This study examines the role of early diagnosis in managing inborn errors of immunity, which are often characterised by early onset and high mortality. By analysing data from 15 patients with a positive family history, the research evaluated the utilization of pre-implantation and prenatal diagnostic tools. While prenatal diagnosis was performed in five cases, all families chose to continue the pregnancy. Early identification, whether prenatal or within the first month of life, enabled the immediate implementation of critical preventive measures, such as protective isolation, strict aseptic techniques and specific antimicrobial prophylaxis. The findings demonstrate that timely diagnosis and prompt prophylactic intervention are essential to reducing infection rates and improving the overall prognosis for infants with hereditary immune disorders.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis and a growing global incidence. Despite its clinical relevance, the processes driving PDAC tumorigenesis and progression remain poorly understood, challenging traditional molecular models. Here, we address the biological and evolutionary mechanisms underpinning PDAC development and growth, integrating cellular and molecular aspects with an ecological perspective. This multidisciplinary approach offers broader insights into the analysis of PDAC pathogenesis, starting from precancerous lesions and moving through to the primary tumor and its metastatic spread. Indeed, cancer cells may be considered as existing within a dynamic ecological context and are influenced by evolutionary pressures similarly to individuals, populations, and species in nature. Genetic and epigenetic changes form the foundation for the emergence of intratumoral heterogeneity, which contributes to tumor progression, metastasis, and resistance to therapy. These processes in PDAC progression are governed by both clonal expansion and the ecological fitness of neoplastic cells within the tumor microenvironment, which represents their ecological niches. Hence, by integrating evolutionary principles into the study of PDAC, we have analyzed the conceptual advantages and potential applications of this approach on the understanding of the complex interactions between tumor cells and their environments. This research emphasizes the contribution of a holistic, evolutionary perspective to cancer biology and underlines the importance of considering genetic, cellular, and ecological factors in the management and development of treatments, and for guiding future research and therapeutic strategies.

Children exhibit unique biological and developmental susceptibility to environmental exposures, characterized by rapid growth, immature organ systems, and a proportionally higher pollutant intake per unit of body weight. Compared to adults, their capacity for detoxification is limited, and the developing brain and immune systems are particularly prone to neurotoxic and inflammatory harms during critical windows of growth. This comprehensive review aims to synthesize current evidence on current and emerging environmental threats for the design of effective strategies for pediatric health policy and clinical practice. The diverse health impacts from key hazards, including outdoor and household air pollution, chemical contaminants, and water quality issues are discussed. Attention is also given to novel stressors such as endocrine-disrupting chemicals (EDCs), microplastics (MPs), and nanoplastics (NPs), as well as the multifaceted threats posed by climate change. Exposure to these factors is robustly linked to a wide spectrum of pediatric morbidities, notably acute respiratory infections, asthma, impaired neurodevelopment, and cardiometabolic disorders. Critically, this health burden is highly inequitable, disproportionately affecting vulnerable populations, particularly those in low- and middle-income settings. The safeguard of children's health requires urgent, evidence-based, child-centered prevention strategies and robust intersectoral collaboration to mitigate these pervasive global challenges.

