Whole Exome Sequencing Identifies Epithelial and Immune Dysfunction-Related Biomarkers in Food Protein-Induced Enterocolitis Syndrome.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a food allergy primarily affecting infants, often leading to vomiting and shock. Due to its poorly understood pathophysiology and lack of specific biomarkers, diagnosis is frequently delayed. Understanding FPIES genetics can shed light on disease susceptibility and pathophysiology-key to developing diagnostic, prognostic, preventive and therapeutic strategies. Using a well-characterised cohort of patients we explored the potential genome-wide susceptibility factors underlying FPIES. METHODS: Blood samples from 41 patients with oral food challenge-proven FPIES were collected for a comprehensive whole exome sequencing association study. RESULTS: Notable genetic variants, including rs872786 (RBM8A), rs2241880 (ATG16L1) and rs2289477 (ATG16L1), were identified as significant findings in FPIES. A weighted SKAT model identified six other associated genes including DGKZ and SIRPA. DGKZ induces TGF-ß signalling, crucial for epithelial barrier integrity and IgA production; RBM8A is associated with thrombocytopenia absent radius syndrome, frequently associated with cow's milk allergy; SIRPA is associated with increased neutrophils/monocytes in inflamed tissues as often observed in FPIES; ATG16L1 is associated with inflammatory bowel disease. Coexpression correlation analysis revealed a functional correlation between RBM8A and filaggrin gene (FLG) in stomach and intestine tissue, with filaggrin being a known key pathogenic and risk factor for IgE-mediated food allergy. A transcriptome-wide association study suggested genetic variability in patients impacted gene expression of RBM8A (stomach and pancreas) and ATG16L1 (transverse colon). CONCLUSIONS: This study represents the first case-control exome association study of FPIES patients and marks a crucial step towards unravelling genetic susceptibility factors underpinning the syndrome. Our findings highlight potential factors and pathways contributing to FPIES, including epithelial barrier dysfunction and immune dysregulation. While these results are novel, they are preliminary and need further validation in a second cohort of patients.

SNPs of the IFNL favour spontaneous clearance of HCV infection in children.

BACKGROUND: Both spontaneous and treatment-induced clearance of hepatitis C virus (HCV) in adults have been associated with genetic polymorphisms in the interferon-λ genes. The aim of the present study was to confirm the association between the rs12979860 and evaluate the association between the rs368234815 and the rs4803217 single-nucleotide polymorphisms (SNPs) of the interferon-λ genes and the outcome of the infection in children. METHODS: Alleles and genotypes frequencies of 32 children, who presented spontaneous clearance of the virus and 135 children, with viral persistence were compared with ethnically matched controls obtained from the 1000 Genomes Project and the International HapMap Project databases. RESULTS: The frequencies of the C/C genotype of rs12979860, the TT/TT of the rs368234815 and the A/C of the rs4803217 were higher in the clearance group than in children with viral persistence (C/C versus T/T + C/T odds ratio (OR): 2.6; 90% confidence intervals (CI): 1.3-5; p = 0.01; TT/TT versus ΔG/TT + ΔG/ΔG OR: 2.8; 90% CI: 1.4-5.5; p = 0.01; and A/A versus A/C OR: 8.3; 90% CI: 1.5-45.9; p = 0.017, respectively) and with the ethnically matched controls. CONCLUSIONS: The rs12979860, the rs368234815 and the rs4803217 SNPs are associated with spontaneous clearance of HCV in children. IMPACT: Innate immune system response has a key role in the outcome of vertically acquired HCV infection in children. The rs12979860, the rs368234815 and the rs4803217 SNPs are associated with spontaneous clearance of HCV in children. Interferons-λ activate the Janus kinase-Stat pathway, which in turn induces several interferon-stimulated genes, leading to suppression of HCV replication both in vivo and in vitro.

Interleukin 28B rs12979860 single-nucleotide polymorphism predicts spontaneous clearance of hepatitis C virus in children.

OBJECTIVE: Recent genome-wide association studies performed in adults correlated single-nucleotide polymorphisms (SNPs rs12979860 and rs8099917) located on chromosome 19, upstream of the interleukin 28B gene, with spontaneous clearance of hepatitis C virus and with response to treatment with paginated interferon and ribavirin. The aim of the present collaborative study was to evaluate the rs12979860 SNP in a large cohort of Italian children with perinatal acquisition of hepatitis C. METHODS: Children were prospectively enrolled in 2 Italian centers. The interleukin 28B rs12979860 SNP was studied according to the diagnosis of chronic infection or spontaneous clearance. RESULTS: One hundred thirty children (86.7%) with chronic infection and 23 (13.3%) with spontaneous clearance of the virus were enrolled. Overall, the interleukin 28B C/C and C/T-T/T genotypes were found in 57 (37.3%) and 96 (62.7%) children, respectively. The proportion of C/C genotype was higher among children who cleared infection (14/23; 60.9%) compared with children with chronic infection (43/130; 33.1%; P = 0.01; odds ratio 3.15; 90% confidence intervals 1.34-7.53). CONCLUSIONS: The present study showed that, as already demonstrated in adults, children with the rs12979860 C/C SNP of the interleukin 28B gene have a higher probability of spontaneous clearance of hepatitis C virus.

