A Genetic Risk Score for the Estimation of Weight Loss After Bariatric Surgery.

BACKGROUND: Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) are the most frequent bariatric surgery procedures worldwide. In this prospective study, we examined the association of a genetic risk score (GRS) with loss of excess weight after bariatric surgery. METHODS: A total of forty-seven morbidly obese Greek patients who underwent SG (81%) or RYGB were recruited, followed up for 2 years and genotyped. Weight loss after surgery was reported as the percentage of excess weight that was lost (%EWL) at 12 and 24 months after surgery. A GRS was constructed based on previously BMI- and WHR-related single nucleotide polymorphisms (SNPs) that were found significantly correlated with weight loss after bariatric surgery in our population. The level of post-surgery %EWL after 12 and 24 months was estimated through two multiple linear regression models that considered the effects of relevant genetic risk variants. RESULTS: The first proposed model suggested that the predictor variables of GRS, age, and BMI had a significant effect on %EWL12m. GRS was significantly associated with %EWL12m, indicating a 4.618% decrease of %EWL12m per score unit. The second model indicated a positive correlation between %EWL24m and %EWL12m, suggesting that while post-surgery weight loss increased during the first 12 months, an increase was expected in the next 12 months as well. GRS was also significantly associated with %EWL24m, indicating approximately 3% decrease of %EWL24m per score unit. CONCLUSION: GRS can be used in the future together with other preoperative parameters in order to predict the outcome of bariatric surgery.

Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival.

Correction to: Oncogene (2015) 34, 4482­4490; doi:10.1038/onc.2014.378; published online 24 November 2014. Following the online publication of this article, the authors have noticed a misspelt surname: S Hider should read S Haider. There is also an addition to the acknowledgements to read 'This study makes use of data generated by the Molecular Taxonomy of Breast Cancer International Consortium, which was funded by Cancer Research UK and the British Columbia Cancer Agency Branch'. The corrected article appears in this issue. The authors would like to apologise for any inconvenience this may cause.

Contribution of IL-12A and IL-12B polymorphisms to Chlamydia trachomatis-specific cell-mediated immune responses.

Inherited variance in the IL-12B gene is associated with susceptibility to Chlamydia trachomatis-induced tubal factor infertility and disease severity. In this study, our aim was to discover how polymorphisms in IL-12-coding genes influence C. trachomatis-induced immune responses and IL-12 production. The study population consisted of 240 women. IL-12A and IL-12B single nucleotide polymorphisms (SNPs) were determined from isolated DNA using the Sequenom system with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. We studied lymphocyte proliferative (LP) responses to C. trachomatis strains E and F elementary bodies (EBs) and recombinant chlamydial heat-shock protein 60 (CHSP60) antigen. IL-12p40 and IL-12p70 levels were measured using the BD Flex Set method. We found a statistically significant association between the C. trachomatis EB antigen-specific LP response and the rs2853694 SNP (P = 0.02). Our study demonstrates that the IL-12 cytokine family is involved in C. trachomatis-specific immune responses. Moreover, C. trachomatis-induced IL-12 production and the IL-12B rs2853694 SNP partially explain individual variation in the C. trachomatis LP response.

Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival.

Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

miR-210 is a target of hypoxia-inducible factors 1 and 2 in renal cancer, regulates ISCU and correlates with good prognosis.

BACKGROUND: Clear cell renal cancer frequently harbours von Hippel-Lindau (VHL) gene mutations, leading to stabilisation of the hypoxia-inducible factors (HIFs) and expression of their target genes. We investigated HIF-1 and HIF-2 in the regulation of microRNA-210 (miR-210), and its clinical relevance in renal tumours. METHODS: RCC4 and 786-O renal cancer cell lines transfected with either an empty vector or functional VHL and incubated in normoxia or hypoxia were examined for miR-210 expression. Hypoxia-inducible factor siRNAs were used to examine their regulation of miR-210. Seventy-one clear cell renal tumours were sequenced for VHL mutations. Expression of miR-210, VHL, CA9, ISCU and Ki-67 were determined by immunohistochemistry and qRT-PCR. RESULTS: In addition to HIF-1 regulating miR-210 in renal cancer, HIF-2 can regulate this microRNA in the absence of HIF-1. MicroRNA-210 is upregulated in renal cancer compared with normal renal cortex tissue. MicroRNA-210 correlates negatively with its gene target ISCU at the protein and mRNA level. MicroRNA-210 correlated with positive outcome variables and negatively with Ki-67. CONCLUSION: We provide further evidence of miR-210 activity in vivo, and show that high miR-210 expression is associated with better clinico-pathological prognostic factors.

Effect of IL12A and IL12B polymorphisms on the risk of Chlamydia trachomatis-induced tubal factor infertility and disease severity.

