Genome-Wide Search for SNP Interactions in GWAS Data: Algorithm, Feasibility, Replication Using Schizophrenia Datasets.

In this study, we looked for potential gene-gene interaction in susceptibility to schizophrenia by an exhaustive searching for SNP-SNP interactions in 3 GWAS datasets (phs000021:phg000013, phs000021:phg000014, phs000167) using our recently published algorithm. The search space for SNP-SNP interaction was confined to 8 biologically plausible ways of interaction under dominant-dominant or recessive-recessive modes. First, we performed our search of all pair-wise combination of 729,454 SNPs after filtering by SNP genotype quality. All possible pairwise interactions of any 2 SNPs (5 × 1011) were exhausted to search for significant interaction which was defined by p-value of chi-square tests. Nine out the top 10 interactions, protein coding genes were partnered with non-coding RNA (ncRNA) which suggested a new alternative insight into interaction biology other than the frequently sought-after protein-protein interaction. Therefore, we extended to look for replication among the top 10,000 interaction SNP pairs and high proportion of concurrent genes forming the interaction pairs were found. The results indicated that an enrichment of signals over noise was present in the top 10,000 interactions. Then, replications of SNP-SNP interaction were confirmed for 14 SNPs-pairs in both replication datasets. Biological insight was highlighted by a potential binding between FHIT (protein coding gene) and LINC00969 (lncRNA) which showed a replicable interaction between their SNPs. Both of them were reported to have expression in brain. Our study represented an early attempt of exhaustive interaction analysis of GWAS data which also yield replicated interaction and new insight into understanding of genetic interaction in schizophrenia.

Reference intervals of spot urine copper excretion in preschool children and potential application in pre-symptomatic screening of Wilson disease.

The objectives were to determine the reference intervals of spot urine copper excretion indexes in pre-school children and to evaluate their utility in screening for Wilson disease (WD). With spot urine collected from a control sample of preschool children (aged 3-7 years, n=153), the reference intervals of spot urine copper excretion indexes and their biological variation were defined. In order to investigate their utility performance in screening for WD in this age group, multiple spot urine samples from six WD patients who were diagnosed at presymptomatic stage were also analysed and compared. Cut-off values useful for detection of WD were defined by receiver operator curve (ROC) analysis. Biological (inter-individual) variation of spot urine copper indexes expressed as coefficient of variation (CVg) were around 60% at this age group, which was moderate and similar to other clinically useful urine tests, such as urine albumin excretion ratio. Spot urine copper excretion strongly correlated with both urine creatinine and osmolality. Linear regression against both creatinine and osmolality showed that ∼94% of data points in healthy preschool children fell within the prediction interval, suggesting that both were useful normalisation factors. ROC showed that copper to osmolality ratio was the best index with an area under curve (AUC) greater than 0.98. Cut-off values of 0.5 µmol/L, 0.1 µmol/mmol and 0.00085 µmol/mOsmol (32 µg/L, 56 µg/g creatinine and 0.054 µg/mOsmol, respectively, in conventional units) for spot urine copper concentration, copper to creatinine ratio and copper to osmolality ratio, respectively, have potential application in the differentiation of WD patients. Based on the data, a new WD screening strategy targeting preschool children is proposed. Application of a bivariate screening strategy using spot urine copper concentration and urine osmolality may be useful in a population-wide screening program for WD among preschool children.

Transcriptome evidence reveals enhanced autophagy-lysosomal function in centenarians.

Centenarians (CENs) are excellent subjects to study the mechanisms of human longevity and healthy aging. Here, we analyzed the transcriptomes of 76 centenarians, 54 centenarian-children, and 41 spouses of centenarian-children by RNA sequencing and found that, among the significantly differentially expressed genes (SDEGs) exhibited by CENs, the autophagy-lysosomal pathway is significantly up-regulated. Overexpression of several genes from this pathway, CTSB, ATP6V0C, ATG4D, and WIPI1, could promote autophagy and delay senescence in cultured IMR-90 cells, while overexpression of the Drosophila homolog of WIPI1, Atg18a, extended the life span in transgenic flies. Interestingly, the enhanced autophagy-lysosomal activity could be partially passed on to their offspring, as manifested by their higher levels of both autophagy-encoding genes and serum beclin 1 (BECN1). In light of the normal age-related decline of autophagy-lysosomal functions, these findings provide a compelling explanation for achieving longevity in, at least, female CENs, given the gender bias in our collected samples, and suggest that the enhanced waste-cleaning activity via autophagy may serve as a conserved mechanism to prolong the life span from Drosophila to humans.

