Investigation of the genetic aetiology and pathogenetic mechanism of disease in patients with late-onset FGD
Abstract
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.
Authors
Hughes, ClaireCollections
- Theses [3822]