A genome-wide association search for type 2 diabetes genes in African Americans.

African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.

Glutathione Precursor, N-Acetyl-Cysteine, Modulates EEG Synchronization in Schizophrenia Patients

Background: Glutathione (GSH) dysregulation at the gene, protein and functional levels observed in schizophrenia patients, and schizophrenia-like anomalies in GSH deficit experimental models, suggest that genetic glutathione synthesis impairments represent one major risk factor for the disease (Do et al., 2009). In a randomized, double blind, placebo controlled, add-on clinical trial of 140 patients, the GSH precursor N-Acetyl-Cysteine (NAC, 2g/day, 6 months) significantly improved the negative symptoms and reduced sideeffects due to antipsychotics (Berk et al., 2008). In a subset of patients (n=7), NAC (2g/day, 2 months, cross-over design) also improved auditory evoked potentials, the NMDA-dependent mismatch negativity (Lavoie et al, 2008). Methods: To determine whether increased GSH levels would modulate the topography of functional brain connectivity, we applied a multivariate phase synchronization (MPS) estimator (Knyazeva et al, 2008) to dense-array EEGs recorded during rest with eyes closed at the protocol onset, the point of crossover, and at its end. Results: The whole-head imaging revealed a specific synchronization landscape in NAC compared to placebo condition. In particular, NAC increased MPS over frontal and left temporal regions in a frequency-specific manner. The topography and direction of MPS changes were similar and robust in all 7 patients. Moreover, these changes correlated with the changes in the Liddle's score of disorganization, thus linking EEG synchronization to the improvement of the clinical picture. Conclusions: The data suggest an important pathway towards new therapeutic strategies that target GSH dysregulation in schizophrenia. They also show the utility of MPS mapping as a marker of treatment efficacy.

Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis.

BACKGROUND: Histologic grade in breast cancer provides clinically important prognostic information. However, 30%-60% of tumors are classified as histologic grade 2. This grade is associated with an intermediate risk of recurrence and is thus not informative for clinical decision making. We examined whether histologic grade was associated with gene expression profiles of breast cancers and whether such profiles could be used to improve histologic grading. METHODS: We analyzed microarray data from 189 invasive breast carcinomas and from three published gene expression datasets from breast carcinomas. We identified differentially expressed genes in a training set of 64 estrogen receptor (ER)-positive tumor samples by comparing expression profiles between histologic grade 3 tumors and histologic grade 1 tumors and used the expression of these genes to define the gene expression grade index. Data from 597 independent tumors were used to evaluate the association between relapse-free survival and the gene expression grade index in a Kaplan-Meier analysis. All statistical tests were two-sided. RESULTS: We identified 97 genes in our training set that were associated with histologic grade; most of these genes were involved in cell cycle regulation and proliferation. In validation datasets, the gene expression grade index was strongly associated with histologic grade 1 and 3 status; however, among histologic grade 2 tumors, the index spanned the values for histologic grade 1-3 tumors. Among patients with histologic grade 2 tumors, a high gene expression grade index was associated with a higher risk of recurrence than a low gene expression grade index (hazard ratio = 3.61, 95% confidence interval = 2.25 to 5.78; P < .001, log-rank test). CONCLUSIONS: Gene expression grade index appeared to reclassify patients with histologic grade 2 tumors into two groups with high versus low risks of recurrence. This approach may improve the accuracy of tumor grading and thus its prognostic value.

The future is genome-wide.

A report of the annual meeting of the European Society of Human Genetics, Amsterdam, 6-9 May 2006.

Identification of structural variation in mouse genomes.

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Infrequent promoter methylation of the MGMT gene in liver metastases from uveal melanoma.

Uveal melanoma is associated with a high mortality rate once metastases occur, with over >90% of metastatic patients dying within less than 1 year from metastases to the liver. The intraarterial hepatic (iah) administration of the alkylating agent fotemustine holds some promise with response rates of 36% and median survival of 15 months. Here, we investigated whether the DNA-repair-protein MGMT may be involved in the variability of response to fotemustine and temozolomide in uveal melanoma. Epigenetic inactivation of MGMT has been demonstrated to be a predictive marker for benefit from alkylating agent therapy in glioblastoma. We found a methylated MGMT promoter in 6% of liver metastases from 34 uveal melanoma patients. The mean MGMT activity measured in liver metastases with negligible liver tissue content was significantly lower than in liver tissue (146 versus 523 fmol/mg protein, p = 0.002). Expression of the MGMT protein was detectable in 50% of 88 metastases by immunohistochemistry on a tissue microarray. Expression was heterogeneous, and in accordance with MGMT activity data, usually lower than in the surrounding liver. Differential MGMT activity/expression between metastasis and liver tissue and more efficient depletion of MGMT with higher doses of alkylating agent therapy using iah delivery may provide the pharmacologic window for the higher response rate. However, these results do not support MGMT methylation status or protein expression as predictive markers for treatment outcome to iah chemotherapy with alkylating agents.

Evaluation of the antitumoral effect sunitinib eluting beads in VX2 liver tumour in a rabbit model.

