Metabolic drives affecting Th17/Treg gene expression changes and differentiation: impact on immune-microenvironment regulation.

The CD4+ T-cell population plays a vital role in the adaptive immune system by coordinating the immune response against different pathogens. A significant transformation occurs in CD4+ cells during an immune response, as they shift from a dormant state to an active state. This transformation leads to extensive proliferation, differentiation, and cytokine production, which contribute to regulating and coordinating the immune response. Th17 and Treg cells are among the most intriguing CD4+ T-cell subpopulations in terms of genetics and metabolism. Gene expression modulation processes rely on and are linked to metabolic changes in cells. Lactylation is a new model that combines metabolism and gene modulation to drive Th17/Treg differentiation and functional processes. The focus of this review is on the metabolic pathways that impact lymphocyte gene modulation in a functionally relevant manner.

Non-Invasive Prenatal Test Analysis Opens a Pandora's Box: Identification of Very Rare Cases of SRY-Positive Healthy Females, Segregating for Three Generations Thanks to Preferential Inactivation of the XqYp Translocated Chromosome.

The translocation of the testis-determining factor, the SRY gene, from the Y to the X chromosome is a rare event that causes abnormalities in gonadal development. In all cases of males and females carrying this translocation, disorder of sex development is reported. In our study, we described a peculiar pedigree with the first evidence of four healthy females from three generations who are carriers of the newly identified t(X;Y)(q28;p11.2)(SRY+) translocation with no evidence of ambiguous genitalia or other SRY-dependent alterations. Our study was a consequence of a Non-Invasive Prenatal Test (NIPT) showing a sexual chromosomal abnormality (XXY) followed by a chorionic villus analysis suggesting a normal karyotype 46,XX and t(X;Y) translocation detected by FISH. Here, we (i) demonstrated the inheritance of the translocation in the maternal lineage via karyotyping and FISH analysis; (ii) characterised the structural rearrangement via chromosomal microarray; and (iii) demonstrated, via Click-iT® EdU Imaging assay, that there was an absolute preferential inactivation of the der(X) chromosome responsible for the lack of SRY expression. Overall, our study provides valuable genetic and molecular information that may lead personal and medical decisions.

Fifteen Years of Iodine Prophylaxis in Italy: Results of a Nationwide Surveillance (Period 2015-2019).

CONTEXT: In 2005, a nationwide program of iodine prophylaxis on a voluntary basis was implemented in Italy by law. However, recent data on iodine status are lacking. OBJECTIVE: The aim of this study was to evaluate efficiency, effectiveness, and possible adverse effects (increased occurrence of thyroid autoimmunity and hyperthyroidism) of the Italian iodine prophylaxis program. METHODS: From 2015 to 2019, a nationwide survey was performed. The use of iodized salt was evaluated in a sample of 164 593 adults and in 998 school canteens. A sample of 4233 schoolchildren (aged 11-13 years) was recruited to assess urinary iodine concentration, prevalence of goiter, and thyroid hypoechogenicity on ultrasound, with the latter being an indirect indicator of thyroid autoimmunity. Neonatal TSH values of 197 677 infants screened in regions representative of Northern, Central, and Southern Italy were analyzed to investigate the percentage of TSH values >5.0 mIU/L. Data on methimazole prescriptions were analyzed as indirect indicators of new cases of hyperthyroidism. RESULTS: The prevalence of the use of iodized salt was 71.5% in adult population and 78% in school canteens. A median urinary iodine concentration of 124 µg/L, a prevalence of goiter of 2.2%, and a prevalence of thyroid hypoechogenicity of 5.7% were observed in schoolchildren. The percentage of neonatal TSH values >5.0 mIU/L resulted still higher (5.1%) than the World Health Organization threshold of 3.0%, whereas the prescriptions of methimazole showed a reduction of 13.5%. CONCLUSION: Fifteen years of iodine prophylaxis have led to iodine sufficiency in Italy, although there still is concern about iodine nutritional status during pregnancy.

Inhibition of serum- and glucocorticoid-induced kinase 1 ameliorates hydrocephalus in preclinical models.

