Genotypic and phenotypic characterization of a cohort of patients affected by rod CNG channel-associated retinitis pigmentosa.

INTRODUCTION: Retinitis pigmentosa (RP), a heterogeneous inherited retinal disorder causing gradual vision loss, affects over 1 million people worldwide. Pathogenic variants in CNGA1 and CNGB1 genes, respectively accounting for 1% and 4% of cases, impact the cyclic nucleotide-gated channel in rod photoreceptor cells. The aim of this study is to describe and compare genotypic and clinical characteristics of a cohort of patients with CNGA1- or CNGB1-related RP and to explore potential genotype-phenotype correlations. METHODS: The following data from patients with CNGA1- or CNGB1-related RP, followed in five Italian inherited retinal degenerations services, were retrospectively collected: genetic variants in CNGA1 and CNGB1, best-corrected visual acuity (BCVA), ellipsoid zone (EZ) width, fundus photographs and short-wavelength fundus autofluorescence (SW-AF) images. Comparisons and correlation analyses were performed by first dividing the cohort in two groups according to the gene responsible for the disease (CNGA1 and CNGB1 groups). In parallel, the whole cohort of RP patients was divided into two other groups, according to the expected impact of the variants at protein level (Low and High group). RESULTS: In total, 29 patients were recruited, 11 with CNGA1- and 18 with CNGB1-related RP. In both CNGA1 and CNGB1, 5 novel variants in CNGA1 and 5 in CNGB1 were found. BCVA was comparable between CNGA1 and CNGB1 groups, as well as between Low and High groups. CNGA1 group had a larger mean EZ width compared to CNGB1 group, albeit not statistically significant, while EZ width did not differ between Low and High groups A statistically significant correlation between EZ width and BCVA as well as between EZ width and age were observed in the whole cohort of RP patients. Fundus photographs of all patients in the cohort showed classic RP pattern, and in SW-AF images an hyperautofluorescent ring was observed in 14/21 patients. CONCLUSION: Rod CNG channel-associated RP was demonstrated to be a slowly progressive disease in both CNGA1- and CNGB1-related forms, making it an ideal candidate for gene augmentation therapies.

A novel homozygous splice site variant in ARL2BP causes a syndromic autosomal recessive rod-cone dystrophy with situs inversus, asthenozoospermia, unilateral renal agenesis and microcysts.

BACKGROUND: This report presents a clinical case of syndromic rod-cone dystrophy due to a splice site variant in the ARL2BP gene causing situs inversus, asthenozoospermia, unilateral renal agenesis and microcysts. The presence of renal agenesis and cryptorchidism expands the clinical manifestations due to ARL2BP variants. The detailed, long-term follow-up contributes valuable insights into disease progression, aiding clinical diagnosis and patient management. CASE PRESENTATION: The male patient complained of photophobia as the first symptom when he was 20 years old followed by nyctalopia, loss of central visual acuity and peripheral visual field ten years later. Genetic analysis identified a likely pathogenic homozygous variant (c.294-1G > C) involving the splicing acceptor site of intron 4. Reported symptoms together with full-field stimulus threshold testing, electroretinogram and advanced multimodal imaging allowed us to recognize the typical characteristics of a mixed retinal dystrophy. Despite the end-stage retinal disease, this patient still retained a useful residual vision at 63 years and had a slow disease progression during the last 5 years of evaluation. DISCUSSION AND CONCLUSIONS: Our findings underscore the variable clinical presentation of ARL2BP variants, emphasizing the importance of a nuanced approach in diagnosing and managing patients. The presence of renal cysts warrants consideration of a differential diagnosis, particularly with Senior-Loken (SLS), Bardet-Biedl (BBS) and Joubert syndromes (JS) but also with Short Rib Thoracic Dysplasia 9, highlighting the need for careful phenotypic evaluation in these cases.

Targeted next-generation sequencing analysis in Italian patients with keratoconus.

