Allelic expression imbalance of PIK3CA mutations is frequent in breast cancer and prognostically significant.

PIK3CA mutations are the most common in breast cancer, particularly in the estrogen receptor-positive cohort, but the benefit of PI3K inhibitors has had limited success compared with approaches targeting other less common mutations. We found a frequent allelic expression imbalance between the missense mutant and wild-type PIK3CA alleles in breast tumors from the METABRIC (70.2%) and the TCGA (60.1%) projects. When considering the mechanisms controlling allelic expression, 27.7% and 11.8% of tumors showed imbalance due to regulatory variants in cis, in the two studies respectively. Furthermore, preferential expression of the mutant allele due to cis-regulatory variation is associated with poor prognosis in the METABRIC tumors (P = 0.031). Interestingly, ER-, PR-, and HER2+ tumors showed significant preferential expression of the mutated allele in both datasets. Our work provides compelling evidence to support the clinical utility of PIK3CA allelic expression in breast cancer in identifying patients of poorer prognosis, and those with low expression of the mutated allele, who will unlikely benefit from PI3K inhibitors. Furthermore, our work proposes a model of differential regulation of a critical cancer-promoting gene in breast cancer.

Germline allelic expression of genes at 17q22 locus associates with risk of breast cancer.

INTRODUCTION: Translation of genome-wide association study (GWAS) findings into preventive approaches is challenged by the identification of the causal risk variants and the understanding of the biological mechanisms by which they act. We present using allelic expression (AE) ratios to perform quantitative case-control analysis as a novel approach to identify risk associations, causal regulatory variants, and target genes. METHODS: Using the breast cancer (BC) risk locus 17q22 to validate this approach, we measured AE ratios in normal breast tissue samples from controls and cases, as well as from unmatched blood samples. Then we used in-silico and in-vitro analysis to map and functionally characterised candidate causal variants. RESULTS: We found a significant shift in the AE patterns of STXBP4 (rs2628315) and COX11 (rs17817901) in the normal breast tissue of cases and healthy controls. Preferential expression of the G-rs2628315 and A-rs17817901 alleles, more often observed in cases, was associated with an increased risk for BC. Analysis of blood samples from cases and controls found a similar association. Furthermore, we identified two putative cis-regulatory variants - rs17817901 and rs8066588 - that affect a miRNA and a transcription factor binding site, respectively. CONCLUSION: We propose causal variants and target genes for the 17q22 BC risk locus and show that using AE ratios in case-control association studies is helpful in identifying risk and mapping causal variants.

Allele-specific miRNA-binding analysis identifies candidate target genes for breast cancer risk.

Most breast cancer (BC) risk-associated single-nucleotide polymorphisms (raSNPs) identified in genome-wide association studies (GWAS) are believed to cis-regulate the expression of genes. We hypothesise that cis-regulatory variants contributing to disease risk may be affecting microRNA (miRNA) genes and/or miRNA binding. To test this, we adapted two miRNA-binding prediction algorithms-TargetScan and miRanda-to perform allele-specific queries, and integrated differential allelic expression (DAE) and expression quantitative trait loci (eQTL) data, to query 150 genome-wide significant ( P ≤ 5 × 10 - 8 ) raSNPs, plus proxies. We found that no raSNP mapped to a miRNA gene, suggesting that altered miRNA targeting is an unlikely mechanism involved in BC risk. Also, 11.5% (6 out of 52) raSNPs located in 3'-untranslated regions of putative miRNA target genes were predicted to alter miRNA::mRNA (messenger RNA) pair binding stability in five candidate target genes. Of these, we propose RNF115, at locus 1q21.1, as a strong novel target gene associated with BC risk, and reinforce the role of miRNA-mediated cis-regulation at locus 19p13.11. We believe that integrating allele-specific querying in miRNA-binding prediction, and data supporting cis-regulation of expression, improves the identification of candidate target genes in BC risk, as well as in other common cancers and complex diseases.

IL10 low-frequency variants in Behçet's disease patients.