BACKGROUND: Hydrocephalus is a pathological accumulation of cerebrospinal fluid (CSF), leading to ventriculomegaly. Hydrocephalus may be primary or secondary to traumatic brain injury, infection, or intracranial hemorrhage. Regardless of cause, current treatment involves surgery to drain the excess CSF. Importantly, there are no long-term, effective pharmaceutical treatments and this represents a clinically unmet need. Many forms of hydrocephalus involve dysregulation in water and electrolyte homeostasis, making this an attractive, druggable target. METHODS: In vitro, a combination of electrophysiological and fluid flux assays was used to elucidate secretory transepithelial electrolyte and fluid flux in a human cell culture model of the choroid plexus epithelium and to determine the involvement of serum-, glucocorticoid-induced kinase 1 (SGK1). In vivo, MRI studies were performed in a genetic rat model of hydrocephalus to determine effects of inhibition of SGK1 with a novel inhibitor, SI113. RESULTS: In the cultured cell line, SI113 reduced secretory transepithelial electrolyte and fluid flux. In vivo, SI113 blocks the development of hydrocephalus with no effect on ventricular size of wild-type animals and no overt toxic effects. Mechanistically, the development of hydrocephalus in the rat model involves an increase in activated, phosphorylated SGK1 with no change in the total amount of SGK1. SI113 inhibits phosphorylation with no changes in total SGK1 levels in the choroid plexus epithelium. CONCLUSION: These data provide a strong preclinical basis for the use of SGK1 inhibitors in the treatment of hydrocephalus.

RANBP1, a member of the nuclear-cytoplasmic trafficking-regulator complex, is the terminal-striking point of the SGK1-dependent Th17+ pathological differentiation.

The Th17+ arrangement is critical for orchestrating both innate and acquired immune responses. In this context, the serum and glucocorticoid regulated kinase 1 (SGK1) exerts a key role in the governance of IL-23R-dependent Th17+ maturation, through the phosphorylation-dependent control of FOXO1 localization. Our previous work has shown that some of the SGK1-key functions are dependent on RAN-binding protein 1 (RANBP1), a terminal gene in the nuclear transport regulation. Here, we show that RANBP1, similarly to SGK1, is modulated during Th17+ differentiation and that RANBP1 fluctuations mediate the SGK1-dependent effects on Th17+ maturation. RANBP1, as the final effector of the SGK1 pathway, affects FOXO1 transport from the nucleus to the cytoplasm, thus enabling RORγt activation. In this light, RANBP1 represents the missing piece, in an essential and rate-limiting manner, underlying the Th17+ immune asset.

Kinase Inhibitors in Genetic Diseases.

Over the years, several studies have shown that kinase-regulated signaling pathways are involved in the development of rare genetic diseases. The study of the mechanisms underlying the onset of these diseases has opened a possible way for the development of targeted therapies using particular kinase inhibitors. Some of these are currently used to treat other diseases, such as cancer. This review aims to describe the possibilities of using kinase inhibitors in genetic pathologies such as tuberous sclerosis, RASopathies, and ciliopathies, describing the various pathways involved and the possible targets already identified or currently under study.

RANBP1 (RAN Binding Protein 1): The Missing Genetic Piece in Cancer Pathophysiology and Other Complex Diseases.

RANBP1 encoded by RANBP1 or HTF9A (Hpall Tiny Fragments Locus 9A), plays regulatory functions of the RAN-network, belonging to the RAS superfamily of small GTPases. Through this function, RANBP1 regulates the RANGAP1 activity and, thus, the fluctuations between GTP-RAN and GDP-RAN. In the light of this, RANBP1 take actions in maintaining the nucleus-cytoplasmic gradient, thus making nuclear import-export functional. RANBP1 has been implicated in the inter-nuclear transport of proteins, nucleic acids and microRNAs, fully contributing to cellular epigenomic signature. Recently, a RANBP1 diriment role in spindle checkpoint formation and nucleation has emerged, thus constituting an essential element in the control of mitotic stability. Over time, RANBP1 has been demonstrated to be variously involved in human cancers both for the role in controlling nuclear transport and RAN activity and for its ability to determine the efficiency of the mitotic process. RANBP1 also appears to be implicated in chemo-hormone and radio-resistance. A key role of this small-GTPases related protein has also been demonstrated in alterations of axonal flow and neuronal plasticity, as well as in viral and bacterial metabolism and in embryological maturation. In conclusion, RANBP1 appears not only to be an interesting factor in several pathological conditions but also a putative target of clinical interest.

Th17-Gene Expression Profile in Patients with Chronic Venous Disease and Venous Ulcers: Genetic Modulations and Preliminary Clinical Evidence.

