Deep phenotyping to improve understanding of causal mechanisms and underlying gene mutations in primary lymphoedema and lymphatic malformations

Lymphoedema is swelling of any body part caused by a fault or obstruction in the lymphatic system, and is one of the most neglected areas in healthcare. Data suggest it is twice as common as type 1 diabetes (which is estimated at 400,000 in the UK) but much less recognised. Primary lymphoedema is often considered genetic in origin, whereas secondary lymphoedema has an identifiable cause such as the surgical removal of lymph glands for cancer. In the last 5 years a number of genes have been identified which, when faulty, cause inherited forms of lymphoedema. Finding the causal gene means a specific diagnosis can be made by a blood test through examination of the DNA of the patient suspected of having that particular type of lymphoedema. This helps inform the patient about their condition and what might happen to them in their lifetime.

We have been studying lymphoedema for over 30 years. Genes have been discovered through investigating the DNA of patients who have as closely matched lymphoedema as it is possible to test. Detailed clinical histories and examination findings are recorded but clinical appearances alone are often not sufficient to distinguish one type of Primary Lymphoedema from another. This proposed research study is designed to provide better methods of investigation that will help distinguish one type of Primary Lymphoedema from another and give us a better insight into the mechanisms that produce the lymphoedema (and so help to design new treatments).

Current tools for investigation of lymphoedema are very limited. Lymphoscintigraphy is the only widely available method within the NHS for the diagnosis of lymphoedema but it does not enable direct visualisation of the lymph vessels. Two methods are proposed to overcome this. The first is Magnetic Resonance Lymphangiography (MRL) using injected contrast, which enables distinction of lymph vessels from blood vessels. MRL will also be used to see the malformed lymph vessels inside the body. The second method of investigation is Indocyanine Green Lymphography (ICGL). This involves the injection of a dye that is seen by a camera in the near infrared spectrum wavelength of light. ICGL has been used to image lymph vessels just under the skin of an arm or leg prior to lymphatic microsurgery but has never been used to study Primary Lymphoedema. MRL will enable imaging of deeper lymph vessels whereas ICGL will provide information on lymph vessel pumping and valve function. Unlike lymphoscintigraphy neither MRL nor ICGL involve radiation.

To study the smaller lymph capillaries in the skin we will perform biopsies but analyse them using a revolutionary, state of the art, 3D imaging technique. This will tell us much more about the structure and function of malfunctioning small lymphatic vessels in the patient groups.

Infection can be a devastating consequence of lymphoedema because the lymph system is part of our immune system, so when the lymph system goes wrong so does immune function. We have already shown that in some genetic forms of lymphoedema, the white cells of the lymph system (lymphocytes) are low in number. There are virtually no studies in humans to explain why this is. We plan to study the numbers, trafficking and function of lymphocytes in order to understand if the immune dysfunction is a result of the genes or secondary to disturbed movement of the cells throughout the body from the lymphoedema.

By developing improved investigation techniques we will be able to categorise our patients more clearly and discover more genes and how those genes make the lymph system grow and work. This may have relevance to diseases other than lymphoedema, which hitherto may not have been known to have a lymphatic contribution; for example the recovery of cardiac muscle after a heart attack may be dependent on lymphatic function. Discovering genes in primary lymphoedema will inform on their wide-ranging roles in human biology and pathology.

Lymphoedema is a chronic debilitating condition characterized by disabling swelling and life threatening infections. Primary lymphoedema often results from a genetic fault whereas secondary lymphoedema develops from an identifiable cause. There is no evidence that drug or surgical therapy is effective and treatment is largely palliative. Improved treatment will only come through better understanding of disease pathophysiology. The finding of causal genes has allowed specific diagnoses to be made. Further study of these genes in animals has informed on their roles in lymphatic development and function. Our group has discovered 8 of the 12 known causal genes for human lymphoedema through gathering cohorts of patients with similar physical characteristics (phenotypes).

