K. G. Burnand

Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24

Introduction: Lymphoedema-distichiasis syndrome (LD) (OMIM 153400) is a rare, primary lymphoedema of pubertal onset, associated with distichiasis. Causative mutations have now been described in FOXC2, a forkhead transcription factor gene. Numerous clinical associations have been reported with this condition, including congenital heart disease, ptosis, varicose veins, cleft palate, and spinal extradural cysts. Subjects: We report clinical findings in 74 affected subjects from 18 families and six isolated cases. All of them were shown to have mutations in FOXC2 with the exception of one family who had two affected subjects with lymphoedema and distichiasis and linkage consistent with the 16q24 locus. Results: The presence of lymphoedema was highly penetrant. Males had an earlier onset of lymphoedema and a significantly increased risk of complications. Lymphatic imaging confirmed the earlier suggestion that LD is associated with a normal or increased number of lymphatic vessels rather than the hypoplasia or aplasia seen in other forms of primary lymphoedema. Distichiasis was 94.2% penetrant, but not always symptomatic. Associated findings included ptosis (31%), congenital heart disease (6.8%), and cleft palate (4%). Other than distichiasis, the most commonly occurring anomaly was varicose veins of early onset (49%). This has not been previously reported and suggests a possible developmental role for FOXC2 in both venous and lymphatic systems. This is the first gene that has been implicated in the aetiology of varicose veins. Conclusion: Unlike previous publications, the thorough clinical characterisation of our patients permits more accurate prediction of various phenotypic abnormalities likely to manifest in subjects with FOXC2 mutations.

The hemodynamics and diagnosis of venous disease

The venous system is, in many respects, more complex than the arterial system and a thorough understanding of venous anatomy, pathophysiology, and available diagnostic tests is required in the management of acute and chronic venous disorders. The venous system develops through several stages, which may be associated with a number of development anomalies. A thorough knowledge of lower extremity venous anatomy, anatomic variants, and the recently updated nomenclature is required of all venous practitioners. Effective venous return from the lower extremities requires the interaction of the heart, a pressure gradient, the peripheral muscle pumps of the leg, and competent venous valves. In the absence of pathology, this system functions to reduce venous pressure from approximately 100 mm Hg to a mean of 22 mm Hg within a few steps. The severe manifestations of chronic venous insufficiency result from ambulatory venous hypertension, or a failure to reduce venous pressure with exercise. Although the precise mechanism remains unclear, venous hypertension is thought to induce the associated skin changes through a number of inflammatory mechanisms. Several diagnostic tests are available for the evaluation of acute and chronic venous disease. Although venous duplex ultrasonography has become the standard for detection of acute deep venous thrombosis, adjuvant modalities such as contrast, computed tomographic, and magnetic resonance venography have an increasing role. Duplex ultrasonography is also the most useful test for detecting and localizing chronic venous obstruction and valvular incompetence. However, it provides relatively little quantitative hemodynamic information and is often combined with measurements of hemodynamic severity determined by a number of plethysmographic methods. Finally, critical assessment of venous treatment modalities requires an understanding of the objective clinical outcome and quality of life instruments available.

Venous lipodermatosclerosis: treatment by fibrinolytic enhancement and elastic compression.

The value of fibrinolytic enhancement with an anabolic steroid (stanozolol) combined with elastic stockings in treating venous lipodermatosclerosis was assessed in a six-month double-blind cross-over trial. Thirty-four legs of 23 patients in whom other treatments had failed were studied. The patients were randomly divided into two groups who were treated with either stanozolol plus elastic stockings or placebo plus elastic stockings for three months, and then vice versa. Treatment with or without stanozolol caused the area of lipodermatosclerosis to decrease, but the rate of healing when patients took stanozolol was double that when they took the placebo, and this was assumed to be biologically important. Stanozolol also reduced the incidence of extravascular fibrin detected in skin biopsy specimens. The elastic stocking with placebo produced significant decreases in leg volume, ankle circumference, and skin thickness. Stanozolol is valuable in treating intractable lipodermatosclerosis, giving relief of pain and reducing induration, inflammation, tenderness, and pigmentation.