Chronic venous disease is a condition globally widespread, resulting in a disabling pathological disorder. The CD4 + Th17+ (Cluster Differentiation 4) lymphocytes represent a regulative factor for innate immunity related to the development of complex diseases. Recently, these mechanisms have been associated with vascular disease. The aim of this work is to validate whether the Th17 response correlates with the development of CVI (Chronic venous insufficiency)and CVLUs (chronic venous limbs ulcers) and whether Th17 markers can be used, both as intrinsic risk factors and diagnostic markers, for disease development. PBL derived from peripheral blood samples of patients and controls were subjected to gene expression analysis for IL23R, IL17, SGK1, TGFß, RORγ, FOXO1, and RANBP1 by qRT-PCR and immunoblot. A post hoc correlation, the diagnostic performance of the target genes, and multivariable analyses were properly conducted. The main expression markers of the CD4 + Th17+ switch were strongly activated in chronic venous insufficiency and in advanced ulceration. The correlation analysis demonstrated the inter-dependence on Th17's signature modulation. ROC (Receiver Operating Characteristic) analysis defined, for the examined genes, a clinical value as the potential diagnostic markers. Multi-logistic regression studies showed that Th17 markers behave as empirical risk factors for CVD (chronic venous disease) development. Taken together, the present data provide a new hypothesis for the TH17-dependent pathogenesis of CVD, favoring the possibility for the development of new diagnostic, preventive, and therapeutic approaches.

Germline Testing in a Cohort of Patients at High Risk of Hereditary Cancer Predisposition Syndromes: First Two-Year Results from South Italy.

Germline pathogenic variants (PVs) in oncogenes and tumour suppressor genes are responsible for 5 to 10% of all diagnosed cancers, which are commonly known as hereditary cancer predisposition syndromes (HCPS). A total of 104 individuals at high risk of HCPS were selected by genetic counselling for genetic testing in the past 2 years. Most of them were subjects having a personal and family history of breast cancer (BC) selected according to current established criteria. Genes analysis involved in HCPS was assessed by next-generation sequencing (NGS) using a custom cancer panel with high- and moderate-risk susceptibility genes. Germline PVs were identified in 17 of 104 individuals (16.3%) analysed, while variants of uncertain significance (VUS) were identified in 21/104 (20.2%) cases. Concerning the germline PVs distribution among the 13 BC individuals with positive findings, 8/13 (61.5%) were in the BRCA1/2 genes, whereas 5/13 (38.4%) were in other high- or moderate-risk genes including PALB2, TP53, ATM and CHEK2. NGS genetic testing showed that 6/13 (46.1%) of the PVs observed in BC patients were detected in triple-negative BC. Interestingly, the likelihood of carrying the PVs in the moderate-to-high-risk genes calculated by the cancer risk model BOADICEA was significantly higher in pathogenic variant carriers than in negative subjects. Collectively, this study shows that multigene panel testing can offer an effective diagnostic approach for patients at high risk of hereditary cancers.

The Variant p.Ala84Pro Is Causative of X-Linked Hypophosphatemic Rickets: Possible Relationship with Burosumab Swinging Response in Adults.

Loss of function mutations in the PHEX gene could determine X-linked dominant hypophosphatemia. This is the most common form of genetic rickets. It is characterized by renal phosphate wasting determining an increase in fibroblast growth factor 23 (FGF-23), growth retard, bone deformities and musculoskeletal manifestations. In recent decades, analysis of the PHEX gene has revealed numerous different mutations. However, no clear genotype-phenotype correlations have been reported in patients with hypophosphatemic rickets (XLH). We report two cases of a 28-year-old-male (patient 1) and a 19-year-old male (patient 2) affected by XLH initially treated with phosphate and 1,25-dihydroxyvitamin-D admitted to the Endocrinology unit because of the persistence of muscle weakness, bone pain and fatigue. After phosphate withdrawal, both patients started therapy with burosumab and symptoms ameliorated in three months. However, patient 1's biochemical parameters did not improve as expected so we decided to investigate his genetic asset. We herein describe a possible clinical implication for the missense "de novo" mutation, c.250G>C (p.Ala84Pro) in the PHEX gene, reported in the PHEX database and classified as a variant of uncertain significance (VUS). The clinical implication of this mutation on disease burden and quality of life in adults is still under investigation.

A Familial Form of Epidermolysis Bullosa Simplex Associated with a Pathogenic Variant in KRT5.