OBJECTIVE: To report variants in 26 candidate genes and describe the clinical features of Italian patients with keratoconus (KC). SUBJECTS/METHODS: Sixty-four patients with a confirmed diagnosis of KC were enrolled in this genetic association study. Patients were classified into two study groups according to whether they had a confirmed diagnosis of progressive or stable KC. A purpose-developed Next Generation Sequencing (NGS) panel was used to identify and analyse the coding exons and flanking exon/intron boundaries of 26 genes known to be associated with KC and corneal dystrophies. Interpretation of the pathogenic significance of variants was performed using in silico predictive algorithms. RESULT: The targeted NGS research identified a total of 167 allelic variants of 22 genes in the study population; twenty-four patients had stable keratoconus (n. 54 variants) and forty patients had progressive disease (n. 113 variants). We identified genetic variants of certain pathogenic significance in five patients with progressive KC; in addition, eight novel genetic variants were found in eight patients with progressive KC. Mutations of FLG, LOXHD1, ZNF469, and DOCK9 genes were twice more frequently identified in patients with progressive than stable disease. Filaggrin gene variants were found in 49 patients (76% of total), of whom 32 patients (80% of progressive KC group) had progressive disease. CONCLUSIONS: Targeted NGS research provided new insights into the causative effect of candidate genes in the clinical phenotype of keratoconus. Filaggrin mutations were found to represent a genetic risk factor for development of progressive disease in Italy.

Correction to "Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications".

[This corrects the article DOI: 10.1021/acsomega.3c04563.].

RP1 Dominant p.Ser740* Pathogenic Variant in 20 Knowingly Unrelated Families Affected by Rod-Cone Dystrophy: Potential Founder Effect in Western Sicily.

Background and Objectives. Retinitis pigmentosa (RP) is the most common inherited rod-cone dystrophy (RCD), resulting in nyctalopia, progressive visual field, and visual acuity decay in the late stages. The autosomal dominant form (ADRP) accounts for about 20% of RPs. Among the over 30 genes found to date related to ADRP, RP1 pathogenic variants have been identified in 5-10% of cases. In a cohort of RCD patients from the Palermo province on the island of Sicily, we identified a prevalent nonsense variant in RP1, which was associated with ADRP. The objective of our study was to analyse the clinical and molecular data of this patient cohort and to evaluate the potential presence of a founder effect. Materials and Methods. From 2005 to January 2023, 84 probands originating from Western Sicily (Italy) with a diagnosis of RCD or RP and their relatives underwent deep phenotyping, which was performed in various Italian clinical institutions. Molecular characterisation of patients and familial segregation of pathogenic variants were carried out in different laboratories using Sanger and/or next-generation sequencing (NGS). Results. Among 84 probands with RCD/RP, we found 28 heterozygotes for the RP1 variant c.2219C>G, p.Ser740* ((NM_006269.2)*, which was therefore significantly prevalent in this patient cohort. After a careful interview process, we ascertained that some of these patients shared the same pedigree. Therefore, we were ultimately able to define 20 independent family groups with no traceable consanguinity. Lastly, analysis of clinical data showed, in our patients, that the p.Ser740* nonsense variant was often associated with a late-onset and relatively mild phenotype. Conclusions. The high prevalence of the p.Ser740* variant in ADRP patients from Western Sicily suggests the presence of a founder effect, which has useful implications for the molecular diagnosis of RCD in patients coming from this Italian region. This variant can be primarily searched for in RP-affected subjects displaying compatible modes of transmission and phenotypes, with an advantage in terms of the required costs and time for analysis. Moreover, given its high prevalence, the RP1 p.Ser740* variant could represent a potential candidate for the development of therapeutic strategies based on gene editing or translational read-through therapy for suppression of nonsense variants.

DEXAMETHASONE IMPLANT VERSUS TOPICAL CARBONIC ANHYDRASE INHIBITORS IN PATIENTS WITH BILATERAL RETINITIS PIGMENTOSA-RELATED CYSTOID MACULAR EDEMA: A Prospective, Paired-Eye Pilot Study.

