A Copernican revolution of multigenic analysis: A retrospective study on clinical exome sequencing in unclear genetic disorders.

Despite the inevitable shift in medical practice towards a deeper understanding of disease etiology and progression through multigenic analysis, the profound historical impact of Mendelian diseases cannot be overlooked. These diseases, such as cystic fibrosis and thalassemia, are characterized by a single variant in a single gene leading to clinical conditions, and have significantly shaped our medical knowledge and treatments. In this respect, the monogenic approach inevitably results in the underutilization of Next-Generation Sequencing (NGS) data. Herein, a retrospective study was performed to assess the diagnostic value of the clinical exome in 32 probands with specific phenotypic characteristics (patients with autoinflammation and immunological dysregulation, N = 20; patients diagnosed with Hemolytic uremic syndrome N = 9; and patients with Waldenström macroglobulinemia, N = 3). A gene enrichment analysis was performed using the *. VCF file generated by SOPHiA-DDM-v4. This analysis selected a subset of genes containing pathogenic or likely pathogenic variants with autosomal dominant (AD) inheritance. In addition, all variants of uncertain significance (VUS) were included, filtered by AD inheritance mode, the presence of compound heterozygotes, and a minor allele frequency (MAF) cutoff of 0.05 %. The aim of the pipeline described here is based on a perspective shift that focuses on analyzing patients' gene assets, offering new light on the complex interplay between genetics and disease presentation. Integrating this approach into clinical practices could significantly enhance the management of patients with rare genetic disorders.

Topical anesthesia versus topical and subconjunctival anesthesia combined in diabetic retinopathy photocoagulation.

PURPOSE: comparison between two anesthetic techniques on the ability to reduce pain during panretinal photocoagulation (PRP) treatment. METHODS: Observational retrospective single center study. Medical charts of patients who underwent PRP for proliferative diabetic retinopathy were revised. Patients were included if they had the first eye treated with oxybuprocaine hydrochloride drops, and in case of severe pain, the fellow eye received topical anesthesia in combination with 2% subconjunctival lidocaine. The groups were compared for pain perception using an analog visual scale (VAS), number of laser spots, number of interruptions, and laser session duration. RESULTS: Forty-two eyes of 21 patients (mean age: 58.3 ± 7.6 years) were analyzed. The mean number of laser spots was significantly higher under combined anesthesia (+84.2 ± 155.9 spots, p = 0.01), with a reduced time for laser execution (-2.5 ± 3.12, p = 0.0008). The use of combined anesthesia significantly decreased the number of interruptions (-40.8%, p < 0.0001) into a single session. On the pain grading scale, the pain perception was significantly lower in the combined anesthesia group (p < 0.0001). In eyes receiving topical anesthesia the treatment was stopped for pain in 5 eyes (23.8%), while 5 eyes under combined anesthesia presented subconjunctival hemorrhage (23.8%). CONCLUSION: Using combined anesthesia in patients subjected to PRP appeared to reduce pain perception limiting the treatment duration and the interruptions for pain without significant complications. Further studies on a larger scale would be desirable to replicate such findings and standardize the analgesic procedures in ophthalmology.

Gender-related differences in patients with carcinoid syndrome: new insights from an Italian multicenter cohort study.

BACKGROUND: The incidence of neuroendocrine neoplasm (NEN) and related carcinoid syndrome (CaS) has increased markedly in recent decades, and women appear to be more at risk than men. As per other tumors, gender may be relevant in influencing the clinical and prognostic characteristics of NEN-associated CS. However, specific data on carcinoid syndrome (CaS) are still lacking. PURPOSE: To evaluate gender differences in clinical presentation and outcome of CaS. METHODS: Retrospective analysis of 144 CaS patients from 20 Italian high-volume centers was conducted. Clinical presentation, tumor characteristics, therapies, and outcomes (progression-free survival, PFS, overall survival, OS) were correlated to gender. RESULTS: Ninety (62.5%) CaS patients were male. There was no gender difference in the site of primary tumor, tumor grade and clinical stage, as well as in treatments. Men were more frequently smokers (37.2%) and alcohol drinkers (17.8%) than women (9.5%, p = 0.002, and 3.7%, p = 0.004, respectively). Concerning clinical presentation, women showed higher median number of symptoms (p = 0.0007), more frequent abdominal pain, tachycardia, and psychiatric disorders than men (53.3% vs 70.4%, p = 0.044; 6.7% vs 31.5%, p = 0.001; 50.9% vs. 26.7%, p = 0.003, respectively). Lymph node metastases at diagnosis were more frequent in men than in women (80% vs 64.8%; p = 0.04), but no differences in terms of PFS (p = 0.51) and OS (p = 0.64) were found between gender. CONCLUSIONS: In this Italian cohort, CaS was slightly more frequent in males than females. Gender-related differences emerged in the clinical presentation of CaS, as well as gender-specific risk factors for CaS development. A gender-driven clinical management of these patients should be advisable.