Epidermolysis bullosa simplex is a disease that belongs to a group of genodermatoses characterised by the formation of superficial bullous lesions caused by minor mechanical trauma to the skin. The skin fragility observed in the EBS is mainly caused by pathogenic variants in the KRT5 and KRT14 genes that compromise the mechanical stability of epithelial cells. By performing DNA sequencing in a female patient with EBS, we found the pathogenic variant c.967G>A (p.Val323Met) in the KRT5 gene. This variant co-segregated with EBS in the family pedigree and was transmitted in an autosomal dominant inheritance manner. This is the first report showing a familial form of EBS due to this pathogenic variant.

Case Report: Identification of a Novel Pathogenic Germline TP53 Variant in a Family With Li-Fraumeni Syndrome.

Li-Fraumeni syndrome (LFS) is an inherited autosomal dominant disease characterized by a predisposition to many cancers. Germline pathogenic variants in TP53 are primarily responsible for LFS. By performing a targeted sequencing panel in a proband with liver carcinoma having a deceased son affected by osteosarcoma, we found the novel heterozygous frameshift variant c.645del (p.Ser215Argfs*32) in the TP53 gene. This variant co-segregated with typical LFS cancers in the family pedigree, consistent with the pathogenicity of this novel and previously undescribed TP53 variant.

A Novel Splicing Variant of COL2A1 in a Fetus with Achondrogenesis Type II: Interpretation of Pathogenicity of In-Frame Deletions.

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.

Si113-prodrugs selectively activated by plasmin against hepatocellular and ovarian carcinoma.

Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian carcinoma models. But the drawback is the low water solubility which prevents its further development. In this context, we successfully overcame this limitation by synthesizing two novel prodrugs introducing the amino acid sequence D-Ala-Leu-Lys (TP). Moreover, TP sequence has a high affinity with plasmin, a protease recognized as overexpressed in many solid cancers, including HCC and ovarian carcinoma. The prodrugs were synthesized and fully characterized in terms of in vitro ADME properties, plasma stability and plasmin-induced release of the parent drug. The inhibitory activity against Sgk1 was evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the presence and absence of human plasmin. In vivo pharmacokinetic properties and preliminary tissue distribution confirmed a better profile highlighting the importance of the prodrug approach. Finally, the prodrug antitumor efficacy was evaluated in an HCC xenografted murine model, where a significant reduction (around 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with paclitaxel in a paclitaxel-resistant ovarian carcinoma xenografted murine model, resulted in an impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of Si113 prodrugs and pave the way for their further development against resistant cancer.

De novo and bi-allelic variants in AP1G1 cause neurodevelopmental disorder with developmental delay, intellectual disability, and epilepsy.

Adaptor protein (AP) complexes mediate selective intracellular vesicular trafficking and polarized localization of somatodendritic proteins in neurons. Disease-causing alleles of various subunits of AP complexes have been implicated in several heritable human disorders, including intellectual disabilities (IDs). Here, we report two bi-allelic (c.737C>A [p.Pro246His] and c.1105A>G [p.Met369Val]) and eight de novo heterozygous variants (c.44G>A [p.Arg15Gln], c.103C>T [p.Arg35Trp], c.104G>A [p.Arg35Gln], c.229delC [p.Gln77Lys∗11], c.399_400del [p.Glu133Aspfs∗37], c.747G>T [p.Gln249His], c.928-2A>C [p.?], and c.2459C>G [p.Pro820Arg]) in AP1G1, encoding gamma-1 subunit of adaptor-related protein complex 1 (AP1γ1), associated with a neurodevelopmental disorder (NDD) characterized by mild to severe ID, epilepsy, and developmental delay in eleven families from different ethnicities. The AP1γ1-mediated adaptor complex is essential for the formation of clathrin-coated intracellular vesicles. In silico analysis and 3D protein modeling simulation predicted alteration of AP1γ1 protein folding for missense variants, which was consistent with the observed altered AP1γ1 levels in heterologous cells. Functional studies of the recessively inherited missense variants revealed no apparent impact on the interaction of AP1γ1 with other subunits of the AP-1 complex but rather showed to affect the endosome recycling pathway. Knocking out ap1g1 in zebrafish leads to severe morphological defect and lethality, which was significantly rescued by injection of wild-type AP1G1 mRNA and not by transcripts encoding the missense variants. Furthermore, microinjection of mRNAs with de novo missense variants in wild-type zebrafish resulted in severe developmental abnormalities and increased lethality. We conclude that de novo and bi-allelic variants in AP1G1 are associated with neurodevelopmental disorder in diverse populations.

Generation of human induced pluripotent stem cell lines (UNIMGi003-A and UNIMGi004-A) from two Italian siblings affected by Unverricht-Lundborg disease.

