The aim of this project was to identify novel mutations in proteins causing immunodeficiency. This was achieved by confirming whole exome sequencing (WES) results of potential mutations and genes of interest through Sanger sequencing and allelic orientation of mutations. Functional experiments were then designed and performed on patient samples and stable cell lines expressing patient mutants. The experiments examined the known functions of the proteins of interest and cell types that appeared to be affected in the patient immunological profile.
The main topic of this thesis is centered around an index case who presented at age 17 with a life-long history of recurrent sinopulmonary infections and established bronchiectasis. Immunological investigations showed severe panhypogammaglobulinaema, marginally reduced CD4 count and reduced T cell proliferative responses.
WES identified potentially damaging mutations in Ca2+-release activated Ca2+ regulator 2A (CRACR2A): c.430 A>G (p.144R>G) and c.898 G>T (p.300E>*) which were inherited via the maternal line; c.834 G>T (p.278E>D) in the paternal allele. In primary patient samples, T cells had reduced calcium flux, cytokine production and p-JNK.
The two mutant patient alleles were expressed using retroviral transduction in the CRACR2A knock-out Jurkat cell line. Both alleles resulted in reduced calcium flux and IL-2 expression when compared to wild-type CRACR2A. The maternal mutant allele expression produced a truncated protein (resulting from the p.300E>* mutation), with abnormal localisation and significantly reduced p-JNK. This truncated protein was not seen in the primary cells, and so is not likely to be expressed in the patient.
The data suggests that biallelic mutations in CRACR2A can lead to primary immunodeficiency, which affects T cell related function. This is the first time CRACR2A has been linked to disease.
The identification of novel mutations in patient with primary immunodeficiency and using functional experiments to see their pathogenic effect in patient samples was also done in patients with STIM1 and STING mutations