PURPOSE: To compare within-subject efficacy and safety of intravitreal dexamethasone implant and topical carbonic anhydrase inhibitors in the treatment of retinitis pigmentosa-related cystoid macular edema. METHODS: Patients with bilateral retinitis pigmentosa-related cystoid macular edema were treated with intravitreal dexamethasone implant in one eye and topical carbonic anhydrase inhibitors in the contralateral eye. The primary endpoint was a change in central macular thickness. Secondary endpoints were changes in best-corrected visual acuity and microperimetric central retinal sensitivity. Intraocular pressure and other ocular complications were evaluated for safety assessment. RESULTS: Nine patients were recruited for this 12-month follow-up study. Central macular thickness was significantly lower in intravitreal dexamethasone implant-treated eyes than in topical carbonic anhydrase inhibitors-treated eyes at Months 1 and 7, whereas mean best-corrected visual acuity was better in eyes treated with topical carbonic anhydrase inhibitors at Month 12 (borderline significant P = 0.0510). There was no difference in microperimetric sensitivity between the two treatments. Three patients developed ocular hypertension after intravitreal dexamethasone implant. Intravitreal dexamethasone implant showed an effect on the contralateral eye in five of nine patients. CONCLUSION: Intravitreal dexamethasone implant was more effective than topical carbonic anhydrase inhibitors in reducing retinitis pigmentosa-related cystoid macular edema 1 month after treatment. Corticosteroids can play a key role in the management of retinitis pigmentosa-related cystoid macular edema; however, their routes, timing, and modes of administration should be further explored.

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants.

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

Clinical, Genotypic, and Imaging Characterization of the Spectrum of ABCA4 Retinopathies.

PURPOSE: To investigate the clinical and genotypic differences in the spectrum of ABCA4-associated retinopathies (ABCA4Rs). DESIGN: Observational, cross sectional case series. PARTICIPANTS: Sixty-six patients (132 eyes) carrying biallelic ABCA4 variants. METHODS: Patients underwent visual acuity measurement and multimodal imaging. Clinical records were reviewed for age at onset, presenting symptoms, genetic variants, and electroretinogram (ERG). Each eye was assigned to a phenotype based on age at onset, imaging and ERG: cone dystrophy-bull's-eye maculopathy (CD-BEM, 40 eyes), cone-rod dystrophy (CRD, 12 eyes), Stargardt disease (SD, 28 eyes), late-onset SD (LO-SD, 38 eyes), and fundus flavimaculatus (14 eyes). Images were analyzed for: peripapillary sparing, retinal pigment epithelium (RPE) atrophy (definitely decreased autofluorescence, DDAF), flecks patterns using autofluorescence; type of atrophy according to Classification of Atrophy Meeting reports, macular and choroidal thickness on OCT; and choriocapillaris flow deficits on OCT angiography. MAIN OUTCOME MEASURES: Primary outcome was to report the demographic, genotypic, and imaging characteristics of the different ABCA4R phenotypes. Secondary objectives included the assessment of imaging biomarkers as outcome measures for clinical trials. RESULTS: Age at onset was lower in CRD (12 ± 8 years) and higher in patients with LO-SD (59 ± 9 years) (all P < 0.01). Central vision loss was a common presenting symptom in CD-BEM and SD, whereas patients with LO-SD primarily complained of difficult dark adaptation. Missense variants were more frequent in CD-BEM, and splice site in CRD and LO-SD (P < 0.05). Peripapillary sparing was absent in 3 eyes with LO-SD (8%). Cone dystrophy-bull's-eye maculopathy eyes typically had complete outer retinal atrophy alterations (98%), whereas CRD and SD eyes showed both complete outer retinal atrophy and complete RPE and outer retinal atrophy (cRORA) (71%-100%). Patients with LO-SD had larger areas of DDAF (100% cRORA) and of choriocapillaris flow deficits (all P < 0.01). Repeatability of DDAF measurements was low for some phenotypes (CD-BEM and CRD) and atrophic areas <7.5 mm2. Resorbed flecks were significantly associated with CRD and LO-SD (P < 0.01). CONCLUSIONS: This research provides a thorough evaluation of the spectrum of ABCA4R. Our findings suggest that certain phenotypes show preferential photoreceptor degeneration (e.g., CD-BEM), whereas others have substantial RPE and choriocapillaris alterations (e.g., LO-SD). We recommend that clinical trial end points take into consideration these imaging features to improve the interpretation of their results. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications.

