LIM Homeobox Transcription Factor 1 Alpha Research Articles

Cerebral-Organoid-Derived Exosomes Alleviate Oxidative Stress and Promote LMX1A-Dependent Dopaminergic Differentiation.

The remarkable advancements related to cerebral organoids have provided unprecedented opportunities to model human brain development and diseases. However, despite their potential significance in neurodegenerative diseases such as Parkinson's disease (PD), the role of exosomes from cerebral organoids (OExo) has been largely unknown. In this study, we compared the effects of OExo to those of mesenchymal stem cell (MSC)-derived exosomes (CExo) and found that OExo shared similar neuroprotective effects to CExo. Our findings showed that OExo mitigated H2O2-induced oxidative stress and apoptosis in rat midbrain astrocytes by reducing excess ROS production, antioxidant depletion, lipid peroxidation, mitochondrial dysfunction, and the expression of pro-apoptotic genes. Notably, OExo demonstrated superiority over CExo in promoting the differentiation of human-induced pluripotent stem cells (iPSCs) into dopaminergic (DA) neurons. This was attributed to the higher abundance of neurotrophic factors, including neurotrophin-4 (NT-4) and glial-cell-derived neurotrophic factor (GDNF), in OExo, which facilitated the iPSCs' differentiation into DA neurons in an LIM homeobox transcription factor 1 alpha (LMX1A)-dependent manner. Our study provides novel insight into the biological properties of cerebral organoids and highlights the potential of OExo in the treatment of neurodegenerative diseases such as PD.

CeRNA network of lncRNA MIR210HG/miR-377-3p/LMX1A in malignant proliferation of glioma cells.

Glioma represents the most heterogeneous and malignant form of brain tumor with a poor prognosis. The long non-coding RNA (LncRNA)-mediated competing endogenous RNA (ceRNA) network plays a regulatory role in cancer progression. The present study was conducted to expound on the role of lncRNA MIR210 host gene (MIR210HG)-mediated ceRNA mechanism in the malignant proliferation of glioma cells and provide a novel theoretical basis for the treatment of glioma. Expression levels of lncRNA MIR210HG, microRNA (miR)-377-3p, and LIM homeobox transcription factor 1 alpha (LMX1A) in glioma tissues and cells were determined by reverse-transcription quantitative polymerase chain reaction. Then, cell proliferation was assessed by cell counting kit-8 and colony formation assays. After that, the subcellular localization of lncRNA MIR210HG was analyzed by subcellular fractionation assay and the bindings of miR-377-3p to lncRNA MIR210HG and LMX1A were analyzed by the dual-luciferase assay. Glioma cells were transfected with si-MIR210HG, miR-377-3p inhibitor, or overexpressed-LMX1A vectors to evaluate their effects on the malignant proliferation of glioma cells. LncRNA MIR210HG was elevated in glioma tissues and cells and inhibition of lncRNA MIR210HG reduced the proliferation potential of glioma cells. LncRNA MIR210HG targeted and inhibited miR-377-3p and miR-377-3p targeted and inhibited LMX1A transcription. miR-377-3p downregulation or LMX1A overexpression reversed the inhibition of silencing lncRNA MIR210HG on glioma cell proliferation. LncRNA MIR210HG was upregulated in glioma tissues and cells and inhibition of lncRNA MIR210HG suppressed glioma cell proliferation through promoting miR-377-3p and repressing LMX1A.

MiR-142-5p promotes cervical cancer progression by targeting LMX1A through Wnt/β-catenin pathway.

