Characterization of Novel StAR (Steroidogenic Acute Regulatory Protein) Mutations Causing Non-Classic Lipoid Adrenal Hyperplasia

Context Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH). Objective StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. Design To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. Setting Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. Patients Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. Results StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (~30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. Conclusions StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed

Characterization of Novel StAR (Steroidogenic Acute Regulatory Protein) Mutations Causing Non-Classic Lipoid Adrenal Hyperplasia

Context Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH). Objective StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. Design To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. Setting Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. Patients Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. Results StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (~30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. Conclusions StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed

Human growth hormone (GH1) gene polymorphism map in a normal-statured adult population

OBJECTIVE: GH1 gene presents a complex map of single nucleotide polymorphisms (SNPs) in the entire promoter, coding and noncoding regions. The aim of the study was to establish the complete map of GH1 gene SNPs in our control normal population and to analyse its association with adult height. DESIGN, SUBJECTS AND MEASUREMENTS: A systematic GH1 gene analysis was designed in a control population of 307 adults of both sexes with height normally distributed within normal range for the same population: −2 standard deviation scores (SDS) to +2 SDS. An analysis was performed on individual and combined genotype associations with adult height. RESULTS: Twenty-five SNPs presented a frequency over 1%: 11 in the promoter (P1 to P11), three in the 5′UTR region (P12 to P14), one in exon 1 (P15), three in intron 1 (P16 to P18), two in intron 2 (P19 and P20), two in exon 4 (P21 and P22) and three in intron 4 (P23 to P25). Twenty-nine additional changes with frequencies under 1% were found in 29 subjects. P8, P19, P20 and P25 had not been previously described. P6, P12, P17 and P25 accounted for 6·2% of the variation in adult height (P = 0·0007) in this population with genotypes A/G at P6, G/G at P6 and A/G at P12 decreasing height SDS (−0·063 ± 0·031, −0·693 ± 0·350 and −0·489 ± 0·265, Mean ± SE) and genotypes A/T at P17 and T/G at P25 increasing height SDS (+1·094 ± 0·456 and +1·184 ± 0·432). CONCLUSIONS: This study established the GH1 gene sequence variation map in a normal adult height control population confirming the high density of SNPs in a relatively small gene. Our study shows that the more frequent SNPs did not significantly contribute to height determination, while only one promoter and two intronic SNPs contributed significantly to it. Studies in larger populations will have to confirm the associations and in vitro functional studies will elucidate the mechanisms involved. Systematic GH1 gene analysis in patients with growth delay and suspected GH deficiency/insufficiency will clarify whether different SNP frequencies and/or the presence of different sequence changes may be associated with phenotypes in them

Missense mutations identified in patients manifesting with non-classic StAR deficiency.

<p>*Mutation on partner allele is given in brackets (p.).</p><p>**Loss of function mutation manifesting clinically at birth with signs of classic StAR deficiency. Data given for comparison.</p><p>***<i>In vitro</i> activity (% of WT) is assessed by pregnenolone production (immunoassay) in COS cells transfected with expression vectors for wild-type or mutant StAR and F2 (the fusion protein P450 side-chain cleavage/adrenodoxin/adrenodoxin reductase). Note that data derive from different laboratories employing similar assay systems.</p

Exit of cholesterol from StAR as studied by steered MD simulations.

<p>StAR protein is shown as a ribbons model colored in a rainbow gradient from the amino terminus in blue to carboxy terminus in red. The A174–V179 loop that was observed to move and make way for the exit of cholesterol is shown in grey. Major amino acids involved in interaction of cholesterol with StAR and formation of cholesterol binding pocket are shown as stick models. The exit route of cholesterol observed during simulation is shown as a solid model in red. In case of S221-StAR a delay in exit of cholesterol was observed, potentially due to altered binding pattern caused by shift in H220 side chain and additional interactions with R188.</p

A closeup of the cholesterol binding pocket of StAR.

<p>After docking of cholesterol to both WT (A) and S221-StAR (B), we calculated potential residues interacting with cholesterol molecule during docking as well as exit of cholesterol from the binding cavity. In case of S221-StAR a loss of interaction with H220 was observed due to a shift in the H220 side chain. Cholesterol is shown as a ball and stick model in magenta. Amino acids interacting with cholesterol are shown as stick models.</p

Genetic analysis of the StAR gene.

<p>Upper panel, scheme of the identified mutations at the nucleotide (c.DNA) and protein (p.) level. Lower panel, family tree with individual electropherograms showing the novel StAR mutations.</p

Model of StAR protein showing reported, non-classic StAR mutations.

<p>Mutations manifesting clinically with NCLAH and having >10% WT StAR activity in a direct functional assay (V187M, R188C, R192C, G221S) are depicted in red. Mutations A218V, M225T, F267S, L275P, for which clinical and functional results are conflicting, are given in blue. StAR loss-of-function mutation L260P is given in black for comparison.</p

Functional testing of the identified StAR mutations <i>in vitro.</i>

<p>COS-1 cells were transiently transfected with expression vectors for the side chain cleavage system (F2) and wild-type (WT) or mutant StAR. A) Protein expression was assessed by Western blot. B) The ability to produce pregnenolone (Preg) was measured in the cell supernatants after 5–600 min using a commercially available ELISA assay. Results are given as mean ± SD. StAR independent Preg production of the cell system was assessed by using empty vector/F2 and adding 22(R)-hydroxycholesterol (22R OH-Chol) to the cell medium.</p

Secondary structural and amino acid conservation of human StAR.

<p>(A) A secondary structure prediction was performed to start the model building of human StAR. Locations of amino acids variants of StAR found in UniProt database and pubmed are indicated. (B) A partial sequencing alignment of human StAR amino acid sequence with a range of StAR proteins from other species found in the UniProt database. Overall, StAR amino acid sequence is very well conserved across species with only occasional difference showing up across whole alignment. All major residues at cholesterol binding pocket, E169, R188, R192 and T223 are conserved in all species studied in this analysis, while H220 was found to be replaced with an asparagine in Chicken and Zebrafish, potentially conserving the core structural elements.</p