HDAC6 inhibition restores TDP-43 pathology and axonal transport defects in human motor neurons with TARDBP mutations.

TDP-43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP-43, are one of the common causes of familial ALS. In this study, we investigate TDP-43 protein behavior in induced pluripotent stem cell (iPSC)-derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP-43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP-43, C-terminal fragments, and phospho-TDP-43. By generating iPSC lines with allele-specific tagging of TDP-43, we find that mutant TDP-43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP-43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP-43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP-43 pathology.

Genetic variability in sporadic amyotrophic lateral sclerosis.

With the advent of gene therapies for amyotrophic lateral sclerosis (ALS), there is a surge in gene testing for this disease. Although there is ample experience with gene testing for C9orf72, SOD1, FUS and TARDBP in familial ALS, large studies exploring genetic variation in all ALS-associated genes in sporadic ALS (sALS) are still scarce. Gene testing in a diagnostic setting is challenging, given the complex genetic architecture of sALS, for which there are genetic variants with large and small effect sizes. Guidelines for the interpretation of genetic variants in gene panels and for counselling of patients are lacking. We aimed to provide a thorough characterization of genetic variability in ALS genes by applying the American College of Medical Genetics and Genomics (ACMG) criteria on whole genome sequencing data from a large cohort of 6013 sporadic ALS patients and 2411 matched controls from Project MinE. We studied genetic variation in 90 ALS-associated genes and applied customized ACMG-criteria to identify pathogenic and likely pathogenic variants. Variants of unknown significance were collected as well. In addition, we determined the length of repeat expansions in C9orf72, ATXN1, ATXN2 and NIPA1 using the ExpansionHunter tool. We found C9orf72 repeat expansions in 5.21% of sALS patients. In 50 ALS-associated genes, we did not identify any pathogenic or likely pathogenic variants. In 5.89%, a pathogenic or likely pathogenic variant was found, most commonly in SOD1, TARDBP, FUS, NEK1, OPTN or TBK1. Significantly more cases carried at least one pathogenic or likely pathogenic variant compared to controls (odds ratio 1.75; P-value 1.64 × 10-5). Isolated risk factors in ATXN1, ATXN2, NIPA1 and/or UNC13A were detected in 17.33% of cases. In 71.83%, we did not find any genetic clues. A combination of variants was found in 2.88%. This study provides an inventory of pathogenic and likely pathogenic genetic variation in a large cohort of sALS patients. Overall, we identified pathogenic and likely pathogenic variants in 11.13% of ALS patients in 38 known ALS genes. In line with the oligogenic hypothesis, we found significantly more combinations of variants in cases compared to controls. Many variants of unknown significance may contribute to ALS risk, but diagnostic algorithms to reliably identify and weigh them are lacking. This work can serve as a resource for counselling and for the assembly of gene panels for ALS. Further characterization of the genetic architecture of sALS is necessary given the growing interest in gene testing in ALS.

CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

The Effect of SMN Gene Dosage on ALS Risk and Disease Severity.

OBJECTIVE: The role of the survival of motor neuron (SMN) gene in amyotrophic lateral sclerosis (ALS) is unclear, with several conflicting reports. A decisive result on this topic is needed, given that treatment options are available now for SMN deficiency. METHODS: In this largest multicenter case control study to evaluate the effect of SMN1 and SMN2 copy numbers in ALS, we used whole genome sequencing data from Project MinE data freeze 2. SMN copy numbers of 6,375 patients with ALS and 2,412 controls were called from whole genome sequencing data, and the reliability of the calls was tested with multiplex ligation-dependent probe amplification data. RESULTS: The copy number distribution of SMN1 and SMN2 between cases and controls did not show any statistical differences (binomial multivariate logistic regression SMN1 p = 0.54 and SMN2 p = 0.49). In addition, the copy number of SMN did not associate with patient survival (Royston-Parmar; SMN1 p = 0.78 and SMN2 p = 0.23) or age at onset (Royston-Parmar; SMN1 p = 0.75 and SMN2 p = 0.63). INTERPRETATION: In our well-powered study, there was no association of SMN1 or SMN2 copy numbers with the risk of ALS or ALS disease severity. This suggests that changing SMN protein levels in the physiological range may not modify ALS disease course. This is an important finding in the light of emerging therapies targeted at SMN deficiencies. ANN NEUROL 2021;89:686-697.

HNRNPK alleviates RNA toxicity by counteracting DNA damage in C9orf72 ALS.

