Aldo-Keto Reductase 1C1 (AKR1C1) as the First Mutated Gene in a Family with Nonsyndromic Primary Lipedema.

Lipedema is an often underdiagnosed chronic disorder that affects subcutaneous adipose tissue almost exclusively in women, which leads to disproportionate fat accumulation in the lower and upper body extremities. Common comorbidities include anxiety, depression, and pain. The correlation between mood disorder and subcutaneous fat deposition suggests the involvement of steroids metabolism and neurohormones signaling, however no clear association has been established so far. In this study, we report on a family with three patients affected by sex-limited autosomal dominant nonsyndromic lipedema. They had been screened by whole exome sequencing (WES) which led to the discovery of a missense variant p.(Leu213Gln) in AKR1C1, the gene encoding for an aldo-keto reductase catalyzing the reduction of progesterone to its inactive form, 20-α-hydroxyprogesterone. Comparative molecular dynamics simulations of the wild-type vs. variant enzyme, corroborated by a thorough structural and functional bioinformatic analysis, suggest a partial loss-of-function of the variant. This would result in a slower and less efficient reduction of progesterone to hydroxyprogesterone and an increased subcutaneous fat deposition in variant carriers. Overall, our results suggest that AKR1C1 is the first candidate gene associated with nonsyndromic lipedema.

TIE1 as a Candidate Gene for Lymphatic Malformations with or without Lymphedema.

TIE1 is a cell surface protein expressed in endothelial cells. Involved in angiogenesis and lymphangiogenesis, including morphogenesis of lymphatic valves, TIE1 is important for lymphatic system functional integrity. The main purpose of this study was to identify different variants in the TIE1 gene that could be associated with lymphatic malformations or dysfunction and predisposition for lymphedema. In a cohort of 235 Italian lymphedema patients, who tested negative for variants in known lymphedema genes, we performed a further test for new candidate genes, including TIE1. Three probands carried different variants in TIE1. Two of these segregated with lymphedema or lymphatic dysfunction in familial cases. Variants in TIE1 could contribute to the onset of lymphedema. On the basis of our findings, we propose TIE1 as a candidate gene for comprehensive genetic testing of lymphedema.

Increasing evidence of hereditary lymphedema caused by CELSR1 loss-of-function variants.

A whole exome sequencing approach was recently used to detect a CELSR1 truncating variant associated with lymphedema in a large pedigree. Since this first report, no other similar associations have been reported in the literature. Here, we present the genetic results of 95 probands tested using a next generation sequencing panel that covered all known lymphedema-associated genes, including CELSR1. Five out of 95 probands (5.3%) were found to carry novel loss-of-function variants in CELSR1. Family segregation studies were possible in four out of five probands and showed possible sex-specific differences: CELSR1 variants showed almost complete penetrance in females and were associated with early-onset lymphedema, whereas in males they showed incomplete penetrance and were associated with late onset of the condition. Since the percentage of lymphedema patients carrying CELSR1 variants is not negligible, we do not hesitate to recommend including this gene in routine genetic testing.

CDH5, a Possible New Candidate Gene for Genetic Testing of Lymphedema.

Background: Expressed by endothelial cells, CDH5 is a cadherin involved in vascular morphogenesis and in the maintenance of vascular integrity and lymphatic function. The main purpose of our study was to identify distinct variants of the CDH5 gene that could be associated with lymphatic malformations and predisposition for lymphedema. Methods and Results: We performed Next Generation Sequencing of the CDH5 gene in 235 Italian patients diagnosed with lymphedema but who tested negative for variants in known lymphedema genes. We detected six different variants in CDH5 five missense and one nonsense. We also tested available family members of the probands. For family members who carried the same variant as the proband, we performed lymphoscintigraphy to detect any lymphatic system abnormalities. Variants were modeled in silico. The results showed that CDH5 variants may contribute to the onset of lymphedema, although further in vitro studies are needed to confirm this hypothesis. Conclusions: Based on our findings, we propose CDH5 as a new gene that could be screened in patients with lymphedema to gather additional evidence.

FOXC2 disease-mutations identified in lymphedema-distichiasis patients cause both loss and gain of protein function.

Dominant mutations in the FOXC2 gene cause a form of lymphedema primarily of the limbs that usually develops at or after puberty. In 90-95% of patients, lymphedema is accompanied by distichiasis. FOXC2 is a member of the forkhead/winged-helix family of transcription factors and plays essential roles in different developmental pathways and physiological processes. We previously described six unrelated families with primary lymphedema-distichiasis in which patients showed different FOXC2 mutations located outside of the forkhead domain. Of those, four were missense mutations, one a frameshift mutation, and the last a stop mutation. To assess their pathogenic potential, we have now examined the subcellular localization and the transactivation activity of the mutated FOXC2 proteins. All six FOXC2 mutant proteins were able to localize into the nucleus; however, the frameshift truncated protein appeared to be sequestered into nuclear aggregates. A reduction in the ability to activate FOXC1/FOXC2 response elements was detected in 50% of mutations, while the remaining ones caused an increase of protein transactivation activity. Our data reveal that either a complete loss or a significant gain of FOXC2 function can cause a perturbation of lymphatic vessel formation leading to lymphedema.

