Endocrine Abstracts (2019) 65 CC2 | DOI: 10.1530/endoabs.65.CC2

Splice-site mutations in the melanocortin-2 receptor accessory protein that lead to early-onset familial glucocorticoid deficiency type 2

Younus Qamar1,2, Avinaash Maharaj2, Li Chan2, Asma Deeb3 & Louise Metherell2

1Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK; 2Centre for Endocrinology, William Harvey Research Institute, Queen Mary University of London, London, UK; 3Paediatric Endocrinology Department, Mafraq Hospital, Abu Dhabi, UAE

Background: Familial glucocorticoid deficiency (FGD) is characterised by isolated glucocorticoid deficiency, with preserved mineralocorticoid production. FGD type 2 is caused by mutations in MRAP encoding the melanocortin-2 receptor accessory protein. MRAP has a single transmembrane domain essential to its function in trafficking the MC2R/ACTH receptor. 15 mutations in MRAP have been described, five of which are within the canonical donor splice-site of intron 3 and predicted to cause complete exon skipping.

Patients and methods: Proband 1 (family 1) was diagnosed at 13 months. His sibling was hyperpigmented but was neither formally diagnosed nor treated and died neonatally after a febrile illness. Proband 2 (family 2), diagnosed at birth, was hyperpigmented with elevated ACTH levels. Both patients have responded to hydrocortisone replacement therapy. The coding exons of MC2R and MRAP were Sanger sequenced from patient DNA. The effect of splice-site mutations was assessed in silico by Human Splicing Finder (HSF3.0) and in vitro by a splicing assay, comparing wild type and mutant heterologous minigenes.

Results: Homozygous mutations, c.106+1delG (family 1) and c.106+2dupT (family 2) were identified in the donor splice-site of intron 3 of MRAP. HSF3.0 predicted c.106+1delG destroys the splice site and c.106+2dupT weakens it. However, there is another high-scoring donor site 23 bp downstream which, if utilised, would result in the addition of frame-shifting 22 bp for c.106+1delG BUT the possibility of in-frame insertion of 24 bp for the c.106+2dupT mutation. An in vitro splicing assay utilised to assess this demonstrated that both variants resulted in complete skipping of exon 3.

Conclusion: Both splice-site mutations lead to complete skipping of exon 3 of MRAP, containing the translational start site, the next methionine from which translation could start is after the essential transmembrane domain. Hence, this is likely to result in the absence of any functional protein which is in keeping with the severe, early presentation in these two families.

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