AIM: To explain the missing heritability after the genome-wide association studies era, sequencing studies allow the identification of low-frequency variants with a stronger effect on disease risk. Common variants in the interleukin 10 gene (IL10) have been consistently associated with Behçet's disease (BD) and the goal of this study is to investigate the role of low-frequency IL10 variants in BD susceptibility. METHODS: To identify IL10 low-frequency variants, a discovery group of 50 Portuguese BD patients were Sanger-sequenced in a 7.7 kb genomic region encompassing the complete IL10 gene, 0.9 kb upstream and 2 kb downstream, and two conserved regions in the putative promoter. To assess if the novel variants are BD- and/or Portuguese-specific, they were assayed in an additional group of BD patients (26 Portuguese and 964 Iranian) and controls (104 Portuguese and 823 Iranian). RESULTS: Rare IL10 coding variants were not detected in BD patients, but we identified 28 known single nucleotide polymorphisms with minor allele frequencies ranging from 0.010 to 0.390, and five novel non-coding variants in five heterozygous cases. ss836185595, located in the IL10 3' untranslated region, was also detected in one Iranian control individual and therefore is not specific to BD. The remaining novel IL10 variants (ss836185596 and ss836185602 in intron 3, ss836185598 and ss836185604 in the putative promoter region) were not found in the replication dataset. CONCLUSION: This study highlights the importance of screening the whole gene and regulatory regions when searching for novel variants associated with complex diseases, and the need to develop bioinformatics tools to predict the functional impact of non-coding variants and statistical tests which incorporate these predictions.

Mitochondrial genome association study with peripheral arterial disease and venous thromboembolism.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) and venous thromboembolism (VTE) are vascular traits sharing common modifiable and non-modifiable risk factors. These vascular pathologies have known nuclear-encoded genetic risk factors and the mitochondrial DNA may account for part of the missing heritability. To determine if PAD and VTE have a dual genetic control (mitochondrial and nuclear), we hereby investigated the association of mitochondrial DNA polymorphisms and haplogroups with these vascular traits. METHODS: The association of mitochondrial single nucleotide polymorphisms (mtSNPs) and haplogroups was tested in 1652 PAD cases and 1629 controls from the eMERGE PAD genome-wide association study (GWAS), and 1241 VTE cases and 1278 controls from the GENEVA GWAS of venous thrombosis (dbGaP accession numbers phs000203.v1.p1 and phs000289.v2.p1, respectively). RESULTS: 66 and 72 mtSNPs passed quality control filters and were tested for association with PAD and VTE, respectively. Significant evidence of population stratification could not be detected in both datasets. Three mtSNPs (m.477T > C, m.9667A > G, and m.10915T > C) were nominally associated (3.01 × 10(-3) ≤ pa ≤ 3.96 × 10(-2)) with PAD in the logistic regression adjusted for confounding factors, and m.11914G > A was nominally associated (pa = 4.14 × 10(-2)) with VTE. None of the nine major mitochondrial haplogroups were associated with either PAD or VTE. CONCLUSION: Unlike other vascular diseases such as stroke and diabetes, these results suggest that common mitochondrial variants individually or in combination do not play a major role in PAD and VTE susceptibility.

Ulcerative Colitis Is Under Dual (Mitochondrial and Nuclear) Genetic Control.

BACKGROUND: Cellular oxidative stress and genetic susceptibility have been implicated in the multifactorial etiology of ulcerative colitis (UC). The nuclear genome association with UC has been intensely investigated, but the role of the mitochondrial DNA (mtDNA) has received far less attention and may account for part of the missing heritability. This study is a comprehensive analysis of the mtDNA contribution to UC susceptibility. METHODS: The association of mitochondrial single-nucleotide polymorphisms (mtSNPs) and haplogroups with UC was tested in 488 cases and 833 controls of European ancestry from the NIDDK IBD Genetics Consortium Ulcerative Colitis Genome-Wide Association Study available through dbGaP and from the Illumina Genotype Control Database (studies 64 and 65). RESULTS: No evidence of population stratification could be detected using 218 ancestry informative markers for European Americans. Seven of the 58 tested mtSNPs were nominally associated with UC, and A10550G in MT-ND4L withstands the Bonferroni correction (P = 1.29E-06, odds ratio [ORG] [95% confidence interval (CI)] = 4.80 [2.54-9.05], 10550G allele: 8.1% of patients and 1.9% of controls). A10550G remains equally associated after conditional analyses on the 11 UC genome-wide association studies (GWAS) top SNPs (6.35E-07 < Pcond < 4.58E-06), which suggests that it constitutes an independent risk factor from nuclear-encoded susceptibility loci. We detected additive (but not multiplicative) epistatic interactions between A10550G and all 11 top GWAS hits. Subhaplogroup K1 (P = 0.021, OR [95% CI] = 1.71 [1.08-2.69]) increased the risk for UC, whereas the U5b lineage conferred protection (P = 0.016, OR [95% CI] = 0.34 [0.14-0.82]). CONCLUSIONS: These results suggest that UC has a dual mitochondrial and nuclear genetic control that warrants further replication in independent data sets and reinforces its etiopathogenic complexity.

