Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing.

BACKGROUND: The filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated. METHODS AND RESULTS: For the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification. CONCLUSIONS: Taken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders.

Dietary supplements in lymphedema.

Lymphedema is a chronic inflammatory disorder resulting from ineffective fluid uptake by the lymphatic system, and the effects are principally felt in the lower limbs. The condition is said to be primary when caused by genetic mutations and secondary when caused by injuries, infections, or surgery. Lymphedema, a worldwide pathology, does not have an effective therapy so far. Leukotriene B4 has recently been identified as a key molecule in lymphedema pathogenesis. Surgical, nonsurgical, and pharmacological treatments have been proposed; however, they do not cure the disease and only ameliorate the symptoms. Nutrition and nutritional status are extremely important in lymphedema physiopathology. Obesity is a comorbidity that exacerbates the risk for secondary lymphedema and constitutes a negative prognostic factor. Indeed, anti-inflammatory foods and their effects on the inflammatory state and on oxidative stress are now being investigated for their possible therapeutic role in lymphedema. Although no special diet has so far been proven to be very effective, specific dietary tips could help in alleviating the edematous state of patients with lymphedema. A few supplements have been tested for lymphedema treatment. Among them, GARLIVE® containing hydroxytyrosol, hesperidin, spermidine and vitamin A, exhibited promising effects in the animal model. Hydroxytyrosol, a polyphenol from olives, showed anti-inflammatory effects and reduced leukotriene B4 synthesis, thus holding promise as a potential natural candidate for lymphedema treatment.

Dietary supplements for lipedema.

Lipedema is a chronic disease that mostly manifests in females as the abnormal distribution of subcutaneous adipose connective tissue, usually coupled with bruising, pain, and edema. Lipedema molecular pathophysiology is currently not clear, but several studies suggest that genetics and hormonal imbalance participate in lipedema pathogenesis. Women with lipedema present in some cases with elevated body mass index, and the appearance of obesity in addition to lipedema, where the obesity can cause serious health issues as in lipedema-free individuals with obesity, such as diabetes and cardiovascular disorders. Unlike obesity, lipedema tissue does not respond well to diet or physical exercise alone. Therefore, in this review we discuss the effect of various dietary supplements that, along with diet and physical exercise, cause fat burning and weight loss, and which could potentially be important in the treatment of lipedema. Indeed, an effective fat burner should convert stored fats into energy, mobilize and break down triglycerides in adipocytes, boost metabolism and inhibit lipogenesis. Common ingredients of fat burning supplements are green tea, caffeine, chromium, carnitine, and conjugated linoleic acid. The use of fat burners could act synergistically with a healthy diet and physical exercise for decreasing adipose tissue deposition in patients with lipedema and resolve related health issues. The effects of fat burners in human studies are sometimes contradictory, and further studies should test their effectiveness in treating lipedema.

Low Efficacy of Genetic Tests for the Diagnosis of Primary Lymphedema Prompts Novel Insights into the Underlying Molecular Pathways.

Lymphedema is a chronic inflammatory disorder caused by ineffective fluid uptake by the lymphatic system, with effects mainly on the lower limbs. Lymphedema is either primary, when caused by genetic mutations, or secondary, when it follows injury, infection, or surgery. In this study, we aim to assess to what extent the current genetic tests detect genetic variants of lymphedema, and to identify the major molecular pathways that underlie this rather unknown disease. We recruited 147 individuals with a clinical diagnosis of primary lymphedema and used established genetic tests on their blood or saliva specimens. Only 11 of these were positive, while other probands were either negative (63) or inconclusive (73). The low efficacy of such tests calls for greater insight into the underlying mechanisms to increase accuracy. For this purpose, we built a molecular pathways diagram based on a literature analysis (OMIM, Kegg, PubMed, Scopus) of candidate and diagnostic genes. The PI3K/AKT and the RAS/MAPK pathways emerged as primary candidates responsible for lymphedema diagnosis, while the Rho/ROCK pathway appeared less critical. The results of this study suggest the most important pathways involved in the pathogenesis of lymphedema, and outline the most promising diagnostic and candidate genes to diagnose this disease.

LIMPRINT in Italy.

