Lymphedema-distichiasis syndrome without FOXC2 mutation: evidence for chromosome 16 duplication upstream of FOXC2.

A patient with the classical phenotype of Lymphedema-Distichiasis syndrome (OMIM 153400) is described who showed no mutations in the sequence of FOXC2. Accordingly, a Gene Chip 250k array analysis was undertaken with dense SNP genotyping of the genomic region surrounding the FOXC2 locus on Chromosome 16 followed by copy number evaluation by real time PCR. The latter assay showed evidence of a duplicated region 5' of FOXC2 that could be causative for the patient's striking phenotype, which included both distichiasis and a hyperplastic refluxing lymphatic vascular and lymph node phenotype associated with pubertal onset lymphedema, scoliosis and strabismus.

Syndromic classification of hereditary lymphedema.

Since the late 1800's, the familial occurrence of peripheral lymphedema has been well-documented in Milroy and Meige syndromes. However, the presence of lymphedema in many other hereditary dysmorphic syndromes has not been fully appreciated. In order to establish more standardized and detailed clinical phenotypic criteria as the basis for rational classification and for greater precision in screening and genetic linkage studies, we conducted a comprehensive literature search and review of OMIM-identified and non-identified hereditary syndromes in which lymphedema was reported as a feature. Modes of inheritance, associated clinical features and images, and specific organ involvement were inventoried and suggested pathophysiologic mechanisms noted. The findings support the recommendation that when peripheral lymphedema of undetermined etiology is found, further careful, comprehensive clinical, including detailed dysmorphic, evaluation along with lymphatic imaging with subsequent syndromic classification is warranted. This information can provide clues to underlying pathogenesis and form the basis for genetic counseling and prognostication as well as offer guidance to the clinical investigator translating research at the molecular level into new approaches for evaluation and therapy.

Traumatic injury of the thoracic duct.

Injuries to the thoracic duct are infrequent but may become life-threatening when chylous leakage persists. This report describes 6 patients with such injuries in whom the leakage resolved spontaneously in one, was corrected using microsurgical lymphatic repair or lymphatic-venous anastomosis in two, successfully treated either by ligation of the thoracic duct or insertion of a peritoneovenous shunt in two, and was eventually controlled after bilateral pleurodesis and thoracic duct ligation by insertion of a peritoneo-venous shunt in one. Conventional lymphography is superior to lymphoscintigraphy and is usually required to document disruption of the thoracic duct.

Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology.

The lymph vascular system parallels the blood vasculature and as one of its key functions returns liquid and solutes to the bloodstream, including macromolecules that have escaped from blood capillaries and entered the interstitium. In conjunction with interspersed lymph nodes and lymphoid organs, the lymphatic vasculature also acts as a conduit for trafficking immune cell populations. Echoing the explosion of knowledge about blood vessel angiogenesis (properly termed "hemangiogenesis"), the past two decades have also witnessed a series of significant, yet less-noticed discoveries bearing on "lymphangiogenesis," along with delineation of the spectrum of lymphedema-angiodysplasia syndromes. Failure of lymph transport promotes a brawny proteinaceous edema of the affected limb, organ, or serous space that is disfiguring, disabling, and on occasion even life-threatening. Key members of the vascular endothelial growth factor (VEGF) and angiopoietin families of vascular growth factors (and their corresponding tyrosine kinase endothelial receptors) have been identified which preferentially influence lymphatic growth and, when manipulated in genetically engineered murine models, produce aberrant "lymphatic phenotypes." Moreover, mutations in VEGF receptor and forkhead family developmental genes have now been linked and implicated in the pathogenesis of two familial lymphedema-angiodysplasia syndromes. Thus, recent advances in "molecular lymphology" are elucidating the poorly understood development, physiology, and pathophysiology of the neglected lymphatic vasculature. In combination with fresh insights and refined tools in "clinical lymphology," these advances should lead not only to earlier detection and more rational classification of lymphatic disease but also to better therapeutic approaches, including designer drugs for lymphangiostimulation and lymphangioinhibition and gene therapy to modulate lymphatic growth.

Segregation analyses and a genome-wide linkage search confirm genetic heterogeneity and suggest oligogenic inheritance in some Milroy congenital primary lymphedema families.

We previously described six families with Milroy congenital lymphedema, only one of which showed possible linkage to a candidate locus on chromosome 5 [Witte et al., 1998]. We have now performed a complex segregation analysis of these families, and performed linkage analyses with the other 387 markers used in our genome-wide search. Our results confirm that Milroy lymphedema is generally inherited as a dominant condition. However, this mode of inheritance, as elucidated from the segregation analyses, did not account for all observed familial correlations. The segregation analysis also suggested that shared environmental or additional genetic factors are important in explaining the observed familial aggregation. The finding of linkage to multiple locations in the largest family studied by multipoint parametric mapping (one of which was confirmed by sib-pair non-parametric mapping), suggests that Milroy congenital lymphedema may be oligogenic in this family.

