The Frequency of Intermediate Alleles in Patients with Cerebellar Phenotypes.

Cerebellar syndromes are clinically and etiologically heterogeneous and can be classified as hereditary, neurodegenerative non-hereditary, or acquired. Few data are available on the frequency of each form in the clinical setting. Growing interest is emerging regarding the genetic forms caused by triplet repeat expansions. Alleles with repeat expansion lower than the pathological threshold, termed intermediate alleles (IAs), have been found to be associated with disease manifestation. In order to assess the relevance of IAs as a cause of cerebellar syndromes, we enrolled 66 unrelated Italian ataxic patients and described the distribution of the different etiology of their syndromes and the frequency of IAs. Each patient underwent complete clinical, hematological, and neurophysiological assessments, neuroimaging evaluations, and genetic tests for autosomal dominant cerebellar ataxia (SCA) and fragile X-associated tremor/ataxia syndrome (FXTAS). We identified the following diagnostic categories: 28% sporadic adult-onset ataxia, 18% cerebellar variant of multiple system atrophy, 9% acquired forms, 9% genetic forms with full-range expansion, and 12% cases with intermediate-range expansion. The IAs were six in the FMR1 gene, two in the gene responsible for SCA8, and one in the ATXN2 gene. The clinical phenotype of patients carrying the IAs resembles, in most of the cases, the one associated with full-range expansion. Our study provides an exhaustive description of the causes of cerebellar ataxia, estimating for the first time the frequency of IAs in SCAs- and FXTAS-associated genes. The high percentage of cases with IAs supports further screening among patients with cerebellar syndromes.

Large scale genome-wide association analyses identify novel genetic loci and mechanisms in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an important cause of morbidity and mortality with both monogenic and polygenic components. We here report results from the largest HCM genome-wide association study (GWAS) and multi-trait analysis (MTAG) including 5,900 HCM cases, 68,359 controls, and 36,083 UK Biobank (UKB) participants with cardiac magnetic resonance (CMR) imaging. We identified a total of 70 loci (50 novel) associated with HCM, and 62 loci (32 novel) associated with relevant left ventricular (LV) structural or functional traits. Amongst the common variant HCM loci, we identify a novel HCM disease gene, SVIL, which encodes the actin-binding protein supervillin, showing that rare truncating SVIL variants cause HCM. Mendelian randomization analyses support a causal role of increased LV contractility in both obstructive and non-obstructive forms of HCM, suggesting common disease mechanisms and anticipating shared response to therapy. Taken together, the findings significantly increase our understanding of the genetic basis and molecular mechanisms of HCM, with potential implications for disease management.

Genetic epidemiology of inherited retinal diseases in a large patient cohort followed at a single center in Italy.

Inherited retinal diseases (IRDs) are the leading cause of vision loss in the working-age population. We performed a retrospective epidemiological study to determine the genetic basis of IRDs in a large Italian cohort (n = 2790) followed at a single referral center. We provided, mainly by next generation sequencing, potentially conclusive molecular diagnosis for 2036 patients (from 1683 unrelated families). We identified a total of 1319 causative sequence variations in 132 genes, including 353 novel variants, and 866 possibly actionable genotypes for therapeutic approaches. ABCA4 was the most frequently mutated gene (n = 535; 26.3% of solved cases), followed by USH2A (n = 228; 11.2%) and RPGR (n = 102; 5.01%). The other 129 genes had a lower contribution to IRD pathogenesis (e.g. CHM 3.5%, RHO 3.5%; MYO7A 3.4%; CRB1 2.7%; RPE65 2%, RP1 1.8%; GUCY2D 1.7%). Seventy-eight genes were mutated in five patients or less. Mitochondrial DNA variants were responsible for 2.1% of cases. Our analysis confirms the complex genetic etiology of IRDs and reveals the high prevalence of ABCA4 and USH2A mutations. This study also uncovers genetic associations with a spectrum of clinical subgroups and highlights a valuable number of cases potentially eligible for clinical trials and, ultimately, for molecular therapies.

Positive predictive values and outcomes for uninformative cell-free DNA tests: An Italian multicentric Cytogenetic and cytogenomic Audit of diagnOstic testing (ICARO study).