BackgroundPrevious work has shown that miR-142-5p in cervical cancer tissues increased significantly compared with adjacent normal tissues. However, the function and the mechanism of miR-142-5p in cervical cancer have not been reported.MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the gene expression levels. MTT, flow cytometry, and transwell assays were performed to explore the functions of miR-142-5p in HeLa cells. The potential target gene of miR-142-5p was investigated via luciferase reporter assays. The protein expression levels were analyzed by Western blotting.ResultsWe found that miR-142-5p expression was elevated but LIM homeobox transcription factor 1 alpha (LMX1A) was decreased in cervical cancer tissues and cells. Overexpression of miR-142-5p or knockdown of LMX1A inhibited cell apoptosis, promoted cell proliferation, migration, invasion abilities, and activated the Wnt/β-catenin pathway. However, knockdown of miR-142-5p or overexpression of LMX1A showed opposite results. LMX1A was identified as a direct target of miR-142-5p by luciferase reporter assays. Finally, rescue experiments demonstrated that LMX1A overexpression attenuated the carcinogenic effect of miR-142-5p mimic on HeLa cells.ConclusionsThese findings suggested that miR-142-5p might be a cervical cancer oncogene and could serve as a potential therapeutic target for the treatment of cervical cancer.

Injury to dopaminergic neurons development via the Lmx1a/Wnt1 autoregulatory loop induced by simazine

Simazine is a kind of persistent organic pollutant that is detected in both ground and water and has several routes of exposure. Here, we explored the mechanisms underlying simazine-related effects on dopaminergic neurons via development-related factors using mouse embryos and embryonic mesencephalic hybrid cell line (MN9D cells). We treated pregnant mice with 50 μg/kg bw, 200 μg/kg bw simazine from the 0.5 day to the 10.5 day of embryonic phase and MN9D cells with 600 μM simazine for 24 h to research the mechanism of dopaminergic neurons acute respond to simazine through preliminary experiments. Protein expressions of LIM homeobox transcription factor 1-alpha (Lmx1a) and LIM homeobox transcription factor 1-beta (Lmx1b) displayed a dose- and time-dependent increase after the exposure to simazine. In the 200 μg/kg bw of embryos and the 24h-600 μM of MN9D cells, protein levels of dopaminergic developmental factors were significantly upregulated, and dopaminergic function was significantly damaged for the abnormal expression of Dyt5b. We demonstrated simazine induced the injury to dopaminergic neurons via the Lmx1a/wingless-related integration site 1 (Wnt1) and Lmx1b pathways. In the transfection experiments, we knocked down Lmx1a and Lmx1b of cells to verify the potential target of simazine-induced injury to dopaminergic neurons, respectively. We detected the protein and mRNA levels of development-related genes of dopaminergic neurons and intracellular dopamine levels in different treatment groups. Based on our experiments’ results, we demonstrated an acute response to 24 h–600 μM simazine treatment, the simazine-induced injury to dopaminergic neuronal which leads to abnormal dopamine levels and dopaminergic impairment is via the activation of the Lmx1a/Wnt1 autoregulatory loop. Lmx1a is a promising target in the search for the mechanisms underlying simazine-induced dopaminergic injury.

LMX1A inhibits C-Myc expression through ANGPTL4 to exert tumor suppressive role in gastric cancer

Our research group has showed that the LIM homeobox transcription factor 1 alpha (LMX1A) is inactivated in gastric cancers. Overexpression of LMX1A inhibits tumor growth. However, the mechanisms remains unclear. Considering LMX1A as a transcription factor, a comparison of RNA-seq between gastric cancer cells (GCCs) and GCCs with LMX1A overexpressed was performed to identify genes transcriptionally activated by LMX1A. Among the potential LMX1A target genes, angiopoietin-like 4 (ANGPTL4) has been reported to be an important tumor suppressor and thus was selected for further validation and research. Both LMX1A and ANGPTL4 showed downregulated expression in gastric cancer samples. More importantly, the expression of LMX1A is positively correlated with ANGPTL4, without including other family members in gastric cancer cell lines. What’s more, knockdown of ANGPTL4 rescued the tumor suppressive phenotype of LMX1A overexpression, which indicated that LMX1A upregulates ANGPTL4 to exert its role. Mechanistically, we found that LMX1A inhibited the expression of the oncogene C-Myc, which is alleviated by ANGPTL4 knockdown. In general, our results showed that LMX1A exerts its tumor suppressive role by activating ANGPTL4 to inhibit C-Myc.