We report the first detailed immunological characterization of a DEGCAGS patient, showing that biallelic ZNF699 loss-of-function variants can cause syndromic combined immunodeficiency and that DNA methylation profiling improves diagnostic precision in selected inborn errors of immunity.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease for which there is currently no cure. Dominant mutations in the TARDBP gene are causative of ALS. In particular, the p. G376D substitution in TDP-43 causes familial ALS and it is associated with TDP-43 mislocalization in the cytosol, increased presence of cytoplasmic aggregates, and lysosomal and mitochondrial dysfunction. We previously designed a small interfering RNA (siRNA) that specifically targets and silences the mutant allele and we demonstrated that, in patient-derived fibroblasts, it can reduce TDP-43 aggregation, decrease oxidative stress, and improve cell viability. Here, we investigated the ability of this siRNA to revert some ALS-associated pathological phenotypes in motor neurons derived from induced pluripotent stem cells (iPSCs), as motor neurons are the primary cells affected in ALS. siRNA treatment reduced TDP-43 mislocalization, enhanced lysosomal function and cell viability, and decreased oxidative stress. These findings indicate that this allele-specific siRNA effectively reverses key ALS-related cellular deficits in motor neurons, representing a promising candidate for targeted therapy in patients carrying the TDP-43 G376D mutation.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder strongly associated with mitochondrial dysfunction and impaired proteostasis. Mutations in TARDBP, encoding TAR DNA-binding protein 43 (TDP-43), contribute to disease pathogenesis through cytoplasmic mislocalization and aggregation. Among these, the ALS-linked TDP-43G376D mutation has been previously associated with oxidative stress, mitochondrial fragmentation, and impaired oxidative phosphorylation. Here, we investigated the impact of TDP-43G376D on Mitochondrial Quality Control (MQC) pathways using patient-derived dermal fibroblasts carrying the mutation at early and advanced disease stages, complemented by HEK293T and Neuro2a cellular models expressing mutant TDP-43. We show that TDP-43G376D impairs mitophagic flux, as evidenced by reduced delivery of damaged mitochondria to lysosomes. This was accompanied by pronounced disruption of mitochondrial cristae architecture and accumulation of mitochondrial DNA damage, indicating compromised mitochondrial genome integrity. Furthermore, TDP-43G376D induces sustained activation of the mitochondrial Unfolded Protein Response (UPRmt), consistent with persistent mitochondrial stress, while selectively impairing the sirtuin-dependent antioxidant branch. In parallel, activation of the Endoplasmic Reticulum UPR (UPRER) was observed, indicating a coordinated engagement of cellular stress pathways. Collectively, our findings identify coordinated alterations in multiple MQC pathways associated with TDP-43G376D rather than isolated mitochondrial defects, supporting further investigation of these pathways in larger and disease-relevant ALS models.

Autoimmune thyroid diseases (AITDs), including Hashimoto thyroiditis and Graves’ disease, are the most common autoimmune endocrinopathies, affecting up to 5% of the population. Pathogenetic pathways have not yet been fully elucidated, even though different immune-genetic alterations have been proposed. Specific immune defects presenting with AITDs may serve as an experimentum naturae to study the involvement of a specific pathway in the pathogenesis of the disease. In fact, since immune dysregulation with autoimmunity frequently characterize inborn errors of immunity (IEIs), understanding the mechanisms of immune tolerance breakdown leading to autoimmunity in these conditions may provide useful insight to understand the pathogenesis of AITDs. In this review, we will highlight the main immunological aspects of AITDs and their pathogenesis in IEIs.

TDP-43 has many functions, including the regulation of autophagy and stress granule dynamics, and mutations in TDP-43 are causative of amyotrophic lateral sclerosis (ALS). Among these, the G376D mutation is associated with TDP-43 cytoplasmic mislocalization and mitochondrial and lysosomal dysfunctions. We discovered that upon TDP-43 silencing, the abundance of RILP, a RAB7A effector, decreases. Similarly, expression of the ALS-causative G376D mutant protein reduces RILP intracellular levels, compromising the autophagic flux. RILP overexpression restores the autophagic flux and reduces stress granule marker levels, demonstrating that RILP can overcome TDP-43 malfunctioning. Mechanistically, we show that TDP-43 controls RILP expression mainly at a translational level. L-leucine restored RILP levels in TDP-43-depleted and TDP-43G376D-expressing cells, whereas inhibition of mTOR signaling or protein synthesis abolished this effect. Polysome profiling further demonstrated that TDP-43 depletion reduces the association of RILP mRNA with heavy polysomes, supporting impaired translational efficiency. RILP downregulation and defective autophagic flux were confirmed in iPSC-derived TDP-43G376D motor neurons, where RILP overexpression improved autophagy and cell viability. In addition, TDP-43 co-immunoprecipitates with both RILP and RAB7A, although only RILP interacts directly with TDP-43. The interaction occurs between the RILP C-terminus and amino acids 320–346 of TDP-43. Interestingly, the TDP-43 mutant displays a stronger interaction with RILP, and its expression reduces RILP-RAB7A interaction, suggesting that pathogenic TDP-43 perturbs the functional coupling between them. These data identify RILP as a new TDP-43 interactor and reveal that disruption of the TDP-43/RILP/RAB7A axis, induced by TDP-43G376D, contributes to defective autophagy that characterizes ALS and possibly other neurodegenerative disorders.

