The role of APC in cellular and tissue organisation in intestinal tumorigenesis

The tumour suppressor adenomatous polyposis coli (APC) is a multifunctional protein regulating a diverse array of effector pathways essential for cellular homeostasis. In most sporadic colon cancers, truncating mutations in APC lead to the loss of the Wnt pathway and microtubule regulatory domains. Studies have established key roles of mutant APC in malignant growth via deregulation of Wnt pathway activity. However, the consequence of the loss of the microtubule regulatory domains of APC in intestinal tumorigenesis has not been determined. In addition, it is widely believed that intestinal tumorigenesis is initiated from a stem cell, with limited studies addressing the potential for adenomas originating from non-stem cells. I hypothesise that the stem cell supporting Paneth cells act as an intrinsic organising centre for the intestinal epithelia, thereby providing a barrier for tumorigenesis. I utilise intestinal tissue and generate various genetically modified 3-dimensional organoid models to study the role of APC in malignant transformation of the intestinal epithelia. I show that APC inactivation leads to alterations in tissue morphology. Intriguingly, my results reveal a novel phenotype upon loss of APC resulting in compromised intracellular organisation that is linked to the deregulation of microtubules. Sufficiency experiments suggest that the different effector roles of APC in the intestinal epithelia are domain-specific. The generation of a novel Paneth cell specific promoter allowed me to demonstrate that the loss of APC specifically in Paneth cells does not compromise intestinal epithelial tissue morphology, a key characteristic of intestinal tumorigenesis. The results presented in here demonstrate that solely the loss of APC in a Paneth cell is not sufficient to initiate intestinal tumorigenesis, suggesting that further insults could be necessary for this non-stem cell to acquire the potential to initiate tumorigenesis. My findings indicate that APC has distinct domain-specific roles in the intestinal epithelia and reveal its novel role in regulating intracellular organisation. I conclude that there are additional consequences for APC mutational inactivation in intestinal tumorigenesis beyond deregulation of the Wnt pathway activity.AstraZenec

Mutational inactivation of Apc in the intestinal epithelia compromises cellular organisation.

The adenomatous polyposis coli (Apc) protein regulates diverse effector pathways essential for tissue homeostasis. Truncating oncogenic mutations in Apc removing its Wnt pathway and microtubule regulatory domains drives intestinal epithelia tumorigenesis. Exuberant cell proliferation is one well-established consequence of oncogenic Wnt pathway activity; however, the contribution of other deregulated molecular circuits to tumorigenesis has not been fully examined. Using in vivo and organoid models of intestinal epithelial tumorigenesis we found that Wnt pathway activity controls intestinal epithelial villi and crypt structure, morphological features lost upon Apc inactivation. Although the Wnt pathway target gene c-Myc (also known as Myc) has critical roles in regulating cell proliferation and tumorigenesis, Apc specification of intestinal epithelial morphology is independent of the Wnt-responsive Myc-335 (also known as Rr21) regulatory element. We further demonstrate that Apc inactivation disrupts the microtubule cytoskeleton and consequently localisation of organelles without affecting the distribution of the actin cytoskeleton and associated components. Our data indicates the direct control over microtubule dynamics by Apc through an independent molecular circuit. Our study stratifies three independent Apc effector pathways in the intestinal epithelial controlling: (1) proliferation, (2) microtubule dynamics and (3) epithelial morphology.This article has an associated First Person interview with the first author of the paper

Frequency and phenotype associations of rare variants in 5 monogenic cerebral small vessel disease genes in 200,000 UK Biobank participants