A 'GGGGCC' repeat expansion in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.

RNF170 mutation causes autosomal dominant sensory ataxia with variable pyramidal involvement.

BACKGROUND: Although hereditary ataxias are a group of clinically and genetically heterogeneous disorders, specific clinical clues can sometimes incriminate certain genes. This can trigger genetic testing in sporadic patients or prompt dissecting certain genes more thoroughly when initial genetic testing is negative. Also for the assembly of gene panels and interpretation of the results, genotype-phenotype correlations remain important to establish. METHODS: We clinically evaluated a Belgian family with autosomal dominant inherited sensory ataxia and variable pyramidal involvement and performed targeted clinical exome sequencing. Secondly, we retrospectively screened sequencing data of an in-house cohort of 404 patients with neuromuscular disorders for variants in the identified gene RNF170. RESULTS: All affected family members showed sensory ataxia on examination. Pyramidal involvement, and sometimes slow-pursuit abnormalities and/or a sensory neuropathy, were more variable findings. We identified the heterozygous variant p.Arg199Cys in RNF170 in all three affected siblings of our family. We did not find additional pathogenic variants in RNF170 in our in-house neuromuscular cohort. CONCLUSIONS: We confirm the heterozygous variant p.Arg199Cys in RNF170 in a Belgian family with autosomal dominant sensory ataxia and variable pyramidal involvement. This constitutes a rare but clinically recognizable phenotype that warrants testing of RNF170. Unlike the distinctive bi-allelic loss of function variants in RNF170 associated with hereditary spastic paraplegia (HSP), the p.Arg199Cys variant is the only one reported in sensory ataxia. It is important for neurologists to be aware of this characteristic phenotype and to include this gene in gene panels for ataxia and HSP.

Histone Deacetylase Inhibition Regulates Lipid Homeostasis in a Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disorder of the motor system. While the etiology is still incompletely understood, defects in metabolism act as a major contributor to the disease progression. Recently, histone deacetylase (HDAC) inhibition using ACY-738 has been shown to restore metabolic alterations in the spinal cord of a FUS mouse model of ALS, which was accompanied by a beneficial effect on the motor phenotype and survival. In this study, we investigated the specific effects of HDAC inhibition on lipid metabolism using untargeted lipidomic analysis combined with transcriptomic analysis in the spinal cord of FUS mice. We discovered that symptomatic FUS mice recapitulate lipid alterations found in ALS patients and in the SOD1 mouse model. Glycerophospholipids, sphingolipids, and cholesterol esters were most affected. Strikingly, HDAC inhibition mitigated lipid homeostasis defects by selectively targeting glycerophospholipid metabolism and reducing cholesteryl esters accumulation. Therefore, our data suggest that HDAC inhibition is a potential new therapeutic strategy to modulate lipid metabolism defects in ALS and potentially other neurodegenerative diseases.

Investigating the Endo-Lysosomal System in Major Neurocognitive Disorders Due to Alzheimer's Disease, Frontotemporal Lobar Degeneration and Lewy Body Disease: Evidence for SORL1 as a Cross-Disease Gene.

Dysfunctions in the endo-lysosomal system have been hypothesized to underlie neurodegeneration in major neurocognitive disorders due to Alzheimer's disease (AD), Frontotemporal Lobar Degeneration (FTLD), and Lewy body disease (DLB). The aim of this study is to investigate whether these diseases share genetic variability in the endo-lysosomal pathway. In AD, DLB, and FTLD patients and in controls (948 subjects), we performed a targeted sequencing of the top 50 genes belonging to the endo-lysosomal pathway. Genetic analyses revealed (i) four previously reported disease-associated variants in the SORL1 (p.N1246K, p.N371T, p.D2065V) and DNAJC6 genes (p.M133L) in AD, FTLD, and DLB, extending the previous knowledge attesting SORL1 and DNAJC6 as AD- and PD-related genes, respectively; (ii) three predicted null variants in AD patients in the SORL1 (p.R985X in early onset familial AD, p.R1207X) and PPT1 (p.R48X in early onset familial AD) genes, where loss of function is a known disease mechanism. A single variant and gene burden analysis revealed some nominally significant results of potential interest for SORL1 and DNAJC6 genes. Our data highlight that genes controlling key endo-lysosomal processes (i.e., protein sorting/transport, clathrin-coated vesicle uncoating, lysosomal enzymatic activity regulation) might be involved in AD, FTLD and DLB pathogenesis, thus suggesting an etiological link behind these diseases.

