Abstracts of the 52nd Workshop for Pediatric Research

Rhea van den Bruck and Patrick P Weil are joint first authors

A male dizygotic twin was delivered at a gestational age of 36 weeks in the Children’s Hospital of the Helios Medical Centre Wuppertal, Witten/Herdecke University Hospital. Contrary to his brother the boy presented dystrophic at birth (1.715 g birth weight; 150 g below 3rd percentile) and developed adverse gastrointestinal conditions within the first 2 months of life. These included chronic mucosal inflammation and oedematous lamina propria in the intestine, which contributed to intractable diarrhoea. At an age of 7 months the infant eventually died of enteral haemorrhages and liver failure. Further anamnesis revealed several similar fatalities in the familial clan with reportedly frequent parental consanguinity. Intractable chronic diarrhoea in infancy are heterogeneous disorders challenging for diagnostics and therapy. Despite extensive diagnostic approaches the etiology of many cases remains elusive. Investigating putatively underlying genetic disorders might clarify many cases.

To contribute to the diagnosis we performed whole exome sequencing of the affected infant as well as his twin brother and parents. We identified a suspicious nonsynonymous single nucleotide polymorphism (SNP) in the integrin beta-6 gene (ITGB6G1312A) entailing a V438M substitution. This SNP is very rare in the G1000 cohort and predicted being potentially harmful. The allelic distribution in the genotyped family members fit well with an autosomal recessive inheritance scheme. We performed computational biological and molecular biological analyses on the α_Vβ6 integrin receptor function suggesting that the integrin α_Vβ6 dimerization could be impaired, potentially causing a loss of α_Vβ6 function in wound healing and epithelial tissue integrity.

Our study provides a starting point for elucidating integrin α_Vβ6 function and for understanding a pathomechanistical relevance of ITGB6V438M.

The authors have written informed consent from the patients’ guardian/parent.

Inflammation-induced FGF10 protein deficiency is associated with bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurely born infants characterized by arrested alveolar development. So far, experimental evidence for a direct role of FGF10 in BPD is lacking.

Using the hyperoxia-induced neonatal lung injury as a mouse model of BPD, the impact of Fgf10 deficiency in Fgf10(+/−) vs. Fgf10(+/+) pups was investigated. In normoxia, no lethality of Fgf10(+/+) and Fgf10(+/−) pups is observed. By contrast, 100% of Fgf10(+/−) pups died within 8 days of hyperoxic injury, with lethality starting at day 5, while Fgf10(+/+) pups were all alive. Lungs of pups from the two genotypes were collected on postnatal day 3 following normoxia or hyperoxia exposure for further analysis. In hyperoxia, Fgf10(+/−) lungs exhibited increased hypoalveolarization. Analysis by FACS of the Fgf10(+/−) vs. control lungs in normoxia, revealed decreased alveolar epithelial type II (AECII) cells over total EpCAM-positive cells ratio. In addition, gene array analysis indicated reduced AECII and increased AECI transcriptomic signatures in isolated AECII cells from Fgf10(+/−) lungs. Such imbalance in differentiation is also seen in hyperoxia and associated with reduced mature surfactant protein B and C expression. Attenuation of FGFR2b ligands activity postnatally in the context of hyperoxia also leads to increased lethality with decreased surfactant expression.

In addition to IL-10, the immune-regulatory cytokine IL-19 and the proinflammatory cytokine IL-20 are dysregulated in monocytes from CRMO patients. Forcing or knocking down gene expression and applying ChIP, we demonstrated that IL10 and IL19 undergo trans-activation by Sp-1. Using ChIP and MeDIP, we showed that IL10, IL19, and IL20 undergo epigenetic (dys-)regulation. Co-culturing primary human monocytes with cytokines, we demonstrated that reduced expression of IL-10, and IL-19 in CRMO results in increased inflammasome activity and IL-1β expression and release. Increased expression of IL-20 had no effect on inflammasome activity.

Altered expression of IL-10 and IL-19 contributes to inflammasome activation and a pro-inflammatory phenotype of monocytes in CNO. Since IL-10-deficient mice develop severe inflammatory bone-loss in arthritis models, which is mediated by inflammasome activation and IL-1 release, our observations contribute to a better understanding of the molecular pathophysiology of CNO. Cytokine dysregulation and inflammasome activation provide promising molecular targets in the search for disease biomarkers and future therapeutic interventions.