BACKGROUND: Interleukin-12 (IL-12) and related cytokines induce activation and differentiation of T cells. Our aim was to investigate the associations between genetic differences in IL-12-family cytokines and the pathogenesis of chlamydial disease. METHODS: The final study population consisted of 100 women with Chlamydia trachomatis-induced tubal factor infertility (TFI) and 125 pregnant women as controls. Three single nucleotide polymorphisms (SNPs) of IL12A and seven SNPs of IL12B genes were determined from isolated DNA using the Sequenom system with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. RESULTS: We found that the IL12B SNP rs3212227 was associated with both susceptibility and severity of TFI. The minor allele C was rare and only one CC homozygote was found among the controls. AC heterozygotes were more common among TFI cases than among controls (P = 0.009) and were associated with increased risk of TFI [odds ratios (OR) = 2.44, 95% confidence intervals (CI) = 1.23-4.87]. Carrying the minor allele C was also associated with disease severity (P for trend = 0.008) and moderate (OR = 2.51, 95% CI = 1.06-5.95) and severe tubal damage (OR = 2.73, 95% CI = 1.15-6.52). CONCLUSIONS: The results suggest that variation in the IL12B gene partly explains inter-individual differences in disease susceptibility and severity.

The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis.

BACKGROUND: To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer. METHODS: Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis. RESULTS: Small-nucleolar RNA expression was as variable as miRNA expression (miR-21, miR-210, miR-10b). Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer. CONCLUSION: Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression.

An oncogenic role of eIF3e/INT6 in human breast cancer.

Altered expression of the eukaryotic translation initiation factor 3 (eIF3) subunit eIF3e/INT6 has been described in various types of human cancer, but the nature of its involvement in tumorigenesis is not yet clear. Using immunohistochemical analysis of 81 primary breast cancers, we found that high tumor grade correlated significantly with elevated cytoplasmic eIF3e level in epithelial tumor cells. Analysis of protein synthesis after siRNA-mediated knockdown in breast cancer cell lines indicated that eIF3e is not required for bulk translation. Microarray analysis of total and polysomal RNAs nonetheless identified distinct sets of mRNAs regulated either positively or negatively by eIF3e; functional classification of these revealed a marked enrichment of genes involved in cell proliferation, invasion and apoptosis. Validated mRNA targets regulated positively at the translational level by eIF3e included urokinase-type plasminogen activator and apoptotic regulator BCL-XL, whereas synthesis of proteins including the mitotic checkpoint component MAD2L1 was negatively regulated. Finally, eIF3e-depleted breast carcinoma cells showed reduced in vitro invasion and proliferation. Taken together, our study data suggest that eIF3e has a positive role in breast cancer progression. It regulates the translation, and in some cases abundance, of mRNAs involved in key aspects of cancer cell biology.

Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions.

Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

Detection, breakpoint identification and detailed characterisation of a CNV at the FRA16D site using SNP assays.

Copy Number Variants (CNV) and other submicroscopic structural changes are now recognised to be widespread across the human genome. We show that SNP data generated for association study can be utilised for the identification of deletion CNVs. During analysis of data for an SNP association study for Specific Language Impairment (SLI) a deletion was identified. SLI adversely affects the language development of children in the absence of any obvious cause. Previous studies have found linkage to a region on chromosome 16. The deletion was located in a known fragile site FRA16D in intron 5-6 of the WWOX gene (also known as FOR). Changes in the FRA16D site have been previously linked to cancer and are often characterised in cell lines. A long-range PCR assay was used to confirm the existence of the deletion. We also show the breakpoint identification and large-scale characterisation of this CNV in a normal human sample set.

LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia.

Left-right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Schizophrenia in an adult with 6p25 deletion syndrome.

Chromosomal deletions at 6p25-p24 are rare findings in patients with developmental delay. There is limited information about the adult phenotype. We present a 36-year-old patient with schizophrenia, mild mental retardation, progressive hearing deficits, and characteristic facial features. Ocular (Axenfeld-Rieger anomaly) abnormalities were diagnosed in infancy; vision, however, has remained unimpaired. There were no other major congenital anomalies. Brain imaging showed only minor changes. There was no family history of intellectual deficits or psychosis. Karyotyping revealed a 6p25 deletion, and detailed fluorescence in situ hybridization (FISH) analyses using 23 probes confirmed a 6.7 Mb 6p25-pter deletion. The breakpoint is near a possible 6p25-p24 locus for schizophrenia. Psychotic illness may be part of the neurodevelopmental abnormalities and long-term outcome of patients with 6p terminal deletions. Other similarly affected patients likely remain to be diagnosed in adult populations of schizophrenia and/or mental retardation.

Evaluation of silicon and polymer substrates for fabrication of integrated microfluidic microsystems for DNA extraction and amplification.