Advances in imaging of lymph flow disorders.

Conventional oil-contrast lymphography has long been the mainstay for lymphatic imaging. However, the emergence of computed tomography (CT) and magnetic resonance (MR) imaging has severely curtailed its use. Because of recent improvements and refinements, lymphangioscintigraphy now permits high-resolution imaging of peripheral lymphatic vessels and provides insight into lymph flow dynamics. It is indispensable for patients with known or suspected lymphatic circulatory disorders in confirming the diagnosis and delineating the pathogenesis and evolution of lymphedema. In addition, lymphangioscintigraphy helps evaluate lymphatic truncal anatomy and radiotracer transport. It can also be used to evaluate the efficacy of various treatment options designed to facilitate lymph flow or reduce lymph formation. The procedure is essentially noninvasive, can easily be repeated, and does not adversely affect the lymphatic vascular endothelium. MR imaging complements lymphangioscintigraphy in the monitoring and treatment of more complex lymphatic circulatory disorders, whereas CT facilitates catheter-guided percutaneous sclerosis or obliteration of specific lymphangiectasia or lymphangioma syndromes. Ultrasonography has proved useful in the setting of filariasis. Patients with a provisional diagnosis of peripheral lymphatic dysfunction or idiopathic edema should undergo diagnostic lymphangioscintigraphy and, in some cases, MR imaging to verify diagnostic accuracy, pinpoint the specific abnormality, and help guide subsequent therapy.

Ignorance in infectious diseases: the case of AIDS, Kaposi sarcoma, and lymphology.

From the perspective of The University of Arizona's innovative Curriculum on Medical (and Other) Ignorance focusing on "what we know we don't know, don't know we don't know, and think we know but don't," the shifting terrain of information-knowledge-ignorance of AIDS (a disorder involving, to various incompletely understood degrees, the four components of the lymphatic system-lymph, lymphatics, lymphocytes, and lymph nodes) and Kaposi sarcoma (a lymphedemogenic lesion thought to arise from trans-differentiated lymphatic endothelium) is surveyed by pinpointing some key unanswered questions that have been raised over the course of the epidemic and pointedly in past International Congresses of Lymphology. These questions are placed in the context of general ignorance about infectious diseases and the relationship of "germ" to "terrain" through the "blood-tissue-lymph loop." A framework is suggested for an "ignorance agenda" encompassing basic biology, clinical management, and societal issues.

Radionuclide lymphangioscintigraphy in the evaluation of peripheral lymphedema.

PURPOSE: The primary difficulty in evaluating and treating peripheral lymphedema is visualization of the lymphatics. Functional lymphatic studies have been performed on patients with peripheral edema to diagnose lymphedema, to determine its severity, and to understand the varied drainage patterns. METHODS: After intradermal injection in the hands or feet, initial flow and whole-body images were taken using Tc-99m human serum albumin in more than 700 patients with possible lymphedema. RESULTS: Clear images of truncal lymph transport and draining lymph nodes were obtained, and pattern differences between primary and secondary lymphedema were seen. Follow-up studies showed any functional change in lymphatic dynamics. CONCLUSION: Peripheral lymphatics can now be easily visualized. Because lymphangioscintigraphy can be performed before and after medical treatment, follow-up evaluation of patients with lymphedema is possible. The procedure is noninvasive, repeatable, easy to perform, and harmless to the lymphatic endothelium.

An imaging evaluation of angiodysplasia syndromes.

Current imaging techniques such as magnetic resonance, magnetic resonance angiography, computer tomography, ultrasound, plain x-rays, and lymphangioscintigraphy have enhanced the ability to define blood and lymph vascular malformations in more precise pathophysiologic terms. Not only can these imaging modalities distinguish arterial anomalies from lymphatic and venous angiodysplasia, but they also readily differentiate edema in the epifascial as opposed to the subfascial peripheral compartments. Moreover, visceral lymphangiectasia (e.g., chylous and non-chylous reflux), bone and muscle overgrowth, agenesis, and fat deposits can also be delineated. Clinical examples are provided including an algorithm for approaching these conditions.

Refinement of a rodent model of peripheral lymphedema.