OBJECTIVES: To establish the positive predictive values (PPV) of cfDNA testing based on data from a nationwide survey of independent clinical cytogenetics laboratories. METHODS: Prenatal diagnostic test results obtained by Italian laboratories between 2013 and March 2020 were compiled for women with positive non-invasive prenatal tests (NIPT), without an NIPT result, and cases where there was sex discordancy between the NIPT and ultrasound. PPV and other summary data were reviewed. RESULTS: Diagnostic test results were collected for 1327 women with a positive NIPT. The highest PPVs were for Trisomy (T) 21 (624/671, 93%) and XYY (26/27, 96.3%), while rare autosomal trisomies (9/47, 19.1%) and recurrent microdeletions (8/55, 14.5%) had the lowest PPVs. PPVs for T21, T18, and T13 were significantly higher when diagnostic confirmation was carried out on chorionic villi (97.5%) compared to amniotic fluid (89.5%) (p < 0.001). In 19/139 (13.9%), of no result cases, a cytogenetic abnormality was detected. Follow-up genetic testing provided explanations for 3/6 cases with a fetal sex discordancy between NIPT and ultrasound. CONCLUSIONS: NIPT PPVs differ across the conditions screened and the tissues studied in diagnostic testing. This variability, issues associated with fetal sex discordancy, and no results, illustrate the importance of pre- and post-test counselling.

Choroidal Caverns in Stargardt Disease.

Purpose: To report choroidal caverns in patients affected by recessive Stargardt disease (STGD1) and to investigate its clinical features. Methods: Retrospective analysis of STGD1 patients recruited at the Regional Reference Center for Hereditary Retinal Degenerations at the Eye Clinic in Florence from 2012 to 2017. Patients included in the study underwent a complete ophthalmic examination including best-corrected visual acuity, color fundus photography, fundus autofluorescence, optical coherence tomography (OCT) and OCT angiography. Results: Eighty-six patients (172 eyes) were included in the study. Twenty-three eyes (13.3%) of 21 patients presented choroidal caverns. The total number of detected choroidal caverns was 63. Choroidal caverns were only present in patients with stage III and IV STGD. Interestingly, patients with choroidal caverns presented larger macular atrophy (20.53 ± 16.9 mm2 vs. 18.11 ± 20.39 mm2), worse visual acuity (1.03 ± 0.29 vs. 0.83 ± 0.26), and a thinner choroidal thickness (245.9 ± 88.7 vs. 266.0 ± 110.5 µm). Conclusions: Choroidal caverns are present only in the advanced stage of STGD1, and a possible degenerative origin of the finding has been hypothesized.

Genetic analysis of suicide: a sample study in Tuscany (Central Italy).

Many studies have examined the genetic contribution to suicide. However, data on suicide in the Italian population are scarce. We therefore aimed to address this gap by investigating a cohort of 111 Italians for whom a verdict of suicide had been declared in court in Florence, Italy between 2007 and 2017. This cohort included 86 men and 25 women. DNA samples were obtained from tissues or blood, and 22 genes from multiple neurobiological pathways previously shown to be associated with the pathogenesis of suicide were analysed. Next-generation sequencing was used to compare these gene sequences with those from a large, normal population. In this study, we identified 19 gene variants that were present at significantly lower frequencies in our Italian cohort than in the general population. In addition, four missense mutations were identified in four different genes: Monoamine Oxidase A (MAOA), 5-Hydroxytryptamine Receptor 2 A (HTR2A), Sodium Voltage-Gated Channel Alpha Subunit 8 (SCN8A), and Nitric Oxide Synthase 3 (NOS3). Our study identified several potential genetic links with suicide in a cohort of Italians and supports a relationship between specific genetic variants and suicidal behaviour in this population.

Nanopore sequencing for the screening of myeloid and lymphoid neoplasms with eosinophilia and rearrangement of PDGFRα, PDGFRß, FGFR1 or PCM1-JAK2.

Eosinophilia represents a group of diseases with heterogeneous pathobiology and clinical phenotypes. Among the alterations found in primary Eosinophilia, gene fusions involving PDGFRα, PDGFRß, FGFR1 or JAK2 represent the biomarkers of WHO-defined "myeloid and lymphoid neoplasms with eosinophilia". The heterogeneous nature of genomic aberrations and the promiscuity of fusion partners, may limit the diagnostic accuracy of current cytogenetics approaches. To address such technical challenges, we exploited a nanopore-based sequencing assay to screen patients with primary Eosinophilia. The comprehensive sequencing approach described here enables the identification of genomic fusion in 60 h, starting from DNA purified from whole blood.

A new GNPAT variant of foetal rhizomelic chondrodysplasia punctata.

