Two novel CYP11B1 mutations in congenital adrenal hyperplasia due to steroid 11ß hydroxylase deficiency in a Tunisian family.

Steroid 11ß hydroxylase deficiency (11ß-OHD) (OMIM # 202010) is the second most common form of congenital adrenal hyperplasia (CAH), accounting for 5-8% of all cases. It is an autosomal recessive enzyme defect impairing the biosynthesis of cortisol. The CYP11B1 gene encoding this enzyme is located on chromosome 8q22, approximately 40kb from the highly homologous CYP11B2 gene encoding for the aldosterone synthase. Virilization and hypertension are the main clinical characteristics of this disease. In Tunisia, the incidence of 11ß-OHD appears higher due to a high rate of consanguinity (17.5% of congenital adrenal hyperplasia). The identical presentation of genital ambiguity (females) and pseudo-precocious puberty (males) can lead to misdiagnosis with 21 hydroxylase deficiency. The clinical hallmark of 11ß hydroxylase deficiency is variable, and biochemical identification of elevated precursor metabolites is not usually available. In order to clarify the underlying mechanism causing 11ß-OHD, we performed the molecular genetic analysis of the CYP11B1 gene in a female patient diagnosed as classical 11ß-OHD. The nucleotide sequence of the patient's CYP11B1 revealed two novel mutations in exon 4: a missense mutation that converts codon AGT (serine) to ATT (isoleucine) (c.650G>T; p.S217I) combined with an insertion of a thymine at the c.652-653 position (c.652_653insT). This insertion leads to a reading frame shift, multiple incorrect codons, and a premature stop in codon 258, that drastically affects normal protein function leading to a severe phenotype with ambiguous genitalia of congenital adrenal hyperplasia due to 11ß hydroxylase deficiency.

Correction: Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Functional characterization of three CYP21A2 sequence variants (p.A265V, p.W302S, p.D322G) employing a yeast co-expression system.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (CYP21A2) deficiency is the commonest inborn error in steroid hormone biosynthesis. Functional in vitro assessment of mutant activity generally correlates well with clinical phenotype and therefore has contributed greatly to phenotype prediction in this CAH variant. Three CYP21A2 sequence variants (g.1641C>T, p.A265V; g.1752G>C, p.W302S; and g.2012A>G, p.D322G) identified in patients with non-classic and simple virilizing CAH were characterized using a yeast co-expression system and a computational three-dimensional CYP21A2 model. Computational analysis of the mutants in the three-dimensional structural model predicted no relevant effect of p.A265V, while p.W302S and p.D322G were predicted to impact significantly on enzyme function. Consistent with these findings, in vitro mutant analysis revealed enzyme activity similar to wild-type for p.A265V, whereas p.W302S and p.D322G exerted activities compatible with simple virilizing and non-classical CAH, respectively. The results indicate that p.A265V is an allelic variant rather than a disease-causing amino acid change, whilst p.W302S and p.D322G could be confirmed as functionally relevant mutations. These findings emphasize the value of in vitro functional analysis of sequence variations in predicting genotype-phenotype correlations and disease severity.

Revealing a subclinical salt-losing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel.

OBJECTIVE: Pseudohypoaldosteronism type I (PHA1) is a rare inborn disease causing severe salt loss. Mutations in the three coding genes of the epithelial sodium channel (ENaC) are responsible for the systemic autosomal recessive form. So far, no phenotype has been reported in heterozygous carriers. PATIENTS: A consanguineous family from Somalia giving birth to a neonate suffering from PHA1 was studied including clinical and hormonal characteristics of the family, mutational analysis of the SCNN1A, SCNN1B, SCNN1G and CFTR genes and in vitro analysis of the functional consequences of a mutant ENaC channel. RESULTS: CFTR mutations have been excluded. SCNN1A gene analysis revealed a novel homozygous c.1684T > C mutation resulting in a S562P substitution in the alphaENaC protein of the patient. Functional analysis showed a significantly reduced S562P channel function compared to ENaC wild type. Protein synthesis and channel subunit assembly were not altered by the S562P mutation. Co-expression of mutant and wild-type channels revealed a dominant negative effect. In heterozygote carriers, sweat sodium and chloride concentrations were increased without additional hormonal or clinical phenotypes. CONCLUSION: Hence, the novel mutation S562P is causing systemic PHA1 in the homozygous state. A thorough clinical investigation of the heterozygote SCNN1A mutation carriers revealed increased sweat sodium and chloride levels consistent with a dominant effect of the mutant S562P allele. Whether this subclinical phenotype is of any consequence for the otherwise asymptomatic heterozygous carriers has to be elucidated.

Clinical and molecular features of type 1 pseudohypoaldosteronism.

Pseudohypoaldosteronism (PHA) is a rare heterogeneous syndrome of mineralocorticoid resistance causing insufficient potassium and hydrogen secretion. PHA type 1 (PHA1) causes neonatal salt loss, failure to thrive, dehydration and circulatory shock. Two different forms of PHA1 can be distinguished on the clinical and genetic level, showing either a systemic or a renal form of mineralocorticoid resistance. This review provides an overview on transepithelial sodium reabsorption and on clinical features and the underlying molecular pathology of systemic and renal PHA1 caused by mutations in the subunit genes (SCNN1A, SCNN1B, SCNN1G) of the epithelial sodium channel (ENaC) and the mineralocorticoid receptor coding gene NR3C2. The in vitro investigation of several mutants has resulted in important progress in the understanding of the physiology of ENaC and the mineralocorticoid receptor. Some mutations are discussed in more detail to demonstrate some of these findings. A better clinical work-up of the patients suffering from PHA1 may delineate additional associations between the genotype and phenotype in the future.