Knockdown of LncRNA SCAMP1 suppressed malignant biological behaviours of glioma cells via modulating miR-499a-5p/LMX1A/NLRC5 pathway.

Dysregulation of long non‐coding RNAs (lncRNAs) confirm that it plays a crucial role in tumourigenesis and malignant progression of glioma. The present study demonstrated that LncRNA secretory carrier membrane protein 1 (SCAMP1) was up‐regulated and functioned as an oncogene in glioma cells. In addition, miR‐499a‐5p was down‐regulated meanwhile exerted tumour‐suppressive function in glioma cells. Subsequently, inhibition of SCAMP1 significantly restrained the cell proliferation, migration and invasion, as well as promoted apoptosis by acting as a molecular sponge of miR‐499a‐5p. Transcription factor LIM homeobox transcription factor 1, alpha (LMX1A) was overexpressed in glioma tissues and cells. Moreover, miR‐499a‐5p targeted LMX1A 3′‐UTR in a sequence‐specific manner. Hence, down‐regulation of SCAMP1 remarkably reduced the expression level of LMX1A, indicating that LMX1A participated in miR‐499a‐5p‐induced tumour‐suppressive effects on glioma cells. Furthermore, knockdown of LMX1A decreased NLR family, CARD domain containing 5 (NLRC5) mRNA and protein expression levels through directly binding to the NLRC5 promoter region. Down‐regulation of NLRC5 obviously inhibited malignant biological behaviours of glioma cells through attenuating the activity of Wnt/β‐catenin signalling pathway. In conclusion, our study clarifies that SCAMP1/miR‐499a‐5p/LMX1A/NLRC5 axis plays a critical role in modulating malignant progression of glioma cells, which provide a novel therapeutic strategy for glioma treatment.

Pharmacological Transdifferentiation of Human Nasal Olfactory Stem Cells into Dopaminergic Neurons.

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific in vitro models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson's disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establish in vitro models that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

MicroRNA-9 selectively targets LMX1A to promote gastric cancer cell progression

LIM homeobox transcription factor 1, alpha (LMX1A) is downregulated in human gastric cancer (GC), functioning as a tumor suppressor. The current study aims to identify specific microRNA that can regulate LMX1A expression. By sequence analysis of LMX1A mRNA 3′-untranslated region (3′-UTR), we show that microRNA-9 (miR-9) putatively targets human LMX1A. In established (AGS cells) and primary human GC cells, ectopic overexpression of miR-9 by a lentiviral construct decreased LMX1A 3′-UTR activity, causing LMX1A mRNA and protein downregulation. Functional analyses show that miR-9 overexpression enhanced GC cell survival and proliferation. On the contrary, miR-9 inhibition by antagomir-9 lentivirus increased LMX1A 3′-UTR activity to upregulate LMX1A mRNA and protein expression, causing GC cell apoptosis. CRISPR/Cas9-mediated LMX1A knockout promoted AGS cell survival and proliferation. Importantly, miR-9 and antagomiR-9 were ineffective to the function of LMX1A-knockout AGS cells. In human GC tissues miR-9 is upregulated, which is negatively correlated with LMX1A downregulation. Together, we conclude that miR-9 selectively targets LMX1A to promote GC cell progression.

Discovery of Novel Cell Surface Markers for Purification of Embryonic Dopamine Progenitors for Transplantation in Parkinson's Disease Animal Models

Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A+ cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of human-PSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.

Genome-Wide Association Study in Vestibular Neuritis: Involvement of the Host Factor for HSV-1 Replication.