We report the first detailed immunological characterization of a DEGCAGS patient, showing that biallelic ZNF699 loss-of-function variants can cause syndromic combined immunodeficiency and that DNA methylation profiling improves diagnostic precision in selected inborn errors of immunity.

BACKGROUND:RAB7 is a small GTPase with multiple cellular roles, regulating late endocytic trafficking and lysosomal biogenesis, influencing mitochondria-lysosome crosstalk, and contributing to many mitochondrial processes. Mitochondrial dysfunctions are widely reported in cancer and the development of cancer therapeutic strategies targeting mitochondria gained momentum in recent years. Mitochondrial impairment can cause alterations of mitochondria-lysosome crosstalk and can influence lysosomal function. Here, we used cell models of pancreatic cancer, one of the deadliest cancers worldwide, to cause a transient mild mitochondrial deficit lowering NDUFS3 protein levels in order to investigate the consequences on RAB7 and on the late endocytic pathway and, thus, the contribution of the mitochondria-lysosomes communication alterations to cancer progression. METHODS:NDUFS3 and RAB7 downregulation was obtained by RNA interference (RNAi). Seahorse assays, Western blot analysis, mitochondrial staining, and Transmission Electron Microscopy (TEM) were used to assess silencing effects on mitochondrial structure and functioning. Western blotting was used to investigate expression of late endocytic pathway proteins and of the invasion marker vimentin. Confocal microscopy was used to analyze the mitochondrial network and lysosomal assessment. Zymography was performed to evaluate the ability to digest the extracellular matrix linked to cancer migration. SRB and colony assays were performed to assess cancer viability and proliferation. Wound healing assay and FluoroBlok membranes were used to determine migration and invasiveness. RESULTS:In pancreatic cancer cells, transient silencing of the NDUFS3 protein caused mitochondrial deficit, slower oxidative metabolism, and mitochondrial morphology alterations. In this context, we observed RAB7 downregulation and impairment of the late endocytic pathway. In addition, NDUFS3-silenced RAB7-downregulated cells showed less invasive tumorigenic potential revealed by reduced levels of vimentin and other Epithelial-to-Mesenchymal Transition proteins, decreased viability, migration and invasiveness. Moreover, we found that modulation of RAB7 expression may regulate vimentin levels and influence mitochondrial morphology and levels of mitochondrial proteins. CONCLUSIONS:Overall, our data show that mitochondrial deficit determines alterations of the crosstalk with lysosomes, leading to dysfunctions, and that this process is regulated by RAB7 acting as an oncogene. This highlights the synergic role of RAB7 and mitochondrial dysfunction, focusing on a cellular mechanism that may boost the effect of mitochondrial dysfunction in the cells, leading to the reduction of the tumorigenic potential.