Novel Candidate Genes in Unstable Areas of Human Atherosclerotic Plaques

Objective—Comparison of gene expression in stable versus unstable atherosclerotic plaque may be confounded by interpatient variability. The aim of this study was to identify differences in gene expression between stable and unstable segments of plaque obtained from the same patient. Methods and Results—Human carotid endarterectomy specimens were segmented and macroscopically classified using a morphological classification system. Two analytical methods, an intraplaque and an interplaque analysis, revealed 170 and 1916 differentially expressed genes, respectively using Affymetrix gene chip analysis. A total of 115 genes were identified from both analyses. The differential expression of 27 genes was also confirmed using quantitative-polymerase chain reaction on a larger panel of samples. Eighteen of these genes have not been associated previously with plaque instability, including the metalloproteinase, ADAMDEC1 (≈37-fold), retinoic acid receptor responder-1 (≈5-fold), and cysteine protease legumain (≈3-fold). Matrix metalloproteinase-9 (MMP-9), cathepsin B, and a novel gene, legumain, a potential activator of MMPs and cathepsins, were also confirmed at the protein level. Conclusions—The differential expression of 18 genes not previously associated with plaque rupture has been confirmed in stable and unstable regions of the same atherosclerotic plaque. These genes may represent novel targets for the treatment of unstable plaque or useful diagnostic markers of plaque instability.

Mutations in FOXC2 Are Strongly Associated With Primary Valve Failure in Veins of the Lower Limb

Background— Mutations in the FOXC2 gene cause lymphedema distichiasis, an inherited primary lymphedema in which a significant number of patients have varicose veins. Because lymphedema distichiasis is believed to be caused by lymphatic valve failure (reflux), and FOXC2 is highly expressed on venous valves in mouse embryos, we tested the hypothesis that FOXC2 mutations may be linked to venous valve failure and reflux. Methods and Results— The venous system of the leg was investigated with Duplex ultrasound. Pathological reflux was recorded by color Duplex ultrasound in all 18 participants with a FOXC2 mutation, including 3 without lymphedema. Every participant with a mutation in FOXC2 showed reflux in the great saphenous vein (n=18), compared with only 1 of 12 referents (including 10 family members; P<0.0001, Fisher exact test). Deep vein reflux was recorded in 14 of 18 participants. Conclusions— FOXC2 is the first gene in which mutations have been strongly associated with primary venous valve failure in both the superficial and deep veins in the lower limb. This gene appears to be important for the normal development and maintenance of venous and lymphatic valves.

RELATION BETWEEN POSTPHLEBITIC CHANGES IN THE DEEP VEINS AND RESULTS OF SURGICAL TREATMENT OF VENOUS ULCERS

41 patients with venous ulceration, investigated by ascending phlebography, had operations to ligate incompetent calf perforating veins. Recurrent ulceration developed within five years of operation in all 23 patients with evidence of deep-vein damage on the initial phlebograms. Only 1 of the seventeen patients with normal deep veins had a recurrent ulcer during the same period of follow-up. Thus local surgery to the perforating veins in postphlebitic limbs was shown to be ineffective.

Failure of Thrombus to Resolve in Urokinase-Type Plasminogen Activator Gene–Knockout Mice Rescue by Normal Bone Marrow–Derived Cells

Background—Monocytes may have an important role in the resolution of venous thrombosis. Increased expression of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) is associated with an ingress of monocytes into the thrombus. This study was designed to evaluate the importance of these activators in thrombus resolution. Methods and Results—Inferior caval vein thrombosis was induced in cohorts of adult wild-type, uPA gene-knockout (uPA−/−), and tPA gene-knockout (tPA−/−) mice in a flow model. Thrombi were harvested from wild-type and uPA−/− mice (n=60 per group) between 1 and 60 days. Thrombi were also obtained from groups of wild-type and tPA−/− mice (n=24 per group) between 1 and 28 days. Thrombus size and macrophage content were measured by computer-assisted image analysis. Thrombus resolution was significantly impaired in the uPA−/− mice compared with wild-type controls (P <0.0001) but was unaffected in tPA−/− mice. Monocyte content in wild-type mice was highest at 14 days after thrombus induction and was ≈4 times greater than in uPA−/− mice (P =0.0043). Thrombus size in uPA−/− mice transplanted with wild-type marrow (0.29±0.06 mm2) was significantly smaller than in uPA−/− mice given uPA−/− bone marrow (3.9±1.1 mm2) (P =0.0022). Donor bone marrow–derived cells expressing LacZ were present in the thrombus after transplantation. Conclusions—The resolution of experimental venous thrombus is dependent on uPA but is unaffected by the absence of tPA. Absence of uPA is also associated with delayed monocyte recruitment into the thrombus. Transplanting wild-type bone marrow restores thrombus resolution in uPA−/− animals, suggesting an important role for bone marrow–derived cells in this process.

