Identification of candidate genes and physiological pathways involved in gonad deformation in whitefish (Coregonus spp.) from Lake Thun, Switzerland

In 2000, fishermen reported the appearance of deformed reproductive organs in whitefish (Coregonus spp.) from Lake Thun, Switzerland. Despite intensive investigations, the causes of these abnormalities remain unknown. Using gene expression profiling, we sought to identify candidate genes and physiological processes possibly associated with the observed gonadal deformations, in order to gain insights into potential causes. Using in situ-synthesized oligonucleotide arrays, we compared the expression levels at 21,492 unique transcript probes in liver and head kidney tissue of male whitefish with deformed and normally developed gonads, respectively. The fish had been collected on spawning sites of two genetically distinct whitefish forms of Lake Thun. We contrasted the gene expression profiles of 56 individuals, i.e., 14 individuals of each phenotype and of each population. Gene-by-gene analysis revealed weak expression differences between normal and deformed fish, and only one gene, ictacalcin, was found to be up-regulated in head kidney tissue of deformed fish from both whitefish forms, However, this difference could not be confirmed with quantitative real-time qPCR. Enrichment analysis on the level of physiological processes revealed (i) the involvement of immune response genes in both tissues, particularly those linked to complement activation in the liver, (ii) proteolysis in the liver and (iii) GTPase activity and Ras protein signal transduction in the head kidney. In comparison with current literature, this gene expression pattern signals a chronic autoimmune disease in the testes. Based on the recent observations that gonad deformations are induced through feeding of zooplankton from Lake Thun we hypothesize that a xenobiotic accumulated in whitefish via the plankton triggering autoimmunity as the likely cause of gonad deformations. We propose several experimental strategies to verify or reject this hypothesis.

Contiguous ∼16 Mb 1p36 deletion: Dominant features of classical distal 1p36 monosomy with haplo-lethality

Monosomy 1p36 results from heterozygous deletions of the terminal short chromosome 1 arm, the most common terminal deletion in humans. The microdeletion is split in two usually non-overlapping and clinically distinct classical distal and proximal 1p36 monosomy syndromes. Using comparative genome hybridization, MLPA and qPCR we identified the largest contiguous ∼16 Mb terminal 1p36 deletion reported to date. It covers both distal and proximal regions, causes a neonatally lethal variant with virtually exclusive features of distal 1p36 monosomy, highlighting the key importance of the gene-rich distal region for the "compound" 1p36 phenotype and a threshold deletion-size effect for haplo-lethality.

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

Background DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. Methods mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. Results We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. Conclusions mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

CLLU1 expression distinguishes chronic lymphocytic leukemia from other mature B-cell neoplasms

The distinction of CLL from other mature B-cell neoplasms, especially from leukemic forms of mantle cell lymphoma or splenic marginal zone lymphoma, can be difficult but has important prognostic and therapeutic implications. We measured CLLU1 (CLL upregulated gene1) mRNA by qPCR and found a highly significant difference between CLL and other lymphoid neoplasms (AUC 0.96, 95%CI 0.93-0.99). Based on our cut-off values we can predict CLL and other mature B-cell neoplasms with high probability (PPV 99% and 94%). Analysis of CLLU1 expression is a rapid and reliable tool that may facilitate the diagnosis of mature B-cell neoplasms especially in inconclusive cases.

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease

REASONS FOR PERFORMING STUDY: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far. HYPOTHESIS: Given the pathogenic role of BPV-1 and BPV-2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection. METHODS: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid-bearing horses and one donkey. Viral load was estimated via quantitative real-time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV-1/-2 genome for one randomly selected lesion per horse and correlated with disease severity. RESULTS: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0-556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild-type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2. CONCLUSIONS: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity. POTENTIAL RELEVANCE: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy.

Genome-wide analysis of rare copy number variations reveals PARK2 as a candidate gene for attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder. Genetic loci have not yet been identified by genome-wide association studies. Rare copy number variations (CNVs), such as chromosomal deletions or duplications, have been implicated in ADHD and other neurodevelopmental disorders. To identify rare (frequency 1%) CNVs that increase the risk of ADHD, we performed a whole-genome CNV analysis based on 489 young ADHD patients and 1285 adult population-based controls and identified one significantly associated CNV region. In tests for a global burden of large (>500 kb) rare CNVs, we observed a nonsignificant (P=0.271) 1.126-fold enriched rate of subjects carrying at least one such CNV in the group of ADHD cases. Locus-specific tests of association were used to assess if there were more rare CNVs in cases compared with controls. Detected CNVs, which were significantly enriched in the ADHD group, were validated by quantitative (q)PCR. Findings were replicated in an independent sample of 386 young patients with ADHD and 781 young population-based healthy controls. We identified rare CNVs within the parkinson protein 2 gene (PARK2) with a significantly higher prevalence in ADHD patients than in controls (P=2.8 × 10(-4) after empirical correction for genome-wide testing). In total, the PARK2 locus (chr 6: 162 659 756-162 767 019) harboured three deletions and nine duplications in the ADHD patients and two deletions and two duplications in the controls. By qPCR analysis, we validated 11 of the 12 CNVs in ADHD patients (P=1.2 × 10(-3) after empirical correction for genome-wide testing). In the replication sample, CNVs at the PARK2 locus were found in four additional ADHD patients and one additional control (P=4.3 × 10(-2)). Our results suggest that copy number variants at the PARK2 locus contribute to the genetic susceptibility of ADHD. Mutations and CNVs in PARK2 are known to be associated with Parkinson disease.Molecular Psychiatry advance online publication, 20 November 2012; doi:10.1038/mp.2012.161.

