Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

Background

Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.

Methodology/Principal Findings

We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.

Conclusions

These results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.     

Determination of Angptl4 mRNA as a Diagnostic Marker of Primary and Metastatic Clear Cell Renal-Cell Carcinoma

Background

We have previously shown that angiopoietin-like 4 (angptl4) mRNA, a hypoxia-inducible gene, is highly expressed in clear cell renal-cell carcinoma (ccRCC), the most common subtype of RCC for which no specific marker is available. We here investigated whether angptl4 mRNA 1) could be a useful diagnostic and/or prognostic marker of ccRCC in a large and comprehensive retrospective series, 2) induction is dependent on the VHL status of tumors.

Methodology/Principal Findings

Using in situ hybridization, we report that angptl4 mRNA is expressed in 100% of both sporadic (n = 102) and inherited (n = 6) primary ccRCCs, without any statistical association with nuclear grade (p = 0.39), tumor size (p = 0.09), stage grouping (p = 0.17), progression-free survival (p = 0.94), and overall survival (p = 0.80). Angptl4 mRNA was also expressed in 26 (87%) of 30 secondary ccRCCs but neither in any other secondary RCCs (n = 7). In contrast, angptl4 mRNA was neither expressed in 94% non-ccRCC renal tumors (papillary RCCs (n = 46), chromophobe RCCs (n = 28), and oncocytomas (n = 9)), nor in non-renal clear cell carcinomas (n = 39). Angptl4 expression was also examined in tumors associated (n = 23) or not associated (n = 66) with VHL disease. 40 (98%) hemangioblastomas expressed angptl4 whereas all pheochromocytomas (n = 23) and pancreatic tumors (n = 25) were angptl4-negative, whatever their VHL status.

Conclusions/Significance

Angptl4 mRNA expression was highly associated with ccRCC (p = 1.5 10⁻⁴⁹, Chi square test) allowing to define its expression as a diagnosis marker for primary ccRCC. Moreover, angptl4 mRNA allows to discriminate the renal origin of metastases of clear-cell carcinomas arising from various organs. Finally, inactivation of VHL gene is neither necessary nor sufficient for angptl4 mRNA induction.     

miR-141 and miR-200c as Markers of Overall Survival in Early Stage Non-Small Cell Lung Cancer Adenocarcinoma

Background

Several treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established.

Methods

miRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44.

Results

High miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma – but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001).

Conclusion

High miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis.     

The PlA1/A2 Polymorphism of Glycoprotein IIIa as a Risk Factor for Stroke: A Systematic Review and Meta-Analysis

Background

The PlA1/A2 polymorphism of glycoprotein IIIa (GPIIIa) has been reported to be associated with risk of stroke in some studies, although other studies suggest no such association. This meta-analysis and systematic review was conducted to investigate the hypothesis that carriage of the PlA2 allele is a risk factor for stroke.

Methods

Electronic databases (MEDLINE and EMBASE) were searched for all articles evaluating carriage of the PlA2 allele and the incidence of stroke. Pooled odds ratios (ORs) were calculated using fixed-effect and random-effect models.

Findings

A total of 35 articles were eligible for inclusion, of which 25 studies were suitable for statistical analysis. For carriage of the PlA2 allele, OR 1.12 (n = 11,873; 95% CI = 1.03–1.22; p = 0.011) was observed for the incidence of stroke in adults, with subgroup analyses identifying the association driven by stroke of an ischaemic (n = 10,494; OR = 1.15, 95% CI = 1.05–1.27; p = 0.003) but not haemorrhagic aetiology (n = 2,470; OR = 0.90, 95% CI = 0.71–1.14; p = 0.398). This association with ischaemic stroke was strongest in individuals homozygous for the PlA2 allele compared to those homozygous for wild-type PlA1 (n = 5,906; OR = 1.74, 95% CI = 1.34–2.26; p<0.001). Subgroup analysis of ischaemic stroke subtypes revealed an increased association with stroke of cardioembolic (n = 1,271; OR 1.56, 95% CI 1.14–2.12; p = 0.005) and large vessel (n = 1,394; OR = 1.76, 95% CI 1.34–2.31; p<0.001) aetiology, but not those of small vessel origin (n = 1,356; OR = 0.99, 95% CI 0.74–1.33; p = 0.950). Egger''s regression test suggested a low probability of publication bias for all analyses (p>0.05).

