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Background Lysosomal storage diseases (LSDs) are inborn errors of metabolism resulting from 50 different inherited disorders. The increasing availability of treatments and the importance of early intervention have stimulated newborn screening (NBS) to diagnose LSDs and permit early intervention to prevent irreversible impairment or severe disability. We present our experience screening newborns in North East Italy to identify neonates with Mucopolysaccharidosis type I (MPS I) and Pompe, Fabry, and Gaucher diseases. Methods Activities of acid β-glucocerebrosidase (ABG; Gaucher), acid α-glucosidase (GAA; Pompe), acid α-galactosidase (GLA; Fabry), and acid α-L-iduronidase (IDUA; MPS-I) in dried blood spots (DBS) from all newborns during a 17-month period were determined by multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD ® assay system. Enzymatic activity cutoff values were determined from 3500 anonymous newborn DBS. In the screening study, samples were retested if the value was below cutoff and a second spot was requested, with referral for confirmatory testing and medical evaluation if a low value was obtained. Results From September 2015 to January 2017, 44,411 newborns were screened for the four LSDs. We recalled 40 neonates (0.09%) for collection of a second DBS. Low activity was confirmed in 20, who had confirmatory testing. Ten of 20 had pathogenic mutations: two Pompe, two Gaucher, five Fabry, and one MPS-I. The incidences of Pompe and Gaucher diseases were similar (1/22,205), with Fabry disease the most frequent (1/8882) and MPS-I the rarest (1/44411). The combined incidence of the four disorders was 1/4411 births. Conclusions Simultaneously determining multiple enzyme activities by MS/MS, with a focus on specific biochemical markers, successfully detected newborns with LSDs. The high incidence of these disorders supports this screening program.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the GLA gene, resulting in a deficient activity of the enzyme α-galactosidase A (α-Gal A). This deficiency leads to the progressive accumulation of globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (Lyso-Gb3), in various tissues, contributing to a broad spectrum of clinical manifestations. Recent evidence highlights the crucial role of inflammation in the pathophysiology of FD, influencing disease progression and clinical outcomes. This review provides a comprehensive overview of the relationship between inflammation and FD, with a particular focus on the impact of inflammatory processes on disease progression and complications.

VEXAS syndrome (Vacuoles, E1-enzyme, X-linked, Autoinflammation, and Somatic) is a recently identified late-onset autoinflammatory disorder characterized by a unique interplay between hematological and inflammatory manifestations. It results from somatic mutations in the UBA1 gene, located on the short arm of the X chromosome. Initially, females were considered mere carriers, with the syndrome primarily affecting males over 50. However, recent evidence indicates that heterozygous females can exhibit symptoms as severe as those seen in hemizygous males. The disease manifests as systemic inflammation, macrocytic anemia, thrombocytopenia, chondritis, neutrophilic dermatoses, and steroid-dependent inflammatory symptoms. Due to its overlap with autoimmune and hematologic disorders such as relapsing polychondritis, Still’s disease, and myelodysplastic syndromes, misdiagnosis is common. At the molecular level, VEXAS syndrome is driven by impaired ubiquitination pathways, resulting in dysregulated immune responses and clonal hematopoiesis. A key diagnostic marker is the presence of cytoplasmic vacuoles in myeloid and erythroid precursors, though definitive diagnosis requires genetic testing for UBA1 mutations. Traditional immunosuppressants and TNF inhibitors are generally ineffective, while JAK inhibitors and IL-6 blockade provide partial symptom control. Azacitidine and decitabine have shown promise in reducing disease burden, but hematopoietic stem cell transplantation (HSCT) remains the only curative treatment, albeit with significant risks. This review provides a comprehensive analysis of VEXAS syndrome, examining its clinical features, differential diagnoses, diagnostic challenges, and treatment approaches, including both pharmacological and non-pharmacological strategies. By enhancing clinical awareness and optimizing therapeutic interventions, this article aims to bridge emerging genetic insights with practical patient management, ultimately improving outcomes for those affected by this complex and often life-threatening disease.

Inflammation is a physiological process whose deregulation causes some diseases including cancer. Nuclear Factor kB (NF-kB) is a family of ubiquitous and inducible transcription factors, in which the p65/p50 heterodimer is the most abundant complex, that play critical roles mainly in inflammation. Glucocorticoid Receptor (GR) is a ligand-activated transcription factor and acts as an anti-inflammatory agent and immunosuppressant. Thus, NF-kB and GR are physiological antagonists in the inflammation process. Here we show that in mice and humans there is a spliced variant of p65, named p65 iso5, which binds the corticosteroid hormone dexamethasone amplifying the effect of the glucocorticoid receptor and is expressed in the liver of patients with hepatic cirrhosis and hepatocellular carcinoma (HCC). Furthermore, we have quantified the gene expression level of p65 and p65 iso5 in the PBMC of patients affected by SARS-CoV-2 disease. The results showed that in these patients the p65 and p65 iso5 mRNA levels are higher than in healthy subjects. The ability of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid (GC) response in the opposite way of the wild type improves our knowledge and understanding of the anti-inflammatory response and identifies it as a new therapeutic target to control inflammation and related diseases.

