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.

Mitochondrial Dysfunction Causes Cell Death in Patients Affected by Fragile-X-Associated Disorders.

Mitochondria are involved in multiple aspects of neurodevelopmental processes and play a major role in the pathogenetic mechanisms leading to neuro-degenerative diseases. Fragile-X-related disorders (FXDs) are genetic conditions that occur due to the dynamic expansion of CGG repeats of the FMR1 gene encoding for the RNA-binding protein FMRP, particularly expressed in the brain. This gene expansion can lead to premutation (PM, 56-200 CGGs), full mutation (FM, >200 CGGs), or unmethylated FM (UFM), resulting in neurodegeneration, neurodevelopmental disorders, or no apparent intellectual disability, respectively. To investigate the mitochondrial mechanisms that are involved in the FXD patients, we analyzed mitochondrial morphology and bioenergetics in fibroblasts derived from patients. Donut-shaped mitochondrial morphology and excessive synthesis of critical mitochondrial proteins were detected in FM, PM, and UFM cells. Analysis of mitochondrial oxidative phosphorylation in situ reveals lower respiration in PM fibroblasts. Importantly, mitochondrial permeability transition-dependent apoptosis is sensitized to reactive oxygen species in FM, PM, and UFM models. This study elucidated the mitochondrial mechanisms that are involved in the FXD phenotypes, and indicated altered mitochondrial function and morphology. Importantly, a sensitization to permeability transition and apoptosis was revealed in FXD cells. Overall, our data suggest that mitochondria are novel drug targets to relieve the FXD symptoms.

Pathogenic variants in SOX11 mimicking Pitt-Hopkins syndrome phenotype.

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder characterised by severe intellectual disability (ID), distinctive facial features and autonomic nervous system dysfunction, caused by TCF4 haploinsufficiency. We clinically diagnosed with PTHS a 14 6/12 -year-old female, who had a normal status of TCF4. The pathogenic c.667del (p.Asp223MetfsTer45) variant in SOX11 was identified through whole exome sequencing (WES). SOX11 variants were initially reported to cause Coffin-Siris syndrome (CSS), characterised by growth restriction, moderate ID, coarse face, hypertrichosis and hypoplastic nails. However, recent studies have provided evidence that they give rise to a distinct neurodevelopmental disorder. To date, SOX11 variants are associated with a variable phenotype, which has been described to resemble CSS in some cases, but never PTHS. By reviewing both clinically and genetically 32 out of 82 subjects reported in the literature with SOX11 variants, for whom detailed information are provided, we found that 7/32 (22%) had a clinical presentation overlapping PTHS. Furthermore, we made a confirmation that overall SOX11 abnormalities feature a distinctive disorder characterised by severe ID, high incidence of microcephaly and low frequency of congenital malformations. Purpose of the present report is to enhance the role of clinical genetics in assessing the individual diagnosis after WES results.

Transcranial direct current stimulation combined with speech therapy in Fragile X syndrome patients: a pilot study.

Background: Fragile X syndrome (FXS) is the leading cause of genetic intellectual disability. Among the neurobehavioral dysfunctions in FXS individuals, language development and literacy are compromised. Recent evidence hypothesized that the disruption of excitatory glutamatergic and GABAergic inhibitory neurotransmission balance might be responsible for impairment in cognitive function. In this study, we evaluated for the first time, the safety, tolerability, and efficacy of anodal prefrontal transcranial direct current stimulation (tDCS) combined with standard speech therapy to enhance language function in FXS patients. Methods: In total, 16 adult FXS patients were enrolled. Participants underwent 45 min of anodic tDCS combined with speech therapy for 5 weeks (3 times per week). Language function was evaluated using the Test for Reception of Grammar-Version 2 (TROG-2) and subtests of the Italian Language Examination (Esame del Linguaggio - II, EDL-II). Right and left dorsolateral prefrontal cortex transcranial magnetic stimulation and concurrent electroencephalography (TMS-EEG) recordings were collected at baseline and after the treatment to evaluate cortical reactivity and connectivity changes. Results: After 5 weeks of combined therapy, we observed a significant improvement in the writing (7.5%), reading (20.3%), repetition (13.3%), and TROG-2 (10.2%) tests. Parallelly with clinical change, TMS-EEG results showed a significant difference in TMS-evoked potential amplitude over the left frontal cortex after treatment (-0.73 ± 0.87 µV) compared to baseline (0.18 ± 0.84 µV). Conclusion: Our study provides novel evidence that left anodal prefrontal tDCS combined with standard speech therapy could be effective in enhancing language function in FXS patients, mainly by inducing a rebalance of the dysfunctional prefrontal cortical excitability.