VCNet: vector-based gene co-expression network construction and its application to RNA-seq data.

MOTIVATION: Building gene co-expression network (GCN) from gene expression data is an important field of bioinformatic research. Nowadays, RNA-seq data provides high dimensional information to quantify gene expressions in term of read counts for individual exons of genes. Such an increase in the dimension of expression data during the transition from microarray to RNA-seq era made many previous co-expression analysis algorithms based on simple univariate correlation no longer applicable. Recently, two vector-based methods, SpliceNet and RNASeqNet, have been proposed to build GCN. However, they failed to work when sample size is less than the number of exons. RESULTS: We develop an algorithm called VCNet to construct GCN from RNA-seq data to overcome this dimensional problem. VCNet performs a new statistical hypothesis test based on the correlation matrix of a gene-gene pair using the Frobenius norm. The asymptotic distribution of the new test is obtained under the null model. Simulation studies demonstrate that VCNet outperforms SpliceNet and RNASeqNet for detecting edges of GCN. We also apply VCNet to two expression datasets from TCGA database: the normal breast tissue and kidney tumour tissue, and the results show that the GCNs constructed by VCNet contain more biologically meaningful interactions than existing methods. CONCLUSION: VCNet is a useful tool to construct co-expression network. AVAILABILITY AND IMPLEMENTATION: VCNet is open source and freely available from https://github.com/wangzengmiao/VCNet under GNU LGPL v3. CONTACT: dengmh@pku.edu.cn or nelsontang@cuhk.edu.hk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MicroRNAs mediated targeting on the Yin-yang dynamics of DNA methylation in disease and development.

For decades, DNA methylation at the 5 position of cytosine (5mC) catalyzed by DNA methyltransferases (DNMTs) is a well-known epigenetic modification in mammalian genome, where it modulates chromatin remodeling and transcriptional silencing. The discovery of Ten-eleven translocation (TET) enzymes that oxidize 5mC to 5-hydroxymethylcytosine (5hmC) prompts a new era of DNA demethylation research. It is now established that in DNA demethylation pathway 5mC is first converted to 5-hydroxymethylcytosine (5hmC), then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) through TETs. Conversion to unmethylated cytosine (5C) is further facilitated by excision mechanism through thymine-DNA glycosylase (TDG) or base excision repair (BER) pathway. Our understanding of DNMTs and TETs on epigenetic dynamics of cytosine methylation has led to a completion of the methylation (Yin) - demethylation (Yang) cycle on epigenetic modifications on cytosine. However, the regulations on DNA demethylation pathway remain largely unknown. In this review, we provide the recent advances on epigenetic dynamics of DNA demethylation and its potential control from the prespective of small non-coding RNA-mediated regulation. Specifically, we will illustrate how microRNAs contribute to active DNA demethylation control in normal and disease development based on recent findings in stem cells and cancer. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Dynamic changes of DNA epigenetic marks in mouse oocytes during natural and accelerated aging.

Aging is a complex time-dependent biological process that takes place in every cell and organ, eventually leading to degenerative changes that affect normal biological functions. In the past decades, the number of older parents has increased significantly. While it is widely recognized that oocyte aging poses higher birth and reproductive risk, the exact molecular mechanisms remain largely elusive. DNA methylation of 5-cytosine (5mC) and histone modifications are among the key epigenetic mechanisms involved in critical developmental processes and have been linked to aging. However, the impact of oocyte aging on DNA demethylation pathways has not been examined. The recent discovery of Ten-Eleven-Translocation (TET) family proteins, thymine DNA glycosylase (TDG) and the demethylation intermediates 5hmC, 5fC and 5caC has provided novel clues to delineate the molecular mechanisms in DNA demethylation. In this study, we examined the cellular level of modified cytosines (5mC, 5hmC, 5fC and 5caC) and Tet/Tdg expression in oocytes obtained from natural and accelerated oocyte aging conditions. Here we show all the DNA demethylation marks are dynamically regulated in both aging conditions, which are associated with Tet3 over-expression and Tdg repression. Such an aberrant expression pattern was more profound in accelerated aging condition. The results suggest that DNA demethylation may be actively involved in oocyte aging and have implications for development of potential drug targets to rejuvenate aging oocytes. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Estrogen receptor α polymorphisms and the risk of cognitive decline: A 2-year follow-up study.