Conclusions

The totality of published data supports the hypothesis that carriage of the PlA2 polymorphism of GPIIIa is a risk factor for ischaemic strokes, and specifically those of cardioembolic and large vessel origin.     

Role of HLA-DP Polymorphisms on Chronicity and Disease Activity of Hepatitis B Infection in Southern Chinese

Background and Aims

The association between HLA-DP single nucleotide polymorphisms (SNPs) and chronic hepatitis B virus (HBV) infection varies between different populations. We aimed to study the association between HLA-DP SNPs and HBV infection and disease activity in the Chinese population of Hong Kong.

Methods

We genotyped SNPs rs3077 (near HLA-DPA1) and rs9277378 and rs3128917 (both near HLA-DPB1) in 500 HBV carriers (hepatitis B surface antigen [HBsAg]-positive), 245 non-HBV infected controls (HBsAg- and antibody to hepatitis B core protein [anti-HBc]-negative), and 259 subjects with natural HBV clearance (HBsAg-negative, anti-HBc-positive). Inactive HBV carriers state was defined by HBV DNA levels <2,000 IU/ml and persistently normal alanine aminotransferase level for least 12 months.

Results

Compared to the non-HBV infected subjects, the HBV carriers had a significantly lower frequency of the rs3077 T allele (p = 0.0040), rs9277378 A allele (p = 0.0068) and a trend for lower frequency of rs3128917 T allele (p = 0.054). These alleles were associated with an increased chance of HBV clearance (rs3077: OR = 1.41, p = 0.0083; rs9277378: OR = 1.61, p = 0.00011; rs3128917: OR = 1.54, p = 0.00017). Significant associations between HLA-DP genotypes and HBV clearance were also found under different genetic models. Haplotype TAT was associated with an increased chance of HBV clearance (OR = 1.64, p = 0.0013). No association was found between these SNPs and HBV disease activity.

Conclusion

HLA-DP SNPs rs3077, rs9277378 and rs3128917 were associated with chronicity of HBV disease in the Chinese. Further studies are required to determine whether these SNPs influence the disease endemicity in different ethnic populations.     

Activation of the PI3K/mTOR/AKT Pathway and Survival in Solid Tumors: Systematic Review and Meta-Analysis

Background

Aberrations in the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/AKT pathway are common in solid tumors. Numerous drugs have been developed to target different components of this pathway. However the prognostic value of these aberrations is unclear.

Methods

PubMed was searched for studies evaluating the association between activation of the PI3K/mTOR/AKT pathway (defined as PI3K mutation [PIK3CA], lack of phosphatase and tensin homolog [PTEN] expression by immunohistochemistry or western-blot or increased expression/activation of downstream components of the pathway by immunohistochemistry) with overall survival (OS) in solid tumors. Published data were extracted and computed into odds ratios (OR) for death at 5 years. Data were pooled using the Mantel-Haenszel random-effect model.

Results

Analysis included 17 studies. Activation of the PI3K/mTOR/AKT pathway was associated with significantly worse 5-year survival (OR:2.12, 95% confidence intervals 1.42–3.16, p<0.001). Loss of PTEN expression and increased expression/activation of downstream components were associated with worse survival. No association between PIK3CA mutations and survival was observed. Differences between methods for assessing activation of the PI3K/mTOR/AKT pathway were statistically significant (p = 0.04). There was no difference in the effect of up-regulation of the pathway on survival between different cancer sites (p = 0.13).

Conclusion

Activation of the PI3K/AKT/mTOR pathway, especially if measured by loss of PTEN expression or increased expression/activation of downstream components is associated with poor survival. PIK3CA mutational status is not associated with adverse outcome, challenging its value as a biomarker of patient outcome or as a stratification factor for patients treated with agents acting on the PI3K/AKT/mTOR pathway.     

Culture-Independent Evaluation of the Appendix and Rectum Microbiomes in Children with and without Appendicitis

Purpose

The function of the appendix is largely unknown, but its microbiota likely contributes to function. Alterations in microbiota may contribute to appendicitis, but conventional culture studies have not yielded conclusive information. We conducted a pilot, culture-independent 16S rRNA-based microbiota study of paired appendix and rectal samples.