BackgroundAutoimmune polyglandular syndrome type 1 (APS-1) is a rare inborn error of immunity caused by mutations in the AIRE gene, typically associated with chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency. We report the first known case of APS-1 complicated by a life-threatening combination of secondary hemophagocytic lymphohistiocytosis (sHLH) and atypical hemolytic uremic syndrome (aHUS), successfully treated with targeted and supportive therapies.Case reportA 16-year-old female with a diagnosis of APS-1 confirmed by the presence of the nonsense variant c.415C>T (R139X) in exon 3 and the Finnish major mutation c.769C>T (R257X) in exon 6 of the AIRE gene presented with fever, cytopenias, organomegaly, and hyperferritinemia, fulfilling criteria for sHLH. Despite immunosuppressive therapy, she developed acute kidney injury, thrombocytopenia, and microangiopathic hemolytic anemia, consistent with aHUS. Treatment with the IL - 1 receptor antagonist anakinra and the complement inhibitor eculizumab led to rapid resolution of systemic inflammation and progressive renal and hematological recovery.ConclusionsHLH is an exceptionally rare complication in APS-1 and has so far been reported in only one patient with a combined EBV and SARS-CoV-2 infection. aHUS has never been described in patients with APS-1. This case highlights the potential for hyperinflammatory and complement-mediated complications in APS-1, supporting the hypothesis of a cytokine storm syndrome that bridges features of sHLH and aHUS. It broadens the known spectrum of immune dysregulation in APS-1 and underscores the importance of early recognition and combined immunomodulatory treatment in similar clinical scenarios.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the degeneration of upper and lower motor neurons in the brain, brainstem and spinal cord. About 10% of familial ALS cases are linked to pathogenetic substitution in TARDBP, the gene encoding the TDP-43 protein. A novel rare causative variant in TARDBP (p.G376D) was recently reported in ALS patients. It leads to TDP-43 cytoplasmic mislocalization, increased oxidative stress and reduced cell viability. However, functional studies on the effects of this molecular defect have not yet been carried out. Mitochondria are highly dynamic organelles, and their deregulation has emerged as a key factor in many diseases, among which is ALS. Therefore, this study aimed at determining the impact of this causative variant on mitochondria. In cellular models expressing TDP-43G376D and in fibroblasts derived from patients carrying this molecular defect, we observed alterations of mitochondrial functionality. We demonstrated increased localization of the mutated protein to mitochondria and a reduced abundance of subunits of complex I and complex II of the mitochondrial respiratory chain, associated with a decrease in mitochondrial membrane potential, in cellular respiration and in cytochrome C oxidase (COX) activity. Moreover, ALS cells showed increased mitochondrial fragmentation and reduced abundance of antioxidant enzymes causing increased oxidative stress. These results expand our knowledge about the molecular mechanisms underlying ALS pathogenesis associated with TDP-43 p.G376D and could help to identify new therapeutic strategies to counteract this disease.

Background: Inborn errors of immunity (IEIs) comprise more than 500 different rare congenital disorders of the immune system and are characterized by susceptibility to infection and immune dysregulation. The significant overlap of the clinical features among the different forms may lead to diagnostic delay. High-throughput sequencing techniques may allow a timely genetic definition. Guidelines for the use and the interpretation of genetic testing produced by the American College of Medical Genetics and Genomics (ACMG) and the European Society of Human Genetics (ESHG) do not cover specifics for their application to IEIs. Objective: The aim of this consensus study was to define the best approach to genetic testing for IEIs. Methods: A panel of experts in the context of the Italian Primary Immunodeficiency Network (IPINet) composed a list of statements that were evaluated by the Delphi method. Results: The experts recommend that genetic testing for IEIs should be offered to selected patients with warning signs for IEIs and highlight the crucial role of thorough phenotyping and functional tests for the conclusive diagnosis of IEI. Comprehensive educational programs targeted to health care professionals and the public should be developed to increase IEIs awareness and reduce diagnostic delay. Ethical issues should be pondered over the diagnostic advantages of genetic tests requested for diagnostic purposes. Conclusion: Adherence to guidelines on the use and interpretation of genetic tests for diagnosing IEIs should help limit the inappropriate use of these techniques, thereby reducing the risk of misdiagnosis and patient apprehension regarding inconclusive genetic results. (J Allergy Clin Immunol 2025;155:1149-60.)

