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Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the "dysglycaemia panel" composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants. [ABSTRACT FROM AUTHOR]

Simple Summary: Neurofibromatosis type 1 and Moyamoya disease can be associated in Moyamoya syndrome (MMS), which causes cerebral arteriopathy consisting of a progressive steno-occlusion of the intracranial arteries. Here, we investigated if rare variants of RNF213, the most common genetic risk factor for Moyamoya, could act as genetic modifiers of MMS phenotype in a pediatric cohort of patients. Next-generation sequencing of NF1 and RNF213 genes was performed on genomic DNA extracted from patients' blood. We found that in MMS patients, RNF213 does not appear to modify Moyamoya occurrence risk. Rather, it is more probable that the loss of neurofibromin 1, the protein encoded by the NF1 gene, is responsible by itself for the excessive proliferation of vascular smooth muscle cells causing arterial stenosis. Further studies in larger cohorts of patients are necessary to confirm these findings and to identify other genetic factors in order to increase our understanding of MMS pathogenesis. Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder caused by mutations in NF1 gene, coding for neurofibromin 1. NF1 can be associated with Moyamoya disease (MMD), and this association, typical of paediatric patients, is referred to as Moyamoya syndrome (MMS). MMD is a cerebral arteriopathy characterized by the occlusion of intracranial arteries and collateral vessel formation, which increase the risk of ischemic and hemorrhagic events. RNF213 gene mutations have been associated with MMD, so we investigated whether rare variants of RNF213 could act as genetic modifiers of MMS phenotype in a pediatric cohort of 20 MMS children, 25 children affected by isolated MMD and 47 affected only by isolated NF1. By next-generation re-sequencing (NGS) of patients' DNA and gene burden tests, we found that RNF213 seems to play a role only for MMD occurrence, while it does not appear to be involved in the increased risk of Moyamoya for MMS patients. We postulated that the loss of neurofibromin 1 can be enough for the excessive proliferation of vascular smooth muscle cells, causing Moyamoya arteriopathy associated with NF1. Further studies will be crucial to support these findings and to elucidate the possible role of other genes, enhancing our knowledge about pathogenesis and treatment of MMS. [ABSTRACT FROM AUTHOR]

Inborn errors of immunity (IEI) include a large group of inherited diseases sharing either poor, dysregulated, or absent and/or acquired function in one or more components of the immune system. Next-generation sequencing (NGS) has driven a rapid increase in the recognition of such defects, though the wide heterogeneity of genetically diverse but phenotypically overlapping diseases has often prevented the molecular characterization of the most complex patients. Two hundred and seventy-two patients were submitted to three successive NGS-based gene panels composed of 58, 146, and 312 genes. Along with pathogenic and likely pathogenic causative gene variants, accounting for the corresponding disorders (37/272 patients, 13.6%), a number of either rare (probably) damaging variants in genes unrelated to patients' phenotype, variants of unknown significance (VUS) in genes consistent with their clinics, or apparently inconsistent benign, likely benign, or VUS variants were also detected. Finally, a remarkable amount of yet unreported variants of unknown significance were also found, often recurring in our dataset. The NGS approach demonstrated an expected IEI diagnostic rate. However, defining the appropriate list of genes for these panels may not be straightforward, and the application of unbiased approaches should be taken into consideration, especially when patients show atypical clinical pictures. [ABSTRACT FROM AUTHOR]

Simple Summary: Neurofibromatosis type 1 (NF1) is a complex disorder characterized by a multisystem involvement and cancer predisposition. It is caused by genetic variants in NF1, a large tumor suppressor gene encoding a cytoplasmatic protein (neurofibromin) with a regulatory role in essential cellular processes. Genotype–phenotype correlations in NF1 patients are so far elusive. We retrospectively reviewed clinical, radiological, and genetic data of 583 individuals with at least 1 National Institutes of Health (NIH) criterion for NF1 diagnosis, including 365 subjects fulfilling criteria for the diagnosis. Novel genotype–phenotype correlations were identified through uni- and multivariate statistical analysis. Missense variants negatively correlated with neurofibromas. Skeletal abnormalities were associated with frameshift variants and whole gene deletions. The c.3721C>T; p.(R1241*) variant positively correlated with structural brain alterations, whereas the c.6855C>A; p.(Y2285*) variant was associated with a higher prevalence of Lisch nodules and endocrinological disorders. These novel NF1 genotype–phenotype correlations may have a relevant role in the implementation of patients' care. Neurofibromatosis type 1 (NF1) is a proteiform genetic condition caused by pathogenic variants in NF1 and characterized by a heterogeneous phenotypic presentation. Relevant genotype–phenotype correlations have recently emerged, but only few pertinent studies are available. We retrospectively reviewed clinical, instrumental, and genetic data from a cohort of 583 individuals meeting at least 1 diagnostic National Institutes of Health (NIH) criterion for NF1. Of these, 365 subjects fulfilled ≥2 NIH criteria, including 235 pediatric patients. Genetic testing was performed through cDNA-based sequencing, Next Generation Sequencing (NGS), and Multiplex Ligation-dependent Probe Amplification (MLPA). Uni- and multivariate statistical analysis was used to investigate genotype–phenotype correlations. Among patients fulfilling ≥ 2 NIH criteria, causative single nucleotide variants (SNVs) and copy number variations (CNVs) were detected in 267/365 (73.2%) and 20/365 (5.5%) cases. Missense variants negatively correlated with neurofibromas (p = 0.005). Skeletal abnormalities were associated with whole gene deletions (p = 0.05) and frameshift variants (p = 0.006). The c.3721C>T; p.(R1241*) variant positively correlated with structural brain alterations (p = 0.031), whereas Lisch nodules (p = 0.05) and endocrinological disorders (p = 0.043) were associated with the c.6855C>A; p.(Y2285*) variant. We identified novel NF1 genotype–phenotype correlations and provided an overview of known associations, supporting their potential relevance in the implementation of patient management. [ABSTRACT FROM AUTHOR]