THE PROGRAMME OF GENETICS OF VENOUS LEG ULCERS
WITH CLINICAL IMPLICATIONS
Vascular Disease Centre, University of Ferrara, Italy

VENOUS Leg Ulcers are multi-factorial and complex disorders, where genetics has a rôle and gene-gene and gene environment interactions play pivotal functions.

Our staff is involved in researches in order to recognize several and common genetic variants (SNIPS-Single Nucleotide Polymorphisms) with a defined rôle in diagnosis and prognosis of venous leg ulcers (VLU). The lesson learned could help everybody in solving practical clinical problems, currently unsolved in clinical practice. The detection of the polymorphisms described below, from a simple blood sample, represents a new tool for treating and preventing VLU.

The mission of the Vascular Diseases Centre at the University of Ferrara, Italy, is to create a multi-disciplinary research group involved in the study of vascular diseases. The staff includes experts both in basic and in clinical sciences, who collaborate back to back in common and innovative research programs. The improvement of knowledge is in turn transferred in the care of patients affected by arterial, venous and lymphatic disorders, as well as in the education, and in exchanging information concerning basic and clinical research pertaining to the vascular system.

Rôle of HFE Mutation in VLU Pathogenisis
One of the critical objectives for physicians involved in evaluating and treating chronic venous disease (CVD) is to identify a prognostic factor for ulcer onset. Clinical as well as hemodynamic parameters, including duplex scanning and plethysmographic investigations, fail to predict ulcer appearances.

Chronic venous stasis determines red blood cells extra- vasation and either dermal haemosiderin deposits or iron-laden phagocytes. Several Authors suspected that iron could play a role in the pathogenesis of venous leg ulcers. They hypothesized that local iron overload could generate free radicals, or activate a proteolytic hyperactivity on the part of metalloproteinases (MMPs), or else down-regulate tissue inhibitors of MMPs. However, they were unable to explain why iron deposits, visible in the legs of patients with CVD, cause lesions only in some individuals, while in others they do not. We hypothesized that such individual differences could be genetically determined, and investigated the role of the C282Y and H63D mutations of the HFE gene. C282Y mutation significantly increases the risk of ulcer in primary CVD of more than six times (OR = 6.69; 1.45 – 30.8; P = 0.01). Patients carrying the H63D variants anticipate the age of ulcer onset by almost ten years (p > 0.004). The increased risk of skin lesion and the anticipated age of onset of the disease in HFE carriers

Figure 1: Prof Paolo Zamboni, Director of the
Vascular Disease Centre at the
University of Ferrara, Italy.

confirm in a clinical setting that intracellular iron deposits of mutated macrophages have less stability than the wildtypes
(1–2).

Our data are potentially applicable to the broad base of primary CVD patients since, among the many who suffer from varicose veins, only 10% will develop a venous ulcer. This high-risk minority could be identified in advance by means of a simple blood test that would act as a genetic screening device. Then such preventive measures as elastic stockings, superficial venous surgery, and avoidance of iron-rich foods and dietary supplements could be utilized in a targeted program of potentially great effectiveness.

Figure 2: An iron laden macrophage migrated in the ulcer bed.
Macrophages carrying the HFE mutations release more iron and
free radicals so increasing the risk of leg ulceration.

Thus, primary CVD could be treated more appropriately, before any lesions develop in those patients with particular genetic haplotypes. Vascular surgery prac-tice could be strongly influenced by the results of the HFE genetic test; presence of the C282Y mutation would strengthen the indications and priorities for surgical correction of superficial venous insufficiency.

Figure 3: The multidisciplinary staff of the Vascular Diseases Centre,
where surgeons and molecular biologist work together.

Rôle of Factor XIII Polymorphisms
We know very well the capabilities of factor XIII in cross linking molecules of fibrin at the end of the coagulation cascade. Factor XIII is also well known as a natural growth factor, since its cross linking properties in a wound can be also extended to collagen, laminin, tenascin and other matrix components. It has also been proposed for the topical therapy of VLU. However, there are genetic variants of factor XIII more favourable to wound healing. Recently, in our Molecular Biology Lab, we recognized that the risk of developing a larger lesion was directly related to the activity and to a definite FXIII genotype (FXIIIA-V34L, FXIIIA-P5634L).

Similarly, the risk of having delayed wound-healing in chronic venous ulcer after successful superficial venous surgery intervention, is reduced in the presence of particular genotypes singly or in combination (i.e., FXIIIA-V34L and FXIIIA-P5634L have a significant function).

It is evident the possibility of improving our prognosis by detecting the above reported genetic variants of factor XIII (3–4).

Figure 4: Mr Andrea Felloni, nurse responsible for the
wound care programme, with our patients.

Our Laboratory Service
Through a convention between hospitals interested in genetics of wound healing and the Azienda Ospedaliera Universitaria di Ferrara, it is possible to detect the above reported polymorphisms plus another twenty genetic variants still not yet published.

Our service includes not only the laboratory but also some treatment suggestions based on the results of the investigations.

For information please contact Dr Donato Gemmati, responsible of the laboratory at the Vascular Disease Centre of the University of Ferrara, Italy. Coordinates:
telephone +390532237291, email: cet@unife.it

References
1. Zamboni P, Tognazzo S, Izzo M, Pancaldi F, Scapoli GL, Liboni A, Gemmati D. Hemochromatosis C282Y gene mutation increases the risk of venous leg ulceration. J. Vasc. Surg. (2005); 42: 309–14.
2. Zamboni P, Izzo M, Tognazzo S, Carandina S, De Palma Massimiliano, et al. The overlapping of local iron overload and HFE mutations in venous leg ulcers pathogenesis. Free Radical Biology & Medicine (2006); 15; 40: 1869–73.
3. Gemmati D, Tognazzo S, Serino ML, Fogato L, Carandina S, De Palma M, et al. Factor XIII V34L polymorphism modulates the risk of chronic venous leg ulcer progression and extension. Wound Repair & Regen. (2004); 12: 512–7.
4. Gemmati D, Tognazzo S, Catozzi L, Carandina S, De Palma M, Tacconi G, Gianesini S, Scapoli GL, Zamboni P Influence of Genetic Polymorphisms in Ulcer Healing Process after Superficial Venous Surgery. J. Vasc. Surg. (2006); 44: 554–62.