In this study, we dissolved Bombyx mori degummed silk [i.e., silk fibroin (SF)] and salmon sperm deoxyribonucleic acid (DNA) in water and used a bioinspired spinning process to obtain an electrospun nanofibrous SF-based patch (ESF). We investigated the bidirectional macroscale actuation behavior of ESF in response to water vapor and its UV-blocking properties as well as those of ESF/DNA films. Fourier transform infrared (FTIR) results suggest that the formation of ß-sheet-rich structures promotes the actuation effect. ESF/DNA film with high-ordered and ß-sheet-rich structures exhibits higher electrical conductivity and is water-insoluble. Given the intrinsic ability of both SF and DNA to absorb UV radiation, we performed biological experiments on the viability of keratinocyte HaCaT cells after exposure to solar spectrum components. Our findings indicate that the ESF/DNA patch is photoprotective and can increase the cellular viability of keratinocytes after UV exposure. Furthermore, we demonstrated that ESF/DNA patches treated with water vapor can serve as suitable scaffolds for tissue engineering and can improve tissue regeneration when cellularized with HaCaT cells. The 3D shape morphing capability of these patches, along with their potential as UV filters, could offer significant practical advantages in tissue engineering.

MAGI-ACMG: Algorithm for the Classification of Variants According to ACMG and ACGS Recommendations.

We have developed MAGI-ACMG, a classification algorithm that allows the classification of sequencing variants (single nucleotide or small indels) according to the recommendations of the American College of Medical Genetics (ACMG) and the Association for Clinical Genomic Science (ACGS). The MAGI-ACMG classification algorithm uses information retrieved through the VarSome Application Programming Interface (API), integrates the AutoPVS1 tool in order to evaluate more precisely the attribution of the PVS1 criterion, and performs the customized assignment of specific criteria. In addition, we propose a sub-classification scheme for variants of uncertain significance (VUS) according to their proximity either towards the "likely pathogenic" or "likely benign" classes. We also conceived a pathogenicity potential criterion (P_POT) as a proxy for segregation criteria that might be added to a VUS after posterior testing, thus allowing it to upgrade its clinical significance in a diagnostic reporting setting. Finally, we have developed a user-friendly web application based on the MAGI-ACMG algorithm, available to geneticists for variant interpretation.

BRUCH MEMBRANE RUPTURE AND CHOROIDAL NEOVASCULARIZATION COMPLICATING EXTENSIVE MACULAR ATROPHY WITH PSEUDODRUSEN-LIKE APPEARANCE: A CASE REPORT.

PURPOSE: To describe a case of extensive macular atrophy with pseudodrusen-like appearance complicated by Bruch membrane rupture and choroidal neovascularization, treated with intravitreal injection of an antivascular endothelial growth factor drug. METHODS: A 54-year-old woman, affected by extensive macular atrophy with pseudodrusen-like appearance, has developed Bruch membrane rupture and choroidal neovascularization. This report discusses the development of the disease and the improvement after a single intravitreal injection of Avastin. RESULTS: After treatment, the visual acuity increased from 20/400 to 20/200; clinical and anatomical improvement remained stable with a 2-month follow-up. CONCLUSION: Extensive macular atrophy with pseudodrusen-like appearance could be complicated by Bruch membrane rupture and subsequent choroidal neovascularization. Antivascular endothelial growth factor intravitreal injection could have beneficial effects on this entity, but concern could raise in respect of possible increase of chorioretinal atrophy.

Development of Salmon Sperm DNA/Regenerated Silk Bio-Based Films for Biomedical Studies on Human Keratinocyte HaCaT Cells under Solar Spectrum.

In this study, we fabricated adhesive patches from silkworm-regenerated silk and DNA to safeguard human skin from the sun's rays. The patches are realized by exploiting the dissolution of silk fibers (e.g., silk fibroin (SF)) and salmon sperm DNA in formic acid and CaCl2 solutions. Infrared spectroscopy is used to investigate the conformational transition of SF when combined with DNA; the results indicated that the addition of DNA provides an increase in the SF crystallinity. UV-Visible absorption and circular dichroism spectroscopy showed strong absorption in the UV region and the presence of B-form of DNA once dispersed in the SF matrix, respectively. Water absorption measurements as well as thermal dependence of water sorption and thermal analysis, suggested the stability of the fabricated patches. Biological results on cellular viability (MTT assay) of keratinocyte HaCaT cells after exposures to the solar spectrum showed that both SF and SF/DNA patches are photo-protective by increasing the cellular viability of keratinocytes after UV component exposure. Overall, these SF/DNA patches promise applications in wound dressing for practical biomedical purposes.

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.

