Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I.

Neurofibromatosis type I, a genetic condition due to pathogenic variants in the NF1 gene, is burdened by a high rate of complications, including neoplasms, which increase morbidity and mortality for the disease. We retrospectively re-evaluated the NF1 gene variants found in the period 2000-2019 and we studied for genotype/phenotype correlations of disease complications and neoplasms 34 variants, which were shared by at least two unrelated families (range 2-11) for a total 141 of probands and 21 relatives affected by Neurofibromatosis type I. Recurrent variants could be ascribed to the most common mutational mechanisms (C to T transition, microsatellite slippage, non-homologous recombination). In genotype/phenotype correlations, the variants p.Arg440*, p.Tyr489Cys, and p.Arg1947*, together with the gross gene deletions, displayed the highest rates of complications. When considering neoplasms, carriers of variants falling in the extradomain region at the 5' end of NF1 had a lower age-related cancer frequency than the rest of the gene sequence, showing a borderline significance (p = 0.045), which was not conserved after correction with covariates. We conclude that (1) hotspots in NF1 occur via different mutational mechanisms, (2) several variants are associated with high rates of complications and cancers, and (3) there is an initial evidence toward a lower cancer risk for carriers of variants in the 5' end of the NF1 gene although not significant at the multivariate analysis.

Patterns of Novel Alleles and Genotype/Phenotype Correlations Resulting from the Analysis of 108 Previously Undetected Mutations in Patients Affected by Neurofibromatosis Type I.

Neurofibromatosis type I, a genetic disorder due to mutations in the NF1 gene, is characterized by a high mutation rate (about 50% of the cases are de novo) but, with the exception of whole gene deletions associated with a more severe phenotype, no specific hotspots and few solid genotype/phenotype correlations. After retrospectively re-evaluating all NF1 gene variants found in the diagnostic activity, we studied 108 patients affected by neurofibromatosis type I who harbored mutations that had not been previously reported in the international databases, with the aim of analyzing their type and distribution along the gene and of correlating them with the phenotypic features of the affected patients. Out of the 108 previously unreported variants, 14 were inherited by one of the affected parents and 94 were de novo. Twenty-nine (26.9%) mutations were of uncertain significance, whereas 79 (73.2%) were predicted as pathogenic or probably pathogenic. No differential distribution in the exons or in the protein domains was observed and no statistically significant genotype/phenotype correlation was found, confirming previous evidences.

Genomic Prediction and Genome-Wide Association Study for Boar Taint Compounds.

With a perspective future ban on surgical castration in Europe, selecting pigs with reduced ability to accumulate boar taint (BT) compounds (androstenone, indole, skatole) in their tissues seems a promising strategy. BT compound concentrations were quantified in the adipose tissue of 1075 boars genotyped at 29,844 SNPs. Traditional and SNP-based breeding values were estimated using pedigree-based BLUP (PBLUP) and genomic BLUP (GBLUP), respectively. Heritabilities for BT compounds were moderate (0.30-0.52). The accuracies of GBLUP and PBLUP were significantly different for androstenone (0.58 and 0.36, respectively), but comparable for indole and skatole (~0.43 and ~0.47, respectively). Several SNP windows, each explaining a small percentage of the variance of BT compound concentrations, were identified in a genome-wide association study (GWAS). A total of 18 candidate genes previously associated with BT (MX1), reproduction traits (TCF21, NME5, PTGFR, KCNQ1, UMODL1), and fat metabolism (CTSD, SYT8, TNNI2, CD81, EGR1, GIPC2, MIGA1, NEGR1, CCSER1, MTMR2, LPL, ERFE) were identified in the post-GWAS analysis. The large number of genes related to fat metabolism might be explained by the relationship between sexual steroid levels and fat deposition and be partially ascribed to the pig line investigated, which is selected for ham quality and not for lean growth.

Infrared Predictions Are a Valuable Alternative to Actual Measures of Dry-Cured Ham Weight Loss in the Training of Genome-Enabled Prediction Models.