Update on bioethical, medical and fertility issues in gender incongruence during transition age.

PURPOSE: Many issues still remain unresolved in the management of pubertal patients with gender incongruence (GI). The aim of this review is to discuss the main aspects of the treatment of these patients to provide a practical approach for clinicians. METHODS: A comprehensive literature search within PubMed was performed to provide updates of available evidence regarding the impact on bioethical, medical and fertility issues in gender incongruence during transition age. RESULTS: Gender Affirming Hormone Treatment (GAHT) and Gender Affirming Surgery (GAS) can induce unsatisfaction with change, future regrets, and the risk of infertility. This raises ethical issues especially in the management of pubertal patients that remain unresolved. Therapy with GnRH analogues (GnRHa) is intended to delay puberty, so as to give the adolescent a longer period of time to decide whether to continue with the treatments. At the level of physical changes, this therapy may have an effect on bone mineralization and body composition; however, long-term longitudinal data are not yet available. An important feature related to the use of GnRHa is the risk of fertility. Gamete cryopreservation is the most established method of fertility preservation (FP) and should be counselled to transgender adolescents. However, these patients are not always interested in having biological children. CONCLUSION: Based on the current evidence, there is a need to conduct further research to clarify certain issues and to standardize clinical practice and improve counselling in transgender adolescent decision making and avoid regrets in the future.

Neuroendocrine neoplasms in the context of inherited tumor syndromes: a reappraisal focused on targeted therapies.

PURPOSE: Neuroendocrine neoplasms can occur as part of inherited disorders, usually in the form of well-differentiated, slow-growing tumors (NET). The main predisposing syndromes include: multiple endocrine neoplasias type 1 (MEN1), associated with a large spectrum of gastroenteropancreatic and thoracic NETs, and type 4 (MEN4), associated with a wide tumour spectrum similar to that of MEN1; von Hippel-Lindau syndrome (VHL), tuberous sclerosis (TSC), and neurofibromatosis 1 (NF-1), associated with pancreatic NETs. In the present review, we propose a reappraisal of the genetic basis and clinical features of gastroenteropancreatic and thoracic NETs in the setting of inherited syndromes with a special focus on molecularly targeted therapies for these lesions. METHODS: Literature search was systematically performed through online databases, including MEDLINE (via PubMed), and Scopus using multiple keywords' combinations up to June 2022. RESULTS: Somatostatin analogues (SSAs) remain the mainstay of systemic treatment for NETs, and radiolabelled SSAs can be used for peptide-receptor radionuclide therapy for somatostatin receptor (SSTR)-positive NETs. Apart of these SSTR-targeted therapies, other targeted agents have been approved for NETs: the mTOR inhibitor everolimus for lung, gastroenteropatic and unknown origin NET, and sunitinib, an antiangiogenic tyrosine kinase inhibitor, for pancreatic NET. Novel targeted therapies with other antiangiogenic agents and immunotherapies have been also under evaluation. CONCLUSIONS: Major advances in the understanding of genetic and epigenetic mechanisms of NET development in the context of inherited endocrine disorders have led to the recognition of molecular targetable alterations, providing a rationale for the implementation of treatments and development of novel targeted therapies.

Risk factors on testicular function in adolescents.

PURPOSE: Adolescence represents an important window for gonadal development. The aim of this review is to carry out a critical excursus of the most recent literature on endogenous and exogenous risk factors related to testicular function, focusing the research on adolescence period. METHODS: A comprehensive literature search within PubMed was performed to provide a summary of currently available evidence regarding the impact on adolescence of varicocele, cryptorchidism, cancer, diabetes, lifestyle factors, endocrine disruptors, obesity and sexually transmitted diseases. We focused on human studies that evaluated a possible impact of these factors on puberty timing and their effects on andrological health. RESULTS: Evidence collected seems to suggest that andrological health in adolescence may be impaired by several factors, as varicocele, cryptorchidism, and childhood cancer. Despite an early diagnosis and treatment, many adolescents might still have symptoms and sign of a testicular dysfunction in their adult life and at the current time it is not possible to predict which of them will experience andrological problems. Lifestyle factors might have a role in these discrepancies. Most studies point out towards a correlation between obesity, insulin resistance, alcohol, smoking, use of illegal drugs and testicular function in pubertal boys. Also, endocrine disruptors and sexually transmitted diseases might contribute to impair reproductive health, but more studies in adolescents are needed. CONCLUSION: According to currently available evidence, there is an emerging global adverse trend of high-risk and unhealthy behaviors in male adolescents. A significant proportion of young men with unsuspected and undiagnosed andrological disorders engage in behaviors that could impair testicular development and function, with an increased risk for later male infertility and/or hypogonadism during the adult life. Therefore, adolescence should be considered a key time for intervention and prevention of later andrological diseases.