Age-related changes in DNA methylation affect renal histology and post-transplant fibrosis.

During ageing, kidney function decreases due to renal tubular atrophy, interstitial fibrosis, glomerulosclerosis and arteriosclerosis. Recently, changes in DNA methylation were shown to contribute to various ageing processes. However, it is unknown whether such changes also contribute to age-related kidney dysfunction. To assess this, we profiled genome-wide changes in DNA methylation (over 800 000 CpG sites) in 95 renal biopsies obtained prior to kidney transplantation from donors aged 16 to 73 years. Donor age significantly associated with the methylation of 92 778 CpGs (false discovery rate under 0.05), corresponding to 10 285 differentially methylated regions. These regions were most frequently located in genes involved in the Wnt/beta-catenin signaling pathway. Using an independent cohort of 67 biopsies, we autonomously validated these findings. Interestingly, the methylation status of these 92 778 age-related CpGs was associated with glomerulosclerosis (34.4% of CpGs at a false discovery rate under 0.05) and interstitial fibrosis (0.9%) and graft function at one year after transplantation, but not with tubular atrophy and arteriosclerosis. No association was observed with any of these pathologies at the time of transplantation (0% at a false discovery rate under 0.05). Thus, age-associated changes in DNA methylation at the time of transplantation predict future injury of transplanted kidneys. Specifically, our epigenome-wide association study demonstrates that epigenetic renal ageing is implicated in progressive fibrosis in both the glomerulus and the interstitium.

Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo.

Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleus post injury identified cathepsin D (CTSD) as the most upregulated gene. In aged GRN-/- cortices, CTSD was also upregulated, but the relative CTSD activity was reduced and improved upon exogenous GRN addition. Moreover, GRN and its C-terminal granulin domain granulinE (GrnE) both stimulated the proteolytic activity of CTSD in vitro. Pull-down experiments confirmed a direct interaction between GRN and CTSD. This interaction was also observed with GrnE and stabilized the CTSD enzyme at different temperatures. Investigating the importance of this interaction for axonal regeneration in vivo we found that, although individually tolerated, a combined reduction of GRN and CTSD synergistically reduced axonal outgrowth. Our data links the neurotrophic effect of GRN and GrnE with a lysosomal chaperone function on CTSD to maintain its proteolytic capacity.

Integrated genome analysis of uterine leiomyosarcoma to identify novel driver genes and targetable pathways.

Uterine leiomyosarcomas (uLMS) are rare, aggressive malignancies for which limited treatment options are available. To gain novel molecular insights into uLMS and identify potential novel therapeutic targets, we characterized 84 uLMS samples for genome-wide somatic copy number alterations, mutations, gene fusions and gene expression and performed a data integration analysis. We found that alterations affecting TP53, RB1, PTEN, MED12, YWHAE and VIPR2 were present in the majority of uLMS. Pathway analyses additionally revealed that the PI3K/AKT/mTOR, estrogen-mediated S-phase entry and DNA damage response signaling pathways, for which inhibitors have already been developed and approved, frequently harbored genetic changes. Furthermore, a significant proportion of uLMS was characterized by amplifications and overexpression of known oncogenes (CCNE1, TDO2), as well as deletions and reduced expression of tumor suppressor genes (PTEN, PRDM16). Overall, it emerged that the most frequently affected gene in our uLMS samples was VIPR2 (96%). Interestingly, VIPR2 deletion also correlated with unfavorable survival in uLMS patients (multivariate analysis; HR = 4.5, CI = 1.4-14.3, p = 1.2E-02), while VIPR2 protein expression was reduced in uLMS vs. normal myometrium. Moreover, stimulation of VIPR2 with its natural agonist VIP decreased SK-UT-1 uLMS cell proliferation in a dose-dependent manner. These data suggest that VIPR2, which is a negative regulator of smooth muscle cell proliferation, might be a novel tumor suppressor gene in uLMS. Our work further highlights the importance of integrative molecular analyses, through which we were able to uncover the genes and pathways most frequently affected by somatic alterations in uLMS.

Differences in MWCNT- and SWCNT-induced DNA methylation alterations in association with the nuclear deposition.