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.

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.

Exfoliative Cytology and Genetic Analysis for a Non-Invasive Approach to the Diagnosis of White Sponge Nevus: Case Series.

BACKGROUND: White Sponge Nevus (WSN) is a rare benign disorder associated with mutations in genes coding for cytokeratin 4 (KRT4) and 13 (KRT13) characterized by dyskeratotic hyperplasia of mucous membranes. This study was aimed at examining different approaches (cytology, pathology and genetic analysis) to WSN diagnosis. METHODS: A series of four patients with asymptomatic white diffuse oral lesions were evaluated and, before performing an incisional biopsy for pathology, an oral brush Thin Prep was collected for exfoliative liquid-based cytology (LBC). DNA for genetic analysis was also obtained from patients and both their parents, using buccal swabs. RESULTS: Pathology and cytology showed similar results, leading to the same diagnosis of hyperkeratotic epithelium with acanthosis and spongiosis, without atypia, demonstrating the efficiency of LBC for the differential diagnosis. Sequencing analysis revealed at least 6 rare variants in the KRT4 and KRT13 genes in each patient, contributed in part by both unaffected parents. CONCLUSIONS: Thin Prep for oral exfoliative cytology and genetic analysis are sufficient for an accurate diagnosis of WSN. The combination of cytological and genetic analyses could substitute the histologic exam, providing a non-invasive alternative for incisional biopsy.

Clinical Reasoning: A Young Man With Subacute Onset of Spastic Paraparesis.

Leukodystrophies are a group of rare neurodegenerative disorders, usually presenting in infancy with a variable combination of cognitive, motor, and coordination impairment. Adult-onset cases are even more rare, often representing a diagnostic challenge even for experienced neurologists. Here, we present a case of a 44-year-old man with subacute and rapidly progressive spastic paraplegia, whose brain MRI revealed white matter abnormalities compatible with a diagnosis of leukodystrophy. We discuss how to apply a simplified diagnostic algorithm to distinguish acquired leukoencephalopathies from leukodystrophies and how to delve into the maze of genetic testing for white matter diseases. In our patient, we reached the diagnosis of a treatable disorder, whose early recognition is essential to prevent severe neurologic deterioration.

Triple Genetic Diagnosis in a Patient with Late-Onset Leukodystrophy and Mild Intellectual Disability.

We describe the complex case of a 44-year-old man with polycystic kidney disease, mild cognitive impairment, and tremors in the upper limbs. Brain MRI showed lesions compatible with leukodystrophy. The diagnostic process, which included clinical exome sequencing (CES) and chromosomal microarray analysis (CMA), revealed a triple diagnosis: autosomal dominant polycystic kidney disease (ADPKD) due to a pathogenic variant, c.2152C>T-p.(Gln718Ter), in the PKD1 gene; late-onset phenylketonuria due to the presence of two missense variants, c.842C>T-p.(Pro281Leu) and c.143T>C-p.(Leu48Ser) in the PAH gene; and a 915 Kb duplication on chromosome 15. Few patients with multiple concurrent genetic diagnoses are reported in the literature; in this ADPKD patient, genome-wide analysis allowed for the diagnosis of adult-onset phenylketonuria (which would have otherwise gone unnoticed) and a 15q11.2 duplication responsible for cognitive and behavioral impairment with incomplete penetrance. This case underlines the importance of clinical genetics for interpreting complex results obtained by genome-wide techniques, and for diagnosing concurrent late-onset monogenic conditions.

Metabolomics application for the design of an optimal diet.

Precision nutrition is an emerging branch of nutrition science that aims to use modern omics technologies (genomics, proteomics, and metabolomics) to assess an individual's response to specific foods or dietary patterns and thereby determine the most effective diet or lifestyle interventions to prevent or treat specific diseases. Metabolomics is vital to nearly every aspect of precision nutrition. It can be targeted or untargeted, and it has many applications. Indeed, it can be used to comprehensively characterize the thousands of chemicals in foods, identify food by-products in human biofluids or tissues, characterize nutrient deficiencies or excesses, monitor biochemical responses to dietary interventions, track long- or short-term dietary habits, and guide the development of nutritional therapies. Indeed, metabolomics can be coupled with genomics and proteomics to study and advance the field of precision nutrition. Integrating omics with epidemiological and clinical data will begin to define the beneficial effects of human food metabolites. In this review, we present the metabolome and its relationship to precision nutrition. Moreover, we describe the different techniques used in metabolomics and present how metabolomics has been applied to advance the field of precision nutrition by providing notable examples and cases.

