For more than 10 years our International Consortium has been working to find the underlying cause of the various types of lymphoedema. Progress was initially slow due to a lack of large families and problems confirming the diagnosis in some cases. Over the past 7 years there have been significant advances in our knowledge, with the finding of 2 genes which cause lymphoedema. In addition, our group at St. George's and others around the world have found a number of changes (mutations) within these genes which are believed to be responsible for the development of lymphoedema.

The first gene, responsible for congenital lymphoedema (Milroy disease), was discovered in 1998 (Ferrell et al 1998). The gene, called vascular endothelial growth factor receptor 3 (VEGFR-3), appears to be responsible for correct development of the lymph system in the foetus.

Our research group has now found many different mutations in this gene in families with congenital onset lymphoedema. In addition to those with a family history of the condition, gene changes have been found in isolated cases proving that the condition can arise spontaneously via a new genetic change (mutation). Testing of this gene has now moved to the NHS service laboratory based in the Southwest Thames Regional Genetic Service (http://www.southwestthamesgenetics.nhs.uk). Further laboratory-based research by Dr. Russell Mellor of St. George's, University of London, has looked at the function of both the lymphatics and the veins in patients with changes in the VEGFR-3 gene.

Lymphoedema-distichiasis syndrome, in which patients with pubertal onset lymphoedema have an extra row of eyelashes which rub on the cornea, is now known to be caused by changes in a gene on chromosome 16 (Fang et al 2000). This gene, called FOXC2, is active during development of the foetus controlling the function of other genes. This controlling effect on other genes probably explains the other findings sometimes associated with this condition such as cleft palate, varicose veins and ptosis (drooping eyelids). Dr. Rachel Bell, Molecular Genetics Department, St. George's Hospital, initially found mutations in more than 30 different families with this rare condition. Subsequently many more families were ascertained and gene changes found in most. Dr Russell Mellor has also studied the veins of patients with FOXC2 mutations and found them to be abnormal (Mellor et al 2007).

We are continuing our search for a gene for pubertal onset lymphoedema (Meige's disease), the most common form of the condition and are hopeful of a breakthrough in the near future.

Once the gene for a condition is found in a family it offers the opportunity for early diagnosis and the prospect of developing new treatments using our new knowledge of the underlying cause. Tentative steps towards the development of gene therapy for lymphoedema have already been taken (Karkkainen et al 2001).

HOW YOU CAN HELP

We are always eager to include more families in our studies. The more families we are able to include, the better our chance of locating the responsible genes. Ideally, there should be 3 or more living, affected members in the family but in some cases we will include isolated cases for later mutation screening when the genes are described.

If you wish to take part or would like further advice, please contact us:

South West Thames Regional Genetics Service
St. George's University of London
Cranmer Terrace
London
SW17 0RE

(glen.brice@stgeorges.nhs.uk)

REFERENCES

Fang J, Dagenais SL, Erickson RP, Arlt MF, Glynn MW, Gorski JL, Seaver LH, Glover TW. (2000) Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. American Journal of Human Genetics. 67(6):1382-8

Ferrell RE, Levinson KL, Esman JH, Kimak MA, Lawrence EC, Barmada MM, Finegold DN. (1998) Hereditary lymphedema: evidence for linkage and genetic heterogeneity. Human Molecular Genetics. 13:2073-8.

Karkkainen MJ, Saaristo A, Jussila L, Karila KA, Lawrence EC, Pajusola K, Bueler H, Eichmann A, Kauppinen R, Kettunen MI, Yla-Herttuala S, Finegold DN, Ferrell RE, Alitalo K. (2001) A model for gene therapy of human hereditary lymphedema. Proceedings of the National Academy of Science U S A. 98(22):12677-82.

Mellor RH, Brice G, Stanton AW, French J, Smith A, Jeffery S, Levick JR, Burnand KG, Mortimer PS; Lymphoedema Research Consortium (2007) Mutations in FOXC2 are strongly associated with primary valve failure in veins of the lower limb. Circulation. 115(14):1912-20

This study is being conducted by Bernadette Waters, Senior Lecturer in the School of Health Professions & Rehabilitation Sciences at the University of Southampton. Bernadette is a doctoral student at the University of Southampton and this study will contribute to her programme completion requirements. It is a qualitative study which focuses on quality of life issues.