Inborn errors of immunity (IEIs) are rare genetic disorders caused by mutations in genes critical for immune cell function, development, or signaling pathways. Gene therapy represents a potential curative treatment for these conditions, with lentiviral vectors (LVs) emerging as a promising tool in this field. However, one of the significant limitations related to the use of LVs is their cargo capacity of around 8 kilobases. This prevents their application for the treatment of conditions requiring the delivery of larger DNA sequences and the stable expression of the transgene in actively proliferating cells. Recent studies have shown that protein trans-splicing is a powerful tool for expanding the cargo capacity of AAV vectors upon co-infection of a host cell with different vectors, each expressing a split-intein–flanked portion of the full-length protein. The aim of our study is to translate the same technology to the platform of LVs. We developed dual-intein LVs, each expressing either the N- or the C-terminal half of the EGFP reporter protein fused to the N- and C-terminal halves of the DnaE split-inteins from Nostoc punctiforme, under the control of EF1A promoter. We co-infected HEK293 cells and observed the reconstitution of a functional full-length protein of the proper size. This demonstrates that trans-splicing applied to LVs is as efficient as for AAVs. This system could be helpful to design a gene therapy approach for specific IEIs, including LRBA deficiency and ataxia-telangiectasia (A-T), caused by mutations in genes larger than 8 kb. We are currently developing dual-intein LVs expressing either the N- or the C-terminal half of the ATM protein, mutated in A-T patients, that will be tested in vitro for their ability to reconstitute a functional protein of the correct size and for the ability to correct the mutant phenotype. Figure 1. Dual-intein LVs reconstitute EGFP in vitro. Spontaneous fluorescence in HEK293 cells detected 72 hours post-infection. I: HEK293 cells infected with EGFP LV at (A) MOI 5 and (B) MOI 3; II: HEK293 cells co-infected with the dual-intein EGFP LVs at (A) MOI 2.5 each vector and (B) MOI 1.5 each vector; III: HEK293 cells co-infected with the dual-intein EGFP LVs at (A) MOI 5 each vector and (B) MOI 3 each vector. The image is representative of N = 3 independent experiments. Figure 2. Western blot analysis performed on HEK293 cell lysates infected with dual-intein EGFP LVs. Cell lysates from cells transduced with: 1. EGFP full-length LVs at MOI = 5; 2. dual-intein N-term EGFP LV at MOI = 5; 3. dual-intein C-term EGFP LV at MOI = 5; 4. dual-intein N-term and C-term EGFP LVs at MOI = 2.5 each vector; 5. dual-intein N-term and C-term EGFP LVs at MOI = 5 each vector; 6. untreated cells. Black arrows indicate the correct spliced EGFP produced from co-infection; blue and red arrows indicate the single halves produced by LVs expressing for the N-term and C-term EGFP, respectively; red boxes indicate the spliced intein. Figure 3. Schematic representation of dual-intein EGFP pLenti containing the nucleotides surrounding the ATM splitting point. In the pLenti encoding for the EGFP N-term half ATM CDS corresponds to nucleotides 3826…3852. In the pLenti encoding for the C-term EGFP, ATM CDS corresponds to nucleotides 3853…3879 and comprises the triplet encoding for Cys1286. nt: nucleotides. Figure 4. Western blot analysis performed on HEK293 cell lysates transfected with dual-intein pLenti EGFP plasmids with the additional ATM nucleotides surrounding the splitting point. Cell lysates from cells transfected with: 1. pLenti EGFP full-length; 2. dual-intein pLenti N-term; 3. dual-intein pLenti C-term EGFP; 4. dual-intein pLenti N-term + dual-intein pLenti C-term with additional nucleotides from ATM CDS; 5. untransfected cells. Black arrow indicates EGFP full length with additional amino acids from ATM CDS; red and blue arrows indicate the N- and the C-term products, respectively.