OBJECTIVE: The neuroprotective role of estrogen is supported by biochemical studies, but the results from clinical trials of estrogen replacement therapy on cognitive decline are controversial. One possible missing link might be the interindividual difference in estrogen receptor expression. In this study, the association of estrogen receptor α (ESR1) polymorphisms and cognitive decline was investigated. METHODS: Chinese older adults (n = 284) were recruited, and the cognitive profile was follow-up over 2-year period. Twenty ESR1 polymorphisms were investigated and correlated with the cognitive decline for the subjects. RESULTS: Significant association was found between ESR1 polymorphisms (rs9340799 [ESR1+351], rs1801132 [ESR1+975], rs6557171, rs9397456, and rs1884049) and subjects with no dementia (Clinical Dementia Rating, CDR 0) and very mild dementia (CDR 0.5). Several ESR1 polymorphisms were associated with cognitive decline as assessed by Chinese versions of Mini-Mental State Examination and Alzheimer Disease Association Scales-Cognitive Subscale. Different sets of ESR1 polymorphisms were associated with cognitive decline from CDR 0 to 0.5 and CDR 0.5 to 1. ESR1 polymorphisms (rs3853248, rs22334693 [ESR1+397], rs9340799 [ESR1+351], rs9397456, rs1801132 [ESR1+975], rs2179922, rs932477, and rs9341016) were associated with the deterioration of episodic memory among subjects with baseline CDR 0, indicating these polymorphisms might be markers for episodic memory decline at an earlier stage. CONCLUSION: This study showed association between ESR polymorphisms and cognitive decline or specific areas in cognitive profile. These findings might be useful in identifying individuals at risk for early intervention, and more research is required to elucidate the underlying mechanisms.

Genetic associations of type 2 diabetes with islet amyloid polypeptide processing and degrading pathways in asian populations.

Type 2 diabetes (T2D) is a complex disease characterized by beta cell dysfunctions. Islet amyloid polypeptide (IAPP) is highly conserved and co-secreted with insulin with over 40% of autopsy cases of T2D showing islet amyloid formation due to IAPP aggregation. Dysregulation in IAPP processing, stabilization and degradation can cause excessive oligomerization with beta cell toxicity. Previous studies examining genetic associations of pathways implicated in IAPP metabolism have yielded conflicting results due to small sample size, insufficient interrogation of gene structure and gene-gene interactions. In this multi-staged study, we screened 89 tag single nucleotide polymorphisms (SNPs) in 6 candidate genes implicated in IAPP metabolism and tested for independent and joint associations with T2D and beta cell dysfunctions. Positive signals in the stage-1 were confirmed by de novo and in silico analysis in a multi-centre unrelated case-control cohort. We examined the association of significant SNPs with quantitative traits in a subset of controls and performed bioinformatics and relevant functional analyses. Amongst the tag SNPs, rs1583645 in carboxypeptidase E (CPE) and rs6583813 in insulin degrading enzyme (IDE) were associated with 1.09 to 1.28 fold increased risk of T2D (P Meta = 9.4×10(-3) and 0.02 respectively) in a meta-analysis of East Asians. Using genetic risk scores (GRS) with each risk variant scoring 1, subjects with GRS≥3 (8.2% of the cohort) had 56% higher risk of T2D than those with GRS = 0 (P = 0.01). In a subcohort of control subjects, plasma IAPP increased and beta cell function index declined with GRS (P = 0.008 and 0.03 respectively). Bioinformatics and functional analyses of CPE rs1583645 predicted regulatory elements for chromatin modification and transcription factors, suggesting differential DNA-protein interactions and gene expression. Taken together, these results support the importance of dysregulation of IAPP metabolism in T2D in East Asians.

A single nucleotide polymorphism in microRNA-146a is associated with the risk for nasopharyngeal carcinoma.

A common GC polymorphism within miRNA-146a precursor region (rs2910164) has been associated with the risk of various cancers despite the underlying mechanism is unclear. In the current study, we aimed to examine the role of rs2910164 in the pathogenesis and predisposition to nasopharyngeal carcinoma (NPC). The GC polymorphism in 233 NPC patients, 173 matched controls and 3613 healthy elderly subjects in our locality were first determined using melting temperature (T(m))-shift allele-specific genotyping method. Results in our case-control study indicated that CC genotype was associated with the risk effect of NPC (adjusted odds ratio of GC + GG vs. CC, 0.49; 95% confidence interval, 0.35-0.69; P < 0.0001). Using real-time polymerase chain reaction (PCR) assay, we subsequently revealed that expressions of both miR-146a and its passenger strand (miR-146a*C or miR-146a*G) were increased in NPC samples (P < 0.001), albeit expression of miR-146a was not linked to the genotype. Furthermore, miR-146a*C in NPC was significantly increased in CC genotype (CC vs. GC, P = 0.038). Finally, we demonstrated by co-immunoprecipitation and luciferase reporter assays that all three miR-146a precursor-derived mature miRNAs interacted with Argonaute2 (Ago2) protein complex and could function as gene silencers. Taken together, our results showed that the variant C in rs2910164 was associated with the predisposition of NPC in Chinese population. This polymorphism may influence the risk of NPC by producing active mature miR-146a*C that regulate distinct set of target genes. These findings may enrich our understanding of how miRNA single nucleotide polymorphism affect NPC pathogenesis, and may have potential implications to improve NPC treatment in the future.