DNA Fragmentation in Viable and Non-Viable Spermatozoa Discriminates Fertile and Subfertile Subjects with Similar Accuracy.

Sperm DNA fragmentation (sDF) negatively affects reproduction and is traditionally detected in total sperm population including viable and non-viable spermatozoa. Here, we aimed at exploring the ability of DNA fragmentation to discriminate fertile and subfertile men when detected in viable (viable sDF), non-viable (non-viable sDF), and total spermatozoa (total sDF). We revealed sDF in 91 male partners of infertile couples and 71 fertile men (max 1 year from natural conception) with LiveTUNEL coupled to flow cytometry, able to reveal simultaneously DNA fragmentation and cell viability. We found that the three sDF parameters discriminated fertile and subfertile men with similar accuracy and independently from age and basal semen parameters: AUCs (area under the curves) (95% CI) were: 0.696 (0.615-0.776), p < 0.001 for total sDF; 0.718 (0.640-0.797), p < 0.001 for viable sDF; 0.760 (0.685-0.835), p < 0.001 for non-viable sDF. We also found that total and non-viable but not viable sDF significantly correlated to age and semen quality. In conclusion, the three sDF parameters similarly discriminated fertile and subfertile men. Viable spermatozoa with DNA fragmentation are likely cells able to fertilize the oocyte but failing to properly support subsequent embryo development. Non-viable sDF could be a sign of a subtler damage extended beyond the non-viable cells.

Early infantile epileptic-dyskinetic encephalopathy due to biallelic PIGP mutations.

OBJECTIVE: To describe clinical, biochemical, and molecular genetic findings in a large inbred family in which 4 children with a severe early-onset epileptic-dyskinetic encephalopathy, with suppression burst EEG, harbored homozygous mutations of phosphatidylinositol glycan anchor biosynthesis, class P (PIGP), a member of the large glycosylphosphatidylinositol (GPI) anchor biosynthesis gene family. METHODS: We studied clinical features, EEG, brain MRI scans, whole-exome sequencing (WES), and measured the expression of a subset of GPI-anchored proteins (GPI-APs) in circulating granulocytes using flow cytometry. RESULTS: The 4 affected children exhibited a severe neurodevelopmental disorder featuring severe hypotonia with early dyskinesia progressing to quadriplegia, associated with infantile spasms, focal, tonic, and tonic-clonic seizures and a burst suppression EEG pattern. Two of the children died prematurely between age 2 and 12 years; the remaining 2 children are aged 2 years 7 months and 7 years 4 months. The homozygous c.384del variant of PIGP, present in the 4 patients, introduces a frame shift 6 codons before the expected stop signal and is predicted to result in the synthesis of a protein longer than the wild type, with impaired functionality. We demonstrated a reduced expression of the GPI-AP CD16 in the granulocytic membrane in affected individuals. CONCLUSIONS: PIGP mutations are consistently associated with an epileptic-dyskinetic encephalopathy with the features of early infantile epileptic encephalopathy with profound disability and premature death. CD16 is a valuable marker to support a genetic diagnosis of inherited GPI deficiencies.

Impact of the 13-Valent Pneumococcal Conjugate Vaccine on Severe Invasive Disease Caused by Serotype 3 Streptococcus Pneumoniae in Italian Children.

The effectiveness and impact of the 13-valent pneumococcal conjugate vaccine (PCV13) against invasive pneumococcal diseases (IPD) due to serotype 3 (ser3) has been questioned. However, the impact of PCV13 on different clinical presentations of ser3-IPD has not been studied so far. The impact of PCV13 on different clinical presentations of ser3-IPD in a population of Italian children aged 0-8 years was evaluated, comparing pre- and post-PCV13 introduction period. Real-time polymerase chain reaction (PCR) was used for the diagnosis and serotyping of IPD. During the observation period (1 January 2006-1 August 2018), ser3 was detected in 60/284 (21.1%) children under 8 with serotyped IPD. The incidence of sepsis and meningitis was 0.24 per 1,000,000 person-years (p-y) in pre-PCV13 and 0.02 per 1,000,000 p-y in post-PCV13. No cases occurred in vaccinated children. In the post-PCV13 period, case reduction was 13% for all ser3 IPD and 92% for sepsis and meningitis. Vaccination impact may be underestimated due to significant improvement in pneumococcal surveillance in post-PCVC13. Our data suggest a significant impact of PCV13 on meningitis and sepsis due to ser3 and a lower impact against pneumonia. While waiting for increasingly effective anti-pneumococcal vaccines, PCV13, which guarantees protection against the most severe clinical presentations of ser3-IPD, is currently the most effective prevention option available.