Selection to reduce ham weight losses during dry-curing (WL) requires individual traceability of hams throughout dry-curing, with high phenotyping costs and long generation intervals. Infrared spectroscopy enables cost-effective, high-throughput phenotyping for WL 24 h after slaughter. Direct genomic values (DGV) of crossbred pigs and their purebred sires were estimated, for observed (OB) and infrared-predicted WL (IR), through models developed from 640 and 956 crossbred pigs, respectively. Five Bayesian models and two pseudo-phenotypes (estimated breeding value, EBV, and adjusted phenotype) were tested in random cross-validation and leave-one-family-out validation. The use of EBV as pseudo-phenotypes resulted in the highest accuracies. Accuracies in leave-one-family-out validation were much lower than those obtained in random cross-validation but still satisfactory and very similar for both traits. For sires in the leave-one-family-out validation scenario, the correlation between the DGV for IR and EBV for OB was slightly lower (0.32) than the correlation between the DGV for OB and EBV for OB (0.38). While genomic prediction of OB and IR can be equally suggested to be incorporated in future selection programs aiming at reducing WL, the use of IR enables an early, cost-effective phenotyping, favoring the construction of larger reference populations, with accuracies comparable to those achievable using OB phenotype.

Concentrations of Boar Taint Compounds Are Weakly Associated with Sexual Behavior of Young Boars.

Strategies to control boar taint (BT) in meat relies on the reduction of skatole, indole, and androstenone concentration. This might have unfavorable effects on the libido of breeding boars. The association between BT compound concentration in backfat and libido was investigated in 391 commercial breeding boars. Six sexual behavior traits (SBT; sexual arousal, salivation, mounting performance, interest in the dummy sow, penis unsheathing, and overall libido score) were scored during the training of the boars with the dummy sow. Variation in SBT was analyzed by proportional-odds cumulative logistic models. Overall, indole, skatole, and androstenone concentrations were weakly associated with libido. Farm of origin, age at training or body weight, and BT compound levels were poor predictors of boar performance (the area under the ROC curve ranged from 0.60 to 0.69). This indicates that BT compound concentrations were weakly associated with libido, even though the probability of observing good SBT scores increased with high levels of androstenone, intermediate or low levels of skatole, and intermediate to high levels of indole. Hence, practices aiming at reducing androstenone, and controlling the concentrations of skatole and indole to intermediate levels are not expected to impair the libido of young boars.

Role of adipose-derived stem cells in chronic cutaneous wound healing.

AIM: Adipose tissue represent an alternative source of multipotent stem cells with characteristics similar to bone marrowderived mesenchymal stem cells (BMSCs), easier to isolate and effective in wound healing enhancement. MATERIAL OF STUDY: Each patient being considered for stem cells graft was subjected to a conventional liposuction procedure (local anaesthesia, aspirated volume about 80cc), in order to isolate a pellet of adipose-derived stem cells (ASCs), which was then mixed with the platelet-rich plasma (PRP) previously collected, in order to obtain an enhanced-ASCs- PRP (e-PRP), now ready for grafting in the context of skin edges as well as at the bottom of the lesion itself. RESULTS: Flow cytometric analysis performed on the pellet obtained with our isolation process, showed that a mean of 5 x 105 ASCs (range: 4,0-6,0 x 105, SD: ± 1 x 105) were collected from 80 ml of adipose tissue, harvested with standard wet liposuction procedure. It represented the 5% of all sample cells (1 x 107), while the others 95% were mostly being blood-derived and endothelial cells. DISCUSSION: By now, the most used isolation protocols take about two hours due to the complex isolation procedure, requiring both animal-derived reagents and collagenase. The amount of ASCs obtained with our isolation process is sufficient to be directly engrafted in the wound without the need of in vitro expansion but, neither serum nor animalderived reagents are used, and it takes only 15' minutes. CONCLUSION: ASCs application is an innovative, effective approach in the treatment of chronic wounds.

Antiangiogenic versus cytotoxic therapeutic approaches to human pancreas cancer: an experimental study with a vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor and gemcitabine.