Sudden Increase and Regrowth of Fecal Coliforms and Escherichia Coli in Wastewater Biosolids After High Solids Centrifugation

Treatment plants that operate either thermophilic or mesophilic anaerobic digesters with centrifugal dewatering processes have consistently observed densities of fecal coliform and Escherichia coli, both indicator bacteria, that decrease during digestion but then increase after dewatering and storage. The increases have been characterized as two separate phenomena to explain this observation: 1) “Sudden Increase,” or SI, which is defined as the increase that occurs immediately after dewatering and 2) “regrowth,” which is defined as an increase during storage of cake samples over a period of hours or days. The SI observation appears to be more prevalent with biosolids that are generated with thermophilic processes and dewatered by centrifugation. Both thermophilic and mesophilic digesters with centrifuge dewatering processes have observed the regrowth phenomena. This research hypothesizes that the SI phenomenon is due to the presence of viable nonculturable (VNC) bacteria that are reactivated during dewatering. In other words, the bacteria were always present but were not enumerated by standard culturing methods (SCM). Analysis of the E. coli density in thermally treated solids by SCMs and quantitative real-time polymerase chain reaction (qPCR) indicated that E. coli densities are often underestimated by SCM. When analyzed with qPCR, the E. coli density after digestion can be 4-5 orders of magnitude greater than the non-detect levels identified by SCMs, which supports the non-culturable hypothesis. The VNC state describes a condition where bacteria are alive but unable to sustain the metabolic process needed for cellular division. Supplements added to culturing media were investigated to determine if the resuscitation of VNC bacteria could be enhanced. The autoinducer molecules Nhexanoyl- L-Homoserine lactone (C6-HSL), 3-oxo-N-octanoyl-L-Homoserine lactone (3-oxo- C8-HSL), and norepinephrine were unable to induce the resuscitation of VNC E. coli. Additional sampling was performed to determine if autoinducer molecules, peroxides, or other as of yet unknown inhibitory agents and toxins could be removed from biosolids during SCM. Culture media supplemented with the peroxide degrading compounds catalase, α-ketoglutaric acid, and sodium pyruvate was unable to resuscitate non-culturable E. coli. The additions of bentonite and exponential growth phase E. coli cell-free supernatant to culturing media were also unable to increase the culturability of E. coli. To remove inhibitory agents and toxins, a cell washing technique was employed prior to performing SCM; however, this cell washing technique may have increased cellular stresses that inhibited resuscitation since cell densities decreased. A novel laboratory-scale dewatering process was also investigated to determine if the SI and regrowth phenomena observed in full-scale centrifugal dewatering could be mimicked in the laboratory using a lab shearing device. Fecal coliform and E. coli densities in laboratory prepared cake samples were observed to be an order of magnitude higher than full-scale dewatered cakes. Additionally, the laboratory-scale dewatering process was able to resuscitate fecal coliforms and E. coli in stored sludge such that the density increased by 4-5 orders of magnitude from nondetect values. Lastly, the addition of aluminum sulfate during centrifuge dewatering at a full-scale utility produced an increased regrowth of fecal coliforms and E. coli that was sustained for 5 days.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.

RNA degradation differentially affects quantitative mRNA measurements of endogenous reference genes in human placenta

It has been highlighted that RNA quality and appropriate reference gene selection is crucial for the interpretation of RT-qPCR results in human placental samples. In this context we investigated the effect of RNA degradation on the mRNA abundance of seven frequently used reference genes in 119 human placental samples. Combining RNA integrity measurements, RT-qPCR analysis and mathematical modeling we found major differences regarding the effect of RNA degradation on the measured expression levels between the different reference genes. Furthermore, we demonstrated that a modified RNA extraction method significantly improved RNA quality and consequently increased transcript levels of all reference genes.

Use and relevance of a bovine mammary gland explant model to study infection responses in bovine mammary tissue

Our aim was to develop an explant model to define more precisely the early response of bovine mammary epithelial cells to infection. Therefore we investigated the mRNA expression encoding for some soluble immunological factors in lipopolysaccharide (LPS)-treated bovine mammary gland explants. Explants were taken out from the mammary gland of eight lactating cows after slaughter then incubated with LPS (10 mug/ml) for 6 h. The mRNA expression of alpha-lactalbumin (alpha-la), various cytokines, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and two immunoglobulin receptors, the neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIGR), were assessed with qPCR before and after 3 h and 6 h of LPS challenge. Both immunoglobulin receptors and alpha-la increased at 3 h then recovered their initial level at 6 h whereas IL-1beta, IL-6 and IL-8 increased only after 6 h (P<0.05). Surprisingly, TNF-alpha transcripts did not show any regulation in response to the LPS treatment. We nevertheless concluded that our model was valid to examine the short-term response of mammary epithelial cell challenged with LPS.

Expression profiling with RNA from formalin-fixed, paraffin-embeeded material

Background Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge. Results We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97. Conclusion We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.