Wiskott-Aldrich syndrome (WAS) (MIM #301000) is a rare X-linked primary immunodeficiency due to mutations in the WAS gene, characterized by thrombocytopenia with small platelets, eczema, recurrent infections, and an increased incidence of autoimmunity and malignancies. A wide spectrum of mutations has been identified in the WAS gene responsible for a broad variety of clinical phenotypes. By using targeted next-generation sequencing (t-NGS), we identified in a 2-month-old boy with thrombocytopenia and immunological alterations a 4-nucleotide deletion from position +3 to +6 of intron 8 (c.777 + 3_777 + 6delGAGT) of WAS, currently classified on ClinVar as a variant of uncertain significance. The in-vitro characterization of the variant revealed the complete retention of intron 8 in the mature transcript, suggesting a splicing defect due to the loss of a splice donor site at the 5′-end of intron 8. By sequencing the polymerase chain reaction product, we identified a premature stop at codon 269; thus, consequently, no Wiskott-Aldrich syndrome protein (WASp) was detectable in peripheral blood mononuclear cells from the patient. Due to the total absence of a full-length WASp, it is expected that the patient will develop the severe form of the disease, although further monitoring is needed to better define his phenotype.

This study explores the possibility of using the prebiotic lactobionic acid (LBA), produced through the valorization of cheese whey, a common dairy by-product, for the production of different fermented milks with/without the probiotic Lacticaseibacillus rhamnosus GG. The presence of LBA enhanced the growth and stability of probiotics and starter cultures, improved the antioxidant activity of the samples, and impacted the volatilome of the fermented milk during the 28 days storage at 6 degrees C. However, while LBA remained constant in LBA samples during both fermentation and shelf life, the acid sugar dropped below the detection limit in LBA + LGG samples, leaving higher residual lactose levels. This indicates that the probiotic prioritized LBA over the disaccharide, which could have significant implications for the final product. This work provides new insights into the use of LBA as an ingredient to functionalize fermented milk and its impact on the metabolism of lactic acid bacteria.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Consequent to the loss of these cells, neuromuscular functions decline, causing progressive weakness, muscle wasting, and paralysis, leading to death in 2 to 5 years. More than 90% of ALS cases are sporadic, while the remaining 10% of cases are familial, due to mutations in 40 different genes. One of the most common genes to be mutated in ALS is TARDBP (transactive response DNA binding protein 43), which encodes TDP-43 (TAR DNA-binding protein 43). A mutation in exon 6 of TARDBP causes the aminoacidic substitution G376D in the C-terminal region of TDP-43, leading to its cytoplasmic mislocalization and aggregation. In fibroblasts derived from patients carrying this mutation, we found a strong increase in lysosome number, with overexpression and higher nuclear translocation of the transcription factor TFEB. In contrast, lysosomal functionality was deeply compromised. Interestingly, lysosomal activity was unaffected at an early stage of the disease, worsening in more advanced stages. Moreover, we observed the same pathological phenotype in iPSC (induced pluripotent stem cells)-derived patient motor neurons carrying the G376D mutation. Therefore, this mutation compromises the functionality of lysosomes, possibly contributing to neurodegeneration.

Peripherin belongs to heterogeneous class III of intermediate filaments, and it is the only intermediate filament protein selectively expressed in the neurons of the peripheral nervous system. It has been previously discovered that peripherin interacts with proteins important for the endo-lysosomal system and for the transport to late endosomes and lysosomes, such as RAB7A and AP-3, although little is known about its role in the endocytic pathway. Here, we show that peripherin silencing affects lysosomal abundance but also positioning, causing the redistribution of lysosomes from the perinuclear area to the cell periphery. Moreover, peripherin silencing affects lysosomal activity, inhibiting EGFR degradation and the degradation of a fluorogenic substrate for proteases. Furthermore, we demonstrate that peripherin silencing affects lysosomal biogenesis by reducing the TFEB and TFE3 contents. Finally, in peripherin-depleted cells, the autophagic flux is strongly inhibited. Therefore, these data indicate that peripherin has an important role in regulating lysosomal biogenesis, and positioning and functions of lysosomes, affecting both the endocytic and autophagic pathways. Considering that peripherin is the most abundant intermediate filament protein of peripheral neurons, its dysregulation, affecting its functions, could be involved in the onset of several neurodegenerative diseases of the peripheral nervous system characterized by alterations in the endocytic and/or autophagic pathways.

