Expression of genes in the 16p11.2 locus during human fetal cortical neurogenesis

The process of the brain developing from a single fertilized egg to the most sophisticated known organ requires precise spatial and temporal control to produce the necessary correct brain size and architecture. A particular region of interest is the cerebral cortex, responsible for higher functions such as language, reasoning and conscious thought. Its expansion in size and complexity from smaller mammals, such as mice, to humans is thought to contribute to our higher functions. However, a caveat of this increased complexity is the increased challenge of generating such a complex structure, and the potential for subtle changes during neurodevelopment to manifest in neurodevelopmental disorders such as Autism Spectrum Disorders (ASD). ASD is a spectrum disorder diagnosed early in childhood based on a range of diagnostic criteria. It is frequently characterised by impaired social interaction, repetitive behaviour and delayed development. While ASD patients share some symptoms, the genetic underpinnings of ASD are highly heterogeneous, with mutations to many single genes or larger genetic regions implicated as ASD risk factors. The 593kbp 16p11.2 locus encompasses 29 protein coding genes and its copy number variation (CNV) by heterozygous microduplication or microdeletion is implicated in around 1% of ASD cases, with many patients born with macrocephaly (deletion) or microcephaly (duplication), potentially indicating a possible problem with generating the correct number of brain cells during development. This suggests the hypothesis that some of the 16p11.2 region genes are involved in neural proliferation in early corticogenesis, and changes to the levels of these genes may affect proliferation, contributing to the 16p11.2 patient phenotype. This hypothesis is supported by a 16p11.2 deletion mouse model which exhibits ASD-like symptoms and altered proliferation in the cortex during embryonic development. Given that the 16p11.2 CNV’s 1% autism incidence makes it the most frequent aetiology of ASD, this region is a promising area of study to understand how genetic dysregulation during critical prenatal cortical neurogenesis can contribute to the ASD phenotype. Despite its strong association with ASD, very little is known about the majority of the 16p11.2 genes, especially regarding brain development. In this thesis, focussing on the developing human neocortex, we aimed to identify which, if any, of these 29 genes were expressed in progenitor cells and describe their expression pattern during critical stages of cortical neurogenesis. We first used a bioinformatics approach to identify 16p11.2 genes expressed in progenitors to narrow down from the 29 genes in the region. We analysed a publicly available single-cell RNA sequencing (scRNA-seq) dataset from the proliferative zones, the ventricular zone and subventricular zone, of the 16-18 gestational week (GW) human fetal cortex. We identified six genes as being highly expressed in the cortical progenitor cells, and two as being significantly higher expressed in progenitors compared to post mitotic cells: KIF22 and ALDOA. We described their protein expression pattern in vivo at key stages of human fetal cortex development. We showed KIF22 protein to be expressed in the germinative zones, and its expression to be restricted to proliferating cells, suggesting a role for this protein in proliferation. We showed KIF22 protein levels to vary with the cell cycle, increasing from G1 through S and G2 phases to peak in mitosis. This suggests that changing the KIF22 protein level, as in the microduplication or microdeletion patients, will affect cell cycle and proliferation manifesting in changes to cortical size and architecture contributing to the 16p11.2 phenotype. ALDOA protein was shown to be present throughout the cortex, although higher in the proliferating regions. We demonstrated that ALDOA is predominantly localised to cytoplasm, and its protein levels or sub-cellular localisation do not change in proliferating or non-proliferating cells. ALDOA has a critical role in energy metabolism, and we can hypothesise that due to its expression throughout the cortex any changes to the ALDOA protein by the 16p11.2 CNV will induce a wide range of effects on brain development In conclusion we have identified two genes highly expressed in progenitors and expressed at much lower levels in post-mitotic cells from the 16p11.2 locus. These genes provide interesting targets for future studies to elucidate the mechanism by which they mediate proliferation and the effects of manipulating their protein levels. This is outwith the scope of this PhD thesis however a range of new techniques are emerging such as cerebral organoids, which can be easily manipulated. These will be a powerful tool to address the hypotheses produced by the descriptive work of this PhD thesis

Untargeted LC-HRMS-based metabolomics to identify novel biomarkers of metastatic colorectal cancer

