Pathomolecular mechanisms of intracerebral haemorrhage: functional analysis of collagen IV variants.

Each year, 150,000 people in the UK have a stroke, which is a major cause of death and disability. There are two types of stroke: those caused by blocked blood vessels and those due to rupture of blood vessels resulting in a brain bleed (haemorrhagic stroke). Intracerebral haemorrhage (brain bleeding), accounts for 15% of all adult strokes, carries the worst prognosis and there are no specific treatments for intracerebral haemorrhage, in part because we do not understand the causes of this disease.
The "basement membrane" is a structure that forms outside of cells and provides strength to blood vessels by acting as a permanent scaffold in the walls of blood vessels. Two collagen proteins produced from genes named COL4A1 and COL4A2 are major components of this basement membrane. We have found that defects in these genes, called "mutations," cause rare inherited forms of haemorrhagic stroke that affects multiple people within a family. Collagen is produced and folded inside cells and then moved out of cells. Our work suggests that these rare mutations affect basement membrane integrity but also have their damaging effects by causing incorrect folding of the COL4A1 and COL4A2 proteins. These misfolded collagen proteins then accumulate inside cells and causes these cells to become "stressed", which interferes with their function.
Excitingly, our new data suggest that changes in these collagen genes, which occur frequently in the population, are a risk factor for intracerebral haemorrhage in the general population. This raises several important questions: 1) How many stroke patients are affected by these common gene changes? 2) How do the changes that occur in the general population cause bleeding, and does their mechanism involve the accumulation of protein within cells and cell stress, similar to the rare inherited mutations?
To answer these questions we will analyse the COL4A1 and COL4A2 genes in DNA samples from a large number of patients from the general population that have had an intracerebral haemorrhage. By identifying the changes that occur in these genes we will determine the proportion of patients in whom altered collagen IV genes contribute to stroke. We will use computer analysis to predict whether the identified alterations affect the amount of collagen produced or may lead to changes in protein structure, similar to what occurs with mutations in the rare inherited forms of intracerebral haemorrhage. For some of the changes identified, we will then directly determine whether they lead to accumulation of collagen and "stress" inside blood vessel cells and establish their effects on the composition of the basement membrane. We will then investigate how the cell stress and basement membrane defects affect the health and function of these cells. Finally, we will determine if this cell stress response, basement membrane defects and other identified defects from blood vessels cells occur in brain samples.
By combining DNA analysis with investigations in cells and brains, we will obtain a much better understanding of how intracerebral haemorrhage develops. This will aid in the long term the development of new and/or more effective therapies for intracerebral haemorrhaging. By identifying the proportion of intracerebral haemorrhage patients in which these collagen changes occur and determining if all changes act in the same way, our results can indicate if a future "one version fits all" therapy is likely to be feasible and we will learn for how many patients these treatments may be effective. Interestingly, some drugs that are used to treat other diseases can reduce stress in cells grown in the laboratory, which would allow relative rapid progress to be made in developing treatments for stroke.

15% of stroke cases are due to intracerebral haemorrhage (ICH), and there is a need for specific treatments, indicating increased knowledge of its genetic and pathomolecular basis is required. Collagen IV is an extracellular matrix component that provides structural support to blood vessels and affects cell behaviour. Rare mutations in COL4A1 or COL4A2 (collagen IV alpha chain 1/2) cause familial ICH and importantly we have now established common COL4A2 variants are a risk factor for sporadic ICH, but their prevalence and disease mechanism are unknown.
Here we will use sequence analysis of a large cohort of sporadic deep ICH cases to establish the identity and prevalence of novel rare mutations and common variants in COL4A1/4A2 in ICH. Functional analysis of selected identified common variants and novel rare mutations (e.g. predicted to enhance or decrease expression, alter protein composition) will be used to address our hypothesis that ER stress is a shared mechanism between sporadic and familial ICH: ER stress results from intracellular retention of secreted proteins, can be pathogenic when chronic and be modulated by FDA-approved compounds.
We will address this using a combination of genome editing, molecular cell biology and complementary -omics approaches, and in so doing provide unparalleled insight into the nature of compositional matrix defects and ER stress response, and their effects on downstream cellular pathways, leading to endothelial cell dysfunction. Parallel analysis of "ER stress only" models will inform on the relative contribution of ER stress. Importantly, the relevance of identified mechanisms will be verified in brain samples of mice and patients with ICH. This will transform our knowledge of the genetic and pathomolecular basis of ICH in the general population, inform on patient stratification based on disease mechanism, and highlight future therapeutic avenues including the potential repurposing of FDA-approved compounds.

This project will inform on the molecular genetics and pathomolecular mechanisms of intracerebral haemorrhage (ICH), the mechanistic impacts of common variants and rare mutations in collagen genes, and putative therapeutic avenues for diseases including ICH associated with collagen IV variants.

As such it will impact:
1) Academics: Increased insight into the molecular genetic basis of ICH, the cellular response to COL4A1/4A2 variants, and potential treatment strategies for ICH and matrix diseases will be of interest to those in the fields of vascular and matrix biology, stroke, collagen diseases, protein folding, and drug discovery pathways. Our large genomic and transcriptomic data sets can be mined for secondary information, for instance by those interested in diseases associated with protein folding and ER stress. Results of this project will be used in undergraduate teaching activities and included in an article for A-level students who may later work in academia or pharmaceutical industries.

2) Patients and their families: There is no specific treatment for ICH and familial collagen IV disease. ICH and haemorrhagic stroke (which account for 15% of adult strokes) severely reduces life expectancy and quality of life, and can devastate lives of patients, family members and carers. Identifying COL4A1/4A2 mutations and risk alleles, and delineating their mechanisms can lead to the future identification of a subset of stroke patients unified by their disease mechanism and a future molecular diagnosis. Understanding how different variants relate to disease will allow for more accurate prognosis during counselling for families in which mutations have been identified and for potential pre- or post-natal testing, empowering family members to make informed choices e.g. elective caesarean to reduce ICH during labour. In the long term, identifying putative therapeutic strategies can positively impact longevity and quality of life for patients and their families.

3) Clinical community: This project will raise awareness in the clinical community of genetic risk factors underlying ICH and of familial collagen IV disease. Our results (see 2) can lead to the identification of a subgroup of ICH patients unified by their disease mechanism, and will improve the ability to distinguish between non-pathogenic polymorphisms and causal variants. This will enhance the accuracy of molecular diagnosis of patients in which collagen IV variants have been detected and will impact positively on patient counselling, management and prognosis prediction. Our work may lead in the long term to ICH patient stratification which would aid the development of more personalised and efficient treatment strategies.

4) UK economy: Stroke costs the UK economy ~£9 billion per annum (including 50% direct care costs; £1.5 billion lost productivity due to death or disability). Our work may highlight putative therapeutic avenues and in the long term lead to improved management (see 2 and 3) and/or treatment strategies (see 3) that would reduce the burden of stroke and familial collagen IV disease to the NHS and UK economy. Moreover, post-doctoral researchers and technical staff will receive high quality multi-disciplinary training that can be transferred to other settings, benefiting the academic, clinical or pharmacological communities which are important to the UK economy. Trained staff may also provide expert advice to UK policy makers.

5) Industry can benefit from secondary mining of the large transcriptomic data sets. Identifying pathways that can be targeted pharmacologically has potential pharmacological implications by highlighting putative therapeutic strategies and may lead to potential repurposing of compounds.