Pancreatic adenocarcinoma is a leading cause of cancer death in the United States and represents a challenging chemotherapeutic problem. The pharmacological control of angiogenesis might represent a novel approach to the management of pancreas cancer, since the pathological development of vascular supply is a critical step for tumor growth and may affect its prognosis. In order to test this hypothesis, SU5416 ([3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one]) a selective inhibitor of the vascular endothelial growth factor receptor-2 tyrosine kinase, and gemcitabine (2', 2'-difluorodeoxycytidine) were tested on endothelial (HUVEC) and pancreatic tumor cells (MIA PaCa-2) in vitro and in vivo alone and in simultaneous association. SU5416 inhibited HUVEC cells stimulated to proliferate by vascular endothelial growth factor but not MIA PaCa-2 cells; the drug concentration that decreased cell growth by 50% (IC50) was 0.14 microM. Furthermore, SU5416 reduced the development of microvessels from placental explants (IC50, 0.23 microM). Gemcitabine inhibited the growth of both HUVEC and MIA PaCa-2 cells with an IC50 of 0.08 and 0.1 microM, respectively. A synergistic effect (combination index <1 and dose reduction index >1) on anti-proliferative and pro-apoptotic activity was calculated with the simultaneous combination of the two drugs on endothelial cells. A marked in vivo antitumor effect on MIA PaCa-2 xenografts was observed with SU5416 at a protracted schedules, as well as with gemcitabine; furthermore, the combination between the two drugs resulted in an almost complete suppression of tumor growth and relapse. In conclusion, the present results provide the evidence of an effective anti-endothelial/antitumor activity of protracted administration of SU5416 on human pancreas cancer xenografts, which is comparable with the one obtained by gemcitabine; moreover, the synergistic combination between these drugs on endothelial cells and the promising association in pancreatic cancer xenografts could be used in future studies and translated into the clinical setting.

Angiogenesis and nerve regeneration in a model of human skin equivalent transplant.

The angiogenesis and reinnervation were studied in a porcine model of human skin equivalent (SE) graft and the relationship between the two processes was investigated. Confocal laser scanning microscopy was used to monitor, during the healing process, the pattern of vascularization and reinnervation at different time points. The SE was obtained by co-culturing fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan biopolymer and grafted on dorsal wounds generated by full-thickness resection in 25/30 Kg Large white pigs. Frozen sections were obtained from biopsies performed in autograft and xenograft, then were immunolabeled by using the endothelial marker lectin Lactifolia and with the neuronal marker gene product PGP9.5. Cajal staining was also used to visualize the nerve fibers. The results show that the vascularization precedes the innervation process. These data are consistent with the view that the development of nervous tissue is driven by nutritional and trophic factors provided by the vascular system. The arborization of the two systems observed during the third week from the graft might play a key role in maintaining the healing process and the graft survival.

Cutaneous tissue engineering and lower extremity wounds (part 1).

Tissue-engineered skin is a novel therapeutic with which difficult-to-heal lower extremity wounds may be treated. Such skins are products of cutaneous tissue engineering that provide an alternative for autologous or allogeneic tissue transplantation, thereby avoiding problems associated with donor site availability, the risk of infection, and scarring. Recently developed tissue-engineered skin equivalents have shown to be superior in certain ways to compression therapy for refractory venous ulcers and acute wounds. These biologic products behave similarly to autografts.

Endoscopic forehead surgery for migraine therapy Personal technique.

AIM: The aim of this study is to prove the therapeutic effectiveness of nerve decompression, performed endoscopically for frontal migraine and by open surgery for occipital migraine. MATERIALS AND METHODS: Twenty patients were enrolled and underwent surgery for endoscopic resection of the glabellar muscle group, including the corrugator supercilii, depressor supercilii, and procerus muscles, while the occipital decompression was performed in open surgery through decompression of occipital nerves from occipital, semispinalis capitis, trapezius and sternocleidomastoid muscles. Every patient was diagnosed with: migraine without aura, chronic tensiontype headache and new daily persistent headache, refractory to medical management. RESULTS: Analyzing the answers given by the patients to validated questionnaires, 9 referred alleviation of migraine symptoms (45%), 8 described elimination of their migraine headaches (40%) while 3 didn't report any improvement. DISCUSSION: Our data confirmed the results of previous studies, pointing out the effectiveness of trigeminal branches and occipital nerves (trigger points) decompression from the surrounding muscles. Moreover, our technique has the same results but it's less invasive and has less collateral effects. CONCLUSIONS: Our results highlight migraine surgery as an effective treatment for patients with migraine headaches who do not tolerate or do not wish to continue medical interventions. KEY WORDS: Endoscopic surgery, Headache migraine.