Correlation of telomere length shortening with TP53 somatic mutations, polymorphisms and allelic loss in breast tumors and esophageal cancer.

Genomic instability caused by telomere erosion is an important mechanism of tumorigenesis. p53 plays a key role in cellular senescence and/or apoptosis associated with telomere erosion which positions p53 as a guard against tumorigenesis. The present study was undertaken to investigate the potential interactions between p53 functional mutations, polymorphisms, allelic loss and telomere erosion in 126 breast tumor patients and 68 esophageal cancer patients. Telomere length (TL) was measured by real-time quantitative PCR. Somatic mutations, polymorphisms and allelic loss in the TP53 gene were detected by direct sequencing of both tumor and normal tissue samples. Our results showed that telomeres were significantly shorter in tumors with somatic p53 mutations compared with tumors with wild-type p53 in both breast tumors (P=0.007) and esophageal cancer (P=0.001). Telomeres of patients with minor genotype CC of rs12951053 and GG of rs1042522 were significantly shorter compared to patients with other genotypes of this single nucleotide polymorphism in esophageal cancer tissue. Furthermore, TP53 allelic loss was detected and significantly associated with somatic mutations in both types of tumor tissues. These findings suggest that somatic p53 mutations, rs12951053 genotype CC and rs1042522 genotype GG contribute to erosion of telomeres, and TP53 allelic loss may be one of the representations of chromosomal instability caused by telomere erosion combined with somatic p53 mutations. These results support that the TP53 gene has a strong interaction with TL erosion in tumorigenesis.

Polarized secretion of interleukin (IL)-6 and IL-8 by human airway epithelia 16HBE14o- cells in response to cationic polypeptide challenge.

BACKGROUND: The airway epithelium participates in asthmatic inflammation in many ways. Target cells of the epithelium can respond to a variety of inflammatory mediators and cytokines. Damage to the surface epithelium occurs following the secretion of eosinophil-derived, highly toxic cationic proteins. Moreover, the surface epithelium itself is responsible for the synthesis and release of cytokines that cause the selective recruitment, retention, and accumulation of various inflammatory cells. To mimic the damage seen during asthmatic inflammation, the bronchial epithelium can be challenged with highly charged cationic polypeptides such as poly-L-arginine. METHODOLOGY/PRINCIPAL FINDINGS: In this study, human bronchial epithelial cells, 16HBE14o- cells, were "chemically injured" by exposing them to poly-l-arginine as a surrogate of the eosinophil cationic protein. Cytokine antibody array data showed that seven inflammatory mediators were elevated out of the 40 tested, including marked elevation in interleukin (IL)-6 and IL-8 secretion. IL-6 and IL-8 mRNA expression levels were elevated as measured with real-time PCR. Cell culture supernatants from apical and basolateral compartments were collected, and the IL-6 and IL-8 production was quantified with ELISA. IL-6 and IL-8 secretion by 16HBE14o- epithelia into the apical compartment was significantly higher than that from the basolateral compartment. Using specific inhibitors, the production of IL-6 and IL-8 was found to be dependent on p38 MAPK, ERK1/2 MAPK, and NF-kappaB pathways. CONCLUSIONS/SIGNIFICANCE: The results clearly demonstrate that damage to the bronchial epithelia by poly-L-arginine stimulates polarized IL-6 and IL-8 secretion. This apically directed secretion of cytokines may play an important role in orchestrating epithelial cell responses to inflammation.

A relook into the association of the estrogen receptor [alpha] gene (PvuII, XbaI) and adolescent idiopathic scoliosis: a study of 540 Chinese cases.