Colorectal cancer is one of the main causes of cancer death worldwide, and novel biomarkers are urgently needed for its early diagnosis and treatment. The utilization of metabolomics to identify and quantify metabolites in body fluids may allow the detection of changes in their concentrations that could serve as diagnostic markers for colorectal cancer and may also represent new therapeutic targets. Metabolomics generates a pathophysiological ‘fingerprint’ that is unique to each individual. The purpose of our study was to identify a differential metabolomic signature for metastatic colorectal cancer. Serum samples from 60 healthy controls and 65 patients with metastatic colorectal cancer were studied by liquid chromatography coupled to high-resolution mass spectrometry in an untargeted metabolomic approach. Multivariate analysis revealed a separation between patients with metastatic colorectal cancer and healthy controls, who significantly differed in serum concentrations of one endocannabinoid, two glycerophospholipids, and two sphingolipids. These findings demonstrate that metabolomics using liquid-chromatography coupled to high-resolution mass spectrometry offers a potent diagnostic tool for metastatic colorectal cancer.This study was supported by a grant (n° 15CC056/DTS17/00081- ISCIII-FEDER) from the Fundación para la Investigación Biosanitaria de Andalucía Oriental (FIBAO) and Roche Pharma S.L. Authors from the Fundación MEDINA acknowledge the receipt of financial support from this public-private partnership of Merck Sharp & Dohme de España S.A. with the University of Granada and Andalusian Regional Government (PIN-0474-2016)

Cost Effectiveness of Screening Individuals With Cystic Fibrosis for Colorectal Cancer

Background & Aims: Individuals with cystic fibrosis are at increased risk of colorectal cancer (CRC) compared with the general population, and risk is higher among those who received an organ transplant. We performed a cost-effectiveness analysis to determine optimal CRC screening strategies for patients with cystic fibrosis. Methods: We adjusted the existing Microsimulation Screening Analysis-Colon model to reflect increased CRC risk and lower life expectancy in patients with cystic fibrosis. Modeling was performed separately for individuals who never received an organ transplant and patients who had received an organ transplant. We modeled 76 colonoscopy screening strategies that varied the age range and screening interval. The optimal screening strategy was determined based on a willingness to pay threshold of $100,000 per life-year gained. Sensitivity and supplementary analyses were performed, including fecal immunochemical test (FIT) as an alternative test, earlier ages of transplantation, and increased rates of colonoscopy complications, to assess if optimal screening strategies would change. Results: Colonoscopy every 5 years, starting at an age of 40 years, was the optimal colonoscopy strategy for patients with cystic fibrosis who never received an organ transplant; this strategy prevented 79% of deaths from CRC. Among patients with cystic fibrosis who had received an organ transplant, optimal colonoscopy screening should start at an age of 30 or 35 years, depending on the patient's age at time of transplantation. Annual FIT screening was predicted to be cost-effective for patients with cystic fibrosis. However, the level of accuracy of the FIT in this population is not clear. Conclusions: Using a Microsimulation Screening Analysis-Colon model, we found screening of patients with cystic fibrosis for CRC to be cost effective. Because of the higher risk of CRC in these patients, screening should start at an earlier age with a shorter screening interval. The findings of this study (especially those on FIT screening) may be limited by restricted evidence available for patients with cystic fibrosis

Refining molecular analysis in the pathways of colorectal carcinogenesis

BACKGROUND & AIMS: In the stepwise model, specific genetic and epigenetic changes accumulate as colorectal adenomas progress to carcinomas (CRCs). CRCs also acquire global phenotypes, particularly microsatellite instability (MSI) and aneuploidy/polyploidy (chromosomal instability, CIN). Few changes specific to MSI-low or CIN+ cancers have been established. METHODS: We investigated 100 CRCs for: mutations and loss of heterozygosity (LOH) where appropriate, of APC, K-ras, BRAF, SMAD4, and p53; deletion on 5q around APC and 18q around SMAD4; total chromosomal-scale losses and gains; MSI; and CIN. RESULTS: As expected, CIN- cancers had fewer chromosomal changes overall than CIN+ lesions, but after correcting for this, 5q deletions alone predicted CIN+ status. 5q deletions were not, however, significantly associated with APC mutations, which were equally frequent in CIN+ and CIN- tumors. We therefore found no evidence to show that mutant APC promotes CIN. p53 mutations/LOH were more common in CIN+ than CIN- lesions, and all chromosomal amplifications were in CIN+ tumors. CIN- cancers could be subdivided according to the total number of chromosomal-scale changes into CIN-low and CIN-stable groups; 18q deletion was the best predictor, being present in nearly all CIN-low lesions and almost no CIN-stable tumors. MSI-low was not associated with CIN, any specific mutation, a mutational signature, or clinicopathologic characteristic. CONCLUSIONS: Overall, the components of the stepwise model (APC, K-ras, and p53 mutations, plus 18q LOH) tended to co-occur randomly. We propose an updated version of this model comprising 4 pathways of CRC pathogenesis, on the basis of 5q/18q deletions, MSI (high/low), and CIN (high/low/stable)