Familial Glucocorticoid Deficiency (FGD) is an autosomal recessive form of adrenal failure characterized by isolated glucocorticoid deficiency with preserved mineralocorticoid secretion. I studied two cohorts of patients who presented with late onset FGD. Firstly I investigated 2 families of Turkish ethnicity who presented with delayed onset adrenal insufficiency and identified two novel missense mutations in the melanocortin 2 receptor accessory protein (MRAP). I characterised both these mutations in vitro and confirmed that both mutant MRAPs demonstrated reduced rather than absent function consistent with the phenotype of delayed presentation. Secondly I studied 3 families from a genetically isolated Irish population who had an interesting variant of adrenal failure. Patients had typical biochemical features of FGD with isolated glucocorticoid deficiency, raised ACTH and normal renin and aldosterone levels. Unlike other forms of FGD, cortisol deficiency was often not as severe and onset was usually in childhood following a period of normal adrenal function. Affected children develop hypocortisolaemia but also growth failure, increased chromosomal breakage and natural killer (NK) cell deficiency. Targeted exome sequencing identified a variant (c.71-1insG) in mini chromosome maintenance homologue 4 (MCM4) that segregated with the disease in all 3 families. RT-PCR of patient leucocyte RNA revealed this mutation leads to aberrant splicing of 3 exon 2 and a foreshortened ORF encoding a prematurely terminated translation product (p. Pro24ArgfsX4). Western blotting of patient lymphocytes revealed loss of the full length MCM4 protein but two smaller MCM4 isoforms were preserved. Histological examination of the adrenals of an MCM4 depletion mouse model revealed an abnormal adrenal morphology. Small, spindle-shaped cells were present throughout the adrenal cortex. These cells did not express either CYP11A1 or CYP11B1 and significantly reduced the number of steroidogenic cells in the zona fasciculata. Further staining showed these cells expressed GATA4, a transcription factor expressed in foetal but not adult adrenals, and capsular markers, indicating that they may be non-steroidogenic capsular cells infiltrating the cortex. MCM4 is one part of a heterohexameric complex essential for normal DNA replication and genome stability in all eukaryotes and no MCM mutation has ever been described in humans. I have identified a mutation in MCM4 that results in adrenal failure, growth retardation, increased chromosomal fragility and NK cell deficiency. Animal models indicate that loss of MCM4 is lethal, but it is likely that the smaller isoforms I observe may rescue the patients from a lethal phenotype. This research has revealed a novel mechanism of adrenal failure and potentially a novel function of MCM4. The seemingly specific impact on adrenal function may reflect a defect in adrenal stem cell differentiation and the inability of capsular cells to differentiate into steroidogenic cells.

Background - The alternatively spliced insulin receptor (IR) exon 11 (36 nucleotides) and the constitutively spliced Apolipoprotein B (APOB) exon 26 (7572 nucleotides) are examples of the shortest and longest exons in the genome. Aim - The aim of this study was to investigate the regulation of splicing of these 2 exons in cell culture using 2′-O-methyl RNA antisense oligonucleotides (ASOs) and peptide nucleic acid (PNA)-peptide hybrid ASOs. Methods - ASOs were designed to target key sequences involved in the splicing of the IR exon 11 and exonic splicing silencer sequences (ESS) in APOB exon 26. HepG2 cells were reverse-transfected with the ASOs for 48 hours, mRNA harvested and RT-PCR was performed to amplify the IR isoform and APOB cDNAs which were separated by PAGE and quantified. Results Insulin receptor exon 11 - 2′-O-methyl RNA ASOs targeted to two intronic sites, the 3′ half of exon 11 and spanning the entire exon caused significant exon skipping. PNA-peptide hybrids predicted to increase exon 11 splicing, paradoxically caused exon skipping. PNA-peptide hybrids with 3′ tails caused exon 11 skipping more effectively than hybrids with 5′ tails. Apolipoprotein B exon 26 - Only combinations of 2′-O-methyl RNA ASOs targeting multiple ESSs in APOB exon 26 caused a small proportion of aberrant splicing. This consisted of complete exon 26 skipping and the selection of a downstream cryptic 3′ splice site in preference to the native 3′ splice site. Discussion - Exclusion of the IR exon 11 can be induced by targeting a combination of intronic or exonic sequences. PNA-peptide hybrid ASOs were unable to increase exon 11 splicing. The aberrant splicing of large constitutive exons such as APOB exon 26 can be induced by targeting multiple ESS sites along its course.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is an early onset neurodegenerative disorder resulting from mutations in the SACS gene that encodes the protein sacsin. Sacsin is a 520kDa multi-domain protein localised at the cytosolic face of the outer mitochondrial membrane with suggested roles in proteostasis and most recently in the regulation of mitochondrial morphology. An excessively interconnected mitochondrial network was observed as a consequence of reduced levels of sacsin protein following SACS knockdown in neuroblastoma cells as well as in an ARSACS patient carrying the common Quebec homozygous SACS mutation 8844delT. Moreover, it was suggested that sacsin has a role in mitochondrial fission as it was found to interact with mitochondrial fission protein Dynamin related protein 1 (Drp1). The aim of this thesis was to explore sacsin’s role in the regulation of mitochondrial morphology and dynamics in non-Quebec ARSACS patients and sacsin knockdown fibroblasts. This study shows that loss of sacsin function promotes a more interconnected mitochondrial network in non-Quebec ARSACS patients and in sacsin knockdown fibroblasts. Moreover, recruitment of the essential mitochondrial fission protein Drp1 to the mitochondria was significantly reduced in ARSACS patient cells and in sacsin knockdown fibroblasts. This reduced recruitment of Drp1 to mitochondria also occurred when cells were treated to induce mitochondrial fission. Furthermore, both the size and intensity of Drp1 foci localised to the mitochondria were significantly reduced in both sacsin knockdown and patient fibroblasts. Finally, reduced ATP production, decreased respiratory capacity of mitochondria and an increase in mitochondrial reactive oxygen species demonstrated impaired mitochondrial function in ARSACS patient and sacsin knockdown fibroblasts. These results suggest a role for sacsin in the stabilisation or recruitment of cytoplasmic Drp1 to prospective sites of mitochondrial fission similar to that observed by other mitochondrial fission accessory proteins.