Polymorphisms, diet and nutrigenomics.

Every human being possesses an exclusive nutritional blueprint inside their genes. Bioactive food components and nutrients affect the expression of such genes. Nutrigenomics is the science that analyzes gene-nutrient interactions (nutrigenetics), which can lead to the development of personalized nutritional recommendations to maintain optimal health and prevent disease. Genomic diversity among various ethnic groups might affect nutrients bioavailability as well as their metabolism. Nutrigenomics combines different branches of science including nutrition, bioinformatics, genomics, molecular biology, molecular medicine, and epidemiology. Genes regulate intake and metabolism of different nutrients, while nutrients positively or negatively influence the expression of a number of genes; testing of specific genetic polymorphisms may therefore become a useful tool to manage weight loss and to fully understand gene-nutrient interactions. Indeed, several approaches are used to study gene-nutrient interactions: epigenetics, the study of genome modification not related to changes in nucleotide sequence; transcriptomics, the study of tissue-specific and time-specific RNA transcripts; proteomics, the study of proteins involved in biological processes; and metabolomics, the study of changes of primary and secondary metabolites in body fluids and tissues. Hence, the use of nutrigenomics to improve and optimize a healthy, balanced diet in clinical settings could be an effective approach for long-term lifestyle changes that might lead to consistent weight loss and improve quality of life.

Dietary supplements in neurological diseases and brain aging.

A healthy diet shapes a healthy mind. Diet quality has a strong association with brain health. Diet influences the onset and consequences of neurological diseases, and dietary factors may influence mental health at individual and population level. The link between unhealthy diet, impaired cognitive function and neurodegenerative diseases indicates that adopting a healthy diet would ultimately afford prevention and management of neurological diseases and brain aging. Neurodegenerative diseases are of multifactorial origin and result in progressive loss of neuronal function in the brain, leading to cognitive impairment and motoneuron disorders. The so-called Mediterranean diet (MedDiet) with its healthy ingredients rich in antioxidant, anti-inflammatory, immune, neuroprotective, antidepressant, antistress and senolytic activity plays an essential role in the prevention and management of neurological diseases and inhibits cognitive decline in neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases. The MedDiet also modulates the gut-brain axis by promoting a diversity of gut microbiota. In view of the importance of diet in neurological diseases management, this review focuses on the dietary components, natural compounds and medicinal plants that have proven beneficial in neurological diseases and for brain health. Among them, polyphenols, omega-3 fatty acids, B vitamins and several ayurvedic herbs have promising beneficial effects.

Mother and Daughter Carrying of the Same Pathogenic Variant in FGFR2 with Discordant Phenotype.

Craniosynostosis are a heterogeneous group of genetic conditions characterized by the premature fusion of the skull bones. The most common forms of craniosynostosis are Crouzon, Apert and Pfeiffer syndromes. They differ from each other in various additional clinical manifestations, e.g., syndactyly is typical of Apert and rare in Pfeiffer syndrome. Their inheritance is autosomal dominant with incomplete penetrance and one of the main genes responsible for these syndromes is FGFR2, mapped on chromosome 10, encoding fibroblast growth factor receptor 2. We report an FGFR2 gene variant in a mother and daughter who present with different clinical features of Crouzon syndrome. The daughter is more severely affected than her mother, as also verified by a careful study of the face and oral cavity. The c.1032G>A transition in exon 8, already reported as a synonymous p.Ala344 = variant in Crouzon patients, also activates a new donor splice site leading to the loss of 51 nucleotides and the in-frame removal of 17 amino acids. We observed lower FGFR2 transcriptional and translational levels in the daughter compared to the mother and healthy controls. A preliminary functional assay and a molecular modeling added further details to explain the discordant phenotype of the two patients.

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Genetics of Inherited Retinal Diseases in Understudied Ethnic Groups in Italian Hospitals.