BACKGROUND: Rhizomelic chondrodysplasia punctata (RCDP) is a clinical entity resulting from defects of peroxisomal metabolism whose clinical phenotype is characterized by rhizomelia, calcified foci in periarticular cartilage, coronal lesions of vertebral bodies, cataracts and severe cognitive delay. Usually, survival does not exceed the first decade of life. Transmission is autosomal recessive and is related to mutations in the PEX7, GNPAT or AGPS. METHODS: A detailed description of the prenatal ultrasound signs of RCDP found in two successive pregnancies in a consanguineous couple is reported. Molecular genetic investigations included the study of the coding regions and the exon-intron junctions of the GNPAT (high-throughput amplification and sequencing performed with Roche NimbleGen SeqCap Target kit on Illumina platform); the confirmation test was carried out by amplification and Sanger sequencing with automatic capillary sequencer. RESULTS: In addition to the typical prenatal ultrasound signs described in the literature in association with RCDP, the presence of prefrontal oedema, never previously described, has been detected in both pregnancies. Moreover, genetic investigations have found a new splicing variant c.924+1G>A of the homozygous GNPAT. CONCLUSION: The role of mutation in the GNPAT suggests a likely association with the clinical phenotype.

RPE65-associated inherited retinal diseases: consensus recommendations for eligibility to gene therapy.

BACKGROUND: This research aimed to establish recommendations on the clinical and genetic characteristics necessary to confirm patient eligibility for gene supplementation with voretigene neparvovec. METHODS: An expert steering committee comprising an interdisciplinary panel of Italian experts in the three fields of medical specialisation involved in the management of RPE65-associated inherited retinal disease (IRD) (medical retina, genetics, vitreoretinal surgery) proposed clinical questions necessary to determine the correct identification of patients with the disease, determine the fundamental clinical and genetics tests to reach the correct diagnosis and to evaluate the urgency to treat patients eligible to receive treatment with voretigene neparvovec. Supported by an extensive review of the literature, a series of statements were developed and refined to prepare precisely constructed questionnaires that were circulated among an external panel of experts comprising ophthalmologists (retina specialists, vitreoretinal surgeons) and geneticists with extensive experience in IRDs in Italy in a two-round Delphi process. RESULTS: The categories addressed in the questionnaires included clinical manifestations of RPE65-related IRD, IRD screening and diagnosis, gene testing and genotyping, ocular gene therapy for IRDs, patient eligibility and prioritisation and surgical issues. Response rates by the survey participants were over 90% for the majority of items in both Delphi rounds. The steering committee developed the key consensus recommendations on each category that came from the two Delphi rounds into a simple and linear diagnostic algorithm designed to illustrate the patient pathway leading from the patient's referral centre to the retinal specialist centre. CONCLUSIONS: Consensus guidelines were developed to guide paediatricians and general ophthalmologists to arrive at the correct diagnosis of RPE65-associated IRD and make informed clinical decisions regarding eligibility for a gene therapy approach to RPE65-associated IRD. The guidelines aim to ensure the best outcome for the patient, based on expert opinion, the published literature, and practical experience in the field of IRDs.

Extramedullary blastic transformation of primary myelofibrosis in the form of disseminated myeloid sarcoma: a case report and review of the literature.

Splenomegaly is a key clinical manifestation of myelofibrosis, and splenectomy is currently indicated in patients with drug refractory, symptomatic splenomegaly or with the aim of improving refractory cytopenias. Transformation to acute myeloid leukemia occurs in up to 20% of patients with myelofibrosis, while cases of myeloid sarcoma have been reported very unfrequently. In this manuscript, we report the case of a 60-year-old man with a history of primary myelofibrosis who underwent splenectomy because of drug-refractory massive splenomegaly, systemic symptoms and anemia. At the opening of the peritoneal cavity, the spleen resulted massively enlarged and tenaciously entrapped by a pervasive neoplastic-like tissue. The extensive involvement of the abdomen fatally complicated the surgical procedure. At postmortem examination, the spleen showed a diffuse infiltration of immature cells that were also found in the peritoneum, bowel, liver, lungs and myocardium. After immunohistochemical, cytogenetic, flow cytometric and molecular characterization of neoplastic population, a diagnosis of disseminated myeloid sarcoma of the spleen was made. This case report highlights a very unusual case of myeloid sarcoma originating from the spleen in a patient with myelofibrosis who had no evidence of bone marrow or peripheral blood involvement by leukemic cells. Molecular characterization showed that leukemic cells originated from the founding clone of the chronic phase. The sarcoma could not be suspected based on clinical findings and was diagnosed only at the time of surgical procedure and autopsy. This case suggests that leukemic transformation of myelofibrosis can originate outside the bone marrow and, presumably rarely, present as a granulocytic sarcoma.

