Endocrine Abstracts

Adrenal insufficiency (AI) is life-threatening and can present alone or in combination with other co-morbidities. Here we describe families with a novel association of AI with porphyria caused by biallelic mutations in protoporphyrinogen oxidase (PPOX) or coproporphyrinogen oxidase (CPOX). The porphyrias are a group of disorders caused by defects in one of eight enzymes within the haem biosynthetic pathway, divided into acute porphyrias, resulting in mainly neurovisceral symptoms, or cutaneous porphyrias, mainly affecting the skin. Acute porphyria attacks can be life-threatening, resulting in permanent disability or death. Variegate porphyria and hereditary coproporphyria are associated with autosomal dominant mutations in PPOX and CPOX respectively, with biallelic inheritance rarely reported and knockout mice that are embryonic lethal. We performed whole exome sequencing (WES), array Comparative Genomic Hybridization (aCGH) and whole genome sequencing (WGS) in 3 families with AI and variegate or hereditary porphyria. In kindred 1, with 4 affected individuals, WES revealed a homozygous mutation, p.Glu339Lys, in PPOX. aCGH and WGS revealed no Copy Number Variants (CNVs) and no other variants, in genes causing AI, common to all affected individuals. In families 2 and 3, homozygous mutations in CPOX p.(Pro367Ala) in one patient and p.(Ser28Ter) in two siblings were identified by WGS and WES respectively, with a clinical picture of AI and hereditary coproporphyria. Unlike other acute porphyria kindreds, the heterozygous parents were asymptomatic, manifesting neither porphyria nor AI, suggesting that the level of enzyme function is key for both phenotypes. Reduced PPOX/CPOX activity could cause AI through either; (i) a dearth of haem for steroidogenic CYP450 enzyme action, (ii) toxicity of intermediate porphyrins or (iii) increased oxidative stress. To investigate the mechanism, we first created H295R, human adrenocortical cells, with differing degrees of PPOX-knockdown (KD) by shRNA. Proliferation, measured by MTT assay or GFP accumulation, was lower in PPOX-KD cells at 72 hours and mitochondrial respiration was diminished, possibly due to toxicity of porphyrin precursors. CYP11A1 expression was unaltered whereas STAR and CYP17A1 were significantly lower in PPOX-KD cells. Finally, and definitively, knockdown of at least 60% PPOX protein in H295R cells reduced cortisol output by 2.2-fold (P<0.01) at baseline and 1.9-fold (P<0.0001) in forskolin stimulated cells. Similar CPOX studies are underway. These studies show that cortisol is reduced in individuals with biallelic PPOX/CPOX mutations and, in conjunction with previous cases of AI in association with other genes in the pathway, suggests adrenal function should be monitored in individuals with porphyria.