Ery-TGN) and DNA-TG in 1009 patients undergoing maintenance therapy for acute lymphoblastic leukaemia (ALL). In discovery analyses (454 patients), the propensity for DNA-TG incorporation (wmDNA-TG/wmEry-TGN ratio) was significantly associated with three intronic SNPs in NT5C2 (top hit: rs72846714; P = 2.09 × 10-10, minor allele frequency 15%). In validation analyses (555 patients), this association remained significant during both early and late maintenance therapy (P = 8.4 × 10-6 and 1.3 × 10-3, respectively). The association was mostly driven by differences in wmEry-TGN, but in regression analyses adjusted for wmEry-TGN (P < 0.0001), rs72846714-A genotype was also associated with a higher wmDNA-TG (P = 0.029). Targeted sequencing of NT5C2 did not identify any missense variants associated with rs72846714 or wmEry-TGN/wmDNA-TG. rs72846714 was not associated with relapse risk, but in a separate cohort of 180 children with relapsed ALL, rs72846714-A genotype was associated with increased occurrence of relapse-specific NT5C2 gain-of-function mutations that reduce cytosol TGN levels (P = 0.03). These observations highlight the impact of both germline and acquired mutations in drug metabolism and disease trajectory.

Transcriptome profiling of fetal Klinefelter testis tissue reveals a possible involvement of long non-coding RNAs in gonocyte maturation.

In humans, the most common sex chromosomal disorder is Klinefelter syndrome (KS), caused by the presence of one or more extra X-chromosomes. KS patients display a varying adult phenotype but usually present with azoospermia due to testicular degeneration, which accelerates at puberty. The timing of the germ cell loss and whether it is caused by dysgenetic fetal development of the testes is not known. We investigated eight fetal KS testes and found a marked reduction in MAGE-A4-positive pre-spermatogonia compared with testes from 15 age-matched controls, indicating a failure of the gonocytes to differentiate into pre-spermatogonia. Transcriptome analysis by RNA-sequencing of formalin-fixed, paraffin-embedded testes originating from four fetal KS and five age-matched controls revealed 211 differentially expressed transcripts in the fetal KS testis. We found a significant enrichment of upregulated X-chromosomal transcripts and validated the expression of the pseudoautosomal region 1 (PAR1) gene, AKAP17A. Moreover, we found enrichment of long non-coding RNAs in the KS testes (e.g. LINC01569 and RP11-485F13.1). In conclusion, our data indicate that the testicular phenotype observed among adult men with KS is initiated already in fetal life by failure of the gonocyte differentiation into pre-spermatogonia, which could be due to aberrant expression of long non-coding RNAs.

Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias.

Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries and performed sequencing of a well-defined and validated 20-member bacterial DNA mock community on five separate occasions and compared results with the expected even distribution. In general the applied method had a median coefficient of variance of 11.8% (range 5.5-73.7%) for all 20 included strains in the mock community across five separate sequencing runs, with underrepresented strains generally showing the largest degree of variation. In terms of accuracy, mock community species belonging to Proteobacteria were underestimated, whereas those belonging to Firmicutes were mostly overestimated. This could be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification from 30 to 120 s resulted in an increased average relative abundance of the three mock community members with the highest genomic GC%, but did not significantly change the overall evenness of the community distribution. Therefore, efforts should be made to optimize the PCR conditions prior to sequencing in order to maximize accuracy.

Meta-analysis of five genome-wide association studies identifies multiple new loci associated with testicular germ cell tumor.