BACKGROUND: Subtle DNA methylation alterations mediated by carbon nanotubes (CNTs) exposure might contribute to pathogenesis and disease susceptibility. It is known that both multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) interact with nucleus. Such, nuclear-CNT interaction may affect the DNA methylation effects. In order to understand the epigenetic toxicity, in particular DNA methylation alterations, of SWCNTs and short MWCNTs, we performed global/genome-wide, gene-specific DNA methylation and RNA-expression analyses after exposing human bronchial epithelial cells (16HBE14o- cell line). In addition, the presence of CNTs on/in the cell nucleus was evaluated in a label-free way using femtosecond pulsed laser microscopy. RESULTS: Generally, a higher number of SWCNTs, compared to MWCNTs, was deposited at both the cellular and nuclear level after exposure. Nonetheless, both CNT types were in physical contact with the nuclei. While particle type dependency was noticed for the identified genome-wide and gene-specific alterations, no global DNA methylation alteration on 5-methylcytosine (5-mC) sites was observed for both CNTs. After exposure to MWCNTs, 2398 genes were hypomethylated (at gene promoters), and after exposure to SWCNTs, 589 CpG sites (located on 501 genes) were either hypo- (N = 493 CpG sites) or hypermethylated (N = 96 CpG sites). Cells exposed to MWCNTs exhibited a better correlation between gene promoter methylation and gene expression alterations. Differentially methylated and expressed genes induced changes (MWCNTs > SWCNTs) at different cellular pathways, such as p53 signalling, DNA damage repair and cell cycle. On the other hand, SWCNT exposure showed hypermethylation on functionally important genes, such as SKI proto-oncogene (SKI), glutathione S-transferase pi 1 (GTSP1) and shroom family member 2 (SHROOM2) and neurofibromatosis type I (NF1), which the latter is both hypermethylated and downregulated. CONCLUSION: After exposure to both types of CNTs, epigenetic alterations may contribute to toxic or repair response. Moreover, our results suggest that the observed differences in the epigenetic response depend on particle type and differential CNT-nucleus interactions.

Genetic modifiers of CHEK2*1100delC-associated breast cancer risk.

PURPOSE: CHEK2*1100delC is a founder variant in European populations that confers a two- to threefold increased risk of breast cancer (BC). Epidemiologic and family studies have suggested that the risk associated with CHEK2*1100delC is modified by other genetic factors in a multiplicative fashion. We have investigated this empirically using data from the Breast Cancer Association Consortium (BCAC). METHODS: Using genotype data from 39,139 (624 1100delC carriers) BC patients and 40,063 (224) healthy controls from 32 BCAC studies, we analyzed the combined risk effects of CHEK2*1100delC and 77 common variants in terms of a polygenic risk score (PRS) and pairwise interaction. RESULTS: The PRS conferred odds ratios (OR) of 1.59 (95% CI: 1.21-2.09) per standard deviation for BC for CHEK2*1100delC carriers and 1.58 (1.55-1.62) for noncarriers. No evidence of deviation from the multiplicative model was found. The OR for the highest quintile of the PRS was 2.03 (0.86-4.78) for CHEK2*1100delC carriers, placing them in the high risk category according to UK NICE guidelines. The OR for the lowest quintile was 0.52 (0.16-1.74), indicating a lifetime risk close to the population average. CONCLUSION: Our results confirm the multiplicative nature of risk effects conferred by CHEK2*1100delC and the common susceptibility variants. Furthermore, the PRS could identify carriers at a high lifetime risk for clinical actions.Genet Med advance online publication 06 October 2016.

Epigenetic effects of carbon nanotubes in human monocytic cells.

Carbon nanotubes (CNTs) are fibrous carbon-based nanomaterials with a potential to cause carcinogenesis in humans. Alterations in DNA methylation on cytosine-phosphate-guanidine (CpG) sites are potential markers of exposure-induced carcinogenesis. This study examined cytotoxicity, genotoxicity and DNA methylation alterations on human monocytic cells (THP-1) after incubation with single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). Higher cytotoxicity and genotoxicity were observed after incubation with SWCNTs than incubation with MWCNTs. At the selected concentrations (25 and 100 µg/ml), DNA methylation alterations were studied. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to assess global DNA methylation, and Illumina 450K microarrays were used to assess methylation of single CpG sites. Next, we assessed gene promoter-specific methylation levels. We observed no global methylation or hydroxymethylation alterations, but on gene-specific level, distinct clustering of CNT-treated samples were noted. Collectively, CNTs induced gene promoter-specific altered methylation and those 1127 different genes were identified to be hypomethylated. Differentially methylated genes were involved in several signalling cascade pathways, vascular endothelial growth factor and platelet activation pathways. Moreover, possible contribution of the epigenetic alterations to monocyte differentiation and mixed M1/M2 macrophage polarisation were discussed.