Gaucher disease is an autosomal recessive disorder caused by dysfunction of the enzyme glucocerebrosidase. The enzyme deficiency is mainly due to mutations in the GBA1 gene, and it is responsible for the accumulation of glucosylceramide within the lysosomes of monocyte macrophage-derived cells; causing the associated symptomatology. In this paper, we describe six new mutations identified in the GBA1 gene, which, in combination with other mutations already documented, lead to absent or reduced glucocerebrosidase activity, resulting in pathological accumulation of the specific substrate and the clinical manifestations associated with Gaucher disease. We have identified three mutations (c.1578_1581dup, c.1308dup, and Y492X) that determine the formation of a premature stop codon in the translation process and three missense mutations (C342F, M280L, and Q247R) that lead to amino acid changes in proteins, resulting in decreased glucocerebrosidase activity. These mutations were never observed in our group of healthy control subjects > 1500 individuals. The patients examined had several clinical manifestations, which included hepatosplenomegaly and bone and hematologic involvement; considering the absence of enzyme activity, this suggests that the new mutations described here are associated with type I Gaucher disease. The identification of new mutations in patients with symptoms referable to Gaucher disease increases the molecular knowledge related to the GBA1 gene and offers to clinicians significant support for the accurate diagnosis of the pathology.

Background: Multiplex Ligation Probe Amplification (MLPA) is a widely used technique for the diagnosis of lysosomal storage diseases (LSDs). It analyses over 40 DNA sequences in a single reaction, identifying copy number variations and large deletions/insertions in genes. The diagnostic process in LSDs starts with analysis of the missing or reduced enzyme, followed by genetic investigation and, if possible, a search for accumulated substrates. However, while genetic analysis using Sanger sequencing is excellent at detecting small genetic variations such as single-nucleotide variants (SNVs) and small insertions or deletions, it cannot detect large deletions or insertions. Methods: In the present study, a total of 800 patients with clinical suspicion of Fabry, Gaucher, or Pompe diseases were investigated. An enzyme assay was carried out on each patient, followed by genetic analysis using PCR, Sanger sequencing, and MLPA. Results: Nine patients with deficient or absent enzyme activity had Sanger sequencing results that could not confirm the molecular genetic diagnosis because either no mutation (Fabry) or only one mutation (Gaucher and Pompe) was identified. Subsequent analysis by MLPA identified two males with a hemizygous deletion and two females with a heterozygous deletion for FD. For PD, one female and two males had a heterozygous deletion. For GD, one male had a homozygous deletion and one female had a heterozygous deletion. The remaining patients were analyzed by MLPA with negative results. Conclusions: The results obtained suggest that MLPA should be used in combination with classical sequencing methods to ensure a correct and timely diagnosis of LSDs.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive, progressive, multisystem hereditary lysosomal storage disease (LSD), which is characterized by the gradual accumulation of dermatan sulphate (DS), heparan sulphate (HS), and glycosaminoglycans (GAGs) in all organs and tissues due to the deficiency of the enzyme α-L-hyduronidase. The multisystem clinical manifestations of varying severities of MPS-I are present in two forms—the “severe form of MPS I” (Hurler type) and the “attenuated form of MPS-I” (Hurler–Scheie or Scheie type). These forms represent the entire case history of the disease. The three phenotypes share common symptoms, including musculoskeletal abnormalities, facial dysmorphisms, hernias, short stature, finger stiffness, carpal tunnel syndrome, and corneal opacities. Abnormalities affecting the internal organs include hepatomegaly, splenomegaly, and valvulopathy. There is some evidence to suggest a similarity and overlap with the clinical symptoms of MPS-I, particularly in cases of another rare LSD that is autosomal and recessively inherited—l’α-mannosidosis. This disorder has been observed to result from a dysfunction of the corresponding α-mannosidase enzyme, which has been shown to lead to the accumulation of mannose-rich N-linked oligosaccharides. This review compares the phenotypic similarities and molecular differences between mucopolysaccharidosis type I (MPS-I) and α-mannosidosis. We review genotype–phenotype correlations, diagnostic difficulties, and the applicability of artificial intelligence for the assistance of differential diagnosis, with the goal of facilitating the earlier and more accurate diagnosis of these rare lysosomal storage diseases.