Mitochondria: Major Regulators of Neural Development.

Mitochondria are organelles derived from primitive symbiosis between archeon ancestors and prokaryotic α-proteobacteria species, which lost the capacity of synthetizing most proteins encoded the bacterial DNA, along the evolutionary process of eukaryotes. Nowadays, mitochondria are constituted by small circular mitochondrial DNA of 16 kb, responsible for the control of several proteins, including polypeptides of the electron transport chain. Throughout evolution, these organelles acquired the capacity of regulating energy production and metabolism, thus becoming central modulators of cell fate. In fact, mitochondria are crucial for a variety of cellular processes, including adenosine triphosphate production by oxidative phosphorylation, intracellular Ca(2+) homeostasis, generation of reactive oxygen species, and also cellular specialization in a variety of tissues that ultimately relies on specific mitochondrial specialization and maturation. In this review, we discuss recent evidence extending the importance of mitochondrial function and energy metabolism to the context of neuronal development and adult neurogenesis.

Genetic Variants Underlying Risk of Intracranial Aneurysms: Insights from a GWAS in Portugal.

Subarachnoid hemorrhage (SAH) is a life-threatening event that most frequently leads to severe disability and death. Its most frequent cause is the rupture of a saccular intracranial aneurysm (IA), which is a blood vessel dilation caused by disease or weakening of the vessel wall. Although the genetic contribution to IA is well established, to date no single gene has been unequivocally identified as responsible for IA formation or rupture. We aimed to identify IA susceptibility genes in the Portuguese population through a pool-based multistage genome-wide association study. Replicate pools were allelotyped in triplicate in a discovery dataset (100 IA cases and 92 gender-matched controls) using the Affymetrix Human SNP Array 6.0. Top SNPs (absolute value of the relative allele score difference between cases and controls |RASdiff|≥13.0%) were selected for technical validation by individual genotyping in the discovery dataset. From the 101 SNPs successfully genotyped, 99 SNPs were nominally associated with IA. Replication of technically validated SNPs was conducted in an independent replication dataset (100 Portuguese IA cases and 407 controls). rs4667622 (between UBR3 and MYO3B), rs6599001 (between SCN11A and WDR48), rs3932338 (214 kilobases downstream of PRDM9), and rs10943471 (96 kilobases upstream of HTR1B) were associated with IA (unadjusted allelic chi-square tests) in the datasets tested (discovery: 6.84E-04≤P≤1.92E-02, replication: 2.66E-04≤P≤2.28E-02, and combined datasets: 6.05E-05≤P≤5.50E-04). Additionally, we confirmed the known association with IA of rs1333040 at the 9p21.3 genomic region, thus validating our dataset. These novel findings in the Portuguese population warrant further replication in additional independent studies, and provide additional candidates to more comprehensively understand IA etiopathogenesis.

Brief report: association of CCR1, KLRC4, IL12A-AS1, STAT4, and ERAP1 With Behçet's disease in Iranians.