Methods

We collected appendix and rectal swabs from 21 children undergoing appendectomy, six with normal appendices and fifteen with appendicitis (nine perforated). After DNA extraction, we amplified and sequenced 16S rRNA genes and analyzed sequences using CLoVR. We identified organisms differing in relative abundance using ANOVA (p<0.05) by location (appendix vs. rectum), disease (appendicitis vs. normal), and disease severity (perforated vs. non-perforated).

Results

We identified 290 taxa in the study''s samples. Three taxa were significantly increased in normal appendices vs. normal rectal samples: Fusibacter (p = 0.009), Selenomonas (p = 0.026), and Peptostreptococcus (p = 0.049). Five taxa were increased in abundance in normal vs. diseased appendices: Paenibacillaceae (p = 0.005), Acidobacteriaceae GP4 (p = 0.019), Pseudonocardinae (p = 0.019), Bergeyella (p = 0.019) and Rhizobium (p = 0.045). Twelve taxa were increased in the appendices of appendicitis patients vs. normal appendix: Peptostreptococcus (p = 0.0003), Bilophila (p = 0.0004), Bulleidia (p = 0.012), Fusobacterium (p = 0.018), Parvimonas (p = 0.003), Mogibacterium (p = 0.012), Aminobacterium (p = 0.019), Proteus (p = 0.028), Actinomycineae (p = 0.028), Anaerovorax (p = 0.041), Anaerofilum (p = 0.045), Porphyromonas (p = 0.010). Five taxa were increased in appendices in patients with perforated vs. nonperforated appendicitis: Bulleidia (p = 0.004), Fusibacter (p = 0.005), Prevotella (p = 0.021), Porphyromonas (p = 0.030), Dialister (p = 0.035). Three taxa were increased in rectum samples of patients with appendicitis compared to the normal patients: Bulleidia (p = 0.034), Dialister (p = 0.003), and Porphyromonas (p = 0.026).

Conclusion

Specific taxa are more abundant in normal appendices compared to the rectum, suggesting that a distinctive appendix microbiota exists. Taxa with altered abundance in diseased and severely diseased (perforated) samples may contribute to appendicitis pathogenesis, and may provide microbial signatures in the rectum useful for guiding both treatment and diagnosis of appendicitis.     

The Associations between Anthropometric Indices and Obstructive Sleep Apnea in a Korean Population

Background

Obesity is a major risk factor for the development of obstructive sleep apnea (OSA). Although clinical and epidemiological studies have shown that OSA and obesity are strongly associated, few Asian studies have examined the associations between anthropometric obesity indices and OSA, especially in the Korean population. The purpose of this study was to evaluate the influence of anthropometric obesity indices on OSA in a Korean population.

Methods

Anthropometric indices, including neck circumference, waist circumference, and body mass index, were assessed in 383 consecutive subjects with suspected OSA.

Results

Of the 383 subjects assessed, 316 (82.5%) were diagnosed with OSA. Neck circumference (r = 0.518), waist circumference (r = 0.570), and body mass index (r = 0.512) were correlated with the apnea-hypopnea index (p<0.001, for all). After adjusting for age, sex, alcohol consumption, and smoking, a logistic regression model showed that neck circumference [odds ratio (OR), 1.414; p<0.001)], waist circumference (OR, 1.114; p<0.001), and body mass index (OR, 1.364; p<0.001) were associated with OSA. The linear regression model showed that neck circumference (β = 3.748, p<0.001), waist circumference (β = 1.272, p<0.001), and body mass index (β = 3.082, p<0.001) were associated with apnea-hypopnea index. The cut-off values for predicting OSA were determined as 34.5 cm for neck circumference, 76.5 cm for waist circumference, and 23.05 kg/m² for body mass index for females, and 38.75 cm for neck circumference, 88.5 cm for waist circumference, and 24.95 kg/m² for body mass index for males.

Conclusion

Increased anthropometric indices were significantly associated with the presence and severity of OSA in a Korean population. In addition, this study demonstrated the cut-off values for body mass index, waist circumference, and neck circumference for increased OSA risk.     

Pharmacogenetics of Efficacy and Safety of HCV Treatment in HCV-HIV Coinfected Patients: Significant Associations with IL28B and SOCS3 Gene Variants

Background and Aims

This was a safety and efficacy pharmacogenetic study of a previously performed randomized trial which compared the effectiveness of treatment of hepatitis C virus infection with pegylated interferon alpha (pegIFNα) 2a vs. 2b, both with ribavirin, for 48 weeks, in HCV-HIV coinfected patients.

