Clinical, genetic, and functional characterization of four patients carrying partial loss-of-function mutations in the steroidogenic acute regulatory protein (StAR)

Abstract

Context: Nonclassic congenital lipoid adrenal hyperplasia (lipoid CAH) is a recently recognized disorder caused by mutations in the steroidogenic acute regulatory protein (StAR) that retain partial function. Affected individuals can present with a phenotype of late onset adrenal insufficiency with only mild or minimally disordered sexual development.

Objectives: The aim was to delineate the clinical spectrum of StAR mutations and correlate phenotype with StAR activity.

Patients: Four patients had nonclassic/atypical lipoid CAH. Adrenal insufficiency was manifested at birth in two patients and at 11 months and 4 yr in the other two. Three were 46,XY with underdeveloped genitalia.

Methods: The StAR gene was sequenced, mutations were recreated in expression vectors, and StAR activity was measured as pregnenolone production in COS-1 cells cotransfected with the cholesterol side-chain cleavage system. StAR mutants were expressed as N-62 StAR in bacteria, and purified proteins were tested for activity with isolated steroidogenic mitochondria and for cholesterol-binding capacity.

Results: DNA sequencing identified mutations on all alleles. Missense mutations were R188C, G221D, L260P, and F267S; we also tested R192C described by others. The respective activities of R188C, R192C, G221D, L260P, and F267S were 8.0, 39.4, 2.4, 3.1, and 6.1% of wild-type in transfected cells, and 12.8, 54.8, 6.3, 1.8, and 9.5% with isolated mitochondria. Cholesterol binding capacities of R188C, R192C, G221D, L260P, and F267S were 6.7, 55.3, 10.2, 4.6, and 20.9%. These data are correlated to the three-dimensional structure of StAR.

Conclusions: There is a broad clinical spectrum of StAR mutations; StAR activities in vitro correlate well with clinical phenotypes.

Figure 1

Quantification of StAR protein. Top panel, The indicated amounts of purified wild-type N-62 StAR protein and mutant purified N-62 StAR proteins were separated by 12% SDS-PAGE. The band intensity was measured by Scion Image software. Bottom panel, The standard curve of StAR protein. The amount of mutant N-62 StAR proteins was plotted against its band intensity.

Figure 2

Activities of StAR mutants. A, Activity of full-length StAR in whole cells. COS-1 cells were cotransfected with expression vectors for the cholesterol side-chain cleavage system (F2) and either wild-type (WT) or mutant StAR, and pregnenolone was measured 48 h later by immunoassay. The StAR-independent substrate 22(R)-hydroxycholesterol (22R-OH) was added to the cell culture media to determine the maximum steroidogenic capacity of the cells. Data are expressed as the mean ± sem from at least three independent experiments, each performed in triplicate. B, Activity of isolated N-62 StAR on mitochondria in vitro. Purified proteins were added to mitochondria from steroidogenic mouse MA-10 Leydig cells, and pregnenolone production from endogenous mitochondrial cholesterol was measured. Data are expressed as mean ± sem from four experiments, each performed in duplicate. C, Cholesterol binding assay. Binding of various concentrations of NBD-cholesterol by wild-type and mutant StAR was measured by fluorescence; control is buffer without protein. Data are expressed as mean ± sem for three experiments, each performed in triplicate.