H. pylori infection may increase the risk of developing reproductive disorders or worsen the clinical expression of this syndrome.

Rett syndrome (OMIM#312750) is a monogenic disorder that may manifest as a large variety of phenotypes ranging from very severe to mild disease. Since there is a weak correlation between the mutation type in the Xq28 disease-gene MECP2/X-inactivation status and phenotypic variability, we used this disease as a model to unveil the complex nature of a monogenic disorder. Whole exome sequencing was used to analyze the functional portion of the genome of two pairs of sisters with Rett syndrome. Although each pair of sisters had the same MECP2 (OMIM*300005) mutation and balanced X-inactivation, one individual from each pair could not speak or walk, and had a profound intellectual deficit (classical Rett syndrome), while the other individual could speak and walk, and had a moderate intellectual disability (Zappella variant). In addition to the MECP2 mutation, each patient has a group of variants predicted to impair protein function. The classical Rett girls, but not their milder affected sisters, have an enrichment of variants in genes related to oxidative stress, muscle impairment and intellectual disability and/or autism. On the other hand, a subgroup of variants related to modulation of immune system, exclusive to the Zappella Rett patients are driving toward a milder phenotype. We demonstrate that genome analysis has the potential to identify genetic modifiers of Rett syndrome, providing insight into disease pathophysiology. Combinations of mutations that affect speaking, walking and intellectual capabilities may represent targets for new therapeutic approaches. Most importantly, we demonstrated that monogenic diseases may be more complex than previously thought.

Cytokines that regulate bone turnover (tumor necrosis factor-alpha, interleukin-6, etc.) may influence the pathogenesis of skeleton disorders, such as osteoporosis. Since Helicobacter pylori infection increases the systemic levels of inflammatory cytokines, we investigated the possibility that this infection increases the risk of developing osteoporosis and affects the bone metabolism in a group of male patients with osteoporosis. We examined 80 osteoporotic male patients and 160 controls for serum antibodies to H. pylori and the CagA protein and determined, in patients alone, the most important biochemical and instrumental parameters of the disease. Fifty-one patients (63.7%) and 107 controls (66.8%) were seropositive for H. pylori infection (nonsignificant); 30 infected patients (58.8%) and 43 infected controls (40.1%) were positive for anti-CagA antibodies (P = 0.028; OR = 2.13). Levels of estradiol in infected CagA-positive patients were significantly lower than in infected CagA-negative patients (28.5 [SD = 10.18] vs. 39.5 [SD = 14.50] pg/ml; P = 0.002) and uninfected patients (35.2 [SD = 12.7] pg/ml; P = 0.028). Levels of urinary cross-laps(a marker of bone resorption) were increased in patients infected by CagA-positive strains compared to patients infected by CagA-negative strains (282.9 [SD = 103.8] vs. 210.5 [SD = 150.1]microg/mmol; P = 0.048) and uninfected patients (204.3 [SD = 130.1] microg/mmol; P = 0.016). Differences among uninfected and infected patients, independent of CagA status, were observed for other markers of bone turnover, but they did not reach statistical significance. Infection by CagA-positive H. pylori strains is more prevalent in men with osteoporosis, who show reduced systemic levels of estrogens and increased bone turnover. H. pylori infection by strains expressing CagA may therefore be considered a risk factor for osteoporisis in men.

Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

Mutations in MECP2 gene have been identified in more than 95% of patients with classic Rett syndrome, one of the most common neurodevelopmental disorders in females. Taking advantage of the breakthrough technology of genetic reprogramming, we investigated transcriptome changes in neurons differentiated from induced Pluripotent Stem Cells (iPSCs) derived from patients with different mutations. Profiling by RNA-seq in terminally differentiated neurons revealed a prominent GABAergic circuit disruption along with a perturbation of cytoskeleton dynamics. In particular, in mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with selective inhibitors of HDAC6, the main α-tubulin deacetylase. These findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the treatment of Rett-associated epileptic behavior as well as for the definition of new therapeutic strategies for Rett syndrome.