Simon J. Furney

Association of Plasma Clusterin Concentration With Severity, Pathology, and Progression in Alzheimer Disease

CONTEXT Blood-based analytes may be indicators of pathological processes in Alzheimer disease (AD). OBJECTIVE To identify plasma proteins associated with AD pathology using a combined proteomic and neuroimaging approach. DESIGN Discovery-phase proteomics to identify plasma proteins associated with correlates of AD pathology. Confirmation and validation using immunodetection in a replication set and an animal model. SETTING A multicenter European study (AddNeuroMed) and the Baltimore Longitudinal Study of Aging. PARTICIPANTS Patients with AD, subjects with mild cognitive impairment, and healthy controls with standardized clinical assessments and structural neuroimaging. MAIN OUTCOME MEASURES Association of plasma proteins with brain atrophy, disease severity, and rate of clinical progression. Extension studies in humans and transgenic mice tested the association between plasma proteins and brain amyloid. RESULTS Clusterin/apolipoprotein J was associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in AD. Increased plasma concentration of clusterin was predictive of greater fibrillar amyloid-beta burden in the medial temporal lobe. Subjects with AD had increased clusterin messenger RNA in blood, but there was no effect of single-nucleotide polymorphisms in the gene encoding clusterin with gene or protein expression. APP/PS1 transgenic mice showed increased plasma clusterin, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques. CONCLUSIONS These results demonstrate an important role of clusterin in the pathogenesis of AD and suggest that alterations in amyloid chaperone proteins may be a biologically relevant peripheral signature of AD.

SF3B1 Mutations Are Associated with Alternative Splicing in Uveal Melanoma

UNLABELLED Uveal melanoma, the most common eye malignancy, causes severe visual morbidity and is fatal in approximately 50% of patients. Primary uveal melanoma can be cured by surgery or radiotherapy, but the metastatic disease is treatment refractory. To understand comprehensively uveal melanoma genetics, we conducted single-nucleotide polymorphism arrays and whole-genome sequencing on 12 primary uveal melanomas. We observed only approximately 2,000 predicted somatic single-nucleotide variants per tumor and low levels of aneuploidy. We did not observe an ultraviolet radiation DNA damage signature, but identified SF3B1 mutations in three samples and a further 15 mutations in an extension cohort of 105 samples. SF3B1 mutations were associated with good prognosis and were rarely coincident with BAP1 mutations. SF3B1 encodes a component of the spliceosome, and RNA sequencing revealed that SF3B1 mutations were associated with differential alternative splicing of protein coding genes, including ABCC5 and UQCC, and of the long noncoding RNA CRNDE. SIGNIFICANCE Our data show that despite its dismal prognosis, uveal melanoma is a relatively simple genetic disease characterized by recurrent chromosomal losses and gains and a low mutational burden. We show that SF3B1 is recurrently mutated in uveal melanoma, and the mutations are associated with aberrant alternative splicing.

Whole genome sequencing of matched primary and metastatic acral melanomas

Next generation sequencing has enabled systematic discovery of mutational spectra in cancer samples. Here, we used whole genome sequencing to characterize somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Our data show that the somatic mutational rates in this acral melanoma sample pair were more comparable to the rates reported in cancer genomes not associated with mutagenic exposure than in the genome of a melanoma cell line or the transcriptome of melanoma short-term cultures. Despite the perception that acral skin is sun-protected, the dominant mutational signature in these samples is compatible with damage due to ultraviolet light exposure. A nonsense mutation in ERCC5 discovered in both the primary and metastatic tumors could also have contributed to the mutational signature through accumulation of unrepaired dipyrimidine lesions. However, evidence of transcription-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. The primary and the metastasis are highly similar at the level of global gene copy number alterations, loss of heterozygosity and single nucleotide variation (SNV). Furthermore, the majority of the SNVs in the primary tumor were propagated in the metastasis and one nonsynonymous coding SNV and one splice site mutation appeared to arise de novo in the metastatic lesion.

Ultraviolet radiation accelerates BRAF-driven melanomagenesis by targeting TP53

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear. The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event. To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a BRAF(V600E) mouse model. In mice expressing BRAF(V600E) in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden. Here we show that sunscreen (UVA superior, UVB sun protection factor (SPF) 50) delayed the onset of UVR-driven melanoma, but only provided partial protection. The UVR-exposed tumours showed increased numbers of single nucleotide variants and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in approximately 40% of cases. TP53 is an accepted UVR target in human non-melanoma skin cancer, but is not thought to have a major role in melanoma. However, we show that, in mice, mutant Trp53 accelerated BRAF(V600E)-driven melanomagenesis, and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans. Furthermore, we identify TP53/Trp53 as a UVR-target gene that cooperates with BRAF(V600E) to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis. Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

Genome-wide association with MRI atrophy measures as a quantitative trait locus for Alzheimer's disease.