BACKGROUND AND OBJECTIVES: Based on previous case reports and disease-based cohorts, a minority of patients with cerebral small vessel disease (cSVD) have a monogenic cause, with many also manifesting extracerebral phenotypes. We investigated the frequency, penetrance, and phenotype associations of putative pathogenic variants in cSVD genes in the UK Biobank (UKB), a large population-based study. METHODS: We used a systematic review of previous literature and ClinVar to identify putative pathogenic rare variants in CTSA, TREX1, HTRA1, and COL4A1/2. We mapped phenotypes previously attributed to these variants (phenotypes-of-interest) to disease coding systems used in the UKB's linked health data from UK hospital admissions, death records, and primary care. Among 199,313 exome-sequenced UKB participants, we assessed the following: the proportion of participants carrying ≥1 variant(s); phenotype-of-interest penetrance; and the association between variant carrier status and phenotypes-of-interest using a binary (any phenotype present/absent) and phenotype burden (linear score of the number of phenotypes a participant possessed) approach. RESULTS: Among UKB participants, 0.5% had ≥1 variant(s) in studied genes. Using hospital admission and death records, 4%–20% of variant carriers per gene had an associated phenotype. This increased to 7%–55% when including primary care records. Only COL4A1 variant carrier status was significantly associated with having ≥1 phenotype-of-interest and a higher phenotype score (OR = 1.29, p = 0.006). DISCUSSION: While putative pathogenic rare variants in monogenic cSVD genes occur in 1:200 people in the UKB population, only approximately half of variant carriers have a relevant disease phenotype recorded in their linked health data. We could not replicate most previously reported gene-phenotype associations, suggesting lower penetrance rates, overestimated pathogenicity, and/or limited statistical power

Author Correction: Organoid culture media formulated with growth factors of defined cellular activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Accuracy of routinely-collected healthcare data for identifying motor neurone disease cases: a systematic review

Background: Motor neurone disease (MND) is a rare neurodegenerative condition, with poorly understood aetiology. Large, population-based, prospective cohorts will enable powerful studies of the determinants of MND, provided identification of disease cases is sufficiently accurate. Follow-up in many such studies relies on linkage to routinely-collected health datasets. We systematically evaluated the accuracy of such datasets in identifying MND cases. Methods: We performed an electronic search of MEDLINE, EMBASE, Cochrane Library and Web of Science for studies published between 01/01/1990-16/11/2015 that compared MND cases identified in routinely-collected, coded datasets to a reference standard. We recorded study characteristics and two key measures of diagnostic accuracy—positive predictive value (PPV) and sensitivity. We conducted descriptive analyses and quality assessments of included studies. Results: Thirteen eligible studies provided 13 estimates of PPV and five estimates of sensitivity. Twelve studies assessed hospital and/or death certificate-derived datasets; one evaluated a primary care dataset. All studies were from high income countries (UK, Europe, USA, Hong Kong). Study methods varied widely, but quality was generally good. PPV estimates ranged from 55–92% and sensitivities from 75–93%. The single (UK-based) study of primary care data reported a PPV of 85%. Conclusions: Diagnostic accuracy of routinely-collected health datasets is likely to be sufficient for identifying cases of MND in large-scale prospective epidemiological studies in high income country settings. Primary care datasets, particularly from countries with a widely-accessible national healthcare system, are potentially valuable data sources warranting further investigation

Accuracy of routinely-collected healthcare data for identifying motor neurone disease cases: a systematic review

Background: Motor neurone disease (MND) is a rare neurodegenerative condition, with poorly understood aetiology. Large, population-based, prospective cohorts will enable powerful studies of the determinants of MND, provided identification of disease cases is sufficiently accurate. Follow-up in many such studies relies on linkage to routinely-collected health datasets. We systematically evaluated the accuracy of such datasets in identifying MND cases. Methods: We performed an electronic search of MEDLINE, EMBASE, Cochrane Library and Web of Science for studies published between 01/01/1990-16/11/2015 that compared MND cases identified in routinely-collected, coded datasets to a reference standard. We recorded study characteristics and two key measures of diagnostic accuracy—positive predictive value (PPV) and sensitivity. We conducted descriptive analyses and quality assessments of included studies. Results: Thirteen eligible studies provided 13 estimates of PPV and five estimates of sensitivity. Twelve studies assessed hospital and/or death certificate-derived datasets; one evaluated a primary care dataset. All studies were from high income countries (UK, Europe, USA, Hong Kong). Study methods varied widely, but quality was generally good. PPV estimates ranged from 55–92% and sensitivities from 75–93%. The single (UK-based) study of primary care data reported a PPV of 85%. Conclusions: Diagnostic accuracy of routinely-collected health datasets is likely to be sufficient for identifying cases of MND in large-scale prospective epidemiological studies in high income country settings. Primary care datasets, particularly from countries with a widely-accessible national healthcare system, are potentially valuable data sources warranting further investigation