Symptoms of Orthorexia Nervosa are associated with time spent on social media: a web-based survey in an Italian population sample.

OBJECTIVE: Orthorexia Nervosa (ON) is an eating disorder of growing interest that is characterized by an obsession with healthy eating. Nowadays, people spend an increasing amount of time on social media, which may negatively impact eating behaviors. The aim of this study was to investigate the relationship between social media usage and risk of ON. SUBJECTS AND METHODS: We conducted an online survey using the 10-item Italian-Düsseldorf Orthorexia Scale questionnaire (I-DOS). A total of 4,107 individuals participated and were classified according to sex, age, education level, marital status, BMI, main occupation, and diet. RESULTS: The prevalence of ON was 28.5%. Participants who reported using social media for over 60 minutes per day had a higher prevalence of ON than those using social media for less than 15 minutes per day. CONCLUSIONS: The results of this study suggest that longer time spent on social media is associated with ON.

Short Report - Medical nutrition therapy for critically ill patients with COVID-19.

No Abstract Available.

FTO rs9939609 influence on adipose tissue localization in the Italian population.

OBJECTIVE: Among the genes involved in obesity, the Fat mass and obesity-associated gene (FTO) is certainly one of the most known and the relation between FTO rs9939609 and BMI is highly discussed; nevertheless, data about its influence on body composition are limited. MATERIALS AND METHODS: We carried out a study on a sample of 1066 Italian subjects, whose body composition and FTO rs9939609 were analyzed. RESULTS: We found significant relations between FTO with arm (p=0.01), abdomen (p=0.00), and trunk circumferences (p=0.00), BMI (p=0.01), FM% (p=0.00), and android FM% (p=0.01), whereas no relations were found between FTO and both gynoid fat and lean mass. CONCLUSIONS: To conclude, the relation between FTO and BMI is confirmed and is related specifically with android FM%. These results indicated that FTO rs9939609 may be a genetic etiological factor for obesity. Indeed, the specificity for the android FM% would indicate FTO as an etiological factor in the development of cardiovascular diseases.

The role of IL-6 gene polymorphisms in the risk of lipedema.

OBJECTIVE: Lipedema is a disorder of adipose tissue characterized by abnormal subcutaneous fat deposition, leading to swelling and enlargement of the lower limbs and trunk. The aim of this study was to evaluate the lipedema phenotype by investigating the role of polymorphisms related to IL-6 (rs1800795) gene in people with diagnosis of lipedema. The second aim was to identify indicators of body composition, useful for a differential analysis between subjects with lipedema and the control group. PATIENTS AND METHODS: Two groups are involved in the study, 45 women with lipedema (LIPPY) and 50 women randomly chosen from the population as Control (CTRL). Clinical and demographical variables recorded include weight, height, body mass index (BMI) and circumference measurements. Body composition (Fat mass, FM; lean mass, LM) was assessed by Dual-energy X-ray Absorptiometry (DXA). The genetic tests for IL-6 (rs18oo795) gene were performed for both groups, using a saliva sample. RESULTS: The study of the relationship between the IL-6 (rs1800795) gene polymorphism, the anthropometric values and the body composition indices has provided the following significant results: subjects with diagnosis of lipedema present statistically significant increased values with regard to weight, BMI, waist, abdomen and hip circumferences, arms, legs and whole FM (% and kg), gynoid FM (kg), legs LM (kg) and ASMMI. Moreover, the value of the waist hip ratio was found to be decreased. CONCLUSIONS: For the first time, we suggested that IL-6 gene polymorphism could characterize subjects with lipedema respect to Normal Weight Obese and obese subjects. The intra-group comparisons (LIPPY carriers vs. LIPPY non-carriers and CTRL carriers vs. CTRL non-carriers) showed no statistically significant values. In contrast, the inter-group comparisons (LIPPY non-carriers vs. CTRL non-carriers and LIPPY carriers vs. CTRL carriers) resulted statistically significant. We have identified other indices, such as leg index, trunk index, abdominal index, total index, that could be promising clinical tools for diagnosis of the lipedema phenotype and for predicting the evolution of the disease.

