Genetic Analysis of Patients with Congenital Hypogonadotropic Hypogonadism: A Case Series.

Congenital hypogonadotropic hypogonadism (cHH)/Kallmann syndrome (KS) is a rare genetic disorder with variable penetrance and a complex inheritance pattern. Consequently, it does not always follow Mendelian laws. More recently, digenic and oligogenic transmission has been recognized in 1.5-15% of cases. We report the results of a clinical and genetic investigation of five unrelated patients with cHH/KS analyzed using a customized gene panel. Patients were diagnosed according to the clinical, hormonal, and radiological criteria of the European Consensus Statement. DNA was analyzed using next-generation sequencing with a customized panel that included 31 genes. When available, first-degree relatives of the probands were also analyzed to assess genotype-phenotype segregation. The consequences of the identified variants on gene function were evaluated by analyzing the conservation of amino acids across species and by using molecular modeling. We found one new pathogenic variant of the CHD7 gene (c.576T>A, p.Tyr1928) and three new variants of unknown significance (VUSs) in IL17RD (c.960G>A, p.Met320Ile), FGF17 (c.208G>A, p.Gly70Arg), and DUSP6 (c.434T>G, p.Leu145Arg). All were present in the heterozygous state. Previously reported heterozygous variants were also found in the PROK2 (c.163del, p.Ile55*), CHD7 (c.c.2750C>T, p.Thr917Met and c.7891C>T, p.Arg2631*), FLRT3 (c.1106C>T, p.Ala369Val), and CCDC103 (c.461A>C, p.His154Pro) genes. Molecular modeling, molecular dynamics, and conservation analyses were performed on three out of the nine variants identified in our patients, namely, FGF17 (p.Gly70Arg), DUSP6 (p.Leu145Arg), and CHD7 p.(Thr917Met). Except for DUSP6, where the L145R variant was shown to disrupt the interaction between ß6 and ß3, needed for extracellular signal-regulated kinase 2 (ERK2) binding and recognition, no significant changes were identified between the wild-types and mutants of the other proteins. We found a new pathogenic variant of the CHD7 gene. The molecular modeling results suggest that the VUS of the DUSP6 (c.434T>G, p.Leu145Arg) gene may play a role in the pathogenesis of cHH. However, our analysis indicates that it is unlikely that the VUSs for the IL17RD (c.960G>A, p.Met320Ile) and FGF17 (c.208G>A, p.Gly70Arg) genes are involved in the pathogenesis of cHH. Functional studies are needed to confirm this hypothesis.

Ethical considerations regarding animal experimentation.

Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees' adoption of the 'four Rs' principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued.

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.

A Multi-Gene Panel to Identify Lipedema-Predisposing Genetic Variants by a Next-Generation Sequencing Strategy.

Lipedema is a disabling disease characterized by symmetric enlargement of the lower and/or upper limbs due to deposits of subcutaneous fat, that is easily misdiagnosed. Lipedema can be primary or syndromic, and can be the main feature of phenotypically overlapping disorders. The aim of this study was to design a next-generation sequencing (NGS) panel to help in the diagnosis of lipedema by identifying genes specific for lipedema but also genes for overlapping diseases, and targets for tailored treatments. We developed an NGS gene panel consisting of 305 genes potentially associated with lipedema and putative overlapping diseases relevant to lipedema. The genomes of 162 Italian and American patients with lipedema were sequenced. Twenty-one deleterious variants, according to 3 out of 5 predictors, were detected in PLIN1, LIPE, ALDH18A1, PPARG, GHR, INSR, RYR1, NPC1, POMC, NR0B2, GCKR, PPARA in 17 patients. This extended NGS-based approach has identified a number of gene variants that may be important in the diagnosis of lipedema, that may affect the phenotypic presentation of lipedema or that may cause disorders that could be confused with lipedema. This tool may be important for the diagnosis and treatment of people with pathologic subcutaneous fat tissue accumulation.

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.

Multimodal imaging of retinitis pigmentosa associated with Mainzer-Saldino syndrome.