Fabry disease (FD) is a progressive, multisystemic X-linked disorder caused by mutations in the GLA gene, often leading to renal failure. Although several screening programs have been conducted, the prevalence of FD in patients with chronic kidney patients who are not dependent on dialysis (NDD-CKD) is likely underestimated due to restrictive inclusion criteria and methodological shortcomings. This study aims to assess the prevalence of FD in NDD-CKD patients using an expanded screening approach. Ongoing outpatients attending Italian nephrology clinics were screened by assay of plasma α-galactosidase A (α-Gal A) activity. Genetic testing was also performed in all females and males with low α-Gal A activity. Inclusion criteria were: (1) females ≥18 years old; (2) males aged between 18 and 70 years; (3) NDD-CKD stages 1-5. Patients with histological diagnosis of glomerulonephritis or diagnosis of autosomal dominant polycystic kidney disease (ADPKD) were excluded. Demographic data and laboratory results were also collected. Among 385 NDD-CKD outpatients, 173 underwent screening. One patient with three family members carrying a novel mutation (c.320 A > G, p.Q107R); one patient with three family members carrying a silent mutation (c.48 T > G, p.L16L) and two patients with a missense mutation (c.376A > G, p.S126G), were identified. Overall, the prevalence of FD was 2.3%, increasing to 5.4% (10 in 183) with family screening. FD may be more common than previously believed, particularly within NDD-CKD populations. FD screening should be expanded to include NDD-CKD patients with known causes of CKD, such as hypertension and diabetes mellitus, and genetic testing should be routinely used for female patients.

Fabry disease (FD) is a progressive multisystemic lysosomal storage disease. Early diagnosis by newborn screening (NBS) may allow for timely treatment, thus preventing future irreversible organ damage. We present the results of 5.5 years of NBS for FD by α-galactosidase A activity and globotriaosylsphingosine (lyso-Gb3) assays in dried blood spot through a multiplexed MS/MS assay. Furthermore, we report our experience with long-term follow-up of positive subjects. We screened more than 170,000 newborns and 22 males were confirmed to have a GLA gene variant, with an incidence of 1:7879 newborns. All patients were diagnosed with a variant previously associated with the later-onset phenotype of FD or carried an unclassified variant (four patients) or the likely benign p.Ala143Thr variant. All were asymptomatic at the last visit. Although lyso-Gb3 is not considered a reliable second tier test for newborn screening, it can simplify the screening algorithm when its levels are elevated at birth. After birth, plasma lyso-Gb3 is a useful marker for non-invasive monitoring of all positive patients. Our study is the largest reported to date in Europe, and presents data from long-term NBS for FD that reveals the current incidence of FD in northeastern Italy. Our follow-up data describe the early disease course and the trend of plasma lyso-Gb3 during early childhood.

Background: Tissue evaluation for RAS (KRAS or NRAS) gene status in metastatic colorectal cancer (mCRC) patients represent the standard of care to establish the optimal therapeutic strategy. Unfortunately, tissue biopsy is hampered by several critical limitations due to its invasiveness, difficulty to access to disease site, patient’s compliance and, more recently, neoplastic tissue spatial and temporal heterogeneity. Methods: The authors performed a systematic literature review to identify available trials with paired matched tissue and ctDNA RAS gene status evaluation. The authors searched EMBASE, MEDLINE, Cochrane, www.ClinicalTrials.gov, and abstracts from international meetings. In total, 19 trials comparing standard tissue RAS mutational status matched paired ctDNA evaluated through polymerase chain reaction (PCR), next generation sequencing (NGS) or beads, emulsions, amplification and magnetics (BEAMing) were identified. Results: The pooled sensitivity and specificity of ctDNA were 0.83 (95% CI: 0.80–0.85) and 0.91 (95% CI: 0.89–0.93) respectively. The pooled positive predictive value (PPV) and negative predictive value (NPV) of the ctDNA were 0.87 (95% CI: 0.81–0.92) and 0.87 (95% CI: 0.82–0.92), respectively. Positive likelihood ratio (PLR) was 8.20 (95% CI: 5.16–13.02) and the negative likelihood ratio (NLR) was 0.22 (95% CI: 0.16–0.30). The pooled diagnostic odds ratio (DOR) was 50.86 (95% CI: 26.15–98.76), and the area under the curve (AUC) of the summary receiver operational characteristics (sROC) curve was 0.94. Conclusion: The authors’ meta-analysis produced a complete and updated overview of ctDNA diagnostic accuracy to test RAS mutation in mCRC. Results provide a strong rationale to include the RAS ctDNA test into randomized clinical trials to validate it prospectively.