In Vitro Splicing Assay Proves the Pathogenicity of Intronic Variants in MRAP

Abstract

Introduction: Familial glucocorticoid deficiency (FGD) is characterised by isolated glucocorticoid deficiency in a patient who retains normal mineralocorticoid production. FGD causing mutations in the MC2R accessory protein, MRAP, are often splice-site or nonsense mutations resulting in a truncated protein. Many of these mutations occur at the canonical donor splice-site of intron 3, where it has been shown previously that c.106 + 2_3dupTA, for example, results in skipping of the first coding exon with unknown consequences at the protein level. Patients and methods: DNA was isolated from three consanguineous individuals diagnosed with early onset FGD (0 - 13 months) with high ACTH and/or low cortisol levels and underwent whole exome sequencing. The proband in family 1 (P1) presented at 13 months and had a hyperpigmented sibling who died in neonatal period due to adrenal failure. Patient 2 (P2), who also had a family history of adrenal insufficiency, was noted to be hyperpigmented at birth with markedly raised ACTH, patient 3 (P3) was noted to have diffuse hyperpigmentation in the early neonatal period and on formal testing at 16m was found to have low serum cortisol. Variants were confirmed using Sanger sequencing and predicted splice-site mutations were investigated using an in vitro splicing assay. Results: Homozygous mutations in MRAP were identified in all three cases which were heterozygous in their parents. Previously described mutations, c.106 + 1delG (chr21:33671388delG; rs1476574441; CD050155) in P1 and c.106 + 2dupT (Chr21: 33671390_91insT; rs761576317; CI118288) in P2 at the canonical donor splice-site of intron 3, were identified, with the former predicted to destroy the splice site and the latter to weaken it. These mutations in vitro resulted in the complete skipping of exon 3, which contains the translational start site, and presumably result in no protein product. A novel homozygous mutation in intron 4, c.206 + 5G>T; (chr21:33679055G>T rs1064796398) was identified in P3, but was not predicted to alter splicing. In vitro, this mutation negates the canonical donor splice site and creates two different alternative sites, both resulting in frameshifts and predicted early termination of the protein (p.Val44fs*50, p.Pro72fs*90). Conclusion: All mutations reported here are predicted to produce no protein, either because the start site is excluded (for c.106 + 1delG and c.106 + 2dupT) or because the transcripts are likely to undergo nonsense mediated decay (for c.206 + 5G>T), resulting in the early onset FGD seen in the patients. Splice prediction protocols, although effective for variants within 2bp of exon/intron boundaries may not predict the true outcome of a base change whereas the splice assay conclusively revealed the effect of all three variants allowing us to assign pathogenicity to them.