Color-Doppler sonography in chronic venous insufficiency: what the radiologist should know.

Chronic venous insufficiency (CVI) is a pathologic condition caused by valvular incompetence, with or without associated venous outflow obstruction, which may affect both the superficial and the deep venous system, causing venous hypertension and stasis. The most common form of CVI is primary varicose veins due to the insufficiency of the saphenous system. Color-Doppler sonography (CDS) is actually the main diagnostic technique of imaging for CVI. In this article, we describe the anatomy, the technique, and the information necessary to the radiologist to perform CDS in chronic venous insufficiency. The knowledge of the venous anatomy is the cornerstone for an adequate sonographic examination. The venous network in the lower extremities is divided into three systems: superficial, deep, and perforating veins. Deep veins are "comitantes" to the corresponding arteries and run under the muscular fascia. Superficial veins course into the subcutaneous fat, superficially to the deep muscular fascia; the main superficial veins are the greater and lesser saphenous and their tributaries. Connection between the saphenous veins are defined as communicating veins. Superficial and deep veins are connected by perforating veins, with flow directed, under normal circumstances, from the superficial to the deep system. The main perforating are the Hunter in the mid thigh, the Dodd in the lower thigh, the Boyd in the upper calf, and the Cockett's in the middle and lower calf. Sonographic examination must be performed in the upright and supine position. Compression sonography and color and PW Doppler are systematically employed to assess the absence of deep venous thrombosis. Femoro-popliteal veins are evaluated with color and PW Doppler for valvular insufficiency with reflux by performing Valsalva maneuver and calf compression. The sapheno-femoral and sapheno-popliteal junctions are examined to identify type of junction, continence, accessory saphenous, and incompetent collaterals. Perforating veins are usually identified at the medial aspect of the thigh and at the medial, lateral, and posterior aspects of the leg. Outward flow (lasting more than 500 ms) in the perforating veins should be considered a sign of their incompetence. Several surgical and interventional procedures are now available for the treatment of the CVI, as follows: vein ligation and stripping, stab avulsion, endoluminal occlusion of the saphenous trunks, subfascial endoscopic perforator surgery, and valvuloplasty.

Teaching subfascial perforator veins surgery: survey on a 2-day hands-on course.

BACKGROUND: The present paper describes a training method with objective evaluation to enhance video-assisted surgical skills in subfascial endoscopic perforator veins surgery (SEPS). Training was scheduled during a 2-day intensive course. METHODS: Hands-on exercises were performed (i) on a simulator to assess whether specific training exercises were helpful in attainment of skills; (ii) on a known animal model that uses the swine abdominal wall and which allows practice in endoscopic dissection and perforator veins (PV) using appropriate instrumentation in an environment that is a reasonable surrogate for the human calf; and (iii) assisting a senior surgeon performing SEPS. Thirty surgeons without experience in SEPS were trained to perform a sequence of standardized drills connected with the SEPS technique. The SEPS simulator consisted of an artificially constructed subfascial space of the leg in which false perforator veins had to be localized, and cut. The participants performed a sequence of drills three times in order to improve their dexterity. The same exercises were then performed on a swine model. The model consisted of the arteries and veins penetrating the rectus fascia and passing into the overlying cutaneous trunci muscle and hypodermis on either side of the midline between the arch of the ribs cranially and the umbilicus caudally. Trainees were required to achieve operative space in the animal subcutaneous fat, to reach and identify the "perforating" subcutaneous vessels, and to interrupt some of them with a 5-mm clamp coagulator ultrasonic scalpel. The time required to perform each dexterity drill was recorded in seconds. Finally, the day after, trainees were asked to drive the senior operator during clinical SEPS performed on eight patients, suggesting the following manoeuvres in order to: (i) enter the subfascial space of the leg; (ii) make operative space; (iii) identify the incompetent perforator vein(s); and (iv) coagulate and divide them with the ultrasonic scalpel. Each of these four steps scored 1 point. RESULTS: All the trainees showed a steady improvement in skill acquisition on the SEPS simulator (P < 0.001), and on the animal model with the single-port technique (P < 0.001). These results reflect positively on the animal model using the dual-port technique, and on the scores achieved in the operating theatre during clinical SEPS. CONCLUSIONS: The validity of the 2-day course was demonstrated by significant improvement in performance with increasing skill on the training models, and in clinical practice.