Human Papillomavirus integration: The mechanism(s) behind the high-risk associated with this event and cervical disease progression

Cervical cancer is the second most common cancer among women worldwide. Infection with Human Papillomavirus (HPV) is essential but not the only contributing factor in cervical cancer development. HPV integration is reported to be present in over 80% of cervical cancers and disruption of HPV genome through integration leads to high levels of HPV oncogene expression. DNA damage and repair pathways are thought to induce HPV integration since HPV is detected at fragile sites in the human genome. There is controversy as to whether integration is an early or late event in cervical oncogenesis and there are no published studies to date that have investigated HPV integration using sensitive, DNA based, techniques at the nucleotide level in cervical precancers. This study aimed to test the hypothesis that integration is an early event in cervical neoplasia and episomal loss causes malignant transformation through transcription of integrated HPV. Also, this study served to pilot whether HPV integration can predict high-grade cervical disease in women with cytological abnormalities with an aim to improve current cervical screening methods. Assays to detect integration and E2 as a marker of episomal state were developed for HPV16, HPV18 and HPV45 and applied to cervical smears and biopsies from women with varying disease grades. The data presented in this thesis highlight that integration may not be essential for cervical cancer progression and different modes of disease progression may exist between young women and older women. Integration was detected at chromosome fragile sites but was more prevalent at SINE or LINE repeat elements; this implies a role for retroelements in the mechanism of integration. Finally, the data here suggest that integration induces a unique selective process in each individual and clonal selection may arise due to altered HPV oncogene expression and/or disruption to human gene expression

Searching the dark genome for Alzheimer's disease risk variants

Sporadic Alzheimer’s disease (AD) is a complex genetic disease, and the leading cause of dementia worldwide. Over the past 3 decades, extensive pioneering research has discovered more than 70 common and rare genetic risk variants. These discoveries have contributed massively to our understanding of the pathogenesis of AD but approximately half of the heritability for AD remains unaccounted for. There are regions of the genome that are not assayed by mainstream genotype and sequencing technology. These regions, known as the Dark Genome, often harbour large structural DNA variants that are likely relevant to disease risk. Here, we describe the dark genome and review current technological and bioinformatics advances that will enable researchers to shed light on these hidden regions of the genome. We highlight the potential importance of the hidden genome in complex disease and how these strategies will assist in identifying the missing heritability of AD. Identification of novel protein-coding structural variation that increases risk of AD will open new avenues for translational research and new drug targets that have the potential for clinical benefit to delay or even prevent clinical symptoms of disease

Psychological stress, cognitive decline and the development of dementia in amnestic mild cognitive impairment

To determine the relationship between psychological stress with cognitive outcomes in a multi-centre longitudinal study of people with amnestic mild cognitive impairment (aMCI) we assessed three parameters of psychological stress (Recent Life Changes Questionnaire (RLCQ); the Perceived Stress Scale (PSS) and salivary cortisol) and their relationship with rates of cognitive decline over an 18 month follow up period and conversion to dementia over a 5.5 year period. In 133 aMCI and 68 cognitively intact participants the PSS score was higher in the aMCI compared with control group but neither the RLCQ scores nor salivary cortisol measures were different between groups. In the aMCI group the RLCQ and the PSS showed no significant association with cognitive function at baseline, cognitive decline or with conversion rates to dementia but high salivary cortisol levels were associated with RLCQ scores and poorer cognitive function at baseline and lower rates of cognitive decline. No relationship was found between salivary cortisol levels and conversion rate to dementia. We conclude that psychological stress as measured by the RLCQ or PSS was not associated with adverse cognitive outcomes in an aMCI population and hypothesise that this may reflect diminished cortisol production to psychological stress as the disease progresses.</p

Periodontitis and cognitive decline in Alzheimer's disease.

Periodontitis is common in the elderly and may become more common in Alzheimer's disease because of a reduced ability to take care of oral hygiene as the disease progresses. Elevated antibodies to periodontal bacteria are associated with an increased systemic pro-inflammatory state. Elsewhere raised serum pro-inflammatory cytokines have been associated with an increased rate of cognitive decline in Alzheimer's disease. We hypothesized that periodontitis would be associated with increased dementia severity and a more rapid cognitive decline in Alzheimer's disease. We aimed to determine if periodontitis in Alzheimer's disease is associated with both increased dementia severity and cognitive decline, and an increased systemic pro inflammatory state. In a six month observational cohort study 60 community dwelling participants with mild to moderate Alzheimer's Disease were cognitively assessed and a blood sample taken for systemic inflammatory markers. Dental health was assessed by a dental hygienist, blind to cognitive outcomes. All assessments were repeated at six months. The presence of periodontitis at baseline was not related to baseline cognitive state but was associated with a six fold increase in the rate of cognitive decline as assessed by the ADAS-cog over a six month follow up period. Periodontitis at baseline was associated with a relative increase in the pro-inflammatory state over the six month follow up period. Our data showed that periodontitis is associated with an increase in cognitive decline in Alzheimer's Disease, independent to baseline cognitive state, which may be mediated through effects on systemic inflammation