High throughput sequencing techniques are very effective tools to achieve a timely genetic definition of inborn errors of immunity (IEIs). However, they often reveal variants of uncertain significance (VUS), and in some cases, the identification of a causative genetic alteration may be hampered by technical issues. Recent studies suggest that genome-wide DNA methylation profiling on blood samples may represent an effective tool for reassessing the pathogenic role of VUS in a defined set of disease-causing genes and to increase the diagnostic yield in unresolved cases. The aim of the study is to define the genome-wide DNA methylation profiling in a cohort of patients with known pathogenic STAT1 gain-of-function (GOF) variants. As proof of principle, genome-wide DNA methylation profiling was evaluated on 11 patients carrying 7 different known pathogenic STAT1 GOF variants. We compared the DNA methylation profile (β-values) between all cases with GOF variants in STAT1 and age- and sex-matched controls. Through hierarchical clustering, we were able to differentiate the patients’ and controls’ groups. In total, 2356 differentially methylated CpG probes (DMPs) were identified. Among these, 75 DMPs (64% hypomethylated and 36% hypermethylated) were in genes regulated by STAT1 and 58 DMPs (60% hypomethylated and 40% hypermethylated) were in genes regulated by STAT3. Using the genes covered by the 2356 DMPs, we performed KEGG pathway enrichment analysis revealing genes implicated in antiviral response to CMV and HPV but also genes implicated in cancer, cellular senescence, and thyroid hormone signaling. These preliminary data suggest that patients with different STAT1 GOF variants display a distinctive genome-wide DNA methylation profile. Further data on a larger cohort of patients are necessary to validate these results and to define whether this technique is effective in assessing the pathogenicity of VUS. If confirmed, this study may be extended to other different IEIs.

22q11.2 deletion syndrome (22q11.2DS) is the most common chromosomal microdeletion syndrome in humans. The clinical phenotype is variable among different patients, also when they come from the same family, suggesting that nongenetic factors may be implicated in the pathogenesis. Recently, a specific episignature was defined for patients affected with 22q11.2DS. However, it has not been yet clarified whether these changes may reflect the variability of the phenotype observed among different patients. The study is aimed at defining genome-wide DNA methylation profiling in a large cohort of patients, including 63 carrying a deletion on the 22q11.2 chromosome and 5 with clinical features of DGS in whom genetic analysis did not reveal any alteration on the chromosome 22 (DGS-like). Among patients with 22q11.2 deletion, 12 were identified through FISH, suggesting that the deletion includes the proximal region, but information on the extension of the deletion is not available. For the remaining 51 patients, 38 carried the typical A-D deletion, 3 carried an A-B deletion, 7 carried a C-D deletion, while 3 patients carried a deletion downstream the DGS region. The analysis included 20 familial cases from 8 kindred. By selecting the 160 previously published differentially methylated CpG probes (DMPs), we were able to distinguish a specific methylation profile in the group of 38 patients carrying the typical A-D deletion, in the 3 patients carrying the A-B deletion, and in those diagnosed with FISH. On the contrary, the 7 patients carrying the distal C-D deletion, the 3 patients carrying a deletion downstream the DGS region and the DGS-like clustered with the controls. When the analysis was extended to a larger number of DMPs, we observed a gradient among typical deletion, distal deletions, DGS-like, and controls. Interestingly, most of the DMPs were found in the DGS region on the non-deleted allele. Hierarchical clustering also revealed similarities among affected and unaffected members of the different families, suggesting that epigenetic modifications may be partially inherited. These data showed a gradient of DMPs among typical deletion, distal deletions, DGS-like, and controls. This may suggest a correlation between the severity of the clinical phenotype and the degree of DNA methylation.