Nano-GLADIATOR: real-time detection of copy number alterations from nanopore sequencing data.

MOTIVATION: The past few years have seen the emergence of nanopore-based sequencing technologies which interrogate single molecule of DNA and generate reads sequentially. RESULTS: In this paper, we demonstrate that, thanks to the sequentiality of the nanopore process, the data generated in the first tens of minutes of a typical MinION/GridION run can be exploited to resolve the alterations of a human genome at a karyotype level with a resolution in the order of tens of Mb, while the data produced in the first 6-12 h allow to obtain a resolution comparable to currently available array-based technologies, and thanks to a novel probabilistic approach are capable to predict the allelic fraction of genomic alteration with high accuracy. To exploit the unique characteristics of nanopore sequencing data we developed a novel software tool, Nano-GLADIATOR, that is capable to perform copy number variants/alterations detection and allelic fraction prediction during the sequencing run ('On-line' mode) and after experiment completion ('Off-line' mode). We tested Nano-GLADIATOR on publicly available ('Off-line' mode) and on novel whole genome sequencing dataset generated with MinION device ('On-line' mode) showing that our tool is capable to perform real-time copy number alterations detection obtaining good results with respect to other state-of-the-art tools. AVAILABILITY AND IMPLEMENTATION: Nano-GLADIATOR is freely available at https://sourceforge.net/projects/nanogladiator/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

En face OCT in choroideremia.

Purpose: To describe the outer retinal tubulation (ORT) morphology using En face OCT elaboration in a large group of patients affected by choroideremia (CHM).Material and Methods: We retrospectively reviewed CHM patients examined at the Regional Reference Center for Hereditary Retinal Degenerations at the Eye Clinic in Florence. We took into consideration genetically confirmed CHM patients with ophthalmological, fundus autofluorescence (FAF) and optical coherence tomography (OCT) examinations.Results: We studied en face OCT features of ORTs in 18 CHM patients, for a total of 36 eyes; (average age 33 years; SD 19,2; range 13-77 years). ORTs were found in 30 eyes of 15 patients (15/18; 83,3% of the patients). We identified 3 en face OCT patterns: round lesions with scalloped boundaries which involved the peripapillary area with more or less evident pseudodendritic ORTs (PD-ORT) (pattern p; 26,7%); central islands with PD-ORTs (pattern i; 53,3%); residual outer retinal areas with no ORTs (pattern r; 20,0%).Conclusions: In CHM, en face OCT imaging allows us to observe various morphological features of the ORTs in different stages of disease, not detectable with other imaging techniques. ORTs were not identified in the mildest phenotypes. En face OCT is a non-invasive useful tool in the characterization and monitoring of the disease.

Defining the diagnostic effectiveness of genes for inclusion in panels: the experience of two decades of genetic testing for hypertrophic cardiomyopathy at a single center.

PURPOSE: Genetic testing in hypertrophic cardiomyopathy (HCM) has long relied on Sanger sequencing of sarcomeric genes. The advent of next-generation sequencing (NGS) has catalyzed routine testing of additional genes of dubious HCM-causing potential. We used 19 years of genetic testing results to define a reliable set of genes implicated in Mendelian HCM and assess the value of expanded NGS panels. METHODS: We dissected genetic testing results from 1,198 single-center HCM probands and devised a widely applicable score to identify which genes yield effective results in the diagnostic setting. RESULTS: Compared with early panels targeting only fully validated sarcomeric HCM genes, expanded NGS panels allow the prompt recognition of probands with HCM-mimicking diseases. Scoring by "diagnostic effectiveness" highlighted that PLN should also be routinely screened besides historically validated genes for HCM and its mimics. CONCLUSION: The additive value of expanded panels in HCM genetic testing lies in the systematic screening of genes associated with HCM mimics, requiring different patient management. Only variants in a limited set of genes are highly actionable and interpretable in the clinic, suggesting that larger panels offer limited additional sensitivity. A score estimating the relative effectiveness of a given gene's inclusion in diagnostic panels is proposed.

CFH Y402H polymorphism in Italian patients with age-related macular degeneration, retinitis pigmentosa, and Stargardt disease.