Unverricht-Lundborg disease (ULD) is an inherited form of progressive myoclonus epilepsy caused by mutations in the gene encoding Cystatin B (CSTB), an inhibitor of lysosomal proteases. The most common mutation described in ULD patients is an unstable expansion of a dodecamer sequence located in the CSTB gene promoter. This expansion is causative of the downregulation of CSTB gene expression and, consequently, of its inhibitory activity. Here we report the generation of induced pluripotent stem cell (iPSC) lines from two Italian siblings having a family history of ULD and affected by different clinical and pathological phenotypes of the disease.

Generation of iPSC lines from two patients affected by febrile seizure due to inherited missense mutation in SCN1A gene.

Here, we described the generation of human induced pluripotent stem cell lines (hiPSCs) from fibroblasts isolated by punch biopsies of two siblings carrying inherited mutation (c.434 T > C) in the SCN1A gene, encoding for the neuronal voltage gated sodium channel NaV1.1. The mutation leads to the substitution of a highly conserved methionine with a threonine (M145T) in the protein sequence, leading to infant febrile seizures (FS). The older brother, affected by complex FS, also developed temporal lobe epilepsy (TLE) during adolescence.

The Emerging Role of SGK1 (Serum- and Glucocorticoid-Regulated Kinase 1) in Major Depressive Disorder: Hypothesis and Mechanisms.

Major depressive disorder (MDD) is a heterogeneous psychiatric disease characterized by persistent low mood, diminished interests, and impaired cognitive and social functions. The multifactorial etiology of MDD is still largely unknown because of the complex genetic and environmental interactions involved. Therefore, no established mechanism can explain all the aspects of the disease. In this light, an extensive research about the pathophysiology of MDD has been carried out. Several pathogenic hypotheses, such as monoamines deficiency and neurobiological alterations in the stress-responsive system, including the hypothalamic-pituitary-adrenal (HPA) axis and the immune system, have been proposed for MDD. Over time, remarkable studies, mainly on preclinical rodent models, linked the serum- and glucocorticoid-regulated kinase 1 (SGK1) to the main features of MDD. SGK1 is a serine/threonine kinase belonging to the AGK Kinase family. SGK1 is ubiquitously expressed, which plays a pivotal role in the hormonal regulation of several ion channels, carriers, pumps, and transcription factors or regulators. SGK1 expression is modulated by cell stress and hormones, including gluco- and mineralocorticoids. Compelling evidence suggests that increased SGK1 expression or function is related to the pathogenic stress hypothesis of major depression. Therefore, the first part of the present review highlights the putative role of SGK1 as a critical mediator in the dysregulation of the HPA axis, observed under chronic stress conditions, and its controversial role in the neuroinflammation as well. The second part depicts the negative regulation exerted by SGK1 in the expression of both the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF), resulting in an anti-neurogenic activity. Finally, the review focuses on the antidepressant-like effects of anti-oxidative nutraceuticals in several preclinical model of depression, resulting from the restoration of the physiological expression and/or activity of SGK1, which leads to an increase in neurogenesis. In summary, the purpose of this review is a systematic analysis of literature depicting SGK1 as molecular junction of the complex mechanisms underlying the MDD in an effort to suggest the kinase as a potential biomarker and strategic target in modern molecular antidepressant therapy.

7q35 Microdeletion and 15q13.3 and Xp22.33 Microduplications in a Patient with Severe Myoclonic Epilepsy, Microcephaly, Dysmorphisms, Severe Psychomotor Delay and Intellectual Disability.

Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ∼240 kb microdeletion at the 7q35 inherited from her father, a ∼538 kb microduplication at the 15q13.3 region and a ∼178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 (CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.

A familial t(4;8) translocation segregates with epilepsy and migraine with aura.

Three relatives carrying a t(4;8)(p15.2;p23.2) translocation had juvenile myoclonic epilepsy, self-limited photosensitive occipital epilepsy and migraine with aura. The t(4;8) translocation interrupted the coding sequence of CSMD1 gene and occurred immediately to the 3'UTR of STIM2 gene. STIM2 was overexpressed in the patient carrying the unbalanced translocation, and all three individuals had a single functional copy of CSMD1. Array CGH study disclosed that these three individuals also carried a deletion at 5q12.3 that involves the RGS7BP gene. The overall results favor the view that CSMD1, STIM2, and RGS7BP genes could contribute to epilepsy and migraine phenotypes in our family.