Methods

The study groups were made of 99 patients (efficacy pharmacogenetic substudy) and of 114 patients (safety pharmacogenetic substudy). Polymorphisms in the following candidate genes IL28B, IL6, IL10, TNFα, IFNγ, CCL5, MxA, OAS1, SOCS3, CTLA4 and ITPA were assessed. Genotyping was carried out using Sequenom iPLEX-Gold, a single-base extension polymerase chain reaction. Efficacy end-points assessed were: rapid, early and sustained virological response (RVR, EVR and SVR, respectively). Safety end-points assessed were: anemia, neutropenia, thrombocytopenia, flu-like syndrome, gastrointestinal disturbances and depression. Chi square test, Student''s T test, Mann-Whitney U test and logistic regression were used for statistic analyses.

Results

As efficacy is concerned, IL28B and CTLA4 gene polymorphisms were associated with RVR (p<0.05 for both comparisons). Nevertheless, only polymorphism in the IL28B gene was associated with SVR (p = 0.004). In the multivariate analysis, the only gene independently associated with SVR was IL28B (OR 2.61, 95%CI 1.2–5.6, p = 0.01). With respect to safety, there were no significant associations between flu-like syndrome or depression and the genetic variants studied. Gastrointestinal disturbances were associated with ITPA gene polymorphism (p = 0.04). Anemia was associated with OAS1 and CTLA4 gene polymorphisms (p = 0.049 and p = 0.045, respectively), neutropenia and thromobocytopenia were associated with SOCS3 gene polymorphism (p = 0.02 and p = 0.002, respectively). In the multivariate analysis, the associations of the SOCS3 gene polymorphism with neutropenia (OR 0.26, 95%CI 0.09–0.75, p = 0.01) and thrombocytopenia (OR 0.07, 95%CI 0.008–0.57, p = 0.01) remained significant.

Conclusions

In HCV-HIV coinfected patients treated with PegIFNα and ribavirin, SVR is associated with IL28B rs8099917 polymorphism. HCV treatment-induced neutropenia and thrombocytopenia are associated with SOCS3 rs4969170 polymorphism.     

High-Density SNP Screening of the Major Histocompatibility Complex in Systemic Lupus Erythematosus Demonstrates Strong Evidence for Independent Susceptibility Regions

A substantial genetic contribution to systemic lupus erythematosus (SLE) risk is conferred by major histocompatibility complex (MHC) gene(s) on chromosome 6p21. Previous studies in SLE have lacked statistical power and genetic resolution to fully define MHC influences. We characterized 1,610 Caucasian SLE cases and 1,470 parents for 1,974 MHC SNPs, the highly polymorphic HLA-DRB1 locus, and a panel of ancestry informative markers. Single-marker analyses revealed strong signals for SNPs within several MHC regions, as well as with HLA-DRB1 (global p = 9.99×10⁻¹⁶). The most strongly associated DRB1 alleles were: *0301 (odds ratio, OR = 2.21, p = 2.53×10⁻¹²), *1401 (OR = 0.50, p = 0.0002), and *1501 (OR = 1.39, p = 0.0032). The MHC region SNP demonstrating the strongest evidence of association with SLE was rs3117103, with OR = 2.44 and p = 2.80×10⁻¹³. Conditional haplotype and stepwise logistic regression analyses identified strong evidence for association between SLE and the extended class I, class I, class III, class II, and the extended class II MHC regions. Sequential removal of SLE–associated DRB1 haplotypes revealed independent effects due to variation within OR2H2 (extended class I, rs362521, p = 0.006), CREBL1 (class III, rs8283, p = 0.01), and DQB2 (class II, rs7769979, p = 0.003, and rs10947345, p = 0.0004). Further, conditional haplotype analyses demonstrated that variation within MICB (class I, rs3828903, p = 0.006) also contributes to SLE risk independent of HLA-DRB1*0301. Our results for the first time delineate with high resolution several MHC regions with independent contributions to SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation.     