Retinitis Pigmentosa Associated with EYS Gene Mutations: Disease Severity Staging and Central Retina Atrophy.

BACKGROUND: Eyes shut homolog (EYS) gene mutations are estimated to affect at least 5% of patients with autosomal recessive retinitis pigmentosa. Since there is no mammalian model of human EYS disease, it is important to investigate its age-related changes and the degree of central retinal impairment. METHODS: A cohort of EYS patients was studied. They underwent full ophthalmic examination as well as assessment of retinal function and structure, by full-field and focal electroretinograms (ERGs) and spectral domain optical coherence tomography (OCT), respectively. The disease severity stage was determined by the RP stage scoring system (RP-SSS). Central retina atrophy (CRA) was estimated from the automatically calculated area of the sub-retinal pigment epithelium (RPE) illumination (SRI). RESULTS: The RP-SSS was positively correlated with age, showing an advanced severity score (≥8) at an age of 45 and a disease duration of 15 years. The RP-SSS was positively correlated with the CRA area. LogMAR visual acuity and ellipsoid zone width, but not ERG, were correlated with CRA. CONCLUSIONS: In EYS-related disease, the RP-SSS showed advanced severity at a relative early age and was correlated with the central area of the RPE/photoreceptor atrophy. These correlations may be relevant in view of therapeutic interventions aimed at rescuing rods and cones in EYS-retinopathy.

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.

Analysis of 29 Targeted Genes for Non-Obstructive Azoospermia: The Relationship between Genetic Testing and Testicular Histology.

PURPOSE: To analyze the presence of potentially pathogenic variants of 29 candidate genes known to cause spermatogenic failure (SPGF) in patients with non-obstructive azoospermia (NOA) who underwent testicular histology. MATERIALS AND METHODS: Forty-eight patients with unexplained NOA referred to the Department of Transfusion Medicine and Transplantation Biology, University Hospital Center Zagreb, Zagreb, Croatia for testicular biopsy. They were divided into three groups: those who had cryptorchidism (n=9), those with varicocele (n=14), and those with idiopathic NOA (n=25). All included patients underwent blood withdrawal for next-generation sequencing (NGS) analysis and gene sequencing. RESULTS: We found a possible genetic cause in 4 patients with idiopathic NOA (16%) and in 2 with cryptorchidism (22%). No pathogenic or possibly pathogenic mutations were identified in patients with varicocele. Variants of undetermined significance (VUS) were found in 11 patients with idiopathic NOA (44%), 3 with cryptorchidism (33%), and 8 patients with varicocele (57%). VUSs of the USP9Y gene were the most frequently as they were found in 14 out of 48 patients (29%). In particular, the VUS USP9Y c.7434+14del was found in 11 patients. They showed varied histological pictures, including Sertoli cell-only syndrome, mixed atrophy, and hypospermatogenesis, regardless of cryptorchidism or varicocele. No direct correlation was found between the gene mutation/variant and the testicular histological picture. CONCLUSIONS: Different mutations of the same gene cause various testicular histological pictures. These results suggest that it is not the gene itself but the type of mutation/variation that determines the testicular histology picture. Based on the data presented above, it remains challenging to design a genetic panel with prognostic value for the outcome of testicular sperm extraction in patients with NOA.

Next-Generation Sequencing of a Large Gene Panel for Outcome Prediction of Bariatric Surgery in Patients with Severe Obesity.

Obesity is a chronic disease in which abnormal deposition of fat threatens health, leading to diabetes, cardiovascular diseases, cancer, and other chronic illnesses. According to the WHO, 19.8% of the adult population in Italy is obese, and the prevalence is higher among men. It is important to know the predisposition of an individual to become obese and to respond to bariatric surgery, the most up-to-date treatment for severe obesity. To this purpose, we developed an NGS gene panel, comprising 72 diagnostic genes and 244 candidate genes, and we sequenced 247 adult obese Italian patients. Eleven deleterious variants in 9 diagnostic genes and 17 deleterious variants in 11 candidate genes were identified. Interestingly, mutations were found in several genes correlated to the Bardet-Biedl syndrome. Then, 25 patients were clinically followed to evaluate their response to bariatric surgery. After a 12-month follow-up, the patients that carried deleterious variants in diagnostic or candidate genes had a reduced weight loss, as compared to the other patients. The NGS-based panel, including diagnostic and candidate genes used in this study, could play a role in evaluating, diagnosing, and managing obese individuals, and may help in predicting the outcome of bariatric surgery.