STUDY DESIGN: A genetic association study of estrogen receptor-[alpha] gene (ESR1) with adolescent idiopathic scoliosis (AIS) in Chinese. OBJECTIVES: To investigate whether: 1) PvuII and XbaI polymorphisms in ESR1 are predisposition factor for AIS and 2) these polymorphisms correlate with the severity of curvature in AIS. SUMMARY OF BACKGROUND DATA: A common single nucleotide polymorphism (SNP) in ESR1 (XbaI) was found to be associated with curve severity in Japanese AIS patients recently. The role of ESR1 as a predisposition gene using a case-control design in other ethnic groups is required to confirm the previous associations. METHODS: A total of 540 Chinese AIS girls with Cobb angle above 20 degrees were recruited as cases together with 260 healthy controls. The effect of ESR1 SNPs on severity of scoliosis was analyzed in a subgroup of AIS patients (n = 364) followed up until skeletal maturity with the maximum Cobb angle recorded. Two SNPs in ESR1 were genotyped by PCR-restriction fragment length polymorphism in all subjects. RESULTS: The allelic frequency of X allele was 23% in both case and control groups. The P allele was found at allelic frequency of 40% and 36% in the case and control groups, respectively. No association between the two ESR1 SNPs and the occurrence of AIS by both genotype and haplotype analysis could be established, suggesting that both SNPs were not predisposition alleles for AIS. AIS patients with different genotypes showed no difference in the maximum Cobb angle. No association was found between the genotype and anthropometric measurements in AIS patients. CONCLUSION: The previously reported association with curve severity could not be replicated in our large series of Chinese AIS patients. The current study also did not show any association of the 2 SNPs with increased risk of having AIS.

Renal outcome in type 2 diabetic patients with or without coexisting nondiabetic nephropathies.

OBJECTIVE: To determine the risk factors for adverse renal outcome in type 2 diabetic patients who underwent renal biopsy and were followed-up longitudinally. RESEARCH DESIGN AND METHODS: We examined 68 consecutive patients with type 2 diabetes during the period of 1985-1999 who underwent renal biopsy for proteinuria > or =1 g/day, renal involvement (proteinuria or renal impairment) at the absence of retinopathy, renal involvement with duration of diabetes < 5 years, or unexplained hematuria of glomerular origin. Their clinical features and underlying renal lesion were correlated with the renal outcome after longitudinal follow-up. Three groups of patients were defined based on their renal pathology: group I consisted of 24 patients (35%) with diabetic glomerulosclerosis (DGS) alone, group II consisted of 13 patients (19%) with nondiabetic nephropathy (NDN) superimposed on DGS, and group III consisted of 31 patients (46%) with NDN alone without evidence of DGS. RESULTS: After a mean follow-up of 123 months from the diagnosis of type 2 diabetes (74 months from the time of renal biopsy), univariate analysis showed that risk factors for reaching end-stage renal disease (requiring maintenance dialysis, or a serum creatinine [SCr] > or =700 micromol/l) included proteinuria > or = 2g/day (P = 0.0087), SCr >120 micromol/l (P = 0.0005), presence of retinopathy (P < 0.00001) at the time of biopsy, and biopsy showing DGS (groups I and II) (P = 0.035). On multivariate analysis, retinopathy was the only independent variable correlated with end-stage renal failure. This study also showed that the association of hematuria or proteinuria with the absence of retinopathy constitutes the strongest indication for a nondiabetic lesion (positive predictive values of 94%). CONCLUSIONS: Patients with type 2 diabetes undergoing renal biopsy constitute a heterogeneous group by their clinical presentations and underlying pathology, but longitudinal studies on the renal outcome of these patients remain limited. Our study showed that renal biopsy is indicated in selective diabetic patients because of potentially treatable nephropathy and of a better prognosis than DGS.

Primary IgA nephropathy with low histologic grade and disease progression: is there a "point of no return"?

Histologic low-grade chronic renal lesions in 144 adults with primary immunoglobulin A (IgA) nephropathy were correlated with clinical parameters of disease progression over a median follow-up of 93 months. Using chronicity-based histologic grading, 50, 59, and 35 patients were glomerular grade (GG) 1a, GG1b, and GG2; 83 and 61 patients were tubulointerstitial grade (TIG) 1 and TIG2; and 25 patients had hyaline arteriolosclerosis. On follow-up, GG and TIG were predictive of disease progression by impairment of renal function, development of hypertension, and significant proteinuria (>1 g/d). Hyaline arteriolosclerosis correlated only with the development of hypertension. Histologic lesions GG1a or TIG1 predicted a significant low risk for disease progression compared with other renal lesions, regardless of the renal manifestation at the time of biopsy. Combined GG1a, TIG1, and isolated hematuria at the time of biopsy enhanced the sensitivity to determine early IgA nephropathy and to define a nonearly cohort with a higher risk of disease progression appropriate for recruitment into clinical therapeutic trials within realistic time frames. The significant risk of progression in other low-grade lesions, such as GG1b or TIG2, suggests that the point of no return in IgA nephropathy may occur much earlier than perceived and that delayed biopsy in these patients no longer may be justified.