This paper is presenting two different alternatives for the DNA extraction and amplification that will be carried out by two competitive research projects developing bioanalytical microsystems with microfluidics. The first project will develop the microfluidics part on polymer material and the other one on silicon. The polymer approach is currently under development based on a modular microfluidic architecture aimed to simplify the process of designing and building such a microsystem device. A silicon alternative is about to start and is expected to decrease packaging costs of the microsystem allowing future manufacturability of the device.

Mapping of three translocation breakpoints associated with orofacial clefting within 6p24 and identification of new transcripts within the region.

Orofacial clefting (OFC) is a common congenital malformation. Here we report the refinement of three translocation breakpoints of patients exhibiting OFC within the 6p24 region, and the isolation and characterisation of novel genes, one of which is directly disrupted by the translocation breakpoint of a patient. The gene has been characterized and orthologues identified in bovine, murine and pufferfish.

Association of the androgen receptor CAG repeat polymorphism with Alzheimer's disease in men.

We examined the CAG repeat polymorphism in exon 1 of the androgen receptor (AR) in an Oxford cohort of 150 cases (101 men) of definite or probable Alzheimer's disease (AD) and 190 elderly controls (140 men). We found that short alleles (< or = 20 CAG repeats) were associated with AD (adjusted odds ratio = 2.5, 95% confidence intervals: 1.2-5.0) in men, but not in women. This association appeared stronger in early-onset AD (< 65 years). We conclude that this AR polymorphism is of potential relevance to the risk of AD in men.

Molecularly defined interstitial tandem duplication 6p case with mild manifestations.

An interstitial tandem duplication of 6p21.1-p22.2 was found in a girl at 11 months of age when she was evaluated for developmental delay. Previous cases reported with partial 6p duplication usually have involved terminal duplications, with breakpoints ranging from 6p11 to 6p25. Our patient exhibits a milder phenotype compared to the previously reported cases in the literature. Features that she has in common with the other cases include craniofacial anomalies, such as broad nasal bridge and bulbous tip, thin lips, incomplete development of the scapha helix bilaterally, mild spastic paraparesis of the lower extremities, gross motor delay, and mild cognitive delays.

The minisatellite of the GPI/AMF/NLK/MF gene: interspecies conservation and transcriptional activity.

Minisatellites are tandemly repeated DNA sequences found throughout the genomes of all eukaryotes. They are regions often prone to instability and hence hypervariability; thus repeat unit sequence is generally not conserved beyond closely related species. We have studied the minisatellite located in intron 9 of the human glucose phosphate isomerase (GPI) gene (also known as neuroleukin, autocrine motility factor, maturation and differentiation factor) and have found, by Zoo blotting coupled with PCR amplification and DNA sequencing, that similar repeat units are present in seven other species of mammal. There is also evidence for the presence of the minisatellite in chicken. The repeat unit does not appear to be present at any other locus in these genomes. Minisatellite DNA has been reported to be involved in recombination activity, control of gene expression of nearby gene(s) (both transcriptional and translational), whilst others form protein coding regions. The high level of conservation exhibited by the GPI minisatellite, coupled with the unique location, strongly suggests a functional role. Our results from transient and stable transfections using luciferase reporter constructs have shown that the GPI minisatellite region can act to increase transcription from the SV40 promoter, CMV promoter and the human GPI promoter.

Construction of a high-resolution 2.5-Mb transcript map of the human 6p21.2-6p21.3 region immediately centromeric of the major histocompatibility complex.

We have constructed a 2.5-Mb physical and transcription map that spans the human 6p21.2-6p21.3 region and includes the centromeric end of the MHC, using a combination of techniques. In total 88 transcription units including exons, cDNAs, and cDNA contigs were characterized and 60 were confidently positioned on the physical map. These include a number of genes encoding nuclear and splicing factors (Ndr kinase, HSU09564, HSRP20); cell cycle, DNA packaging, and apoptosis related [p21, HMGI(Y), BAK]; immune response (CSBP, SAPK4); transcription activators and zinc finger-containing genes (TEF-5, ZNF76); embryogenesis related (Csa-19); cell signaling (DIPP); structural (HSET), and other genes (TULP1, HSPRARD, DEF-6, EO6811, cyclophilin), as well as a number of RP genes and pseudogenes (RPS10, RPS12-like, RPL12-like, RPL35-like). Furthermore, several novel genes (a Br140-like, a G2S-like, a FBN2-like, a ZNF-like, and B1/KIAA0229) have been identified, as well as cDNAs and cDNA contigs. The detailed map of the gene content of this chromosomal segment provides a number of candidate genes, which may be involved in several biological processes that have been associated with this region, such as spermatogenesis, development, embryogenesis, and neoplasia. The data provide useful tools for synteny studies between mice and humans, for genome structure analysis, gene density comparisons, and studies of nucleotide composition, of different isochores and Giemsa light and Giemsa dark bands.