Purpose: Describing the clinical and genetic features of an ethnically heterogeneous group of (inherited retinal diseases) IRD patients from different underrepresented countries, referring to specialized Italian Hospitals, and expanding the epidemiological spectrum of the IRD in understudied populations. Methods: The patients' phenotypes underwent were characterized by exhaustive ophthalmological examinations, including morpho-functional testing. Genetic testing was performed using next-generation sequencing (NGS) and gene sequencing panels targeting a specific set of genes, Sanger sequencing and-when necessary-multiplex ligation-dependent probe amplification (MLPA) to better identify the genotype. When possible, segregation analysis was performed in order to confirm unsolved cases. Results: The article reports the results of the phenotypes and genotypes of 123 IRD probands, 69 males and 54 females, mean age 41 (IQR, 54-30) years, disease onset at 13 (IQR, 27.25-5) years. Thirty-three patients out of 123 (26.8%) were Africans (North/Northwest Africa), 21 (17.1%) Asians, 19 (15.4%) Americans (South/Central America) and 50 (40.7%) Europeans (Eastern Europe). Retinitis pigmentosa was the most represented phenotype (56%), followed by cone dystrophy (11%) and Leber congenital amaurosis (7%), while ABCA4 was the most frequently mutated gene (18%), followed by USH2A (9%) and RPGR (5%). About ABCA4 variants found in Stargardt disease, macular and cone dystrophies were predominant in Asian (42%) and European (21%) patients. The most represented inheritance pattern was autosomal recessive, while a higher frequency of homozygous patients versus compound heterozygotes as compared to previous studies on Italian IRD patients was evidenced, reflecting a possible higher frequency of inbreeding marriages. Conclusion: Though limited by the relatively low number of patients, the present paper paints a picture of the clinical and genetic features of IRD patients from understudied ethnic groups referred to Italian specialized hospitals and extended the epidemiological studies on underrepresented world regional areas.

Identification of ultra-rare genetic variants in pediatric acute onset neuropsychiatric syndrome (PANS) by exome and whole genome sequencing.

Abrupt onset of severe neuropsychiatric symptoms including obsessive-compulsive disorder, tics, anxiety, mood swings, irritability, and restricted eating is described in children with Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS). Symptom onset is often temporally associated with infections, suggesting an underlying autoimmune/autoinflammatory etiology, although direct evidence is often lacking. The pathological mechanisms are likely heterogeneous, but we hypothesize convergence on one or more biological pathways. Consequently, we conducted whole exome sequencing (WES) on a U.S. cohort of 386 cases, and whole genome sequencing (WGS) on ten cases from the European Union who were selected because of severe PANS. We focused on identifying potentially deleterious genetic variants that were de novo or ultra-rare (MAF) < 0.001. Candidate mutations were found in 11 genes (PPM1D, SGCE, PLCG2, NLRC4, CACNA1B, SHANK3, CHK2, GRIN2A, RAG1, GABRG2, and SYNGAP1) in 21 cases, which included two or more unrelated subjects with ultra-rare variants in four genes. These genes converge into two broad functional categories. One regulates peripheral immune responses and microglia (PPM1D, CHK2, NLRC4, RAG1, PLCG2). The other is expressed primarily at neuronal synapses (SHANK3, SYNGAP1, GRIN2A, GABRG2, CACNA1B, SGCE). Mutations in these neuronal genes are also described in autism spectrum disorder and myoclonus-dystonia. In fact, 12/21 cases developed PANS superimposed on a preexisting neurodevelopmental disorder. Genes in both categories are also highly expressed in the enteric nervous system and the choroid plexus. Thus, genetic variation in PANS candidate genes may function by disrupting peripheral and central immune functions, neurotransmission, and/or the blood-CSF/brain barriers following stressors such as infection.

Mechanisms of the FMR1 Repeat Instability: How Does the CGG Sequence Expand?

A dynamic mutation in exon 1 of the FMR1 gene causes Fragile X-related Disorders (FXDs), due to the expansion of an unstable CGG repeat sequence. Based on the CGG sequence size, two types of FMR1 alleles are possible: "premutation" (PM, with 56-200 CGGs) and "full mutation" (FM, with >200 triplets). Premutated females are at risk of transmitting a FM allele that, when methylated, epigenetically silences FMR1 and causes Fragile X syndrome (FXS), a very common form of inherited intellectual disability (ID). Expansions events of the CGG sequence are predominant over contractions and are responsible for meiotic and mitotic instability. The CGG repeat usually includes one or more AGG interspersed triplets that influence allele stability and the risk of transmitting FM to children through maternal meiosis. A unique mechanism responsible for repeat instability has not been identified, but several processes are under investigations using cellular and animal models. The formation of unusual secondary DNA structures at the expanded repeats are likely to occur and contribute to the CGG expansion. This review will focus on the current knowledge about CGG repeat instability addressing the CGG sequence expands.