Immunological Features of Neuroblastoma Amplified Sequence Deficiency: Report of the First Case Identified Through Newborn Screening for Primary Immunodeficiency and Review of the Literature.

This is the first case of NBAS disease detected by NBS for primary immunodeficiency. NBS with KRECs is revealing unknown potentialities detecting conditions that benefit from early recognition like NBAS deficiency. Immune phenotyping should be mandatory in patients with NBAS deficiency since they can exhibit severe immunodeficiency with hypogammaglobulinemia as the most frequent finding. Fever during infections is a known trigger of acute liver failure in this syndrome, so immune dysfunction, should never go unnoticed in NBAS deficiency in order to start adequate therapy and prophylaxis.

Pre-diagnosing and managing patients with GM1 gangliosidosis and related disorders by the evaluation of GM1 ganglioside content.

GM1 ganglioside, a monosialic glycosphingolipid and a crucial component of plasma membranes, accumulates in lysosomal storage disorders, primarily in GM1 gangliosidosis. The development of biomarkers for simplifying diagnosis, monitoring disease progression and evaluating drug therapies is an important objective in research into neurodegenerative lysosomal disorders. With this in mind, we established fluorescent imaging and flow-cytometric methods to track changes in GM1 ganglioside levels in patients with GM1 gangliosidosis and in control cells. We also evaluated GM1 ganglioside content in patients' cells treated with the commercially available Miglustat, a substrate inhibitor potentially suitable for the treatment of late-onset GM1 gangliosidosis. The flow-cytometric method proved to be sensitive, unbiased, and rapid in determining variations in GM1 ganglioside content in human lymphocytes derived from small amounts of fresh blood. We detected a strong correlation between GM1 ganglioside content and the clinical severity of GM1 gangliosidosis. We confirm the ability of Miglustat to act as a substrate reduction agent in the patients' treated cells. As well as being suitable for diagnosing and managing patients with GM1 gangliosidosis this method could be useful in the diagnosis and management of other lysosomal diseases, such as galactosialidosis, Type C Niemann-Pick, and any other disease with pathologic variations of GM1 ganglioside.

The utility of the basophil activation test in the diagnosis of immediate amoxicillin or amoxicillin-clavulanate hypersensitivity in children and adults.

BACKGROUND: The basophil activation test (BAT), has been proposed as a possible assay for the diagnosis of immediate-type allergy to beta-lactams (BLs). The aim of this study was to assess the utility of BAT in the diagnosis of amoxicillin (AMX) or AMX-clavulanate (AMX-C) IgE-mediated hypersensitivity in children and adults. MATERIAL AND METHODS: Eighteen children and 21 adults, with clinical history of immediate reactions to AMX or AMX-C, were referred to Anna Meyer Children's Hospital and San Giovanni di Dio Hospital, respectively. They underwent in vivo tests (skin prick test and intradermal test). Moreover, BAT with AMX or AMX-C was performed within 6 months from the reaction. RESULTS: In the pediatric group, the concordance between the skin tests (ST) and BAT results was 83.3%. Upon comparing the symptom grades and ST results to the BAT results, we found that the reaction severity and ST positivity did not correlate with BAT results in children. In the adult group, the concordance between the ST and BAT results was 61.9%. Upon comparing patients with severe reactions and patients with mild reactions in terms of BAT results, we found a BAT sensitivity of 38.5% and a specificity of 100%. When comparing the symptom grades to the BAT results, we found that no patients with mild symptoms had a positive BAT result, whereas 38.5% of patients with severe symptoms had a positive BAT result. CONCLUSIONS: BAT does not seem to be a useful tool to increase the sensitivity of an allergy work-up to diagnose immediate hypersensitivity to AMX or AMX-C.

Pneumococcal serotype distribution in adults with invasive disease and in carrier children in Italy: Should we expect herd protection of adults through infants' vaccination?