Background: To define the profile of patients presenting with chronic edema (CE) in three centers in Italy (Lymphoedema IMpact and PRevalence INTernational). Methods and Results: Data were collected in patients referred for CE between September 2016 and July 2017. A total of 1637 were recruited, 86.7% (1419) outpatients and 13.3% (218) inpatients with 80.6% (1319) female and mean age 54 years. Primary lymphedema occurred in 28.2% (461). In the 71.8% (1176) with secondary CE cancer occurred in 72% (846) and 28% (330) due to other causes. Data showed that 84.2% (226) had full upper body mobility, 15.5% (41) had limited mobility and 0.2% (2) had lost all mobility. Lower limb mobility status: 90.4% (1205) complete mobility, 8.4% (112) reduced mobility and 1.2% (21) wheelchair bound. Concurrent leg ulceration occurred in 32.9% (322) with 3.1% (51) having antibiotics. Treatment patterns varied with only 32.4% (530) receiving instructions in skin care, 61.2% (1002) multilayer compression and a further 67.8% (1110) compression garment with 17.6% (288) having sequential pressure therapy. Only 1.4% (23) had received psychological support. Out of the total 481/1637 (29.4%) were not prescribed any treatment. Only 50.4% (825) had access to subsidized treatments within the National and Regional Health Care System, whereas 49.6% (81) had to pay themselves with only half (50.9%) having access to treatment centers that were near their home. Conclusion: Results from this study and active lobbying have led to changes in reimbursement of care for primary and secondary lymphedema in Italy; this has led to a much more optimistic picture for those affected.

Genetic Determinants of the Effects of Training on Muscle and Adipose Tissue Homeostasis in Obesity Associated with Lymphedema.

It is widely accepted that metabolic changes associated with training are influenced by a person's genetic background. In this review, we explore the polymorphisms underlying interindividual variability in response to training of weight loss and muscle mass increase in obese individuals, with or without lymphedema, and in normal-weight subjects. We searched PubMed for articles in English published up to May 2019 using the following keywords: (((physical training[Title/Abstract] OR sport activity[Title/Abstract]) AND predisposition[Title/Abstract]) AND polymorphism [Title/Abstract]). We identified 38 single-nucleotide polymorphisms that may modulate the genetic adaptive response to training. The identification of genetic marker(s) that improve the beneficial effects of training may in perspective make it possible to assess training programs, which in combination with dietary intervention can optimize body weight reduction in obese subjects, with or without lymphedema. This is particularly important for patients with lymphedema because obesity can worsen the clinical status, and therefore, a personalized approach that could reduce obesity would be fundamental in the clinical management of lymphedema.

Study of a supplement and a genetic test for lymphedema management.

Malformations in the lymphatic vasculature, injury, surgery, trauma or toxic damage may lead to swelling of the limbs caused by inefficient lymphatic uptake and flow (lymphedema). Lymphedema can be congenital or acquired. Primary lymphedema is rare and caused by mutations in single genes, secondary lymphedema is more common and caused by a trauma in association with a genetic predisposition. We decided to develop a genetic test that would determine the genetic predisposition to the onset of lymphedema and to predict the course of the disease by analyzing polymorphisms involved in leukotriene B4 (LTB4) synthetic pathway, and variants involved in the onset of secondary lymphedema. There are not many compounds available for the treatment of the negative effects of lymph accumulation, we therefore designed a food supplement based on the hydroxytyrosol, that has anti-oxidant, anti-bacterial and anti-inflammatory activities.

The direct renin inhibitor aliskiren improves vascular remodelling in transgenic rats harbouring human renin and angiotensinogen genes.

In the present study, we tested the hypothesis that chronic treatment with the direct rennin inhibitor aliskiren improves the remodelling of resistance arteries in dTGR (double-transgenic rats). dTGR (5 weeks) were treated with aliskiren (3 mg/kg of body mass per day) or ramipril (1 mg/kg of body mass per day) for 14 days and compared with age-matched vehicle-treated dTGR. BP (blood pressure) was similarly reduced in both aliskiren-treated and ramipril-treated rats compared with control dTGR (167±1 and 169±2 mmHg compared with 197±4 mmHg respectively; P<0.05). The M/L (media-to-lumen) ratio assessed on pressurized preparations was equally reduced in aliskiren-treated and ramipril-treated rats compared with controls (6.3±0.5 and 6.4±0.2% compared with 9.8±0.4% respectively; P<0.05). Endothelium-dependent and -independent relaxations were similar among the groups. L-NAME (N(G)-nitro-L-arginine methyl ester) significantly reduced acetylcholine-induced dilation in drug-treated dTGR. This effect was significantly more prominent in aliskiren-treated rats. eNOS (endothelial NO synthase) expression showed a 2-fold increase only in aliskiren-treated dTGR as compared with controls (P<0.01) and ramipril-treated dTGR (P<0.05). Plasma nitrite, as an index of NO production, was significantly increased in dTGR treated with either aliskiren or ramipril compared with controls. Only aliskiren induced a 2-fold increase in plasma nitrite, which was significantly greater than that induced by ramipril (P<0.05). gp91(phox) expression and ROS (reactive oxygen species) production in aorta were significantly and similarly reduced by both drugs. In conclusion, equieffective hypotensive doses of aliskiren or ramipril reduced the M/L ratio of mesenteric arteries and improved oxidative stress in dTGR. However, only aliskiren increased further NO production in the vasculature. Hence, in dTGR, direct renin inhibition induces favourable effects similar to that induced by ACE (angiotensin-converting enzyme) inhibition in improving vascular remodelling through different mechanisms.