Proximal Tubule Dysfunction Is Associated with Podocyte Damage Biomarkers Nephrin and Vascular Endothelial Growth Factor in Type 2 Diabetes Mellitus Patients: A Cross-Sectional Study

Background

There is an ongoing debate as to whether early diabetic nephropathy in Type 2 diabetes mellitus may be attributed to the glomerulus or to the proximal tubule. Urinary excretion of nephrin and vascular endothelial growth factor may increase even in the normoalbuminuria stage. In the course of diabetic nephropathy, the proximal tubule may be involved in the uptake of urinary nephrin and vascular endothelial growth factor.

Materials and Methods

Two groups of consecutive Type 2 diabetes mellitus outpatients (38 normo-, 32 microalbuminuric) and 21 healthy subjects were enrolled in a cross-sectional study and evaluated concerning the relation of proximal tubule dysfunction with the podocyte biomarkers excretion, assessed by ELISA methods. The impact of advanced glycation end-products on this relation was also queried.

Results

Urinary alpha₁-microglobulin and kidney injury molecule-1 correlated with urinary albumin:creatinine ratio (R² = 0.269; p<0.001; R² = 0.125; p<0.001), nephrinuria (R² = 0.529; p<0.001; R² = 0.203; p<0.001), urinary vascular endothelial growth factor (R² = 0.709; p<0.001; R² = 0.360; p<0.001), urinary advanced glycation end-products (R² = 0.578; p<0.001; R² = 0.405; p<0.001), serum cystatin C (R² = 0.130; p<0.001; R² = 0.128; p<0.001), and glomerular filtration rate (R² = 0.167; p<0.001; R² = 0.166; p<0.001); nephrinuria and urinary vascular endothelial growth factor correlated with urinary albumin:creatinine ratio (R² = 0.498; p<0.001; R² = 0.227; p<0.001), urinary advanced glycation end-products (R² = 0.251; p<0.001; R² = 0.308; p<0.001), serum cystatin C (R² = 0.157; p<0.001; R² = 0.226; p<0.001), and glomerular filtration rate (R² = 0.087; p = 0.007; R² = 0.218; p<0.001).

Conclusions

In Type 2 diabetes mellitus there is an association of proximal tubule dysfunction with podocyte damage biomarkers, even in the normoalbuminuria stage. This observation suggests a potential role of the proximal tubule in urinary nephrin and urinary vascular endothelial growth factor processing in early diabetic nephropathy, a fact which could be related to advanced glycation end-products intervention. Podocyte damage and proximal tubule dysfunction biomarkers could be validated as a practical approach to the diagnosis of early diabetic nephropathy by further studies on larger cohorts.     

Association of Genetic Variants with Primary Angle Closure Glaucoma in Two Different Populations

Purpose

A recent large genome-wide association study (GWAS) identified multiple variants associated with primary angle-closure glaucoma (PACG). The present study investigated the role of these variants in two cohorts with PACG recruited from Australia and Nepal.

Method

Patients with PACG and appropriate controls were recruited from eye clinics in Australia (n = 232 cases and n = 288 controls) and Nepal (n = 106 cases and 204 controls). Single nucleotide polymorphisms (SNPs) rs3753841 (COL11A1), rs1015213 (located between PCMTD1 and ST18), rs11024102 (PLEKHA7), and rs3788317 (TXNRD2) were selected and genotyped on the Sequenom. Analyses were conducted using PLINK and METAL.

Results

After adjustment for age and sex, SNP rs3753841 was found to be significantly associated with PACG in the Australian cohort (p = 0.017; OR = 1.34). SNPs rs1015213 (p = 0.014; OR 2.35) and rs11024102 (p = 0.039; OR 1.43) were significantly associated with the disease development in the Nepalese cohort. None of these SNPs survived Bonferroni correction (p = 0.05/4 = 0.013). However, in the combined analysis, of both cohorts, rs3753841 and rs1015213 showed significant association with p-values of 0.009 and 0.004, respectively both surviving Bonferroni correction. SNP rs11024102 showed suggestive association with PACG (p-value 0.035) and no association was found with rs3788317.

Conclusion

The present results support the initial GWAS findings, and confirm the SNP’s contribution to PACG. This is the first study to investigate these loci in both Australian Caucasian and Nepalese populations.     