The 7-valent pneumococcal conjugate vaccine (PCV7) produced a significant herd protection in unvaccinated adult population mostly because of pneumococcus carriage decrease in vaccinated children. It is not known if the 13-valent pneumococcal vaccine can give similar effect on adults. Aims of the work were to evaluate whether the 6 additional serotypes are present in nasopharynx of children and serotype distribution in invasive pneumococcal infections (IPD) in adults. Realtime-PCR was used to evaluate pneumococcal serotypes in adults with confirmed IPD and in nasopharyngeal swabs (NP) from 629 children not vaccinated or vaccinated with PCV7 and resident in the same geographical areas. Two hundred twenty-one patients (116 males, median 67.9 years) with IPD were studied (pneumonia n = 103, meningitis n = 61 sepsis n = 50, other n = 7). Two hundred twelve were serotyped. The most frequent serotypes were 3, (31/212; 14.6%), 19A, (19/212; 9.0%), 12 (17/212; 8.0%), 7F, (14/212; 6.6%). In NP of children, the frequency of those serotypes causing over 50% of IPD in adults was very low, ranging from 0.48% for serotype 7F to 7.9% for serotype 19A. On the other side serotype 5, very frequent in NP (18.7%) caused <1% IPD. In conclusion serotypes causing IPD in adults are very rarely found in children NP. We suggest that herd protection obtainable with the additional 6 serotypes included in PCV13 may be more limited than that demonstrated with PCV7 in the past. In order to reduce the burden of disease in adults, adults should be offered a specific vaccination program with highly immunogenic PCV.

Interferon-gamma release assay sensitivity in children younger than 5 years is insufficient to replace the use of tuberculin skin test in western countries.

Tuberculin skin test, QuantiFERON-TB Gold In-Tube and T-SPOT.TB were performed in 338 children at risk for tuberculosis (TB), including 70 active TB cases. In children <5 years of age, QuantiFERON-TB Gold In-Tube sensitivity was 73.3% [95% confidence interval (CI): 57.5-89.1]; and T-SPOT.TB sensitivity was 59.3% (95% CI: 40.1-77.8); both were inferior to tuberculin skin test sensitivity (90.0%; 95% CI: 79.3-100). In children ≥ 5 years QuantiFERON-TB Gold In-Tube sensitivity was 92.5% (95% CI: 84.4-100); T-SPOT.TB sensitivity was 73.0% (95% CI: 58.6-87.3) ; and tuberculin skin test sensitivity was 97.5% (95% CI: 92.6-100).Test specificities were similar in all age groups.

Comparative analysis of rs12979860 SNP of the IFNL3 gene in children with hepatitis C and ethnic matched controls using 1000 Genomes Project data.

The rs12979860 single nucleotide polymorphism located on chromosome 19q13.13 near the interferon L3 gene (formerly and commonly known as interleukin 28B gene) has been associated in adults with both spontaneous and treatment induced clearance of hepatitis C virus. Although the exact mechanism of these associations remains unclear, it suggests that variation in genes involved in the immune response against the virus favours viral clearance. Limited and preliminary data are available on this issue in children. The aim of the present study was to evaluate, in a representative cohort of children with perinatal infection, the potential association between rs12979860 single nucleotide polymorphism and the outcome of hepatitis C virus infection. Alleles and genotypes frequencies were evaluated in 30 children who spontaneously cleared the virus and in 147 children with persistent infection and were compared with a population sample of ethnically matched controls with unknown hepatitis C status obtained using the 1000 Genomes Project data. The C allele and the C/C genotype showed greater frequencies in the clearance group (76.7% and 56.7%, respectively) when compared with both children with viral persistence (C allele 56.5%, p = 0.004; C/C genotype 32.7%, p = 0.02) and with the ethnically matched individuals (C allele 59.7%, p = 0.02; C/C genotype 34.7%, p = 0.03). Children with the C/C genotype were 2 times more likely to clear hepatitis C virus relative to children with the C/T and T/T genotypes combined (odds ratio: 2.7; 90% confidence intervals: 1.3-5.8). The present study provides the evidence that the rs12979860 single nucleotide polymorphism influences the natural history of hepatitis C virus in children.

Serial T-SPOT.TB and quantiFERON-TB-Gold In-Tube assays to monitor response to antitubercular treatment in Italian children with active or latent tuberculosis infection.

We performed a prospective study to investigate T-SPOT.TB and QuantiFERON-TB Gold In-Tube (QFT-G-IT) dynamics during antitubercular treatment in active tuberculosis (TB) or latent TB. Eighteen children with latent TB and 26 with TB were enrolled. At 6 months of follow-up reversion rate was 5.88% (95% CI:0-13.79) for QFT-G-IT; 9.09% (95% CI:0.59-17.58) for T-SPOT.TB (P=0.921) in TB cases. Significant decline in quantitative response was observed exclusively in TB cases. Our results suggest that serial IGRA have limited use in children receiving antitubercular treatment.