BACKGROUND: The complement system has been implicated in the pathogenesis of age-related macular degeneration (AMD) and the CFH Y402H polymorphism has been suggested as a major risk factor for AMD. Recent evidences supported the role of inflammation in the pathogenesis of some retinal dystrophies. Aim of this study was to evaluate the prevalence of CFHY402H polymorphism in a group of Italian patients affected by atrophic AMD, Stargardt disease (STGD), or retinitis pigmentosa(RP). MATERIALS AND METHODS: Our case-control association study included 116 patients with atrophic AMD, 77 with RP, 86 with STGD, and 100 healthy controls. All the patients were evaluated by a standard ophthalmologic examination and OCT. ERG was performed on STGD and RP patients. All the subjects underwent a blood drawing for genetic testing and the CFHY402H polymorphism was genotyped with the TaqMan real-time polymerase chain reaction single nucleotide polymorphism assay. RESULTS: The prevalence of the risk genotype C/C was higher in the AMD group than in controls (p < 0.001). The risk allele C was more frequent in the AMD group than in controls (p < 0.001). The prevalence of the risk genotype was higher in the RP patients than in controls (p < 0.001) and similarly the risk allele C was more frequent in the RP group (p = 0.008). The CFHY402H genotype distribution was not different between patients with STGD and the controls, for the biallelic (p = 0.531) and for the monoallelic (p = 0.318) evaluation. CONCLUSIONS: In our series of Italian patients, the CFHY402H genotype is associated with atrophic AMD and RP, but not with STGD. This result may support the hypothesis of a complement system dysregulation in the pathogenesis of AMD and RP.

Fundus phenotype in retinitis pigmentosa associated with EYS mutations.

PURPOSE: to report phenotypic and genotypic features in a group of autosomal recessive retinitis pigmentosa (arRP) patients associated with EYS mutations. METHODS: we retrospectively reviewed the clinical records and the molecular genetic data of arRP patients carrying mutations in the EYS gene. All the patients underwent a comprehensive opthalmological examination. Additional investigation included color fundus photography, fundus autofluorescence, Goldmann visual field, OCT scans and full-field standard electroretinography. RESULTS: we studied 10 RP patients (20 eyes) characterized by mutations in the EYS gene. Thirteen different sequence variants in the EYS gene were identified. In total, nine mutations found in our series had not previously been reported in the literature. All patients in our series complained of typical RP symptoms at the onset of the disease, namely night blindness and progressive constriction of the visual field. Visual acuity ranged from light perception to 20/20. Relevant findings reported in our series are Interdigitation-zone (IZ band) involvement, present even in the milder phenotypes and an estimated prevalence of 6.2% of arRP associated with EYS mutations. CONCLUSIONS: we reported the mutation spectrum of a group of EYS-related RP patients including nine novel mutations and the associated clinical phenotypes. Our series is the largest group of EYS-related arRP patients in the Italian population.

S737F is a new CFTR mutation typical of patients originally from the Tuscany region in Italy.

BACKGROUND: An increasing number of patients have been described as having a number of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) variants for which it lacks a clear genotype-phenotype correlation. We assesses the clinical features of patients bearing the S737F (p.Ser737Phe) CFTR missense variant and evaluated the residual function of CFTR protein on nasal epithelial cells (NEC). METHODS: A retrospective database was performed from individuals homozygous or compound heterozygous for the S737F variant followed in the Cystic Fibrosis (CF) Centre of Florence. We performed a nasal brushing in cooperating patients and compared the results with those of patients followed in the pediatric CF Centre of Naples. RESULTS: 9/295 (3%) subjects carrying at least S737F CFTR variant on one allele were identified. Patients were diagnosed in 7/9 cases by newborn screening and in two cases for dehydration with hypochloremic metabolic alkalosis; at diagnosis sweat chloride levels (SCL) were in the pathological range in only one case. After a mean follow up of 8,6 years (range 0,5-15,8), SCL were in the pathological range in 8/9 cases (mean age at CF diagnosis: 1,5 years), all patients were pancreatic sufficiency and respiratory function was normal. The gating activity on NEC was 15.6% and 12.7% in two patients compound heterozygous for W1282X and DelE22_24, while it was ranged between 6,2% and 9,8% in CF patients. CONCLUSIONS: S737F is a CFTR mutation associated to hypochloremic alkalosis in childhood, mild CF phenotype in teenage years and a residual function of CFTR protein.