Identification of new genetic susceptibility loci for breast cancer through consideration of gene-environment interactions.

Genes that alter disease risk only in combination with certain environmental exposures may not be detected in genetic association analysis. By using methods accounting for gene-environment (G × E) interaction, we aimed to identify novel genetic loci associated with breast cancer risk. Up to 34,475 cases and 34,786 controls of European ancestry from up to 23 studies in the Breast Cancer Association Consortium were included. Overall, 71,527 single nucleotide polymorphisms (SNPs), enriched for association with breast cancer, were tested for interaction with 10 environmental risk factors using three recently proposed hybrid methods and a joint test of association and interaction. Analyses were adjusted for age, study, population stratification, and confounding factors as applicable. Three SNPs in two independent loci showed statistically significant association: SNPs rs10483028 and rs2242714 in perfect linkage disequilibrium on chromosome 21 and rs12197388 in ARID1B on chromosome 6. While rs12197388 was identified using the joint test with parity and with age at menarche (P-values = 3 × 10(-07)), the variants on chromosome 21 q22.12, which showed interaction with adult body mass index (BMI) in 8,891 postmenopausal women, were identified by all methods applied. SNP rs10483028 was associated with breast cancer in women with a BMI below 25 kg/m(2) (OR = 1.26, 95% CI 1.15-1.38) but not in women with a BMI of 30 kg/m(2) or higher (OR = 0.89, 95% CI 0.72-1.11, P for interaction = 3.2 × 10(-05)). Our findings confirm comparable power of the recent methods for detecting G × E interaction and the utility of using G × E interaction analyses to identify new susceptibility loci.

Genetic variants in VEGF pathway genes in neoadjuvant breast cancer patients receiving bevacizumab: Results from the randomized phase III GeparQuinto study.

Studies assessing the effect of bevacizumab (BEV) on breast cancer (BC) outcome have shown different effects on progression-free and overall survival, suggesting that a subgroup of patients may benefit from this treatment. Unfortunately, no biomarkers exist to identify these patients. Here, we investigate whether single nucleotide polymorphisms (SNPs) in VEGF pathway genes correlate with pathological complete response (pCR) in the neoadjuvant GeparQuinto trial. HER2-negative patients were randomized into treatment arms receiving either BEV combined with standard chemotherapy or chemotherapy alone. In a pre-planned biomarker study, DNA was collected from 729 and 724 patients, respectively from both treatment arms, and genotyped for 125 SNPs. Logistic regression assessed interaction between individual SNPs and both treatment arms to predict pCR. Five SNPs may be associated with a better response to BEV, but none of them remained significant after correction for multiple testing. The two SNPs most strongly associated, rs833058 and rs699947, were located upstream of the VEGF-A promoter. Odds ratios for the homozygous common, heterozygous and homozygous rare rs833058 genotypes were 2.36 (95% CI, 1.49-3.75), 1.20 (95% CI, 0.88-1.64) and 0.61 (95% CI, 0.34-1.12). Notably, some SNPs in VEGF-A exhibited a more pronounced effect in the triple-negative subgroup. Several SNPs in VEGF-A may be associated with improved pCR when receiving BEV in the neoadjuvant setting. Although none of the observed effects survived correction for multiple testing, our observations are consistent with previous studies on BEV efficacy in BC. Further research is warranted to clarify the predictive value of these markers.

Evaluation of efficacy and safety markers in a phase II study of metastatic colorectal cancer treated with aflibercept in the first-line setting.