Quantification of the Leydig cell compartment in testicular biopsies and association with biochemical Leydig cell dysfunction in testicular cancer survivors.

A simple histological method to evaluate the Leydig cell compartment is lacking. We aimed to establish such a method and to investigate if Leydig cell hyperplasia of the biopsy contralateral to the tumour-bearing testicle in patients with testicular germ cell cancer is associated with biochemical signs of Leydig cell dysfunction after long-term follow-up. A case group of 50 long-term testicular germ cell cancer survivors without human chorionic gonadotropin elevation, 10 testicular germ cell cancer patients with elevated human chorionic gonadotropin and 10 controls without testicular malignancy were included. For each subject, 2-4 representative sections from their testicular biopsies were selected for analysis. Using the image processing program ImageJ (V.1.48, NIH), an area with a minimum of 50 tubules was selected and delineated (total selected area) and the total Leydig cell area was calculated by adding up every delineated Leydig cell group within the total selected area. Four different methods were tested for the ability to quantify the Leydig cell compartment. In the 50 testicular germ cell cancer survivors, associations between the area of the Leydig cell compartment and serum levels of testosterone and luteinising hormone were investigated using linear regression analysis. The Leydig cell compartment was best quantified by the total Leydig cell area/total selected area index, which was significantly larger in the human chorionic gonadotropin-positive patients than in controls (P = 0.00001). In the 50 human chorionic gonadotropin-negative testicular germ cell cancer survivors, increasing total Leydig cell area/total selected area was significantly associated with decreased levels of total testosterone and decreased total testosterone/luteinising hormone ratio after a median of 9-year follow-up. In conclusion, a new simple method, total Leydig cell area/total selected area, was established to estimate the Leydig cell compartment in testicular biopsies. The index identified Leydig cell hyperplasia in the contralateral biopsy in patients with testicular germ cell cancer, and it was associated with long-term biochemical Leydig cell dysfunction. Although in testicular germ cell cancer survivors, the clinical value is limited because the contralateral biopsies are not commonly available, we propose a closer andrological follow-up in any patient with an increased total Leydig cell area/total selected area index.

Efeito de dietas contendo plasma sanguíneo desidratado sobre características microbiológicas, imunológicas e histológicas de leitões leves ao desmame / Effect of spray-dried plasma diets on microbiological, immunological and histological parameters of light weight weaned pigs

Foram avaliados os efeitos do plasma sanguíneo desidratado (PSD) sobre desempenho, perfil imunológico, histológico, microbiológico e peso de órgãos de leitões leves, desmamados aos 21 dias de idade. Foram utilizados 24 leitões, com idade média inicial de 21 dias, em delineamento experimental completamente ao acaso. Os tratamentos foram: T1 - animais pesados ao desmame, sem suplementação com PSD; T2 - animais leves ao desmame, suplementados com 10g/animal/dia de PSD; T3 - animais leves ao desmame, suplementados com 20g/animal/dia de PSD; T4 - animais leves ao desmame, sem suplementação com PSD. A adição de 20g de PSD na dieta melhorou o ganho diário de peso, aumentou o peso (g/kg) do baço e o título de IgA no soro entre 21 e 31 dias de idade. A inclusão de 10g de PSD aumentou o comprimento e a largura do linfonodo ileocólico. A inclusão de PSD traz benefícios aos leitões nos primeiros 10 dias pós-desmame, atuando principalmente nos órgãos linfoides e na mucosa intestinal.

The aim of this experiment was to evaluate the effects of spray-dried plasma (SDP) on the growth performance, immunological, histological and microbiological profile and weight of organs of light weight weaned pigs. The trial was done using 24 pigs with an initial mean age of 21 days in a completely randomized experimental design. The treatments were: T1 - heavy weight weaned pigs, without SDP supplementation; T2 - light weight weaned pigs, supplemented with 10g/animal/day of SDP; T3 - light weight weaned pigs, supplemented with 20g/animal/day of SDP; T4 - light weight weaned pigs, without SDP supplementation. The inclusion of 20g of SDP in the diet improved the weight gain, spleen weight (g/kg) and serum IgA title between 21 and 31 days of age. The inclusion of 10g of SDP in the diet improved the length and width of the ileocolic lymph node. In the first 10 days after weaning, SDP improved the development of lymphoid organs and the protection of the intestinal mucosa.