The aim of the research is to explore the biographies of adults who have primary lymphoedema in order to demonstrate how their lives have been affected by the experiences they have had. It is also hoped to uncover the particular challenges of living with a condition for which there is currently no cure and in which self-motivation and self-care are fundamentally important to the long-term management of the condition.

A major limitation of much research focussing on people with chronic conditions like lymphoedema, is that it has not allowed the voice of the affected individual to be easily heard. Rather, researchers have tended to defer to the voice of the healthcare 'experts'. Inevitably, the research conducted has tended to reflect the views of those experts, rather than listening to, and learning from, the personal experiences of those living with chronic disability.

This biographical study of people with lymphoedema is intended to give people an opportunity to tell their own stories. It is anticipated that the exploration of these stories will help to uncover realities and expose & challenge stereotypes. It might also be expected that this exploration could be helpful to the research subjects in that they can begin to have their views of the management of their condition uncovered and debated.

Participants should be over 16 years of age and have primary lymphoedema. They will be helping to increase the knowledge base about lymphoedema by assisting in this research study - but there will be no personal benefit in taking part. All the information gathered will be handled carefully to preserve anonymity. No actual names will be recorded and home locations will not be revealed.

Participants will be asked to reflect on their own life stories, related to their experiences of lymphoedema, in advance of the meeting with the researcher. They will be asked to focus on life-events that have special significance or meaning. The participants will then meet with the researcher on one or two occasions and be asked to recount these stories during an interview. The interview sessions will be audio-taped with the permission of particpants. It is expected that each session will take between one and two hours and will be conducted at a time and place convenient for participants. As each transcript is prepared, it will be sent to partcipants so that they can judge whether their story is being expressed clearly. This gives them the opportunity to reflect on their stories, as told so far, and to reconsider their meanings.

All the data collected will be analysed carefully using a narrative analysis approach. The research will be written up and may be published in a journal or presented at a conference. All data will be stored in accordance with the University's research governance policy.

Participants will be asked to fill out a consent form and would be free to withdraw at any time without giving a reason.

Please note this study is now full.

Bernadette Waters (Researcher)
Senior Lecturer
School of Health Professions & Rehabilitation Sciences
University of Southampton
Southampton SO17 1BJ
Tel: 023 80597638
E-mail: bw3@soton.ac.uk

Dr Russell Mellor & Dr Anthony Stanton, working in Professor Mortimer's Lab in collaboration with Professor Rodney Levick at St George's Hospital Medical School.

Physiology: Oedema (= swelling) is the presence of an abnormally large amount of fluid (the interstitial fluid) surrounding the cells of a tissue. Two systems are involved in fluid turnover, the blood vessels and the lymphatic vessels. Fluid leaks (or filters) continually from the blood capillaries into the surrounding tissue. One function of the lymphatic system is to return excess tissue fluid to the blood stream. The lymphatic system begins as initial lymphatic vessels, very narrow thin walled vessels that join together like the many tributaries of a river, forming larger vessels (pre-collectors, collectors) that eventually form major lymph trunks. The largest trunk empties into the veins in the upper chest.
In a steady state (where tissue volume is constant) the amount of fluid filtering from the capillaries into the tissue must equal the amount of fluid being removed from the tissue by the lymphatic system. Anything that increases the amount of fluid entering the tissue (increased filtration of fluid) or decreases the amount of fluid removed by the lymphatics (reduction of lymph flow) leads eventually to swelling.

Background: Computed tomography (CT) shows that in breast cancer related lymphoedema (BCRL) the oedema is mainly confined to the skin and subcutaneous layer, and not the muscle. This is a finding that we recently confirmed using ultrasound. In the swollen arm, we found there was a close relationship between skin and subcutaneous thickness (in fact, in the opposite nonswollen arm there was a similar but inverse relationship). X-ray lymphography (this involves the injection of a substance into the lymphatic vessels that shows up on X-rays - it is little performed these days because of side-effects) shows blockage of major lymphatic vessels at the axilla, and in some cases lymphatic blockage is evident in women treated for breast cancer who do not have lymphoedema. Some 75% of women who have had breast cancer are spared lymphoedema, and these facts clearly indicate the complexity of the condition.