BACKGROUND: Aflibercept (ziv-aflibercept) is an anti-angiogenic agent recently approved in combination with FOLFIRI for the treatment of metastatic colorectal cancer (mCRC) patients previously treated with oxaliplatin. Despite heterogeneity in response to aflibercept, no biomarkers for efficacy or adverse effects have been identified. Here we present biomarker data from the randomised phase II AFFIRM trial assessing aflibercept in combination with mFOLFOX6 first line in mCRC. METHODS: Ninety-six somatic mutations in key oncogenic drivers of mCRC and 133 common single-nucleotide polymorphisms (SNPs) in vascular endothelial growth factor (VEGF) pathway genes were analysed, and 27 plasma markers measured at baseline, during and after treatment. We assessed correlations of these three classes of biomarkers with progression-free survival (PFS) and adverse events (AEs). RESULTS: Somatic mutations identified in KRAS, BRAF, NRAS, PIK3CA and PIK3R1 did not significantly correlate with PFS (multiple testing-adjusted false discovery rate (FDR) or multiple testing-adjusted FDR>0.3). None of the individual SNPs correlated with PFS (multiple testing-adjusted FDR>0.22), but at the gene level variability in VEGFB significantly correlated with PFS (multiple testing-adjusted FDR=0.0423). Although none of the plasma markers measured at baseline significantly correlated with PFS, high levels of circulating IL8 at baseline together with increased levels of IL8 during treatment were significantly associated with reduced PFS (multiple testing-adjusted FDR=0.0478). No association was found between biomarkers and AEs. CONCLUSIONS: This represents the first biomarker study in mCRC treated with aflibercept. High IL8 plasma levels at baseline and subsequent increases in IL8 were associated with worse PFS, suggesting that IL8 may act as a potentially predictive biomarker of aflibercept treatment outcome.

DNA methylation profiling of non-small cell lung cancer reveals a COPD-driven immune-related signature.

INTRODUCTION: Non-small cell lung cancer (NSCLC) is a heterogeneous disorder consisting of distinct molecular subtypes each characterised by specific genetic and epigenetic profiles. Here, we aimed to identify novel NSCLC subtypes based on genome-wide methylation data, assess their relationship with smoking behaviour, age, COPD, emphysema and tumour histopathology, and identify the molecular pathways underlying each subtype. METHODS: Methylation profiling was performed on 49 pairs of tumour and adjacent lung tissue using Illumina 450 K arrays. Transcriptome sequencing was performed using Illumina HiSeq2000 and validated using expression data from The Cancer Genome Atlas (TCGA). Tumour immune cell infiltration was investigated by immunohistochemistry. RESULTS: Unsupervised hierarchical clustering of tumour methylation data revealed two subgroups characterised by a significant association between cluster membership and presence of COPD (p=0.024). Ontology analysis of genes containing differentially methylated CpGs (false discovery rate, FDR-adjusted p<0.05) revealed that immune genes were strongly enriched in COPD tumours, but not in non-COPD tumours. This COPD-specific immune signature was attributable to methylation changes in immune genes expressed either by tumour cells or tumour-infiltrating immune cells. No such differences were observed in adjacent tissue. Transcriptome profiling similarly revealed that genes involved in the immune response were differentially expressed in COPD tumours (FDR-adjusted p<0.05), an observation that was independently replicated using TCGA data. Immunohistochemistry validated these findings, revealing fewer CD4-positive T lymphocytes in tumours derived from patients with COPD. CONCLUSIONS: Lung tumours of patients with COPD differ from those of patients without COPD, with differentially methylated and expressed genes being mainly involved in the immune response.

Whole-genome sequencing reveals a coding non-pathogenic variant tagging a non-coding pathogenic hexanucleotide repeat expansion in C9orf72 as cause of amyotrophic lateral sclerosis.

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) has a familial cause in 10% of patients. Despite significant advances in the genetics of the disease, many families remain unexplained. We performed whole-genome sequencing in five family members from a pedigree with autosomal-dominant classical ALS. A family-based elimination approach was used to identify novel coding variants segregating with the disease. This list of variants was effectively shortened by genotyping these variants in 2 additional unaffected family members and 1500 unrelated population-specific controls. A novel rare coding variant in SPAG8 on chromosome 9p13.3 segregated with the disease and was not observed in controls. Mutations in SPAG8 were not encountered in 34 other unexplained ALS pedigrees, including 1 with linkage to chromosome 9p13.2-23.3. The shared haplotype containing the SPAG8 variant in this small pedigree was 22.7 Mb and overlapped with the core 9p21 linkage locus for ALS and frontotemporal dementia. Based on differences in coverage depth of known variable tandem repeat regions between affected and non-affected family members, the shared haplotype was found to contain an expanded hexanucleotide (GGGGCC)(n) repeat in C9orf72 in the affected members. Our results demonstrate that rare coding variants identified by whole-genome sequencing can tag a shared haplotype containing a non-coding pathogenic mutation and that changes in coverage depth can be used to reveal tandem repeat expansions. It also confirms (GGGGCC)n repeat expansions in C9orf72 as a cause of familial ALS.