Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency

Abstract

Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available.

Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort.

Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries.

Results: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles.

Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

Fig. 1.

Localization of POR mutations at the DNA and protein level. A, Schematic representation of the POR gene. The untranslated exon U1 is given as a black box and coding exons are numbered. Novel mutations are given in red font, and the most common mutation p.A287P in exon 8 is marked in blue. Mutations cluster in the 3′ region without any additional common mutation apart from the p.A287P mutation. The partial deletion del ex U1–1 and the partial duplication dup ex2–5 that were identified by MLPA are represented by labeled red boxes. B, Schematic representation of the POR protein. Mutant residues are located in various locations all over the three functional domains of the POR protein. C, Three-dimensional POR protein model illustrating the localization of mutant residues in relation to the three functional moieties: nicotinamide adenine dinucleotide phosphate (NADPH; blue), flavin adenine dinucleotide (FAD; yellow), and flavin mononucleotide (FMN; green). Residues affected by missense mutations are given in dark yellow, frame shift mutations in orange, and residues affected by nonsense mutations as well as an in-frame duplication are marked in red.

Fig. 2.

In vivo steroidogenic enzyme activities in PORD patients (n = 23) as determined by urinary steroid profiling. Diagnostic steroid substrate over product ratios reflective of distinct steroidogenic enzyme activities and measured by GC/MS are shown in comparison with an age-matched reference cohort. Box plots represent the interquartile ranges (25th to 75th percentile), whiskers the fifth and 95th percentiles, respectively, of the reference cohort; each PORD case is represented by specific symbols.