Cdh11 Acts as a Tumor Suppressor in a Murine Retinoblastoma Model by Facilitating Tumor Cell Death

CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01). Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8) (p = 0.01) and had fewer multifocal tumors at PND28 (p = 0.016), compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003). In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p<0.03 for all markers), while proliferation in vivo remained unaffected (p = 0.121). Activated caspase-3 was significantly decreased and β-catenin expression increased in Cdh11 knockdown experiments in vitro. These data suggest that Cdh11 displays tumor suppressor properties in vivo and in vitro in murine retinoblastoma through promotion of cell death.     

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer

HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p = 0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR = 0.45 [CI 0.23–0.90], p = 0.023; HR = 0.55 [CI 0.32–0.94], p = 0.028, respectively), with even more pronounced impact in node-positive patients (HR = 0.21 [CI 0.07–0.63], p = 0.006; HR = 0.29 [CI 0.13–0.65], p = 0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n = 2809): HR = 0.79 [CI 0.7–0.9], log-rank p = 0.0003; for OS (n = 971): HR = 0.63 [CI 0.48–0.83], log-rank p = 0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.     

IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses,Fc-Glycans and a Fab-Peptide Sequence

The Fc-glycan profile of IgG₁ anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG₁, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG₄ peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG₄, the ACPA-IgG₄ Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG₁ compared to FT-IgG₁ were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG₁ relative to FT-IgG₁ as well as lower content of IgG₂ (p = 0.0000001) and elevated content of IgG₄ (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG₁ and ACPA-IgG₄ are distinct. Given that IgG₁ and IgG₄ have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.     

A New Specific Succinate-Glutamate Metabolomic Hallmark in Sdhx-Related Paragangliomas

Paragangliomas (PGLs) are frequently associated with germline mutations in genes involved in energy metabolism. The purpose of the present study was to assess whether the tumor metabolomic profile of patients with hereditary and apparently sporadic PGLs enables the distinction of different subtypes of tumors. Twenty-eight unrelated patients with a histological diagnosis of PGLs were included in the present study. Twelve had germline mutations in SDHx genes (5 SDHB, 7 SDHD), 6 VHL, and 10 were apparently sporadic. Intact tumor samples from these patients (one per patient) were evaluated with ¹H high-resolution magic angle spinning (HRMAS) NMR spectroscopy. SDHx-related tumors were characterized by an increase in succinate levels in comparison to other tumor subtypes (p = 0.0001 vs VHL and p = 0.000003 vs apparently sporadic). Furthermore, we found significantly lower values of glutamate in SDHx-related tumors compared to other subtypes (p = 0.0007 vs VHL and p = 0.003 vs apparently sporadic). Moreover, SDHx-tumors also exhibited lower values of ATP/ADP/AMP (p = 0.01) compared to VHL. VHL tumors were found to have the highest values of glutathione (GSH) compared to other tumors. Based on 4 metabolites (succinate, glutamate, GSH, and ATP/ADP/AMP), tumors were accurately distinguished from the other ones on both 3- and 2-class PLS-DA models. The present study shows that HRMAS NMR spectroscopy is a very promising method for investigating the metabolomic profile of various PGLs. The present data suggest the existence of a specific succinate-glutamate hallmark of SDHx PGLs. The relevance of such a metabolomic hallmark is expected to be very useful in designing novel treatment options as well as improving the diagnosis and follow-up of these tumors, including metastatic ones.     

Evidence for STAT4 as a Common Autoimmune Gene: rs7574865 Is Associated with Colonic Crohn's Disease and Early Disease Onset

Background

Recent studies demonstrated an association of STAT4 variants with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), indicating that multiple autoimmune diseases share common susceptibility genes. We therefore investigated the influence of STAT4 variants on the susceptibility and phenotype of inflammatory bowel diseases (IBD) in a large patient and control cohort.

Methodology/Principal Findings

Genomic DNA from 2704 individuals of Caucasian origin including 857 patients with Crohn''s disease (CD), 464 patients with ulcerative colitis (UC), and 1383 healthy, unrelated controls was analyzed for seven SNPs in the STAT4 gene (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694, rs10174238). In addition, a detailed genotype-phenotype analysis was performed. Our analysis revealed an association of the STAT4 SNP rs7574865 with overall decreased susceptibility to CD (p = 0.047, OR 0.86 [95% CI 0.74–0.99]). However, compared to CD patients carrying the wild type genotype, the STAT4 SNP rs7574865 was significantly associated with early CD onset (p = 0.021) and colonic CD (p = 0.008; OR = 4.60, 95% CI 1.63–12.96). For two other STAT4 variants, there was a trend towards protection against CD susceptibility (rs7568275, p = 0.058, OR 0.86 [95% CI 0.74–1.00]; rs10174238, p = 0.057, OR 0.86 [95% CI 0.75–1.00]). In contrast, we did not observe any association with UC susceptibility. Evidence for weak gene-gene interaction of STAT4 with the IL23R SNP rs11209026 was lost after Bonferroni correction.