In children, asthma is one of the most common chronic diseases with increasing prevalence. Human rhinovirus infection (HRVI) plays an important role in asthma exacerbations and is thought to be involved in the asthma development during childhood. McErlean et al. reported that HRVI changes the epigenetic gene regulatory mechanism of DNA methylation in nasal epithelium cells. We hypothesized that HRVI in the epithelium initiate a different methylation pattern in asthmatic and non-asthmatic children.

The overlap of viral modified DNA methylation sites in AST and non-AST is with 211 CpGs (p < 0.05) very limited. In the AST we detected 6922 (p < 0.05) viral altered CpGs which were not modified after HRVI in non-AST. Furthermore 6569 CpGs (p < 0.05) showed a viral induced DNA methylation modification in non-AST but not in AST subjects. Vanin-1 showed a decreased DNA methylation after HRVI specifically in AST (p =0.012).

A subset of patients with autoinflammatory diseases, characterized by recurrent febrile episodes and systemic inflammation of yet unknown origin harbour mutations in the CASP1 gene, resulting in reduced enzymatic activity of caspase-1 and impaired IL-1β secretion. Thus, systemic inflammation in these individuals most likely results from the induction of alternative proinflammatory pathways, which was supported by elevated concentrations of the proinflammatory cytokine TNFα in some of these patients. Previously, we demonstrated that CASP1 variants maintain prolonged interactions with receptor-interacting protein kinase 2 resulting in enhanced activation of NF-κB.

Here we aimed to investigate whether an enzymatically inactive variant caspase-1 (p.C284A) leads to the alteration of speck formation and pyroptosis, a caspase-1 dependent proinflammatory cell death.

Using an in vitro model of transduced immortalized murine macrophages we demonstrate that caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the variant p.C284A caspase-1 as compared to the WT. Using FLIM-FRET and co-immunoprecipitation we additionally show extended and more intense interaction of the variant p.C284A with ASC compared with WT caspase-1. Applying live cell imaging, we for the first time show that pyroptosomes of enzymatically inactive variant caspase-1 spread during cell division.

Proinflammatory signal transduction through propagation of specks during cell division may be a highly efficient and previously not appreciated mechanism during (auto)inflammation. Additionally, macrophages expressing variant p.C284A caspase-1 resist cell death and therefore may cause an initially weaker but altogether more persistent inflammatory stimulus.

The high-affinity choline transporter (CHT, encoded by SLC5A7) is a critical determinant of acetylcholine signalling at the specialized neuromuscular junctions (NMJ) and central cholinergic synapses. Here we describe an autosomal recessive form of severe congenital hypotonia associated with CHT N-terminal missense mutations. The patients presented with severe neurodevelopmental delay and hypotonia indicating the dysfunction of the central synapses. Patient muscle biopsies revealed targetoid-like lesions. Whole exome sequencing revealed co-segregating SLC5A7 mutations in two families with in total 3 patients. Each sequence mutation (NM_021815.2, c.282 T > A / p.Ser94Arg in Family 1, c.335 T > A, p.Val112Glu in Family 2) result in a single amino acid change in the transmembrane spanning domain. Each substitution affects residues which display high evolutionarily sequence conservation show high damage prediction, and alter residues located in the intracellular loop regions located between the third – sixth transmembrane domains. In vivo pan-neuronal expression of the wild type and mutant CHT in Caenorhabditis elegans revealed a complete loss of axonal transport associated with CHT mutation. Consistent with this our choline transport cell transfection assays and immunohistochemical studies demonstrated significant reductions in CHT cell surface levels, as well as a severe reduction in transporter activity associated with CHT mutation. These findings contrast with our previous description of a dominant-negative frameshift mutation at the C-terminus of CHT, associated with significantly reduced but not completely abrogated CHT transporter function leading to autosomal dominant distal hereditary motor neuropathy (dHMN). Together our findings define distinct neurological outcomes, disease processes, and modes of inheritance arising from different classes of CHT mutation and provide invaluable insight into the biological role of CHT as well as NMJ dysfunction.