Dietary supplements for obesity.

Obesity and associated complications including diabetes, cardiometabolic dysfunction, disability, malignancy and premature mortality are considered epidemic. Research on obesity is therefore of worldwide importance. The development of obesity is a multifactorial phenomenon with contributions from biological, behavioral, genetic and environmental factors. Obesity and its associated issues require various lifestyle modifications and treatment options such medication, exercise, diet, surgery, pharmacological therapy and dietary supplements. Dietary supplements are considered an attractive alternative to traditional therapy due to their low toxicity profile and their accessibility to the general population. Dietary supplements may include one or more dietary ingredients. In this narrative review, we analyze the effects on obesity and obesity-related issues of various natural components. For example, there are a myriad of supplements that have been used as dietary supplements for weight loss such as minerals, vitamins, amino acids, metabolites, herbs, and plant extracts. This narrative review aims to present the benefits and side-effects of several ingredients of dietary supplements for weight loss and treatment of obesity. In particular, the mechanism of action, results of clinical trials, and possible side effects will be presented for the following ingredients: ß-Glucans, bitter orange, calcium, vitamin D, chitosan, chromium, cocoa, coleus forskohlii, conjugate linoleic acid, ephedra sinica, fucoxanthin, garcinia cambogia, glucomannan, green coffee, green tea, guar gum, raspberry, hoodia gordonii, irvingia gabonensis, phenylpropylamine, pyruvate, white kidney bean.

Dietary supplements in retinal diseases, glaucoma, and other ocular conditions.

Environmental pollution, inadequate eating habits and unhealthy lifestyles have led to a tremendous increase in ocular diseases worldwide. Given the costly treatments that are currently available for the most common and threatening eye diseases (such as cataract, dry eye disorder, or diabetic retinopathy), curing these diseases or preventing refractive errors by taking nutraceuticals and natural compounds that are present in our daily diet is a very valuable intervention. The eyes are the most important part of our visual system and require micronutrients such as vitamins, carotenoids, trace metals, and omega-3 fatty acids in order to function properly and to protect themselves against light-induced and age-mediated degenerative disorders. The Mediterranean Diet (MedDiet) has been in the limelight since the 1980s because of the several health benefits it provides, including eye health. MedDiet is characterized by the consumption of small amounts of red meat, while emphasizing the intake of fish, eggs, nuts, legumes, citrus fruits, green vegetables, olives and their derivatives, especially olive oil, and dairy products in a proportionate manner, in order to achieve the maximum health benefits. The antioxidant, anti-inflammatory, and neuroprotective properties of these foods - both when used as an ingredient in the dietary regime or as a source of nutritional supplements - have shown promising results in the management of chronic degenerative ocular diseases, both in animal models and in human subjects. In this chapter, we will focus on the importance of MedDiet and natural compounds for the visual system and its role in slowing down age-related ocular degeneration.

Dietary supplements for improving nitric-oxide synthesis.

Nitric oxide (NO) is an essential component of the human body, involved in blood vessel dilation, stimulation of hormone release, signaling and regulation of neurotransmission. Nitric oxide is synthesized by nitric-oxide-synthase-dependent and -independent pathways. Nitric oxide supplementation improves cardiac health, enhances performance during exercise, reduces high blood pressure during pregnancy, reduces erectile dysfunction and improves healing processes and respiratory response. Nitric-oxide-associated benefits are mostly apparent in untrained or moderately trained individuals. L-arginine and L-citrulline supplementation contributes to nitric oxide levels because L-arginine is directly involved in NO synthesis, whereas L-citrulline acts as an L-arginine precursor that is further converted to NO by a reaction catalyzed by NO synthase. L-arginine supplements increase respiratory response and enhance performance during exercise, while L-citrulline with malate and other molecules increase working capacity. Various studies involving beetroot juice have reported a significant increase in plasma nitrite levels, regarded as markers of NO, after intake of beetroot juice. Although NO supplementation may have mild to moderate side-effects, using smaller or divided doses could avoid some of these side-effects. Since nitric oxide supplementation may worsen certain health conditions and may interfere with certain medicines, it should only be taken under medical supervision.