Alzheimers disease (AD) is a progressive neurodegenerative disorder with considerable evidence suggesting an initiation of disease in the entorhinal cortex and hippocampus and spreading thereafter to the rest of the brain. In this study, we combine genetics and imaging data obtained from the Alzheimers Disease Neuroimaging Initiative and the AddNeuroMed study. To identify genetic susceptibility loci for AD, we conducted a genome-wide study of atrophy in regions associated with neurodegeneration in this condition. We identified one single-nucleotide polymorphism (SNP) with a disease-specific effect associated with entorhinal cortical volume in an intron of the ZNF292 gene (rs1925690; P-value=2.6 × 10−8; corrected P-value for equivalent number of independent quantitative traits=7.7 × 10−8) and an intergenic SNP, flanking the ARPP-21 gene, with an overall effect on entorhinal cortical thickness (rs11129640; P-value=5.6 × 10−8; corrected P-value=1.7 × 10−7). Gene-wide scoring also highlighted PICALM as the most significant gene associated with entorhinal cortical thickness (P-value=6.7 × 10−6).

Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt–PCP–JNK pathway

Although the mechanism of Aβ action in the pathogenesis of Alzheimer’s disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt–planar cell polarity (PCP)–c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt–PCP–JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt–PCP–JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.

Application of Sequencing, Liquid Biopsies, and Patient-Derived Xenografts for Personalized Medicine in Melanoma

UNLABELLED Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell-derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. SIGNIFICANCE Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments.

Structural and functional properties of genes involved in human cancer.

BackgroundOne of the main goals of cancer genetics is to identify the causative elements at the molecular level leading to cancer.ResultsWe have conducted an analysis of a set of genes known to be involved in cancer in order to unveil their unique features that can assist towards the identification of new candidate cancer genes.ConclusionWe have detected key patterns in this group of genes in terms of the molecular function or the biological process in which they are involved as well as sequence properties. Based on these features we have developed an accurate Bayesian classification model with which human genes have been scored for their likelihood of involvement in cancer.

Mitochondrial Dysfunction and Immune Activation are Detectable in Early Alzheimer's Disease Blood

Alzheimers disease (AD), like other dementias, is characterized by progressive neuronal loss and neuroinflammation in the brain. The peripheral leukocyte response occurring alongside these brain changes has not been extensively studied, but might inform therapeutic approaches and provide relevant disease biomarkers. Using microarrays, we assessed blood gene expression alterations occurring in people with AD and those with mild cognitive changes at increased risk of developing AD. Of the 2,908 differentially expressed probes identified between the three groups (p < 0.01), a quarter were altered in blood from mild cognitive impairment (MCI) and AD subjects, relative to controls, suggesting a peripheral response to pathology may occur very early. There was strong evidence for mitochondrial dysfunction with decreased expression of many of the respiratory complex I-V genes and subunits of the core mitochondrial ribosome complex. This mirrors changes previously observed in AD brain. A number of genes encoding cell adhesion molecules were increased, along with other immune-related genes. These changes are consistent with leukocyte activation and their increased the transition from circulation into the brain. In addition to expression changes, we also found increased numbers of basophils in people with MCI and AD, and increased monocytes in people with an AD diagnosis. Taken together this study provides both an insight into the functional response of circulating leukocytes during neurodegeneration and also identifies potential targets such as the respiratory chain for designing and monitoring future therapeutic interventions using blood.

Differences in the evolutionary history of disease genes affected by dominant or recessive mutations

BackgroundGlobal analyses of human disease genes by computational methods have yielded important advances in the understanding of human diseases. Generally these studies have treated the group of disease genes uniformly, thus ignoring the type of disease-causing mutations (dominant or recessive). In this report we present a comprehensive study of the evolutionary history of autosomal disease genes separated by mode of inheritance.ResultsWe examine differences in protein and coding sequence conservation between dominant and recessive human disease genes. Our analysis shows that disease genes affected by dominant mutations are more conserved than those affected by recessive mutations. This could be a consequence of the fact that recessive mutations remain hidden from selection while heterozygous. Furthermore, we employ functional annotation analysis and investigations into disease severity to support this hypothesis.ConclusionThis study elucidates important differences between dominantly- and recessively-acting disease genes in terms of protein and DNA sequence conservation, paralogy and essentiality. We propose that the division of disease genes by mode of inheritance will enhance both understanding of the disease process and prediction of candidate disease genes in the future.