Evidence against altered excitatory/inhibitory balance in the posteromedial cortex of young adult APOE E4 carriers: a resting state 1H-MRS study

A strategy to gain insight into early changes that may predispose people to Alzheimer's disease (AD) is to study the brains of younger cognitively healthy people that are at increased genetic risk of AD. The Apolipoprotein (APOE) E4 allele is the strongest genetic risk factor for AD, and several neuroimaging studies comparing APOE E4 carriers with non-carriers at age ∼20–30 years have detected hyperactivity (or reduced deactivation) in posteromedial cortex (PMC), a key hub of the default network (DN), which has a high susceptibility to early amyloid deposition in AD. Transgenic mouse models suggest such early network activity alterations may result from altered excitatory/inhibitory (E/I) balance, but this is yet to be examined in humans. Here we test the hypothesis that PMC fMRI hyperactivity could be underpinned by altered levels of excitatory (glutamate) and/or inhibitory (GABA) neurotransmitters in this brain region. Forty-seven participants (20 APOE E4 carriers and 27 non-carriers) aged 18–25 years underwent resting-state proton magnetic resonance spectroscopy (1H-MRS), a non-invasive neuroimaging technique to measure glutamate and GABA in vivo. Metabolites were measured in a PMC voxel of interest and in a comparison voxel in the occipital cortex (OCC). There was no difference in either glutamate or GABA between the E4 carriers and non-carriers in either MRS voxel, or in the ratio of glutamate to GABA, a measure of E/I balance. Default Bayesian t-tests revealed evidence in support of this null finding. Our findings suggest that PMC hyperactivity in APOE E4 carriers is unlikely to be associated with, or possibly may precede, alterations in local resting-state PMC neurotransmitters, thus informing our understanding of the spatio-temporal sequence of early network alterations underlying APOE E4 related AD risk

Single-nucleus RNA sequencing identifies new classes of proximal tubular epithelial cells in kidney fibrosis

Background Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function and to regeneration versus organ fibrosis following injury. This study used single-nucleus RNA sequencing (snRNAseq) to describe the phenotype of PTCs in renal fibrosis. Methods Kidneys were harvested from naïve mice and from mice with renal fibrosis induced by chronic aristolochic acid administration. Nuclei were isolated using Nuclei EZ Lysis buffer. Libraries were prepared on the 10× platform, and snRNAseq was completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing. Results A total of 23,885 nuclei were analyzed. PTCs were found in five abundant clusters, mapping to S1, S1–S2, S2, S2-cortical S3, and medullary S3 segments. Additional cell clusters (“new PTC clusters”) were at low abundance in normal kidney and in increased number in kidneys undergoing regeneration/fibrosis following injury. These clusters exhibited clear molecular phenotypes, permitting labeling as proliferating, New-PT1, New-PT2, and (present only following injury) New-PT3. Each cluster exhibited a unique gene expression signature, including multiple genes previously associated with renal injury response and fibrosis progression. Comprehensive pathway analyses revealed metabolic reprogramming, enrichment of cellular communication and cell motility, and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signaling to fibroblasts and inflammatory activation to macrophages. Conclusions These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis

Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease

We identified rare coding variants associated with Alzheimer’s disease (AD) in a 3-stage case-control study of 85,133 subjects. In stage 1, 34,174 samples were genotyped using a whole-exome microarray. In stage 2, we tested associated variants (P<1×10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, an additional 14,997 samples were used to test the most significant stage 2 associations (P<5×10-8) using imputed genotypes. We observed 3 novel genome-wide significant (GWS) AD associated non-synonymous variants; a protective variant in PLCG2 (rs72824905/p.P522R, P=5.38×10-10, OR=0.68, MAFcases=0.0059, MAFcontrols=0.0093), a risk variant in ABI3 (rs616338/p.S209F, P=4.56×10-10, OR=1.43, MAFcases=0.011, MAFcontrols=0.008), and a novel GWS variant in TREM2 (rs143332484/p.R62H, P=1.55×10-14, OR=1.67, MAFcases=0.0143, MAFcontrols=0.0089), a known AD susceptibility gene. These protein-coding changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified AD risk genes. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to AD development

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as novel risk factors for Alzheimers Disease

The genetic component of Alzheimer’s disease (AD) has been mainly assessed using Genome Wide Association Studies (GWAS), which do not capture the risk contributed by rare variants. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals —16,036 AD cases and 16,522 controls— in a two-stage analysis. Next to known genes TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Next to these genes, the rare variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential driver genes in AD-GWAS loci. Rare damaging variants in these genes, and in particular loss-of-function variants, have a large effect on AD-risk, and they are enriched in early onset AD cases. The newly identified AD-associated genes provide additional evidence for a major role for APP-processing, Aβ-aggregation, lipid metabolism and microglial function in AD