Background: We demonstrated that DC Bead (Biocompatibles UK, Ltd) could be loaded with sunitinib and injected intra-arterially in the rabbit without unexpected toxicity. The purpose of this study is to evaluate the antitumoral effect of sunitinib eluting beads in the VX2 tumor model of liver cancer. Methods: VX2 tumors were implanted in the left liver lobe of New-Zealand white rabbits. Animals were assigned to 3 groups: Group 1 (n=6) received 1.5mg of sunitinib loaded in 0.05ml of 100-300um DC Bead, group 2 (n=5) received 0.05ml of 100-300um DC Bead, group 3 (n=5) received 0.05ml NaCl 0.9% in the left hepatic artery. One animal in each group was sacrificed at 24 hours and the others were followed for survival until day 15. Liver enzymes were measured daily. In group 1, plasmatic sunitinib concentration were measured daily by LC MS/MS tandem mass spectroscopy. At day 15 all living animals were sacrificed. After sacrifice, the livers were harvested for determination of the VEGF receptor tyrosine kinase activity by western blot and histopathological examination. Results: In group 1, no animals died during follow-up. In group 2, 2 animals died during follow-up on day x. In control group 3, 3 animals died during follow up on day x. In group 1 plasmatic sunitinib levels remained under therapeutic concentration throughout the experiment. Very high concentrations of sunitinib were measured in the liver tissue 24 and 15 days after embolization. Inhibition of the phosphorylation of the RTK was demonstrated at 24h and 15 days in groups 1. Sunitinib eluting beads seemed to penetrate in the tumor more effectively and there was more necrosis around the beads than their bland counterparts. Conclusions: Administration of sunitinib eluting beads in VX2 carrying rabbits resulted invery high drug concentrations at the site of embolization with minimal systemic passage. Despite the very high tissular sunitinib concentration we did not observe any additional toxicity with loaded beads. Sunitinib eluting beads inhibit the activation of RTK's triggered by ischemia and seem to prolong survival of the treated animals. Therefore we consider that local treatment with sunitinib may provide a promising approach for the treatment of liver cancer.

MDM2 and CDK4 immunohistochemistry is a valuable tool in the differential diagnosis of low-grade osteosarcomas and other primary fibro-osseous lesions of the bone.

Low-grade osteosarcoma is a rare malignancy that may be subdivided into two main subgroups on the basis of location in relation to the bone cortex, that is, parosteal osteosarcoma and low-grade central osteosarcoma. Their histological appearance is quite similar and characterized by spindle cell stroma with low-to-moderate cellularity and well-differentiated anastomosing bone trabeculae. Low-grade osteosarcomas have a simple genetic profile with supernumerary ring chromosomes comprising amplification of chromosome 12q13-15, including the cyclin-dependent kinase 4 (CDK4) and murine double-minute type 2 (MDM2) gene region. Low-grade osteosarcoma can be confused with fibrous and fibro-osseous lesions such as fibromatosis and fibrous dysplasia on radiological and histological findings. We investigated MDM2-CDK4 immunohistochemical expression in a series of 72 low-grade osteosarcomas and 107 fibrous or fibro-osseous lesions of the bone or paraosseous soft tissue. The MDM2-CDK4 amplification status of low-grade osteosarcoma was also evaluated by comparative genomic hybridization array in 18 cases, and the MDM2 amplification status was evaluated by fluorescence in situ hybridization or quantitative real-time polymerase chain reaction in 31 cases of benign fibrous and fibro-osseous lesions. MDM2-CDK4 immunostaining and MDM2 amplification by fluorescence in situ hybridization or quantitative real-time polymerase chain reaction were investigated in a control group of 23 cases of primary high-grade bone sarcoma, including 20 conventional high-grade osteosarcomas, two pleomorphic spindle cell sarcomas/malignant fibrous histiocytomas and one leiomyosarcoma. The results showed that MDM2 and/or CDK4 immunoreactivity was present in 89% of low-grade osteosarcoma specimens. All benign fibrous and fibro-osseous lesions and the tumors of the control group were negative for MDM2 and CDK4. These results were consistent with the MDM2 and CDK4 amplification results. In conclusion, immunohistochemical expression of MDM2 and CDK4 is specific and provides sensitive markers for the diagnosis of low-grade osteosarcomas, helping to differentiate them from benign fibrous and fibro-osseous lesions, particularly in cases with atypical radio-clinical presentation and/or limited biopsy samples.

The role of astroglia in neuroprotection.

Astrocytes are the main neural cell type responsible for the maintenance of brain homeostasis. They form highly organized anatomical domains that are interconnected into extensive networks. These features, along with the expression of a wide array of receptors, transporters, and ion channels, ideally position them to sense and dynamically modulate neuronal activity. Astrocytes cooperate with neurons on several levels, including neurotransmitter trafficking and recycling, ion homeostasis, energy metabolism, and defense against oxidative stress. The critical dependence of neurons upon their constant support confers astrocytes with intrinsic neuroprotective properties which are discussed here. Conversely, pathogenic stimuli may disturb astrocytic function, thus compromising neuronal functionality and viability. Using neuroinflammation, Alzheimer's disease, and hepatic encephalopathy as examples, we discuss how astrocytic defense mechanisms may be overwhelmed in pathological conditions, contributing to disease progression.