Milroy disease and the VEGFR-3 mutation phenotype

Primary congenital lymphoedema (Milroy disease) is a rare autosomal dominant condition for which a major causative gene defect has recently been determined. Mutations in the vascular endothelial growth factor receptor 3 (VEGFR-3) gene have now been described in 13 families world-wide. This is a review of the condition based on the clinical findings in 71 subjects from 10 families. All 71 individuals have a mutation in VEGFR-3. Ninety per cent of the 71 individuals carrying a VEGFR-3 mutation showed signs of oedema, which was confined in all cases to the lower limbs. In all but two cases onset of swelling was from birth. Other symptoms and signs included cellulitis (20%), large calibre leg veins (23%), papillomatosis (10%), and upslanting toenails (10%). In males, hydrocoele was the next most common finding after oedema (37%). Thorough clinical examination of these patients indicates that there are few clinical signs in addition to lower limb oedema. Rigorous phenotyping of patients produces a high yield of VEGFR-3 mutations.

Expression and Cellular Localization of the CC Chemokines PARC and ELC in Human Atherosclerotic Plaques

Local immune responses are thought to play an important role in the development of atherosclerosis. Histological studies have shown that human atherosclerotic lesions contain T lymphocytes throughout all stages of development, many of which are in an activated state. A number of novel CC chemokines have been described recently, which are potent chemoattractants for lymphocytes: PARC (pulmonary and activation-regulated chemokine), ELC (EBI1-ligand chemokine), LARC (liver and activation-regulated chemokine), and SLC (secondary lymphoid-tissue chemokine). Using reverse transcriptase-polymerase chain reaction and in situ hybridization, we have found gene expression for PARC and ELC but not for LARC or SLC in human atherosclerotic plaques. Immunohistochemical staining of serial plaque sections with specific cell markers revealed highly different expression patterns of PARC and ELC. PARC mRNA was restricted to CD68+ macrophages (n = 14 of 18), whereas ELC mRNA was widely expressed by macrophages and intimal smooth muscle cells (SMC) in nearly all of the lesions examined (n = 12 of 14). ELC mRNA was also found to be expressed in the medial SMC wall of highly calcified plaques (n = 4). Very low levels of ELC mRNA expression could also be detected in normal mammary arteries but no mRNA expression for PARC was detected in these vessels (n = 4). In vitro, ELC mRNA was found to be up-regulated in aortic SMC stimulated with tumor necrosis factor-a and interferon-gamma but not in SMC stimulated with serum. Both PARC and ELC mRNA were expressed by monocyte-derived macrophages but not monocytes. The expression patterns of PARC and ELC mRNA in human atherosclerotic lesions suggest a potential role for these two recently described CC chemokines in attracting T lymphocytes into atherosclerotic lesions.

Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughout the gene

Abstract. Lymphoedema-distichiasis (LD) is a dominantly inherited form of primary lymphoedema with onset of lower limb swelling at puberty or later. There is variable penetrance of this disorder, but the most consistently inherited feature is distichiasis, viz. fine hairs arising inappropriately from the meibomian glands. We established linkage of this disorder to 16q24.3 and the gene has recently been identified as the forkhead transcription factorFOXC2. We report the mutational analysis of 14 families with LD. All but one of these pedigrees have small insertions or deletions in the gene, which seem likely to produce haploinsufficiency. The mutation sites are scattered throughout the gene. There is one family with a mis-sense mutation in the forkhead domain of the protein. This base alteration is not a common polymorphism, is co-inherited with the disease and produces a non-conservative amino acid change.