OBJECTIVE: To independently replicate the top findings from 4 published genome-wide association studies (GWAS) of susceptibility genes in Behçet's disease (BD). METHODS: We tested 14 single-nucleotide polymorphisms (SNPs) in 13 genomic loci (excluding the major histocompatibility complex [MHC], IL10, and IL23R-IL12RB2, which have already been associated with BD in Iranians) for allelic and genotypic associations with BD in 973 patients and 828 controls from Iran and performed meta-analyses of the significantly associated markers. RESULTS: Six SNPs (in decreasing order of significance, rs7616215 located 38 kb downstream of CCR1, rs2617170 [p.Asn104Ser] in KLRC4, rs17810546 in IL12A-AS1, rs7574070 in STAT4, and rs10050860 [p.Asp575Asn] and rs13154629 in ERAP1) were nominally associated with BD in both allelic association tests (5.05 × 10(-9) ≤ Pallele ≤ 7.55 × 10(-3) ) and sex-adjusted genotypic association tests (6.01 × 10(-9) ≤ adjusted P value ≤ 1.30 × 10(-2) ). For all 6 SNPs tested by meta-analysis (Pmeta ), the association with BD was strengthened, because the direction and magnitude of association were similar across populations (e.g., for rs7574070, odds ratio [OR] for A allele 1.29 [95% confidence interval (95% CI) 1.21-1.37], Pmeta = 2.34 × 10(-16) ; for rs7616215, OR for C allele 0.70 [95% CI 0.65-0.76], Pmeta = 1.54 × 10(-19) ; for rs17810546, OR for A allele 0.60 [95% CI 0.52-0.70], Pmeta = 6.34 × 10(-11) ; for rs2617170, OR for T allele 0.76 [95% CI 0.70-0.81], Pmeta = 2.75 × 10(-14) ; for rs13154629, OR for TT genotype 2.76 [95% CI 2.01-3.80], Pmeta = 3.57 × 10(-10) ). CONCLUSION: This study reinforces the notion that CCR1, KLRC4, IL12A-AS1, STAT4, and ERAP1 are bona fide susceptibility genes for BD, in addition to the MHC, IL10, and IL23R-IL12RB2 loci. Future genetic and functional studies are now warranted to uncover the roles of these genes in the pathogenesis of BD.

Characterization of the major histocompatibility complex locus association with Behçet's disease in Iran.

INTRODUCTION: The aim of this study was to characterize the association of human leukocyte antigen (HLA) B alleles and major histocompatibility complex (MHC) single nucleotide polymorphisms (SNPs) with Behçet's disease (BD) in an Iranian dataset. METHODS: The association of three SNPs in the MHC region previously identified as the most associated in high-density genotyping studies was tested in a case-control study on 973 BD patients and 825 controls from Iran, and the association of HLA-B alleles was tested in a subset of 681 patients and 414 controls. RESULTS: We found that HLA-B*51 (P = 4.11 × 10(-41), OR [95% CI] = 4.63[3.66-5.85]) and B*15 confer risk for BD (P = 2.83 × 10(-2), OR [95% CI] = 1.75[1.08-2.84]) in Iranian, and in B*51 negative individuals, only the B*15 allele is significantly associated with BD (P = 2.51 × 10(-3), OR [95% CI] = 2.40[1.37-4.20]). rs76546355, formerly known as rs116799036, located between HLA-B and MICA (MHC class I polypeptide-related sequence A), demonstrated the same level of association with BD as HLA-B*51 (P adj = 1.78 × 10(-46), OR [95% CI] = 5.46[4.21-7.09], and P adj = 8.34 × 10(-48), OR [95% CI] = 5.44[4.20-7.05], respectively) in the HLA-B allelotyped subset, while rs2848713 was less associated (P adj = 7.14 × 10(-35), OR [95% CI] = 3.73[2.97-4.69]) and rs9260997 was not associated (P adj = 1.00 × 10(-1)). Additionally, we found that B*51 genotype-phenotype correlations do not survive Bonferroni correction, while carriers of the rs76546355 risk allele predominate in BD cases with genital ulcers, positive pathergy test and positive BD family history (2.31 × 10(-4) ≤ P ≤ 1.59 × 10(-3)). CONCLUSIONS: We found that the HLA-B*51 allele and the rs76546355/rs116799036 MHC SNP are independent genetic risk factors for BD in Iranian, and that positivity for the rs76546355/rs116799036 risk allele, but not for B*51, does correlate with specific demographic characteristics or clinical manifestations in BD patients.

FUT2: filling the gap between genes and environment in Behçet's disease?