Background: Syndromic ciliopathies have been variably linked to different retinal dystrophies. However, to date, few reports have characterized by means of multimodal imaging the retinal degeneration occurring in Mainzer-Saldino syndrome (MSS).Methods: Two siblings with history of kidney disease and other systemic abnormalities presented at our eye clinic in October 2017 complaining of night blindness and visual loss. They underwent a complete ophthalmologic examination including visual acuity (VA) assessment, optical coherence tomography (OCT) and blue-light autofluorescence (BAF). A screen for inherited retinal dystrophies was performed in this occasion.Results: At baseline, the youngest sister had slightly worse VA (20/30 vs. 20/20-25 Snellen equivalents). On fundoscopy, both siblings had severe thinning of the peripheral retina, attenuation of retinal vessels and widespread accumulation of pigmented deposits. Significant outer retinal atrophy with apparent foveal sparing was appreciable on OCT.During the 3 years of follow-up, vision remained overall stable in both patients whereas minimal progression of outer retinal atrophy was observed by means of OCT. Genetic analysis revealed compound heterozygosity in the IFT172 gene. Based on these findings, a diagnosis of retinitis pigmentosa (RP) associated with MSS was formulated.Conclusions: Our report describes the cases of two siblings affected by retinitis pigmentosa associated with MSS. Although both carrying the same mutations and a severe RP phenotype, the youngest sister had slightly more advanced retinal degeneration highlighting the remarkable variability related to the IFT172 retinopathy.

Progression of Atrophy and Visual Outcomes in Extensive Macular Atrophy with Pseudodrusen-like Appearance.

Purpose: To report visual outcomes and rate of retinal pigment epithelium (RPE) atrophy progression in patients with extensive macular atrophy with pseudodrusen-like appearance (EMAP). Design: Retrospective, observational study. Participants: Patients with EMAP and symptom onset before 55 years of age, at least 12 months of follow-up using Spectralis blue-light fundus autofluorescence (BAF) and OCT and with no other ocular or systemic conditions. Methods: Best-corrected visual acuity (BCVA), BAF, and OCT images were reviewed at baseline and at each annual visit until the last available follow-up. Atrophy was measured by 2 graders using the region finder software on Heidelberg Explorer and confirmed using OCT scans covering the entire atrophic lesion. The following imaging biomarkers were analyzed at each visit: foveal atrophy, vitreomacular traction, outer retinal tubulations, choroidal caverns and subfoveal choroidal thickness, border autofluorescence pattern (hyper-autofluorescent or iso-autofluorescent), and border irregularity as expressed by circularity index (CI). Main Outcome Measures: Primary outcomes were annual rate of atrophy enlargement and BCVA loss in EMAP patients. Secondary outcomes included the assessment of potential factors able to predict disease progression. Results: Thirty-six eyes from 18 patients with EMAP (6 men [33%]; mean age at symptom onset, 48.1 ± 1.7 years) were included. Mean follow-up lasted 32.8 ± 14.3 months. RPE atrophy increased from 10.8 ± 6.3 mm2 at baseline to 18.1 ± 8.3 mm2 at the end of follow-up, with a rate of 2.91 ± 1.09 mm2/year. Faster progression was associated with smaller CI at baseline (P = 0.02) and with iso-autofluorescent lesion borders (P = 0.01). Visual acuity declined progressively at a rate of 7.4 ± 5.8 letters per year, with 57% of eyes showing vision of 20/200 Snellen or worse at the 4-year follow-up. Worse visual outcomes were observed in patients with early foveal involvement at baseline (P = 0.02). Conclusions: Patients affected by EMAP present a rapid expansion of RPE atrophy that is comparable with the diffuse-trickling form of geographic atrophy. More irregular and iso-autofluorescent lesion borders seem to predict faster progression. Our findings may provide relevant information for patient counseling and future interventional approaches to select the best candidates and proper clinical outcomes.

NOTCH1: Review of its role in lymphatic development and study of seven families with rare pathogenic variants.