The international Testicular Cancer Consortium (TECAC) combined five published genome-wide association studies of testicular germ cell tumor (TGCT; 3,558 cases and 13,970 controls) to identify new susceptibility loci. We conducted a fixed-effects meta-analysis, including, to our knowledge, the first analysis of the X chromosome. Eight new loci mapping to 2q14.2, 3q26.2, 4q35.2, 7q36.3, 10q26.13, 15q21.3, 15q22.31, and Xq28 achieved genome-wide significance (P < 5 × 10-8). Most loci harbor biologically plausible candidate genes. We refined previously reported associations at 9p24.3 and 19p12 by identifying one and three additional independent SNPs, respectively. In aggregate, the 39 independent markers identified to date explain 37% of father-to-son familial risk, 8% of which can be attributed to the 12 new signals reported here. Our findings substantially increase the number of known TGCT susceptibility alleles, move the field closer to a comprehensive understanding of the underlying genetic architecture of TGCT, and provide further clues to the etiology of TGCT.

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology.

Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8-100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.

Klinefelter syndrome comorbidities linked to increased X chromosome gene dosage and altered protein interactome activity.

Klinefelter syndrome (KS) (47,XXY) is the most common male sex chromosome aneuploidy. Diagnosis and clinical supervision remain a challenge due to varying phenotypic presentation and insufficient characterization of the syndrome. Here we combine health data-driven epidemiology and molecular level systems biology to improve the understanding of KS and the molecular interplay influencing its comorbidities. In total, 78 overrepresented KS comorbidities were identified using in- and out-patient registry data from the entire Danish population covering 6.8 million individuals. The comorbidities extracted included both clinically well-known (e.g. infertility and osteoporosis) and still less established KS comorbidities (e.g. pituitary gland hypofunction and dental caries). Several systems biology approaches were applied to identify key molecular players underlying KS comorbidities: Identification of co-expressed modules as well as central hubs and gene dosage perturbed protein complexes in a KS comorbidity network build from known disease proteins and their protein-protein interactions. The systems biology approaches together pointed to novel aspects of KS disease phenotypes including perturbed Jak-STAT pathway, dysregulated genes important for disturbed immune system (IL4), energy balance (POMC and LEP) and erythropoietin signalling in KS. We present an extended epidemiological study that links KS comorbidities to the molecular level and identify potential causal players in the disease biology underlying the identified comorbidities.

Comparison of global gene expression profiles of microdissected human foetal Leydig cells with their normal and hyperplastic adult equivalents.

STUDY QUESTION: Do human adult Leydig cells (ALCs) within hyperplastic micronodules display characteristics of foetal LCs (FLCs)? SUMMARY ANSWER: The gene expression profiles of FLCs and all ALC subgroups were clearly different, but there were no significant differences in expressed genes between the normally clustered and hyperplastic ALCs. WHAT IS KNOWN ALREADY: LCs are the primary androgen producing cells in males throughout development and appear in chronologically distinct populations; FLCs, neonatal LCs and ALCs. ALCs are responsible for progression through puberty and for maintenance of reproductive functions in adulthood. In patients with reproductive problems, such as infertility or testicular cancer, and especially in men with high gonadotrophin levels, LC function is often impaired, and LCs may cluster abnormally into hyperplastic micronodules (defined as clusters of >15 LCs in a cross-section). STUDY DESIGN, SIZE, DURATION: A genome-wide microarray study of LCs microdissected from human foetal and adult tissue samples (n = 12). Additional tissue specimens (n = 15) were used for validation of the mRNA expression data at the protein level. PARTICIPANTS/MATERIALS, SETTING, METHODS: Frozen human tissue samples were used for the microarray study, including morphologically normal foetal (gestational week 10-11) testis samples, and adult testis specimens with normal LC distribution, LC micronodules or LC micronodules adjacent to hCG-producing testicular germ cell tumours. Transcriptome profiling was performed on Agilent whole human genome microarray 4 × 44 K chips. Microarray data pre-processing and statistical analysis were performed using the limma R/Bioconductor package in the R software, and differentially expressed genes were further analysed for gene set enrichment using the DAVID Bioinformatics software. Selected genes were studied at the protein level by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptomes of FLCs and ALCs differed significantly from each other, whereas the profiles of the normally clustered and hyperplastic ALCs were similar despite morphological heterogeneity. The study revealed several genes not known previously to be expressed in LCs during early development, including sulfotransferase family 2A member 1 (SULT2A1), WNT1-inducible signalling pathway protein 2 (WISP2), hydroxyprostaglandin dehydrogenase (HPGD) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), whose expression changes were validated at the protein level. LARGE SCALE DATA: The transcriptomic data are deposited in ArrayExpress (accession code E-MTAB-5453). LIMITATIONS, REASONS FOR CAUTION: The small number of biological replicates and the necessity of RNA amplification due to the scarcity of human tissues, especially foetal specimens, are the main limitations of the study. Heterogeneous subpopulations of LCs within micronodules were not discriminated during microdissection and might have affected the expression profiling. The study was constrained by the lack of availability of truly normal controls. Testis samples used as 'controls' displayed complete spermatogenesis and were from patients with germ cell neoplasia but with undetectable hCG and normal hormone levels. WIDER IMPLICATIONS OF THE FINDINGS: The changes in LC morphology and function observed in patients with reproductive disorders possibly reflect subtle changes in the expression of many genes rather than regulatory changes of single genes or pathways. The study provides new insights into the development and maturation of human LCs by the identification of a number of potential functional markers for FLC and ALC. STUDY FUNDING AND COMPETING INTEREST(S): The study was supported by research grants from the Danish Cancer Society, the Capital Region's Research Fund for Health Research, Rigshospitalet's research funds, the Villum Kann Rasmussen Foundation, the Danish Innovation Fund, ReproUnion, Kirsten and Freddy Johansen's foundation and the Novo Nordisk Foundation. None of the funding agencies had any influence on the study. The authors declare no conflicts of interest.