Objective: In order to identify genetic variants associated with vestibular neuritis, a common cause of peripheral vertigo with a potential causative link to the reactivation of herpes simplex type 1 (HSV-1), we conducted a genome-wide association study.Methods: Association was assessed using approximately 8 million variants. 131 patients with vestibular neuritis and 2,609 controls of European ancestry were included.Results: Genome-wide associations with vestibular neuritis were detected in 4 regions containing protein coding genes assignable to two functional groups: virus hypothesis and insulin metabolism. Genes of set 1 are related to viral processes: nuclear receptor subfamily 3 group C member 2 (NR3C2) is a receptor for mineralocorticoids and glucocorticoids and was shown to be a host factor for HSV-1 replication. Ankyrin repeat domain 30A (ANKRD30A) encodes a host factor for human immunodeficiency virus-1 (HIV-1) infection. It shows rapid evolution and is induced by interferon stimulation. Mediator complex 30 (MED30), an important member of the mediator complex, has been shown to be involved in replication of HIV-1, a knockdown leading to impaired viral replication. The second set of genes LIM homeobox transcription factor 1 alpha (LMX1A), solute carrier family 30 member 8 (SLC30A8) is associated with insulin metabolism and resistance, a feature of some patients in whom type 2 diabetes is an accompanying comorbidity of vestibular neuritis.Conclusions: Using a GWAS approach to evaluate the etiology of vestibular neuritis these findings provide another piece of evidence that it may be caused by a viral inflammation.

LMX1A inhibits metastasis of gastric cancer cells through negative regulation of β-catenin

Previously, we reported that the LIM homeobox transcription factor 1, alpha (LMX1A) presented tumor-suppressing roles in gastric AGS cells. Here, we showed that LMX1A also inhibits metastasis-related behaviors including migration and invasion of gastric cancer cells. Mechanistic study revealed that the role of LMX1A was mediated by β-catenin, as knockdown of LMX1A upregulated the expression of β-catenin and knockdown of β-catenin reversed the effects of LMX1A silencing. β-catenin is essential for the activation of WNT signaling pathway. Indeed, knockdown of LMX1A activated the expressions of WNT signaling target genes T cell-specific transcription factor 4 (TCF4) and matrix metalloproteinase-7 (MMP7). What is more, the expression of LMX1A was negatively correlated with WNT signaling target genes in two datasets of human gastric cancer tissues. Thus, our study revealed an anti-metastatic role of LMX1A in gastric cancer which is mediated by the negative regulation of β-catenin signaling target genes.

Polymorphisms of dopamine pathway genes NRG1 and LMX1A are associated with cognitive performance in bipolar disorder.

LIM homeobox transcription factor 1, alpha (LMX1A) and neuregulin 1 (NRG1) are susceptibility genes for schizophrenia that have been implicated in the dopaminergic pathway and have been associated with altered cognitive functioning. We hypothesized that single nucleotide polymorphisms (SNPs) in LMX1A and NRG1 would be associated with cognitive functioning in bipolar disorder. In total, four SNPs were directly genotyped. Regression models with five aggregated cognitive domains and intelligence quotient (IQ) score were run using risk variants of LMX1A (rs11809911, rs4657412, rs6668493) and NRG1 (rs35753505) as predictors. Models were performed in a clinical sample of patients with bipolar disorder (n = 114) and healthy controls (n = 104). The risk variants of the rs11809911 SNP in LMX1A were negatively associated with IQ score and memory/learning, whereas the risk variants of rs35753505 in NRG1 were positively associated with IQ score (adjusted R(2) = 0.17, Q = 0.006) and memory/learning (adjusted R(2) = 0.24, Q = 0.001). The risk variants of the rs35753505 SNP in NRG1 were positively associated with language (adjusted R(2) = 0.11, Q = 0.006), visuospatial functions (adjusted R(2) = 0.23, Q = 0.001), and attention/speed (adjusted R(2) = 0.25, Q = 0.001). Results could not be replicated in controls. The risk variants of the rs35753505 SNP were associated with increased performance in several cognitive domains and IQ, whereas the risk variants of the rs11809911 SNP in LMX1A was associated with reduced IQ and memory/learning.

