Standing, ASI; (2014) Genetic Mapping for the Discovery of Novel Genes Causing Autoinflammatory Diseases. Doctoral thesis , UCL (University College London).

Abstract

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.

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