OBJECTIVES: To identify new susceptibility loci for Behçet's disease (BD), we performed a genome-wide association study (GWAS) using DNA pooling. METHODS: Two replicate pools of 292 Iranian BD cases and of 294 age- and sex-matched controls were allelotyped in quadruplicate on the Affymetrix Genome-Wide Human SNP Array 6.0. Of the 51 top markers, 47 were technically validated through individually genotyping. Replication of validated single nucleotide polymorphisms (SNPs) was performed in an independent Iranian dataset (684 cases and 532 controls). RESULTS: In addition to the well-established HLA-B locus, rs7528842 in a gene desert on chromosome 1p21.2, and rs632111 at the 3'UTR of FUT2 were associated in both the discovery and replication datasets (individually and in combination). However, only the FUT2 SNP was associated in a previous GWAS for BD in Turkish people. Fine-mapping of FUT2 in the full Iranian dataset showed additional associations in five coding SNPs (2.97E-06

MicroRNA-34a Modulates Neural Stem Cell Differentiation by Regulating Expression of Synaptic and Autophagic Proteins.

We have previously demonstrated the involvement of specific apoptosis-associated microRNAs (miRNAs), including miR-34a, in mouse neural stem cell (NSC) differentiation. In addition, a growing body of evidence points to a critical role for autophagy during neuronal differentiation, as a response-survival mechanism to limit oxidative stress and regulate synaptogenesis associated with this process. The aim of this study was to further investigate the precise role of miR-34a during NSC differentiation. Our results showed that miR-34a expression was markedly downregulated during neurogenesis. Neuronal differentiation and cell morphology, synapse function, and electrophysiological maturation were significantly impaired in miR-34a-overexpressing NSCs. In addition, synaptotagmin 1 (Syt1) and autophagy-related 9a (Atg9a) significantly increased during neurogenesis. Pharmacological inhibition of autophagy impaired both neuronal differentiation and cell morphology. Notably, we showed that Syt1 and Atg9a are miR-34a targets in neural differentiation context, markedly decreasing after miR-34a overexpression. Syt1 overexpression and rapamycin-induced autophagy partially rescued the impairment of neuronal differentiation by miR-34a. In conclusion, our results demonstrate a novel role for miR-34a regulation of NSC differentiation, where miR-34a downregulation and subsequent increase of Syt1 and Atg9a appear to be crucial for neurogenesis progression.

Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling.

The low survival and differentiation rates of stem cells after either transplantation or neural injury have been a major concern of stem cell-based therapy. Thus, further understanding long-term survival and differentiation of stem cells may uncover new targets for discovery and development of novel therapeutic approaches. We have previously described the impact of mitochondrial apoptosis-related events in modulating neural stem cell (NSC) fate. In addition, the endogenous bile acid, tauroursodeoxycholic acid (TUDCA) was shown to be neuroprotective in several animal models of neurodegenerative disorders by acting as an anti-apoptotic and anti-oxidant molecule at the mitochondrial level. Here, we hypothesize that TUDCA might also play a role on NSC fate decision. We found that TUDCA prevents mitochondrial apoptotic events typical of early-stage mouse NSC differentiation, preserves mitochondrial integrity and function, while enhancing self-renewal potential and accelerating cell cycle exit of NSCs. Interestingly, TUDCA prevention of mitochondrial alterations interfered with NSC differentiation potential by favoring neuronal rather than astroglial conversion. Finally, inhibition of mitochondrial reactive oxygen species (mtROS) scavenger and adenosine triphosphate (ATP) synthase revealed that the effect of TUDCA is dependent on mtROS and ATP regulation levels. Collectively, these data underline the importance of mitochondrial stress control of NSC fate decision and support a new role for TUDCA in this process.

Targeting molecular networks for drug research.

The study of molecular networks has recently moved into the limelight of biomedical research. While it has certainly provided us with plenty of new insights into cellular mechanisms, the challenge now is how to modify or even restructure these networks. This is especially true for human diseases, which can be regarded as manifestations of distorted states of molecular networks. Of the possible interventions for altering networks, the use of drugs is presently the most feasible. In this mini-review, we present and discuss some exemplary approaches of how analysis of molecular interaction networks can contribute to pharmacology (e.g., by identifying new drug targets or prediction of drug side effects), as well as list pointers to relevant resources and software to guide future research. We also outline recent progress in the use of drugs for in vitro reprogramming of cells, which constitutes an example par excellence for altering molecular interaction networks with drugs.