PacMAGI: A pipeline including accurate indel detection for the analysis of PacBio sequencing data applied to RPE65.

Third generation sequencing methods, like PacBio, provide information about structural variants, introns, enhancers and promoters. We developed an automated pipeline, called PacMAGI, including quality control, alignment, SNV, INDELs, structural variant calling, phasing, annotation and variant interpretation, for the analysis of PacBio data for any target region. Bi-allelic mutations in the RPE65 gene are associated with different inherited retinal dystrophies, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Diagnostic panel-based NGS analysis is performed on coding regions and intron/exon junctions of genes. To obtain a more conclusive diagnosis, we applied PacMAGI to obtain a second hit on RPE65 in LCA or RP patients who showed a single heterozygous variant by NGS. We used PacBio to sequence the full gene and identify putative second-hits in intronic, problematic and promoter regions. All variants identified in the diagnostic setting with NGS were correctly detected by the pipeline, and thanks to our custom algorithm for INDELs, a previously undetected 'Pathogenic' frameshift variant was found in a RP patient already identified to carry a 'Likely Pathogenic' variant.

Genetic characteristics of 234 Italian patients with macular and cone/cone-rod dystrophy.

Two-hundred and thirty-four Italian patients with a clinical diagnosis of macular, cone and cone-rod dystrophies (MD, CD, and CRD) were examined using next-generation sequencing (NGS) and gene sequencing panels targeting a specific set of genes, Sanger sequencing and-when necessary-multiplex ligation-dependent probe amplification (MLPA) to diagnose the molecular cause of the aforementioned diseases. When possible, segregation analysis was performed in order to confirm unsolved cases. Each patient's retinal phenotypic characteristics were determined using focal and full-field ERGs, perimetry, spectral domain optical coherence tomography and fundus autofluorescence. We identified 236 potentially causative variants in 136 patients representing the 58.1% of the total cohort, 43 of which were unpublished. After stratifying the patients according to their clinical suspicion, the diagnostic yield was 62.5% and 53.8% for patients with MD and for those with CD/CRD, respectively. The mode of inheritance of all cases confirmed by genetic analysis was 70% autosomal recessive, 26% dominant, and 4% X-linked. The main cause (59%) of both MD and CD/CRD cases was the presence of variants in the ABCA4 gene, followed by variants in PRPH2 (9%) and BEST1 (6%). A careful morpho-functional evaluation of the phenotype, together with genetic counselling, resulted in an acceptable diagnostic yield in a large cohort of Italian patients. Our study emphasizes the role of targeted NGS to diagnose MDs, CDs, and CRDs, as well as the clinical usefulness of segregation analysis for patients with unsolved diagnosis.

A next generation sequencing gene panel for use in the diagnosis of anorexia nervosa.

PURPOSE: The aim of this study was to increase knowledge of genes associated with anorexia nervosa (AN) and their diagnostic offer, using a next generation sequencing (NGS) panel for the identification of genetic variants. The rationale underlying this test is that we first analyze the genes associated with syndromic forms of AN, then genes that were found to carry rare variants in AN patients who had undergone segregation analysis, and finally candidate genes intervening in the same molecular pathways or identified by GWAS or in mouse models. METHODS: We developed an NGS gene panel and used it to screen 68 Italian AN patients (63 females, 5 males). The panel included 162 genes. Family segregation study was conducted on available relatives of probands who reported significant genetic variants. RESULTS: In our analysis, we found potentially deleterious variants in 2 genes (PDE11A and SLC25A13) associated with syndromic forms of anorexia and predicted deleterious variants in the following 12 genes: CD36, CACNA1C, DRD4, EPHX2, ESR1, GRIN2A, GRIN3B, LRP2, NPY4R, PTGS2, PTPN22 and SGPP2. Furthermore, by Sanger sequencing of the promoter region of NNAT, we confirmed the involvement of this gene in the pathogenesis of AN. Family segregation studies further strengthened the possible causative role of CACNA1C, DRD4, GRIN2A, PTGS2, SGPP2, SLC25A13 and NNAT genes in AN etiology. CONCLUSION: The major finding of our study is the confirmation of the involvement of the NNAT gene in the pathogenesis of AN; furthermore, this study suggests that NGS-based testing can play an important role in the diagnostic evaluation of AN, excluding syndromic forms and increasing knowledge of the genetic etiology of AN. LEVEL OF EVIDENCE: Level I, experimental study.