Colonic transit time is related to bacterial metabolism and mucosal turnover in the gut.

Little is known about how colonic transit time relates to human colonic metabolism and its importance for host health, although a firm stool consistency, a proxy for a long colonic transit time, has recently been positively associated with gut microbial richness. Here, we show that colonic transit time in humans, assessed using radio-opaque markers, is associated with overall gut microbial composition, diversity and metabolism. We find that a long colonic transit time associates with high microbial richness and is accompanied by a shift in colonic metabolism from carbohydrate fermentation to protein catabolism as reflected by higher urinary levels of potentially deleterious protein-derived metabolites. Additionally, shorter colonic transit time correlates with metabolites possibly reflecting increased renewal of the colonic mucosa. Together, this suggests that a high gut microbial richness does not per se imply a healthy gut microbial ecosystem and points at colonic transit time as a highly important factor to consider in microbiome and metabolomics studies.

Genome-wide assessment of the association of rare and common copy number variations to testicular germ cell cancer.

Testicular germ cell cancer (TGCC) is one of the most heritable forms of cancer. Previous genome-wide association studies have focused on single nucleotide polymorphisms, largely ignoring the influence of copy number variants (CNVs). Here we present a genome-wide study of CNV on a cohort of 212 cases and 437 controls from Denmark, which was genotyped at ∼1.8 million markers, half of which were non-polymorphic copy number markers. No association of common variants were found, whereas analysis of rare variants (present in less than 1% of the samples) initially indicated a single gene with significantly higher accumulation of rare CNVs in cases as compared to controls, at the gene PTPN1 (P = 3.8 × 10(-2), 0.9% of cases and 0% of controls). However, the CNV could not be verified by qPCR in the affected samples. Further, the CNV calling of the array-data was validated by sequencing of the GSTM1 gene, which showed that the CNV frequency was in complete agreement between the two platforms. This study therefore disconfirms the hypothesis that there exists a single CNV locus with a major effect size that predisposes to TGCC. Genome-wide pathway association analysis indicated a weak association of rare CNVs related to cell migration (false-discovery rate = 0.021, 1.8% of cases and 1.1% of controls). Dysregulation during migration of primordial germ cells has previously been suspected to be a part of TGCC development and this set of multiple rare variants may thereby have a minor contribution to an increased susceptibility of TGCCs.