BACKGROUND: We developed a Next-Generation-Sequencing (NGS) protocol to screen the most frequent genetic variants related to lymphedema and a group of candidate genes. The aim of the study was to find the genetic cause of lymphedema in the analyzed patients. METHODS: We sequenced a cohort of 246 Italian patients with lymphatic malformations. In the first step, we analyzed genes known to be linked to lymphedema: 235 out of 246 patients tested negative for the most frequent variants and underwent testing for variants in a group of candidate genes, including the NOTCH1 gene, selected from the database of mouse models. We also performed in silico analysis to observe molecular interactions between the wild-type and the variant amino acids and other protein residues. RESULTS: Seven out of 235 probands, five with sporadic and two with familial lymphedema, were found to carry rare missense variants in the NOTCH1 gene. CONCLUSIONS: Our results propose that NOTCH1 could be a novel candidate for genetic predisposition to lymphedema.

Genetic analysis of genes associated with Mendelian dementia.

BACKGROUND AND AIM: Dementia is a disease associated with cognitive and/or behavioral changes that interfere with the ability to perform daily activities. Alzheimer's disease is the most common type of dementia. The aim of this mini-review is to summarize all the syndromes characterized by dementia and for which the associated gene is known. METHODS: We searched those syndromes in PubMed and OMIM database. RESULTS: Two forms of dementia exist: the multifactorial dementia results from the interaction of different genetic and environmental factors, the hereditary dementia associated with a single gene. Individuals with a family history of dementia and early onset of the disease are more likely to have a hereditary form of dementia. Dementias are mainly autosomal dominant, but they can also be autosomal recessive or X-linked. CONCLUSIONS: Since dementia has high clinical and genetic heterogeneity, the use in diagnostics of a large panel of genes may greatly help to speed up the determination of the molecular diagnosis and/or establish a risk of recurrence in family members for the purpose of planning appropriate preventive and/or therapeutic measures.

Genetic test for Mendelian fatigue and muscle weakness syndromes.

Several inherited disorders involve chronic fatigue, muscle weakness and pain. These conditions can depend on muscle, nerve, brain, metabolic and mitochondrial defects. A major trigger of muscle weakness and fatigue is exercise. The amount of exercise that triggers symptoms and the frequency of symptoms are highly variable. In this review, the genetic causes and molecular pathways involved in these disorders are discussed along with the diagnostic and treatment options available, with the aim of fostering understanding of the disease and exploring therapeutic options.

Genetic test for the personalization of sport training.

Genetic variants may contribute to confer elite athlete status. However, this does not mean that a person with favourable genetic traits would become a champion because multiple genetic interactions and epigenetic contributions coupled with confounding environmental factors shape the overall phenotype. This opens up a new area in sports genetics with respect to commercial genetic testing. The analysis of genetic polymorphisms linked to sport performance would provide insights into the potential of becoming an elite endurance or power performer. This mini-review aims to highlight genetic interactions that are associated with performance phenotypes and their potentials to be used as markers for talent identification and trainability.

Comparison between American and European legislation in the therapeutical and alimentary bacteriophage usage.

Bacteriophages, though discovered a century ago, still lag behind in the race of antimicrobials due to scarce information about their biology, pharmacology, safety and suitability as therapeutic agents. Although they possess several capabilities of practical utility in medicine, they are still unable to satisfy the regulatory standards set by the regulatory authorities in both United States (US) and European Union (EU). Bacteriophages and their products (lysins) are considered as drugs, therefore they should follow the same route of the chemical drugs in order to achieve regulatory approvals for commercial production and application. However, lack of definitive guidelines and regulations has rendered bacteriophages less attractive to pharmaceutical companies and funding agencies, making it difficult for clinicians and researchers to set up wide scale clinical trials in order to prove efficacy, safety and stability of bacteriophages and their products. In this review, we will discuss the current regulations for developing phages and phage-based products for therapeutic purposes in the US and EU.

Bacteriophages in food supplements obtained from natural sources.

Human gastrointestinal tract is colonized by bacteria that constitute the interstinal microbiota. Changes in the microbiota may lead to several chronic disorders. Bacteriophages are viruses that specifically target bacteria. Several food components contain bacteriophages and probiotics. Bacteriophages have a great specificity for harmful bacteria, helping the growth of good bacteria. Because of their qualities, bacteriophages are considered beneficial component of probiotics that target the pathogenic bacteria and support the natural human microbiota.

Bacteriophages presence in nature and their role in the natural selection of bacterial populations.

