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Title: Investigation of the genetic aetiology and pathogenetic mechanism of disease in patients with late-onset FGD
Author: Hughes, Claire
Awarding Body: Queen Mary, University of London
Current Institution: Queen Mary, University of London
Date of Award: 2013
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medicine ; Endocrinology