StemCellNet: an interactive platform for network-oriented investigations in stem cell biology.

Stem cells are characterized by their potential for self-renewal and their capacity to differentiate into mature cells. These two key features emerge through the interplay of various factors within complex molecular networks. To provide researchers with a dedicated tool to investigate these networks, we have developed StemCellNet, a versatile web server for interactive network analysis and visualization. It rapidly generates focused networks based on a large collection of physical and regulatory interactions identified in human and murine stem cells. The StemCellNet web-interface has various easy-to-use tools for selection and prioritization of network components, as well as for integration of expression data provided by the user. As a unique feature, the networks generated can be screened against a compendium of stemness-associated genes. StemCellNet can also indicate novel candidate genes by evaluating their connectivity patterns. Finally, an optional dataset of generic interactions, which provides large coverage of the human and mouse proteome, extends the versatility of StemCellNet to other biomedical research areas in which stem cells play important roles, such as in degenerative diseases or cancer. The StemCellNet web server is freely accessible at http://stemcellnet.sysbiolab.eu.

Mitochondrial translocation of p53 modulates neuronal fate by preventing differentiation-induced mitochondrial stress.

AIMS: Apoptosis regulatory proteins, such as p53, play a pivotal role in neural differentiation, through mechanisms independent of cell death. In addition, p53 has been identified as an important regulator of mitochondrial survival response, maintaining mitochondrial DNA (mtDNA) integrity and oxidative protection. The aim of this study was to determine the role of mitochondrial p53 in organelle damage and neural differentiation. RESULTS: Our results show that mitochondrial apoptotic events such as reactive oxygen species production, mitochondrial membrane permeabilization, and cytochrome c release are typical of early-stage mouse neural stem cell differentiation, which occurs 3-18 h after induction of differentiation, with no evidence of cell death. In addition, decreased mtDNA content, lipidated LC3 (LC3-II), colocalization of mitochondria and LC3-II puncta, and mitochondria-associated Parkin are consistent with activation of mitophagy. Importantly, at early stages of neural differentiation, p53 was actively translocated to mitochondria and attenuated mitochondrial oxidative stress, cytochrome c release, and mitophagy. Forced mitochondrial translocation of p53 increased neurogenic potential and neurite outgrowth. INNOVATION AND CONCLUSION: In conclusion, our results reveal a novel role for mitochondrial p53, which modulates mitochondrial damage and apoptosis-related events in the context of neural differentiation, thus enhancing neuronal fate.

Amyloid ß peptides promote autophagy-dependent differentiation of mouse neural stem cells: Aß-mediated neural differentiation.

Although regarded as neurotoxic, amyloid ß (Aß) peptides may also mediate a wide range of nonpathogenic processes. Autophagy has been implicated in Aß-mediated effects, although its precise function in neural differentiation remains unknown. Here, we addressed the role of different Aß fragments in neural stem cell (NSC) proliferation and differentiation, and investigated whether autophagy is involved in Aß-induced alterations of neural fate. Our results demonstrate that neuronal and glial-specific protein markers are significantly induced by both Aß1-40 and Aß1-42. However, Aß1-40 preferentially enhances neurogenesis of NSCs, as determined by ßIII-tubulin, NeuN, and MAP2 neuronal marker immunoreactivity, while Aß1-42 appears to favor gliogenesis. In contrast, Aß25-35 does not influence NSC fate. The effect of Aß1-40 on neurogenesis is partially dependent on its role in NSC self-renewal as both S-phase of the cell cycle and BrdU labeling were markedly increased. Nevertheless, Aß1-40 resulted also in increased Tuj1 promoter activity. Autophagy, assessed by conversion of endogenous LC3-I/II, fluorescence of pGFP-LC3-transfected cells, and Atg9 protein levels, was evident in both Aß1-40- and Aß1-42-treated NSCs, independently of reactive oxygen species production and apoptosis. Finally, inhibition of autophagy by pharmacologic means abrogated Aß-induced lineage-specific protein markers. These results support distinct roles for different Aß peptides in NSC fate decision and underline the importance of autophagy control of this process.