A reliable, inexpensive experimental counterpart of peripheral lymphedema has been notoriously difficult to reproduce thereby stifling basic and clinical research into this frustrating clinical condition. Accordingly, in 45 adult Wistar-Fuzzy rats, we attempted to produce sustained hindlimb lymphedema by either groin nodal/lymphatic microsurgical ablation (S) (guided by visual blue dye lymphography) or limited field-groin irradiation (R) alone (4500 rads) or combined S followed by R or R followed by S with an additional non-manipulated group serving as controls. Observations were made for 30-100 days thereafter. Hindlimb volumes were determined serially using the truncated cone formula based on multiple circumferential measurements at standardized intervals along the affected hindlimb and the findings compared with similar measurements in the contralateral non-manipulated hindlimb. In randomly selected rats from each group, lymphatic drainage was assessed by lymphangioscintigraphy (LAS), soft tissue swelling by magnetic resonance imaging (MRI), and edema fluid total protein content by refractometry. Whereas S or R alone produced only transient or mild hindlimb edema without associated morbidity or mortality, S-R or R-S induced moderate to severe sustained protein-rich hindlimb lymphedema associated with 9-13% early mortality and notable late local limb morbidity. Lymphatic obstruction was documented by sustained maintenance of increased hindlimb volume, subcutaneous fluid accumulation (MRI), and impaired lymphatic drainage (LAS). This reproducible rodent model of secondary lymphedema reliably simulates a stable clinical condition for a window of up to 100 days and should thereby facilitate standardized testing of therapeutic/preventive protocols and basic research into lymphatic dynamics in secondary lymphedema.

Diagnostic and interventional imaging of lymphatic disorders.

During the past several years, technological innovations in nuclear diagnostics and computer imaging have rekindled enthusiasm for visualising the lymphatic system in peripheral lymphoedema and related disorders of lymph flow. Isotope lymphography or lymphangioscintigraphy has now largely replaced conventional (direct) oil-contrast lymphography for evaluating lymphatic dysplasia as it is much simpler, safe, repeatable, and provides both structural and functional detail of the lymphatic system. Magnetic resonance imaging (MRI), alone or in combination with superparamagnetic contrast agents (lymphangiomagnetograms) or fat subtraction (suppression) has the potential to yield further dividends in understanding a variety of enigmatic oedematous states including lymphoedema. Other imaging techniques of promise include ultrasonography (primarily for filariasis), fluorescent microangiolymphography, and intradermal brominated fluorocarbon (primarily for lymph nodes). Not only can these readily non-invasive imaging techniques be used to monitor and document the efficacy of treatments designed to remedy defective lymph transport and function, but in certain conditions (e.g., chylous reflux syndrome), they can be employed to obliterate incompetent lymphangiectatic/lymphangiomatous truncal elements through sclerosis using percutaneous computer-guided catheters.

Epilogue: what we don't know about cancer.

Despite billions of dollars expended since the "war on cancer" was declared more than 25 years ago, cancer continues to be a leading cause of death worldwide, and its precise pathogenesis remains elusive. Moreover, current treatments, even when successful, are grounded in extensive operations, high energy irradiation, and toxic chemotherapy, which carry substantial short- and long-term sequelae. It is, therefore, appropriate to reconsider current concepts of cancer biology in terms of the vast ignorance that surrounds this ancient scourge. From the perspective of a Curriculum on Medical Ignorance, this epilogue surveys questions raised by authors of this monograph, other experts, and patients afflicted with cancer and related disorders. Further, the focus is on basic biological, clinical, and societal implications of carcinogenesis theories as well as the nature and process of scientific inquiry in the context of the general phenomenon of ignorance in medicine.

A novel type of adhering junction in an epithelioid tumorigenic rat cell culture line.

Two major types of plaque-bearing adhering junctions are commonly distinguished: the actin microfilament-anchoring adhaerens junctions (AJs) and the desmosomes anchoring intermediate-sized filaments (IFs). Both types of junction usually possess the common plaque protein, plakoglobin, whereas the other plaque proteins and the transmembrane cadherins are mutually exclusive. For example, AJs contain E-, N-, or P-cadherin in combination with alpha- and beta-catenin, vinculin and alpha-actinin, whereas in desmosomes, desmogleins and desmocollins are associated with desmoplakin and one or several of the plakophilins (PP1-3). Here we describe a novel type of adhering junction comprising proteins of both AJs and desmosomes and the tight junction (TJ) plaque protein, ZO-1, in a newly established, liver-derived tumorigenic rat cell line (RMEC-1). By immunofluorescence microscopy, cell-cell contacts are characterized by mostly continuous-appearing lines which are usually resolved by electron microscopy as extended arrays of closely spaced small plaque subunits. These plaque-covered regions are positive for plakoglobin, alpha- and beta-catenin, the arm-repeat protein p120, vinculin, desmoplakin and protein ZO-1. They are positive for E-cadherin in cultures early on in passaging, but tend to turn negative for all known cadherins in densely grown cultures. On immunoblotting SDS-PAGE-separated proteins from dense-grown cell monolayers, "pan-cadherin" antibodies have reacted with a band at approximately 140 kDa, identified as N-cadherin by peptide fingerprinting of the immunoprecipitated protein, which for reasons not yet clear is modified or masked in immunolocalization experiments. The exact histological derivation of RMEC-1 cells is not known. However, the observations of several endothelial markers and the fact that all cells are rich in IFs containing vimentin and/or desmin, while only subpopulations also reveal IFs containing CKs 8 and 18, is suggestive of a mesenchymal, probably endothelial origin. We discuss the molecular relationship of this novel type of extended junction with other types of adhering junctions.