PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation--in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations

Abstract

Context: PAPSS2 (PAPS synthase 2) provides the universal sulfate donor PAPS (3'-phospho-adenosine-5'-phosphosulfate) to all human sulfotransferases, including SULT2A1, responsible for sulfation of the crucial androgen precursor dehydroepiandrosterone (DHEA). Impaired DHEA sulfation is thought to increase the conversion of DHEA toward active androgens, a proposition supported by the previous report of a girl with inactivating PAPSS2 mutations who presented with low serum DHEA sulfate and androgen excess, clinically manifesting with premature pubarche and early-onset polycystic ovary syndrome.

Patients and methods: We investigated a family harboring two novel PAPSS2 mutations, including two compound heterozygous brothers presenting with disproportionate short stature, low serum DHEA sulfate, but normal serum androgens. Patients and parents underwent a DHEA challenge test comprising frequent blood sampling and urine collection before and after 100 mg DHEA orally, with subsequent analysis of DHEA sulfation and androgen metabolism by mass spectrometry. The functional impact of the mutations was investigated in silico and in vitro.

Results: We identified a novel PAPSS2 frameshift mutation, c.1371del, p.W462Cfs*3, resulting in complete disruption, and a novel missense mutation, c.809G>A, p.G270D, causing partial disruption of DHEA sulfation. Both patients and their mother, who was heterozygous for p.W462Cfs*3, showed increased 5α-reductase activity at baseline and significantly increased production of active androgens after DHEA intake. The mother had a history of oligomenorrhea and chronic anovulation that required clomiphene for ovulation induction.

Conclusions: We provide direct in vivo evidence for the significant functional impact of mutant PAPSS2 on DHEA sulfation and androgen activation. Heterozygosity for PAPSS2 mutations can be associated with a phenotype resembling polycystic ovary syndrome.

Figure 1.

In silico analysis of the mutant PAPSS2 proteins. A, Either DHEA is converted via to T and DHT, activating the androgen receptor, or DHEA is sulfated by DHEA sulfotransferase (SULT2A1), which requires provision of the universal sulfate donor PAPS, generated by successive ATP sulfurylase and APS kinase activities of PAPSS2. A, androstenedione; APS, adenosine 5′-phosphosulfate; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; PAP, 3′-phosphoadenosine 5′-phosphate; PPi, pyrophosphate. B and C, Homology model of human PAPSS2 based on the 1XNJ structure of PAPSS1 (12), with one high-affinity dimer represented in gray, the other in cyan, visualizing protein truncation by the frameshift mutation p.W462Cfs*3 (B) and the glycine residue affected by the missense mutation p.G270D (C).

Figure 2.

Functional in vitro assessment of the mutant PAPSS2 proteins. A, Residual enzyme activity expressed as percentage of wild-type (WT) activity, defined as 100%, based on measurements of the conversion of DHEA to DHEAS in HEK293 cells cotransfected with SULT2A1 and WT or mutant PAPSS2. Error bars represent the mean ± SEM of three independent triplicate experiments. B, Representative Western blot demonstrating equal loading and equal SULT2A1 protein expression, but significantly lower expression of mutant PAPSS2 proteins in HEK293 cells cotransfected with SULT2A1 and WT or mutant PAPSS2. C, Treatment with the proteasome inhibitor MG-132 enhances protein expression of the PAPSS2 mutants p.T48R and p.G270D, confirming increased ubiquitination of the mutant proteins.

Figure 3.

DHEA sulfation and androgen synthesis after an oral challenge with 100 mg DHEA. A–C, Serum concentrations of DHEAS (A), DHEA (B), and the ratio of serum DHEA/DHEAS (C) in the two brothers with compound PAPSS2 mutations (closed symbols) and their heterozygous parents (mother, empty circle; father, empty square) in comparison to healthy female controls (n = 20). D, Serum androstenedione after DHEA in patients, parents and healthy controls. E, Percentage of 24-hour urinary DHEAS excretion in relation to active androgen metabolite excretion after oral DHEA administration and the percentage of 5α-reduced androsterone to 5β-reduced etiocholanolone excretion, demonstrating reduced DHEAS generation and enhanced production of 5α-reduced androgens in the patients and their mother. The excretion pattern in the father resembled that observed in healthy controls (n = 8).