Changes in the subcellular distribution of NADPH oxidase subunit p47phox in dendrites of rat dorsomedial nucleus tractus solitarius neurons in response to chronic administration of hypertensive agents.

NADPH oxidase-generated superoxide can modulate crucial intracellular signaling cascades in neurons of the nucleus tractus solitarius (NTS), a brain region that plays an important role in cardiovascular processes. Modulation of NTS signaling by superoxide may be linked to the subcellular location of the mobile NADPH oxidase p47(phox) subunit, which is known to be present in dendrites of NTS neurons. It is not known, however, if hypertension can produce changes in the trafficking of p47(phox) in defined NTS subregions, particularly the preferentially barosensitive dorsomedial NTS (dmNTS), or preferentially gastrointestinal medial NTS (mNTS). We used immunogold electron microscopy to determine if p47(phox) localization was differentially affected in dendritic profiles of neurons from these NTS subregions of the rat in response to distinct models of hypertension, namely chronic 7-day subcutaneous administration of angiotensin II (AngII), or phenylephrine. In small (<1 microm) dendritic processes, both AngII and phenylephrine produced a decrease in intracellular p47(phox) labeling selectively in dmNTS neurons. In intermediate-size (1-2 microm) dendritic profiles in the dmNTS region only, there was an increase in p47(phox) labeling in response to each hypertensive agent, although these changes occurred in different subcellular compartments. There was an increase in non-vesicular labeling in response to AngII, but an increase in surface labeling with phenylephrine. Moreover, each of the changes in p47(phox) targeting mentioned above occurred in dendritic profiles with, or without immunoperoxidase labeling for the AngII AT-1A receptor subtype (AT-1A). These results indicate that chronic administration of agents that induce hypertension can also produce changes in the subcellular localization in p47(phox) in dmNTS neurons. Thus, systemic hypertension may produce alterations in the trafficking of proteins associated with superoxide production in central autonomic neurons, thus revealing a potentially important neurogenic component of free radical production and systemic blood pressure elevation.

Subcellular localization of nicotinamide adenine dinucleotide phosphate oxidase subunits in neurons and astroglia of the rat medial nucleus tractus solitarius: relationship with tyrosine hydroxylase immunoreactive neurons.

Superoxide produced by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase mediates crucial intracellular signaling cascades in the medial nucleus of the solitary tract (mNTS), a brain region populated by catecholaminergic neurons, as well as astroglia that play an important role in autonomic function. The mechanisms mediating NADPH oxidase (phagocyte oxidase) activity in the neural regulation of cardiovascular processes are incompletely understood, however the subcellular localization of superoxide produced by the enzyme is likely to be an important regulatory factor. We used immunogold electron microscopy to determine the phenotypic and subcellular localization of the NADPH oxidase subunits p47(phox), gp91(phox,) and p22(phox) in the mNTS in rats. The mNTS contains a large population of neurons that synthesize catecholamines. Significantly, catecholaminergic signaling can be modulated by redox reactions. Therefore, the relationship of NADPH oxidase subunit labeled neurons or glia with respect to catecholaminergic neurons was also determined by dual labeling for the superoxide producing enzyme and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. In the mNTS, NADPH oxidase subunits were present primarily in somatodendritic processes and astrocytes, some of which also contained TH, or were contacted by TH-labeled axons, respectively. Immunogold quantification of NADPH oxidase subunit localization showed that p47(phox) and gp91(phox) were present on the surface membrane, as well as vesicular organelles characteristic of calcium storing smooth endoplasmic reticula in dendritic and astroglial processes. These results indicate that NADPH oxidase assembly and consequent superoxide formation are likely to occur near the plasmalemma, as well as on vesicular organelles associated with intracellular calcium storage within mNTS neurons and glia. Thus, NADPH oxidase-derived superoxide may participate in intracellular signaling pathways linked to calcium regulation in diverse mNTS cell types. Moreover, NADPH oxidase-derived superoxide in neurons and glia may directly or indirectly modulate catecholaminergic neuron activity in the mNTS.

Nox2, Ca2+, and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius.

The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca(2+) currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca(2+) or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',7'-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC(50)=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca(2+) currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca(2+) attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca(2+) currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca(2+) currents that underlie the actions of Ang II on central autonomic regulation.