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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]

Hematologists are frequently involved in the diagnostic pathway of Gaucher disease type 1 (GD1) patients since they present several hematological signs. However, GD1 is mainly underdiagnosed because of a lack of awareness. In this multicenter study, we combine the use of a diagnostic algorithm with a simple test (β-glucosidase activity on Dried Blood Spot) in order to facilitate the diagnosis in a population presenting to the hematologist with splenomegaly and/or thrombocytopenia associated with other hematological signs. In this high-risk population, the prevalence of GD1 is 3.3%. We propose an equation that predicts the probability of having GD1 according to three parameters that are routinely evaluated: platelet count, ferritin, and transferrin saturation. [ABSTRACT FROM AUTHOR]

Metformin (MET) is the drug of choice for patients with type 2 diabetes and has been proposed for use in cancer therapy and for treating other metabolic diseases. More than 14,000 studies have been published addressing the cellular mechanisms affected by MET. However, several in vitro studies have used concentrations of the drug 10-100-fold higher than the plasmatic concentration measured in patients. Here, we evaluated the biochemical, metabolic, and morphologic effects of various concentrations of MET. Moreover, we tested the effect ofMETon Fanconi Anemia (FA) cells, a DNArepair genetic disease with defects in energetic and glucose metabolism, as well as on human promyelocytic leukemia (HL60) cell lines. We found that the response of wild-type cells to MET is concentration dependent. Low concentrations (15 and 150 µM) increase both oxidative phosphorylation and the oxidative stress response, acting on the AMPK/Sirt1 pathway, while the high concentration (1.5 mM) inhibits the respiratory chain, alters cell morphology, becoming toxic to the cells. In FA cells, MET was unable to correct the energetic/respiratory defect and did not improve the response to oxidative stress andDNAdamage. By contrast, HL60 cells appear sensitive also at 150 µM. Our findings underline the importance of the MET concentration in evaluating the effect of this drug on cell metabolism and demonstrate that data obtained from in vitro experiments, that have used high concentrations of MET, cannot be readily translated into improving our understanding of the cellular effects of metformin when used in the clinical setting. [ABSTRACT FROM AUTHOR]

An exonic missense mutation, c.436C>G, in the PLP1 gene of a patient affected by the hypomyelinating leukodystrophy, Pelizaeus–Merzbacher disease, has previously been found to be responsible for the alteration of the canonical alternative splicing profile of the PLP1 gene leading to the loss of the longer PLP isoform. Here we show that the presence of the c.436C>G mutation served to introduce regulatory motifs that appear to be responsible for the perturbed splicing pattern that led to loss of the major PLP transcript. With the aim of disrupting the interaction between the PLP1 splicing regulatory motifs and their cognate splicing factors, we designed an antisense oligonucleotide-based in vitro correction protocol that successfully restored PLP transcript production in oligodendrocyte precursor cells. [ABSTRACT FROM AUTHOR]

Mutational analysis of the GNPTAB gene was performed in 46 apparently unrelated patients with mucolipidosis IIalpha/beta or IIIalpha/beta, characterized by the mistargeting of multiple lysosomal enzymes as a consequence of a UDP-GlcNAc-1-phosphotransferase defect. The GNPTAB mutational spectrum comprised 25 distinct mutant alleles, 22 of which were novel, including 3 nonsense mutations (p.Q314X, p.R375X, p.Q507X), 5 missense mutations (p.I403T, p.C442Y, p.C461G, p.Q926P, p.L1001P), 6 microduplications (c.749dupA, c.857dupA, c.1191_1194dupGCTG, c.1206dupT, c.1331dupG, c.2220_2221dupGA) and 8 microdeletions (c.755_759delCCTCT, c.1399delG, c.1959_1962delTAGT, c.1965delC, c.2550_2554delGAAAA, c.3443_3446delTTTG, c.3487_3490delACAG, c.3523_3529delATGTTCC). All micro-duplications/deletions were predicted to result in the premature termination of translation. A novel exonic SNP (c.303G>A; E101E) was identified which is predicted to create an SFRS1 (SF2/ASF) binding site that may be of potential functional/clinical relevance. This study of mutations in the GNPTAB gene, the largest yet reported, extends our knowledge of the mutational heterogeneity evident in MLIIalpha/beta/MLIIIalpha/beta.