Gene expression profiling and association studies implicate the neuregulin signaling pathway in Behçet's disease susceptibility.

Behçet's disease (BD) is a complex disease with genetic and environmental risk factors implicated in its etiology; however, its pathophysiology is poorly understood. To decipher BD's genetic underpinnings, we combined gene expression profiling with pathway analysis and association studies. We compared the gene expression profiles in peripheral blood mononuclear cells (PBMCs) of 15 patients and 14 matched controls using Affymetrix microarrays and found that the neuregulin signaling pathway was over-represented among the differentially expressed genes. The Epiregulin (EREG), Amphiregulin (AREG), and Neuregulin-1 (NRG1) genes of this pathway stand out as they are also among the top differentially expressed genes. Twelve haplotype tagging SNPs at the EREG-AREG locus and 15 SNPs in NRG1 found associated in at least one published BD genome-wide association study were tested for association with BD in a dataset of 976 Iranian patients and 839 controls. We found a novel association with BD for the rs6845297 SNP located downstream of EREG, and replicated three associations at NRG1 (rs4489285, rs383632, and rs1462891). Multifactor dimensionality reduction analysis indicated the existence of epistatic interactions between EREG and NRG1 variants. EREG-AREG and NRG1, which are members of the epidermal growth factor (EGF) family, seem to modulate BD susceptibility through main effects and gene-gene interactions. These association findings support a role for the EGF/ErbB signaling pathway in BD pathogenesis that warrants further investigation and highlight the importance of combining genetic and genomic approaches to dissect the genetic architecture of complex diseases.

Association study of IL10 and IL23R-IL12RB2 in Iranian patients with Behçet's disease.

OBJECTIVE: Independent replication of the findings from genome-wide association studies (GWAS) remains the gold standard for results validation. Our aim was to test the association of Behçet's disease (BD) with the interleukin-10 gene (IL10) and the IL-23 receptor-IL-12 receptor ß2 (IL23R-IL12RB2) locus, each of which has been previously identified as a risk factor for BD in 2 different GWAS. METHODS: Six haplotype-tagging single-nucleotide polymorphisms (SNPs) in IL10 and 42 in IL23R-IL12RB2 were genotyped in 973 Iranian patients with BD and 637 non-BD controls. Population stratification was assessed using a panel of 86 ancestry-informative markers. RESULTS: Subtle evidence of population stratification was found in our data set. In IL10, rs1518111 was nominally associated with BD before and after adjustment for population stratification (odds ratio [OR] for T allele 1.20, 95% confidence interval [95% CI] 1.02-1.40, unadjusted P [P(unadj) ] = 2.53 × 10(-2) ; adjusted P [P(adj) ] = 1.43 × 10(-2) ), and rs1554286 demonstrated a trend toward association (P(unadj) = 6.14 × 10(-2) ; P(adj) = 3.21 × 10(-2) ). Six SNPs in IL23R-IL12RB2 were found to be associated with BD after Bonferroni correction for multiple testing, the most significant of which were rs17375018 (OR for G allele 1.51, 95% CI 1.27-1.78, P(unadj) = 1.93 × 10(-6) ), rs7517847 (OR for T allele 1.48, 95% CI 1.26-1.74, P(unadj) = 1.23 × 10(-6) ), and rs924080 (OR for T allele 1.29, 95% CI 1.20-1.39, P = 1.78 × 10(-5) ). SNPs rs10489629, rs1343151, and rs1495965 were also significantly associated with BD in all tests performed. Results of meta-analyses of our data combined with data from other populations further confirmed the role of rs1518111, rs17375018, rs7517847, and rs924080 in the risk of BD, but no epistatic interactions between IL10 and IL23R-IL12RB2 were detected. Results of imputation analysis highlighted the importance of IL23R regulatory regions in the susceptibility to BD. CONCLUSION: These findings independently confirm, extend, and refine the association of BD with IL10 and IL23R-IL12RB2. These associations warrant further validation and investigation in patients with BD, as they may have implications for the development of novel therapies (e.g., immunosuppressive therapy targeted at IL-23p19).

Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation.

Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, ß-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.