TRAP1 chaperone protein mutations and autoinflammation

We identified a consanguineous kindred, of three affected children with severe autoinflammation, resulting in the death of one sibling and allogeneic stem cell transplantation in the other two. All three were homozygous for MEFV p.S208C mutation; however, their phenotype was more severe than previously reported, prompting consideration of an oligogenic autoinflammation model. Further genetic studies revealed homozygous mutations in TRAP1, encoding the mitochondrial/ER resident chaperone protein tumour necrosis factor receptor associated protein 1 (TRAP1). Identification of a fourth, unrelated patient with autoinflammation and compound heterozygous mutation of TRAP1 alone facilitated further functional studies, confirming the importance of this protein as a chaperone of misfolded proteins with loss of function, which may contribute to autoinflammation. Impaired TRAP1 function leads to cellular stress and elevated levels of serum IL-18. This study emphasizes the importance of considering digenic or oligogenic models of disease in particularly severe phenotypes and suggests that autoinflammatory disease might be enhanced by bi-allelic mutations in TRAP1

Autoinflammatory periodic fever, immunodeficiency, and thrombocytopenia (PFIT) caused by mutation in actinregulatory gene WDR1

The importance of actin dynamics in the activation of the inflammasome is becoming increasingly apparent. IL-1β, which is activated by the inflammasome, is known to be central to the pathogenesis of many monogenic autoinflammatory diseases. However, evidence from an autoinflammatory murine model indicates that IL-18, the other cytokine triggered by inflammasome activity, is important in its own right. In this model, autoinflammation was caused by mutation in the actin regulatory gene WDR1 We report a homozygous missense mutation in WDR1 in two siblings causing periodic fevers with immunodeficiency and thrombocytopenia. We found impaired actin dynamics in patient immune cells. Patients had high serum levels of IL-18, without a corresponding increase in IL-18-binding protein or IL-1β, and their cells also secreted more IL-18 but not IL-1β in culture. We found increased caspase-1 cleavage within patient monocytes indicative of increased inflammasome activity. We transfected HEK293T cells with pyrin and wild-type and mutated WDR1 Mutant protein formed aggregates that appeared to accumulate pyrin; this could potentially precipitate inflammasome assembly. We have extended the findings from the mouse model to highlight the importance of WDR1 and actin regulation in the activation of the inflammasome, and in human autoinflammation

Genetic Mapping for the Discovery of Novel Genes Causing Autoinflammatory Diseases

The autoinflammatory syndromes are an emerging group of diseases, some with defined genetic cause, characterised by seemingly unprovoked inflammation which derives from a disruption of innate immunity. Novel as yet undefined syndromes are increasingly recognised in consanguineous families who may have normal parents and both affected and unaffected offspring. This type of family is ideal for genetic mapping as both copies of the (presumed recessive) disease causing alleles are likely to have originated in a recent shared common ancestor, and may be linked with markers which will be homozygous in the affected children. In this thesis affected and unaffected offspring and parents in three first-cousin Pakistani families were genotyped with Illumina 610 SNP arrays. This data was used for homozygosity mapping and parametric multipoint linkage analysis. For the first family, a 5Mb region was identified from this mapping. Candidate genes were chosen by members of an expert panel and the exons of these genes were Sanger sequenced. DNA from the entire homozygous region linked to the disease locus (5Mb) was captured using a custom designed 385k array from Nimblegen and then resequenced using the Illumina Genome Analyser II, revealing over 50 coding change variants. These entered a filtering process involving: the selection of rare variants and screening of extended family members, ethnically matched controls and disease controls, and the exclusion of unlikely candidates. This ultimately identified two missense variants of interest in two genes in family A, a novel variant in MEFV the gene affected in Familial Mediterranean Fever, and a variant in TNF Receptor Associated Protein 1 gene TRAP1. The potential contribution of disrupting of the function of these genes to the pathogenesis was assessed using siRNA knockdown in HeLa and THP1 cells. Outcomes assessed include levels of reactive oxygen species (ROS), which are central to many inflammatory processes, and cytokine production. This suggested that TRAP1 may be involved through elevated ROS and TNFα secretion

Polymorphonuclear leucocyte phagocytic function, γδ T-lymphocytes and testosterone as separate stress-responsive markers of prolonged, high-intensity training programs

Excessive exercise with limited recovery may lead to detrimental states of overreaching or the overtraining syndrome. Chronic maladaptation in endocrine and immune mechanisms occur with the incidence of these states. Exercise-induced cortisol and testosterone responses have been proposed as biomarkers of overreaching, with blunted responses following intensified-training periods. Yet, limited information on the effects of overreaching in immunity is available. Healthy individuals completed a 30-min running protocol (the RPETP) before and after a 12-day intensified-training period. Blood and saliva were collected before, after and 30min after RPETP at pre-training and post-training. Plasma and salivary cortisol and testosterone, leucocyte proliferation and polymorphonuclear leucocyte phagocytic activity were examined. Plasma and salivary cortisol were acutely unaffected pre-training (−14% and 0%, p ​> ​0.05) and post-training (−14% and +46%, p ​> ​0.05). Comparing pre-training with post-training, blunted responses were observed in plasma testosterone (43%–19%, p ​ ​0.05). No acute or resting changes in total leucocyte counts or most leucocyte subsets occurred pre-training or post-training. Yet, a 194% acute elevation in γδ T-lymphocyte number occurred pre-training (p ​< ​0.05), and average resting concentrations were 174% higher post-training. Baseline phagocytic activity was 47% lower post-training (p ​< ​0.05). Intensified training was detrimental, significantly reducing phagocytic activity. Testosterone blunted post-training, indicating an excessive training-related hypothalamic-pituitary gonadal dysfunction. The γδ T-lymphocytes sensitivity to exercise was noted, rendering it as a potential stress-responsive cellular marker. The usefulness of the RPETP to track the onset of overreaching is proposed