Phages are the obligate parasite of bacteria and have complex interactions with their hosts. Phages can live in, modify, and shape bacterial communities by bringing about changes in their abundance, diversity, physiology, and virulence. In addition, phages mediate lateral gene transfer, modify host metabolism and reallocate bacterially-derived biochemical compounds through cell lysis, thus playing an important role in ecosystem. Phages coexist and coevolve with bacteria and have developed several antidefense mechanisms in response to bacterial defense strategies against them. Phages owe their existence to their bacterial hosts, therefore they bring about alterations in their host genomes by transferring resistance genes and genes encoding toxins in order to improve the fitness of the hosts. Application of phages in biotechnology, environment, agriculture and medicines demands a deep insight into the myriad of phage-bacteria interactions. However, to understand their complex interactions, we need to know how unique phages are to their bacterial hosts and how they exert a selective pressure on the microbial communities in nature. Consequently, the present review focuses on phage biology with respect to natural selection of bacterial populations.

Neurobiological basis of chiropractic manipulative treatment of the spine in the care of major depression.

BACKGROUND AND AIM: Major depressive disorder is associated with an autonomic nervous system imbalance. All the symptoms of depression (high cortisol, high adrenalin, insomnia, agitation, anxiety) can probably be attributed to over-activation of the sympathetic nervous system. We performed this review in order to highlight the possible links between chiropractic intervention, its potential molecular effects and its possible outcomes on patients with depression. METHODS: We performed a literature search for all the relevant manuscript regarding the effects of chiropractic and depression on the autonomic nervous system. RESULTS: Chiropractic care and spinal manipulation regulate the autonomic nervous system at peripheral level and its projections to the central nervous system. In particular, they may activate the parasympathetic system to counterbalance the activity of the sympathetic system. Vagal parasympathetic stimulation is also considered an effective therapy for major depression as it releases neurotrophins essential for anti-depressive therapies, including brain-derived neurotrophic factor and nerve growth factor. CONCLUSION: Chiropractic and spinal manipulative therapies along with vagal nerve stimulation may therefore be regarded as treatment options for depression.

Genetic and physiological autonomic nervous system factors involved in failed back surgery syndrome: A review of the literature and report of nine cases treated with pulsed radiofrequency.

BACKGROUND AND AIM: failed back surgery syndrome is one of the most important causes of chronic low back pain that involve the physiology of autonomic nervous system factors. Some genetic and molecular factor can be determinant in the development of failed back surgery syndrome and novel therapy are needed. Pulsed radiofrequency treatment could be an innovative treatment option for this syndrome. METHODS: 44 patients classified with failed back surgery syndrome from the Poliambulanza Foundation Hospital of Brescia patients were treated with standard therapy for six months; 9 of these patients who showed no improvement were candidates for pulsed radiofrequency therapy for three months. RESULTS AND CONCLUSIONS: reduction of lumbar and radicular pain, disability and number of drug classes prescribed improved significantly (p <0.001) in patients treated with pulsed radiofrequency compared to whom that follow only the standard therapy. The role of the nervous system is important for understanding how pulsed radiofrequency can improve the health of patients with back pain. We suggest that some genetic and molecular studies are needed for better understand the role of this therapy in back pain.

Natural compounds as inhibitors of SARS-CoV-2 endocytosis: A promising approach against COVID-19.

BACKGROUND AND AIM: The recent COVID-19 pandemic caused by SARS-CoV-2 affected more than six million people and caused thousands of deaths. The lack of effective drugs or vaccines against SARS-CoV-2 further worsened the situation. This review is focused on the identification of molecules that may inhibit viral entry into host cells by endocytosis. METHODS: We performed the literature search for these natural compounds in the articles indexed in PubMed. RESULTS: Natural products against viral infections have been gaining importance in recent years. Specific natural compounds like phytosterols, polyphenols, flavonoids, citrus, galangal, curcuma and hydroxytyrosol are being analyzed to understand whether they could inhibit SARS-CoV-2. CONCLUSIONS: We reviewed natural compounds with potential antiviral activity against SARS-CoV-2 that could be used as a treatment for COVID-19.

A pilot study on the preventative potential of alpha-cyclodextrin and hydroxytyrosol against SARS-CoV-2 transmission.