Capillary density: The amount of fluid filtering into the tissue may be altered by a change in the number of capillaries. We looked at the capillaries in the skin using simultaneous dual-site fluorescence angiography (Mellor et al. 2002). Volunteers lay on a couch with their arms out to the side and a microscope was positioned over each arm. This allowed us to view the skin and the magnified image was recorded on videotape for later analysis. A fluorescent dye (sodium fluorescein) was injected intravenously. Once the dye was injected, it circulated in the blood stream and entered the skin capillaries. Through the microscope, the capillaries appear as fluorescent dots, so that a count of the number of capillaries can be made in a standard area. We showed that capillary density was not decreased in the swollen arm as it might have been because of the increase in skin area. Angiogenesis (growth of new blood vessels) must have occurred in order to maintain capillary density at normal levels. In controls, treated for breast cancer but without BCRL, there was no difference in density between the arms, indicating that any angiogenesis was related to the swelling and not to the breast cancer treatment.

Initial lymphatic vessel density: If lymphatic density alters, the amount of fluid the lymphatic system can remove may also alter. We examined the density of initial lymphatic vessels and the spread of dye in the skin using fluorescence microlymphangiography (FML) (Mellor et al. 2000). While viewing the forearm with the microscope described previously, a dye called FITC-dextran was injected into the skin. This fluorescent molecule is taken up by the lymphatic vessels which appear clearly as white lines spreading from the dye depot. In the swollen arm, lymphatic density was hugely increased compared with the nonswollen arm, as was the spread of dye within the lymphatics horizontally along the skin. Either there is increased recruitment of previously dormant vessels and/or lymphangiogenesis (growth of new lymphatic vessels). Lymph is most likely finding the path of least resistance which in the swollen arm is along the skin, rather than straight through it to the deeper collector vessels. Control experiments once again showed that the abnormalities were related to the swelling, not to the breast cancer treatment.

Initial lymphatic vessel diameter: The diameter of the initial lymphatics in the skin was examined using modified FML. If BCRL was due solely to lymphatic blockage at the axilla then the lymphatic vessels would be dilated. With the help of Professor Michael Duff we used computer analysis to analyse images generated by FML. In controls, lymphatic vessels in the arm on the side of the surgery/radiotherapy and in the opposite arm were of similar diameter. Contrary to expectation, in BCRL the lymphatic vessels in the swollen arm were not dilated, in fact they were narrower than those in the non-swollen arm. Strangely though, when the two groups were compared, the vessels in the swollen arms were the same diameter as those in the control group, and the non-swollen arm was the odd one out, the vessels being wider than in all other arms. This curious finding has excited much interest in our colleagues worldwide, and awaits elucidation.

Lymphoscintigraphy: Measurement of lymph flow would be valuable but, unfortunately, this is not possible in humans. We have used lymphoscintigraphy (frequently used clinically to investigate limb swelling) to measure local lymph drainage at different sites in the arm, to attempt to quantify lymphatic function. This work is in collaboration with Professor Mike Peters at Addenbrooke's Hospital, Cambridge. We have shown that lymph drainage (measured as the removal rate of injected radioactive tracer compound) is decreased in regions of the arm particularly affected by swelling (near the elbow, the hand) when compared with the opposite arm. Interestingly, when the hand is spared, local lymph drainage in the hand is not impaired. This indicates that drainage may fail locally, giving rise to the uneven distribution of swelling often seen.
When comparing groups of women with and without hand involvement it was evident that drainage was not reduced in the swollen hand, rather, it was increased in the opposite 'normal' hand. This was another finding showing that the 'normal' arm may not be normal. We have also shown that lymph drainage from the muscle (a tissue little affected by the oedema) is also reduced in the swollen arm, and that the reduction is greater in patients with greater swelling. We now suspect that muscle lymphatic function somehow governs the overall swelling, and that excess lymph from the muscle is re-routed into the subcutaneous and cutaneous layers. The collector vessels here become overloaded and the tissues swell.

Current and future work: Current work revolves around both BCRL and primary lymphoedema. Dr Stanton is doing a prospective study on women without lymphoedema and is looking at the question of the regionality of swelling. Dr Mellor is now examining the lymphatic system (using FML and biopsy specimens) and the venous systems (using colour Doppler ultrasound) of two types of primary lymphoedema (lymphoedema distichiasis and Milroy's disease), and linking the results to genetic findings already completed.

Funding: Henry Smith Charity, Wellcome Trust, Medical Research Council, Frances and Augustus Newman Foundation.