Enhanced differentiation of neural progenitor cells into neurons of the mesencephalic dopaminergic subtype on topographical patterns

Parkinson's disease (PD) is a neurodegenerative disease attributed to the loss of midbrain dopaminergic (DA) neurons. The current lack of predictive models for this disease has been hampered by the acquirement of robust cells, posing a major barrier to drug development. Differentiation of stem cells into subtype specific cells may be guided by appropriate topographical cues but the role of topography has hitherto not been well understood. We used a Multi-Architecture (MARC) chip with various topographical structures and identified three topographies, which generate DA neurons from murine hippocampal neural progenitor cells with the highest percentage of neuronal (β-III-tubulin positive) and dopaminergic (tyrosine hydroxylase positive) populations. Analysis on single pattern structures showed that 2 μm gratings with 2 μm spacing and 2 μm height (2 μm gratings) and 2 μm gratings with hierarchical structure produced cells with the highest gene expression of TH and PITX3, with the longest neurite and highest percentage of alignment. Quantitative image analysis showed the 2 μm gratings produced cells with the highest expression of pituitary homeobox 3 (PITX3), LIM homeobox transcription factor 1 alpha (LMX1a), aldehyde dehydrogenase 1 family member A1 (ALDH1a1) and microtubule associated protein 2 (MAP2), as compared to nano-gratings and unpatterned controls. These patterns also enhance DA neuron differentiation on different substrate rigidities, as seen on both poly-dimethylsiloxane (PDMS) and tissue culture polystyrene (TCPS) substrates. These results show the use of topographical influence for neuronal subtype specification, which could be translated into a wide range of clinical applications for PD.

Efficient differentiation of human embryonic stem cells toward dopaminergic neurons using recombinant LMX1A factor.

Direct differentiation of dopaminergic (DA) neurons from human pluripotent stem cells (hPSCs) in the absence of gene manipulation is the most desired alternative to clinical treatment of Parkinson disease. Protein transduction-based methods could be efficient, safe approaches to enhance direct differentiation of human embryonic stem cells (hESCs) to DA neurons. In the present study, we compared the differentiation efficiency of DA neurons from hESCs with and without the application of LIM homeobox transcription factor 1 alpha (LMX1A), a master regulatory protein in the development of the midbrain neurons and SHH proteins. The results obtained revealed that the treatment of hESCs with recombinant LMX1A (rLMX1A) protein along with dual SMAD inhibition led to higher expression of LMX1B, LMX1A, FOXA2, PITX3, EN1, and WNT1 effector endogenous genes and two-fold expression of PITX3. Moreover, the highest expression level of PITX3 and TH was observed when rLMX1A was added to the induction medium supplemented with SHH. To our best knowledge, this is the first report demonstrating the application of TAT-LMX1A recombinant protein to enhance hESC differentiation to DA as shown by the expression of DA specific makers. These findings pave the way for enhancing the differentiation of hESCs to DA neurons safely and efficiently without genetic modification.

Recovery of behavioral symptoms in hemi-parkinsonian rhesus monkeys through combined gene and stem cell therapy

The use of adipose mesenchymal stromal cells (ASCs) in cellular and genic therapy has attracted considerable attention as a possible treatment for neurodegenerative disorders, including Parkinson disease. However, the effects of gene therapy combined with intracerebral cell transplantation have not been well defined. Recent studies have demonstrated the respective roles of LIM homeobox transcription factor 1, alpha (LMX1A) and Neurturin (NTN) in the commitment of embryonic stem cells (ESCs) to a midbrain dopaminergic neuronal fate and the commitment of mesenchymal stromal cells to cells supporting the nutrition and protection of neurons. We investigated a novel in vitro neuronal differentiation strategy with the use of LMX1A and Neurturin. We were able to elicit a neural phenotype regarding cell morphology, specific gene/protein expression and physiological function. Neuronal-primed ASCs derived from rhesus monkey (rASCs) combined with adenovirus containing NTN and tyrosine hydroxylase (TH) (Ad-NTN-TH) were implanted into the striatum and substantia nigra of methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored with the use of behavioral tests and health measures until the fourth month after implantation. The differentiated cells transcribed and expressed a variety of dopaminergic neuron-specific genes involved in the SHH/LMX1A pathway. Single-photon emission computed tomography analysis and postmortem analysis revealed that the grafting of rASCs combined with Ad-NTN-TH had neuroprotective effects compared with Ad-NTN-TH or rASCs alone. Behavioral measures demonstrated autograft survival and symptom amelioration. These findings may lead to cellular sources for autologous transplantation of Parkinson disease. Combined transplantation of Ad-NTN-TH and induced rASCs expressing LMX1A and NTN may be a better therapy candidate for the treatment of Parkinson disease.

