Identification of a dna methylation episignature in the 22q11.2 deletion syndrome

The 22q11.2 deletion syndrome (22q11.2DS) is the most common genomic disorder in humans and is the result of a recurrent 1.5 to 2.5 Mb deletion, encompassing approximately 20–40 genes, respectively. The clinical presentation of the typical deletion includes: Velocardiofacial, Di George, Opitz G/BBB and Conotruncalanomaly face syndromes. Atypical deletions (proximal, distal or nested) are rare and characterized mainly by normal phenotype or mild intellectual disability and variable clinical features. The pathogenetic mechanisms underlying this disorder are not completely understood. Because the 22q11.2 region harbours genes coding for transcriptional factors and chromatin remodelers, in this study, we performed analysis of genome‐wide DNA methylation of peripheral blood from 49 patients with 22q11.2DS using the Illumina Infinium Methylation EPIC bead chip arrays. This cohort comprises 43 typical, 2 proximal and 4 distal deletions. We demonstrated the evidence of a unique and highly specific episignature in all typical and proximal 22q11.2DS. The sensitivity and specificity of this signature was further confirmed by comparing it to over 1500 patients with other neurodevelopmental disorders with known episignatures. Mapping the 22q11.2DS DNA methylation episignature provides both novel insights into the molecular pathogenesis of this disorder and an effective tool in the molecular diagnosis of 22q11.2DS

Compound heterozygous missense and deep intronic variants in NDUFAF6 unraveled by exome sequencing and mRNA analysis.

Biallelic mutations in NDUFAF6 have been identified as responsible for cases of autosomal recessive Leigh syndrome associated with mitochondrial complex I deficiency. Here we report two siblings and two unrelated subjects with Leigh syndrome, in which we found the same compound heterozygous missense (c.532G>C:p.A178P) and deep intronic (c.420+784C>T) variants in NDUFAF6. We demonstrated that the identified intronic variant creates an alternative splice site, leading to the production of an aberrant transcript. A detailed analysis of whole-exome sequencing data together with the functional validation based on mRNA analysis may reveal pathogenic variants even in non-exonic regions

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Parent inheritance of RB1 hypomorphic mutations and somatic mosaicism can explain low penetrance in retinoblastoma

Retinoblastoma is the most common eye cancer in children. Numerous families have been described displaying reduced penetrance and expressivity. An extensive molecular characterization of seven families led us to identify the two main mechanisms underlying these phenomena: i) mosaicism of amorphic mutations; and ii) parent-of-origin-effect of hypomorphic mutations. Somatic mosaicism for RB1 splicing mutations (c.1960+5G>C and c.2106+2T>C) leading to a complete loss of function was demonstrated by high-depth NGS in two families. In both cases, the healthy carrier parent (one with retinoma) showed a mutation frequency lower than that expected for a heterozygous individual, indicating a 56-60% mosaicism level. Parent-of-origin-effect is a mechanism by which the phenotypic impact of an allele depends on the parental origin. An imprinting mechanism justifying a ~3 fold excess of the RB1 maternal canonical transcript has been previously described. As a consequence, hypomorphic mutations, if maternally inherited, may retain sufficient suppressor activity to prevent tumor onset. In five low-penetrant families we identified paternally inherited hypomorphic mutations in the affected members, namely a deletion resulting in the loss of 37 aminoacids at the N-terminus (c.608-16_608del), an exonic substitution with a “leaky” splicing effect (c.1331A>G), a partially deleterious substitution (p.Arg661Trp) and a truncating C-terminal mutation (c.2663+2T>C). The identification of these mechanisms changes the genetic/prenatal counseling and the clinical management of families, raising to a 50% the recurrence risk for unaffected carriers, offspring of RB1-mutated mothers, and imposing the need for second tumor surveillance in unaffected carriers at risk of developing adult-onset cancer such as osteosarcoma or leiomyosarcoma

Phallusiasterols A and B: two new sulfated sterols from the Mediterranean tunicate Phallusia fumigata and their effects as modulators of the PXR receptor

Purification of the apolar extracts of the marine ascidian Phallusia fumigata, afforded two new sulfated sterols, phallusiasterols A (1) and B (2)​. The structures of the new compounds have been elucidated using mass spectrometry and NMR experiments. The effects of phallusiasterols A and B as modulators of pregnane-​X-​receptor (PXR) have been investigated. These studies revealed that phallusiasterol A induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target genes CYP3A4 and MDR1 in the same cell line. Molecular docking calculations suggested the theoretical binding mode of phallusiasterol A with hPXR and revealed that phallusiasterol A fitted well in the LBD of PXR

Evidence of predisposing epimutation in retinoblastoma

Retinoblastoma (RB), which represents the most common childhood eye cancer, is caused by biallelic inactivation of RB1 gene. Promoter hypermethylation is quite frequent in RB tissues but conclusive evidence of soma-wide predisposing epimutations is currently scant. Here, 50 patients who tested negative for RB1 germline sequence alterations were screened for aberrant promoter methylation using methylation-specific MLPA. The assay, performed on blood, identified a sporadic patient with methylation of CpG106, absent in parents’ DNA. Bisulfite pyrosequencing accurately quantified CpG methylation in blood DNA (mean ∼49%) and also confirmed the aberration in DNA isolated from oral mucosa although at lower levels (mean ∼34%). Using a tag-SNP, methylation was demonstrated to affect the maternal allele. Real-time qPCR demonstrated RB1 transcriptional silencing. In conclusion, we documented that promoter methylation can act as the first “hit” in Knudson's model. This mosaic epimutation mimics the effect of an inactivating mutation and phenocopies RB onset

Synthesis, structural behaviors and biological properties of thrombin binding aptamers singly modified with chiral m-nitro-benzene glycidoyl moiety

The thrombin binding aptamer (TBA) is a 15-mer oligonucleotide (ON) which folds into a typical chair-like G-quadruplex structure containing one TGT and two TT loops.(1) It binds thrombin acting as an anticoagulant agent.(2) In our ongoing investigation on the molecular bases of the anti-thrombin activity of TBA, (3,4) we expanded TBA structure-activity relationships (SARs) by synthesizing four new analogues, named TBA-7R(Ph-NO2), TBA-7S(Ph-NO2), TBA-12R(Ph-NO2) and TBA-12S(Ph-NO2), in which, the (R) or (S)-3-(3-nitrophenoxy)propane-1,2-diol moiety replaced, one at a time, the thymine residues at positions 7 and 12. The new derivatives were then tested in vitro using the prothrombin time (PT) coagulation assay. In order to rationalize acquired SARs, a multi‐disciplinary approach including biophysical and computational studies was applied. The evaluation of the anti-thrombin activity showed that all the tested analogues are able to increase the prothrombin time of human plasma at both 2 and 20 μM concentrations. Preliminary results obtained by CD and Electrophoretic Mobility Shift Assay experiments, showed that all the synthesized aptamers are able to fold into G-quadruplex structures in buffered solutions containing Na+ or K+ ions. Analyses of CD melting curves, as well as, preliminary results obtained by computational studies, evidenced that the R or S chirality of (3-nitrophenoxy)propane-1,2-diol at 12 position affected the thermal stability of the resulting TBA-G-quadruplex, while the same effect is not observed at 7 position. Similarly, the relation between G-quadruplex thermal stability and antithrombin activity of the new analogues depends on the position (7 or 12) of the introduced glycerol derivative