BACKGROUND AND AIM OF THE WORK: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current pandemics. This virus attacks the cells by binding to the transmembrane angiotensin I converting enzyme 2. In this study, we experimented a food supplement containing alpha-cyclodextrin and hydroxytyrosol for the improvement of the defenses against the SARS-CoV-2. Hydroxytyrosol has anti-viral properties and is able to reduce the serum lipids in mice. α-cyclodextrin has the ability to deplete sphingolipids and phospholipids from the cellular membranes. The aim of the present preliminary open non-controlled interventional study was to evaluate the efficacy of alpha-cyclodextrin and hydroxytyrosol in improving defenses against SARS-CoV-2. METHODS: Fifty healthy volunteers at a higher risk of SARS-CoV-2 infection from Northern Cyprus and six positive individuals for SARS-CoV-2 were enrolled in this study. The in silico prediction was performed using D3DOCKING to evaluate the interactions of hydroxytyrosol and alpha-cyclodextrin with proteins involved in the SARS-CoV-2 endocytosis. RESULTS: The 50 volunteers did not become positive in 15 days for SARS-CoV-2 after the administration of the compound for two weeks, despite they were at higher risk of infection than the general population. Interestingly, in the cohort of six positive patients, two patients were administered the spray and became negative after five days, despite the viral load was higher in the treated subjects than the untreated patients who became negative after ten days. In addition, we identified possible interactions among hydroxytyrosol and alpha-cyclodextrin with the protein Spike and the human proteins ACE2 and TMPRSS2. CONCLUSIONS: We reported on the results of the possible role of alpha-cyclodextrin and hydroxytyrosol in improving defenses against SARS-CoV-2. The next step will be the administration of the compound to a larger cohort in a controlled study to confirm the reduction of the infection rate of SARS-CoV-2 in the treated subjects.

Segregation Analysis of Rare NRP1 and NRP2 Variants in Families with Lymphedema.

Neuropilins are transmembrane coreceptors expressed by endothelial cells and neurons. NRP1 and NRP2 bind a variety of ligands, by which they trigger cell signaling, and are important in the development of lymphatic valves and lymphatic capillaries, respectively. This study focuses on identifying rare variants in the NRP1 and NRP2 genes that could be linked to the development of lymphatic malformations in patients diagnosed with lymphedema. Two hundred and thirty-five Italian lymphedema patients, who tested negative for variants in known lymphedema genes, were screened for variants in NRP1 and NRP2. Two probands carried variants in NRP1 and four in NRP2. The variants of both genes segregated with lymphedema in familial cases. Although further functional and biochemical studies are needed to clarify their involvement with lymphedema and to associate NRP1 and NRP2 with lymphedema, we suggest that it is worthwhile also screening lymphedema patients for these two new candidate genes.

Clinical Evaluation of a Custom Gene Panel as a Tool for Precision Male Infertility Diagnosis by Next-Generation Sequencing.

BACKGROUND: Up to 15% of couples are infertile and male factor infertility accounts for approximately 50% of these cases. Male infertility is a multifactorial pathological condition. The genetic of male infertility is very complex and at least 2000 genes are involved in its etiology. Genetic testing by next-generation sequencing (NGS) technologies can be relevant for its diagnostic value in male infertile patients. Therefore, the aim of this study was to implement the diagnostic offer with the use of an NGS panel for the identification of genetic variants. METHODS: We developed an NGS gene panel that we used in 22 male infertile patients. The panel consisted of 110 genes exploring the genetic causes of male infertility; namely spermatogenesis failure due to single-gene mutations, central hypogonadism, androgen insensitivity syndrome, congenital hypopituitarism, and primary ciliary dyskinesia. RESULTS: NGS and a subsequent sequencing of the positive pathogenic or likely pathogenic variants, 5 patients (23%) were found to have a molecular defect. In particular, pathogenic variants were identified in TEX11, CCDC39, CHD7, and NR5A1 genes. Moreover, 14 variants of unknown significance and 7 novel variants were found that require further functional studies and family segregation. CONCLUSION: This extended NGS-based diagnostic approach may represent a useful tool for the diagnosis of male infertility. The development of a custom-made gene panel by NGS seems capable of reducing the proportion of male idiopathic infertility.