Expression profiling of rectal tumors defines response to neoadjuvant treatment related genes.

To date, no effective method exists that predicts the response to preoperative chemoradiation (CRT) in locally advanced rectal cancer (LARC). Nevertheless, identification of patients who have a higher likelihood of responding to preoperative CRT could be crucial in decreasing treatment morbidity and avoiding expensive and time-consuming treatments. The aim of this study was to identify signatures or molecular markers related to response to pre-operative CRT in LARC. We analyzed the gene expression profiles of 26 pre-treatment biopsies of LARC (10 responders and 16 non-responders) without metastasis using Human WG CodeLink microarray platform. Two hundred and fifty seven genes were differentially over-expressed in the responder patient subgroup. Ingenuity Pathway Analysis revealed a significant ratio of differentially expressed genes related to cancer, cellular growth and proliferation pathways, and c-Myc network. We demonstrated that high Gng4, c-Myc, Pola1, and Rrm1 mRNA expression levels was a significant prognostic factor for response to treatment in LARC patients (p<0.05). Using this gene set, we were able to establish a new model for predicting the response to CRT in rectal cancer with a sensitivity of 60% and 100% specificity. Our results reflect the value of gene expression profiling to gain insight about the molecular pathways involved in the response to treatment of LARC patients. These findings could be clinically relevant and support the use of mRNA levels when aiming to identify patients who respond to CRT therapy.

The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models.

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.

Intronic variants in the NFKB1 gene may influence hearing forecast in patients with unilateral sensorineural hearing loss in Meniere's disease.

Meniere's disease is an episodic vestibular syndrome associated with sensorineural hearing loss (SNHL) and tinnitus. Patients with MD have an elevated prevalence of several autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and psoriasis), which suggests a shared autoimmune background. Functional variants of several genes involved in the NF-κB pathway, such as REL, TNFAIP3, NFKB1 and TNIP1, have been associated with two or more immune-mediated diseases and allelic variations in the TLR10 gene may influence bilateral affectation and clinical course in MD. We have genotyped 716 cases of MD and 1628 controls by using the ImmunoChip, a high-density genotyping array containing 186 autoimmune loci, to explore the association of immune system related-loci with sporadic MD. Although no single nucleotide polymorphism (SNP) reached a genome-wide significant association (p<10(-8)), we selected allelic variants in the NF-kB pathway for further analyses to evaluate the impact of these SNPs in the clinical outcome of MD in our cohort. None of the selected SNPs increased susceptibility for MD in patients with uni or bilateral SNHL. However, two potential regulatory variants in the NFKB1 gene (rs3774937 and rs4648011) were associated with a faster hearing loss progression in patients with unilateral SNHL. So, individuals with unilateral MD carrying the C allele in rs3774937 or G allele in rs4648011 had a shorter mean time to reach hearing stage 3 (>40 dB HL) (log-rank test, corrected p values were p = 0.009 for rs3774937 and p = 0.003 for rs4648011, respectively). No variants influenced hearing in bilateral MD. Our data support that the allelic variants rs3774937 and rs4648011 can modify hearing outcome in patients with MD and unilateral SNHL.

Coactivated platelet-derived growth factor receptor {alpha} and epidermal growth factor receptor are potential therapeutic targets in intimal sarcoma.

Intimal sarcoma (IS) is a rare, malignant, and aggressive tumor that shows a relentless course with a concomitant low survival rate and for which no effective treatment is available. In this study, 21 cases of large arterial blood vessel IS were analyzed by immunohistochemistry and fluorescence in situ hybridization and selectively by karyotyping, array comparative genomic hybridization, sequencing, phospho-kinase antibody arrays, and Western immunoblotting in search for novel diagnostic markers and potential molecular therapeutic targets. Ex vivo immunoassays were applied to test the sensitivity of IS primary tumor cells to the receptor tyrosine kinase (RTK) inhibitors imatinib and dasatinib. We showed that amplification of platelet-derived growth factor receptor α (PDGFRA) is a common finding in IS, which should be considered as a molecular hallmark of this entity. This amplification is consistently associated with PDGFRA activation. Furthermore, the tumors reveal persistent activation of the epidermal growth factor receptor (EGFR), concurrent to PDGFRA activation. Activated PDGFRA and EGFR frequently coexist with amplification and overexpression of the MDM2 oncogene. Ex vivo immunoassays on primary IS cells from one case showed the potency of dasatinib to inhibit PDGFRA and downstream signaling pathways. Our findings provide a rationale for investigating therapies that target PDGFRA, EGFR, or MDM2 in IS. Given the clonal heterogeneity of this tumor type and the potential cross-talk between the PDGFRA and EGFR signaling pathways, targeting multiple RTKs and aberrant downstream effectors might be required to improve the therapeutic outcome for patients with this disease.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.