To understand the mechanisms causing the lymphoedema more informative methods of investigation are needed. We plan 4 lines of enquiry in genotyped human lymphoedema: (1) better imaging to investigate the main collecting vessels within a limb. We have developed MR lymphangiography in breast cancer related lymphoedema and have recently applied ICG Lymphography to study lymphatic vessel pumping. (2) the use of 3D ultramicroscopy of skin biopsies. With our project partners in Germany we have already demonstrated how visualization of the entire vascular network of the samples at the cellular resolution level permits a far more detailed description of initial lymphatic network morphology compared to traditional 2D sections. (3) to investigate the immune dysfunction in genotyped lymphoedema using FACS analysis and proliferation studies using isotope labelling of T cells, as well as primary and secondary immune response rates in vivo. (4) functional studies of causative genes and their mutations in mosaic forms of primary lymphoedema and lymphatic malformations to inform on causal genes and disease mechanisms.

The WHO estimates that around 40 million people in tropical countries have lymphoedema caused by filariasis, making lymphoedema more prevalent than Alzheimer disease, which they estimate affects 36 million worldwide. Patients suffering from lymphoedema and lymphatic malformations are only too well aware of the limited knowledge of healthcare professionals on the lymphatic system and lymphatic disorders. Currently there are no drugs or surgery proven to improve lymphatic function. Pharma are unlikely to fully engage unless potential target mechanisms for drug development are better understood.

The programme meets MRC's strategic priority of 'Living a long and healthy life' as it will increase the understanding of the processes that regulate fluid homeostasis and immunity in the healthy lymphatic system and how factors associated with chronic disease alter the function. We propose to apply novel methods of investigation to understand better the differences between the specific genetic forms of lymphoedema. The methods should prove their value in understanding the mechanisms producing the lymphoedema and so guide direction for patient diagnosis, management and targeting new therapies. The research programme will bring benefit to all sections of society from patients to scientists, the pharmaceutical industry and health care professionals.

Patients will benefit from the improved knowledge that explains their lymphoedema. Finding the causal gene means a specific diagnosis can be made by a blood test through examination of the DNA of the patient suspected of having that particular type of lymphoedema. This helps inform the patient about their condition, what might happen to them in their lifetime and the risk of other family members inheriting the disorder. The national patients' organisation, the Lymphoedema Support Network, is eager to disseminate and exploit advances in knowledge to their members and media.

Healthcare professionals will be advised on better ways of diagnosis and management. For those forms of primary lymphoedema where the causal gene is known a simple DNA examination can confirm the diagnosis. Knowledge of the gene informs genetic counsellors how to advise families of risks to subsequent offspring. Better methods of investigation should encourage radiologists to re-evaluate their choice of diagnostic imaging. Improved understanding of the contribution of the lymphatic system to immune dysfunction should inform scientists and clinicians working in infection and immunity.

Scientists working on organ systems are beginning to realise the importance of the lymphatic vasculature and a more detailed understanding of the molecular mechanisms involved in the regulation of this system is only now emerging from animal research leading to novel insights into lymphatic biology. Evidence suggests that the lymphatic vasculature is not just involved in lymphatic specific disease such as lymphoedema, but in other pathologies such as cardiovascular disease, infection and immunity, cancer, and probably obesity - the four major challenges in healthcare in the 21st century. This novel research will advance academic knowledge and UK competitiveness and will help us influence the international research agenda in this field.

The pharmaceutical industry will benefit from the knowledge generated regarding the mechanisms causing lymphoedema. This new knowledge must be applicable to human lymphoedema before robust targets for drug development can be pursued. This research would be the first step in our quest to delineate novel treatment strategies.

It is anticipated that results will have an immediate impact on these beneficiaries. The programme will also support the training of highly skilled researchers in the field of lymphovascular biology who we hope will start a surge in interest in the emerging discipline of lymphovascular medicine drawing in other physicians and surgeons to this area of clinical practice.