Conclusions/Significance

Our results identified the STAT4 SNP rs7574865 as a disease-modifying gene variant in colonic CD. However, in contrast to SLE and RA, the effect of rs7574865 on CD susceptibility is only weak.     

Human Intelligence and Polymorphisms in the DNA Methyltransferase Genes Involved in Epigenetic Marking

Epigenetic mechanisms have been implicated in syndromes associated with mental impairment but little is known about the role of epigenetics in determining the normal variation in human intelligence. We measured polymorphisms in four DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L) involved in epigenetic marking and related these to childhood and adult general intelligence in a population (n = 1542) consisting of two Scottish cohorts born in 1936 and residing in Lothian (n = 1075) or Aberdeen (n = 467). All subjects had taken the same test of intelligence at age 11yrs. The Lothian cohort took the test again at age 70yrs. The minor T allele of DNMT3L SNP 11330C>T (rs7354779) allele was associated with a higher standardised childhood intelligence score; greatest effect in the dominant analysis but also significant in the additive model (coefficient = 1.40_additive; 95%CI 0.22,2.59; p = 0.020 and 1.99_dominant; 95%CI 0.55,3.43; p = 0.007). The DNMT3L C allele was associated with an increased risk of being below average intelligence (OR 1.25_additive; 95%CI 1.05,1.51; p = 0.011 and OR 1.37_dominant; 95%CI 1.11,1.68; p = 0.003), and being in the lowest 40^th (p_additive = 0.009; p_dominant = 0.002) and lowest 30^th (p_additive = 0.004; p_dominant = 0.002) centiles for intelligence. After Bonferroni correction for the number variants tested the link between DNMT3L 11330C>T and childhood intelligence remained significant by linear regression and centile analysis; only the additive regression model was borderline significant. Adult intelligence was similarly linked to the DNMT3L variant but this analysis was limited by the numbers studied and nature of the test and the association was not significant after Bonferroni correction. We believe that the role of epigenetics in the normal variation in human intelligence merits further study and that this novel finding should be tested in other cohorts.     

Dietary Habits in Patients with Ischemic Stroke: A Case-Control Study

Background and Aims

Diet appears to have some role in stroke development. The objective of our study was to describe the dietary habits in patients admitted with acute ischemic stroke and compare selected dietary components with healthy controls. Adherence to healthy diet behaviors was also assessed.

Methods

A case-control study of consecutive patients with acute ischemic stroke admitted to the Neurology Department of Hospital del Mar from 2007 to 2010. Patients were matched by age and sex with control subjects. A previously validated nutritional survey was administered to patients and controls. Demographic data, vascular risk factors, caloric intake and dietary nutrients were evaluated. Intention to follow a healthy diet was also assessed in both groups.

Results

A total of 300 acute ischemic stroke patients and 300 controls with evaluation of dietary habits. No differences were observed in vascular risk factors, except smoking habit, diabetes and ischemic heart disease. Stroke patients reported a higher caloric intake: 2444.8(1736.8–3244.5) vs 2208.7(1753.1–2860.7) Kcal, p = 0.001. After adjusting for energy intake, patients had higher intake of proteins (p<0.001; OR 1.02), total cholesterol (p = 0.001; OR 1.04), and breaded foods (p = 0.001; OR 1.94) and lower consumption of probiotic yogurt (p = 0.002; OR 0.88). Compared to patients, control participants indicated greater intention to eat vegetables (p = 0.002; OR 1.5) and whole foods (p = 0.000; OR 2.4) and reduce their intake of salt (p = 0.002; OR 1.7), fat (p = 0.000; OR 3.7) and sweets (p = 0.004; OR 1.7) than patients.

Conclusion

We observed different dietary patterns between stroke patients and controls. Stroke patients have a higher caloric intake and are less concerned about maintaining healthy nutritional habits.