The association of osteopontin and LMX1A expression with World Health Organization grade in meningiomas and gliomas

Osteopontin (OPN) and LIM homeobox transcription factor 1, alpha (LMX1A) are important factors related to tumour progression, invasion and metastasis in human cancers. The aim of this study was to test the hypothesis that expression of OPN and of LMX1A correlate with the World Health Organization (WHO) grading system of primary brain tumours. Immunohistochemical analyses of OPN and LMX1A expression were performed in 139 cases of brain tumour, including 65 meningiomas, 71 gliomas, and three central neurocytomas. More than 90% of WHO grade I meningiomas showed negative or weak staining for OPN and LMX1A. However, among all WHO grade II and III meningiomas, 100% and 66.7% showed moderate or strong staining for OPN and LMX1A, respectively. Similarly, higher percentages of WHO grade I and II gliomas than of WHO grade III and IV gliomas showed negative or weak staining for OPN. A higher intensity of immunoreactivity for LMX1A correlated with more advanced grade in WHO grade I-III gliomas, but not in WHO grade IV tumours. Higher immunostaining intensity for OPN and LMX1A correlated with WHO grades for meningiomas and some gliomas. Contrary to our expectations, LMX1A staining in WHO grade IV gliomas was shown to be weaker than in WHO grade III tumours.

Detecting purely epistatic multi-locus interactions by an omnibus permutation test on ensembles of two-locus analyses.

BackgroundPurely epistatic multi-locus interactions cannot generally be detected via single-locus analysis in case-control studies of complex diseases. Recently, many two-locus and multi-locus analysis techniques have been shown to be promising for the epistasis detection. However, exhaustive multi-locus analysis requires prohibitively large computational efforts when problems involve large-scale or genome-wide data. Furthermore, there is no explicit proof that a combination of multiple two-locus analyses can lead to the correct identification of multi-locus interactions.ResultsThe proposed 2LOmb algorithm performs an omnibus permutation test on ensembles of two-locus analyses. The algorithm consists of four main steps: two-locus analysis, a permutation test, global p-value determination and a progressive search for the best ensemble. 2LOmb is benchmarked against an exhaustive two-locus analysis technique, a set association approach, a correlation-based feature selection (CFS) technique and a tuned ReliefF (TuRF) technique. The simulation results indicate that 2LOmb produces a low false-positive error. Moreover, 2LOmb has the best performance in terms of an ability to identify all causative single nucleotide polymorphisms (SNPs) and a low number of output SNPs in purely epistatic two-, three- and four-locus interaction problems. The interaction models constructed from the 2LOmb outputs via a multifactor dimensionality reduction (MDR) method are also included for the confirmation of epistasis detection. 2LOmb is subsequently applied to a type 2 diabetes mellitus (T2D) data set, which is obtained as a part of the UK genome-wide genetic epidemiology study by the Wellcome Trust Case Control Consortium (WTCCC). After primarily screening for SNPs that locate within or near 372 candidate genes and exhibit no marginal single-locus effects, the T2D data set is reduced to 7,065 SNPs from 370 genes. The 2LOmb search in the reduced T2D data reveals that four intronic SNPs in PGM1 (phosphoglucomutase 1), two intronic SNPs in LMX1A (LIM homeobox transcription factor 1, alpha), two intronic SNPs in PARK2 (Parkinson disease (autosomal recessive, juvenile) 2, parkin) and three intronic SNPs in GYS2 (glycogen synthase 2 (liver)) are associated with the disease. The 2LOmb result suggests that there is no interaction between each pair of the identified genes that can be described by purely epistatic two-locus interaction models. Moreover, there are no interactions between these four genes that can be described by purely epistatic multi-locus interaction models with marginal two-locus effects. The findings provide an alternative explanation for the aetiology of T2D in a UK population.ConclusionAn omnibus permutation test on ensembles of two-locus analyses can detect purely epistatic multi-locus interactions with marginal two-locus effects. The study also reveals that SNPs from large-scale or genome-wide case-control data which are discarded after single-locus analysis detects no association can still be useful for genetic epidemiology studies.

An epigenetic marker panel for screening and prognostic prediction of ovarian cancer

Aberrant CpG island hypermethylation is a common finding of cancers, which might be detectable in the tissue or serum of affected patients. We analyzed DNA methylation by methylation-specific polymerase chain reaction of 7 genes, which included secreted frizzled receptor proteins 1, 2, 4, 5 (SFRP1, 2, 4, 5), SRY-box 1 (SOX1), paired box gene 1 (PAX1) and LIM homeobox transcription factor 1, alpha (LMX1A) in primary tumor samples from 126 patients with ovarian cancer, 75 with a benign tumor and 14 with borderline malignancy of an ovarian tumor, and in the serum from 26 patients with ovarian cancer and 20 with a benign tumor. Six of 7 genes had higher methylation rates in patients with ovarian cancer than in borderline malignancy or benign tumor (p<0.001). The methylation of SFRP1, SFRP2, SOX1 and LMX1A genes correlated with recurrence and overall survival of ovarian cancer patients. Combining the data for SFRP1, SFRP2 and SOX1 genes gave a relative risk for recurrence of 3.19 (p=0.013) in patients with at least one gene methylation, and combining the data for SFRP1, SOX1 and LMX1A gave an RR for cancer-related death of 6.09 (p=0.010). Methylation analysis of tissues and serum revealed a significant correlation (kappa values, 0.332-0.598) and a highly sensitivity and specificity rates (73.08 and 75%) as a screening marker. In conclusion, promoter hypermethylation of specific genes in critical pathways is common in ovarian cancer and has potential as a prognostic factor and a promising serum marker for early screening.

1343 Animal model of ischemia of spinal cord using the rat and its protection by systemic moderate hypothermia

The use of adipose mesenchymal stromal cells (ASCs) in cellular and genic therapy has attracted considerable attention as a possible treatment for neurodegenerative disorders, including Parkinson disease. However, the effects of gene therapy combined with intracerebral cell transplantation have not been well defined. Recent studies have demonstrated the respective roles of LIM homeobox transcription factor 1, alpha (LMX1A) and Neurturin (NTN) in the commitment of embryonic stem cells (ESCs) to a midbrain dopaminergic neuronal fate and the commitment of mesenchymal stromal cells to cells supporting the nutrition and protection of neurons.We investigated a novel in vitro neuronal differentiation strategy with the use of LMX1A and Neurturin. We were able to elicit a neural phenotype regarding cell morphology, specific gene/protein expression and physiological function. Neuronal-primed ASCs derived from rhesus monkey (rASCs) combined with adenovirus containing NTN and tyrosine hydroxylase (TH) (Ad-NTN-TH) were implanted into the striatum and substantia nigra of methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored with the use of behavioral tests and health measures until the fourth month after implantation.The differentiated cells transcribed and expressed a variety of dopaminergic neuron-specific genes involved in the SHH/LMX1A pathway. Single-photon emission computed tomography analysis and postmortem analysis revealed that the grafting of rASCs combined with Ad-NTN-TH had neuroprotective effects compared with Ad-NTN-TH or rASCs alone. Behavioral measures demonstrated autograft survival and symptom amelioration.These findings may lead to cellular sources for autologous transplantation of Parkinson disease. Combined transplantation of Ad-NTN-TH and induced rASCs expressing LMX1A and NTN may be a better therapy candidate for the treatment of Parkinson disease.