Congenital adrenal hyperplasia due to 11-hydroxylase deficiency--insights from two novel CYP11B1 mutations (p.M92X, p.R453Q).

BACKGROUND: Steroid 11-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Herein, we describe two novel CYP11B1 mutations (g659_660dupTG, p.M92X; g.4817G>A, p.R453Q) found in a patient diagnosed with classic 11OHD, after presenting with borderline elevated 17-hydroxyprogesterone concentrations in CAH newborn screening. METHODS: A novel CYP11B1 variant (p.R453Q) identified in a patient with classic 11OHD was characterized employing a COS7 cell assay and a computational three-dimensional CYP11B1 model. RESULTS: The in vitro expression analysis revealed an almost complete loss of function of p.R453Q. This finding was consistent with the clinical presentation of classic 11OHD as the patient was compound heterozygous for p.R453Q and a nonsense mutation (p.M92X) on the other allele. Inserting the p.R453Q mutation into our CYP11B1 model provided two potential explanations for the almost complete loss of enzyme activity. Firstly, the heme coordination is most likely disturbed. A second possibility could be an altered interaction with the redox partner adrenodoxin. CONCLUSION: Results indicate that both novel mutations are disease-causing mutations. Proving the pathogenic effect of a missense sequence variation is of particular importance for clinical genetic counseling as this provides essential information on the prediction of recurrence risk and disease severity.

Gonadotropin- and Adrenocorticotropic Hormone-Independent Precocious Puberty of Gonadal Origin in a Patient with Adrenal Hypoplasia Congenita Due to DAX1 Gene Mutation - A Case Report and Review of the Literature: Implications for the Pathomechanism.

BACKGROUND/AIMS: Mutations in the DAX1 gene cause X-linked adrenal hypoplasia congenita (AHC) classically associated with hypogonadotropic hypogonadism. Unexpectedly, precocious puberty (PP) has been reported in some cases, its mechanism remaining unclear. METHODS: We longitudinally studied a boy with AHC due to DAX1 gene mutation who developed peripheral PP at age 4.5 years. Initially he presented pubic hair, penile enlargement, advanced bone age and elevated testosterone levels. PP progressed with acne, body odour and ejaculations. In addition, we summarized reported findings of patients with DAX1 mutations and PP in the literature in a structured manner providing a basis to discuss possible pathomechanisms of PP in DAX1 patients. RESULTS: In our patient, hydrocortisone treatment was increased to 20 mg/m2/day as suggested in similar published cases. However, despite the suppression of adrenocorticotropic hormone (ACTH), this remained without clinical effect or change in laboratory results. The progression of symptoms of pubertal development was well suppressed under cyproterone acetate treatment. Twenty-four-hour steroid urine excretion rate measurements excluded an effect of adrenal androgens and showed a prepubertal rise of excreted testosterone. Testes size remained small. GnRH testing showed peripheral PP. CONCLUSION: We hypothesize that an intrinsic, gonadotropin- and ACTH-independent activation of steroidogenesis in the DAX1 deficient testes leads to PP in AHC patients with DAX1 mutations.

Functional and structural consequences of a novel point mutation in the CYP21A2 gene causing congenital adrenal hyperplasia: potential relevance of helix C for P450 oxidoreductase-21-hydroxylase interaction.

BACKGROUND: Congenital adrenal hyperplasia is caused by insufficient adrenal steroid biosynthesis due to impaired steroidogenic enzymes. The majority of patients suffer from deficiency of 21-hydroxylase (CYP21) coded by the CYP21A2 gene. OBJECTIVE: Our objective was to study the functional and structural consequences of the novel CYP21A2 missense mutation c.364A > C (K121Q) detected in a female patient with nonclassical 21-hydroxylase deficiency. The patient was compound heterozygous for the novel K121Q mutation and the mild P453S mutation. RESULTS: In vitro expression analysis of the mutant K121Q enzyme in transiently transfected COS-7 cells revealed reduced CYP21 activity of 14.0 +/- 5% for the conversion of 17-hydroxyprogesterone and 19.5 +/- 4% for the conversion of progesterone. K121 is located on helix C in the CYP21 protein, which is part of the heme coordinating system. In addition, helix C is involved in the interaction with the electron-providing enzyme P450 oxidoreductase. Protein modeling revealed that the substitution of glutamine for the basic amino acid lysine introduces an electrostatic change on the surface of CYP21 and may additionally change heme coordination. We hypothesize that the electron flux between P450 oxidoreductase and CYP21 is impaired and, moreover, that substrate affinity is altered due to heme dislocation with K121Q. CONCLUSION: Both the interaction of P450 oxidoreductase and CYP21 as well as heme coordination are likely to be disturbed due to the K121Q mutation. Our data exemplify how the combination of in vitro expression and structural protein analysis provide novel insights into molecular mechanisms of reduced CYP21 activity, eventually explaining the patient's phenotype.

Carboxyl-terminal mutations in 3beta-hydroxysteroid dehydrogenase type II cause severe salt-wasting congenital adrenal hyperplasia.

INTRODUCTION: 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) deficiency is a rare cause of congenital adrenal hyperplasia caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. The function of the C terminus of the 3beta-HSD protein is not known. OBJECTIVE: We studied the functional consequences of three novel C-terminal mutations in the 3beta-HSD protein (p.P341L, p.R335X and p.W355X), detected in unrelated 46,XY neonates with classical 3beta-HSD type II deficiency showing different degrees of under-virilization. METHODS AND RESULTS: In vitro expression of the two truncated mutant proteins yielded absent conversion of pregnenolone and dehydroepiandrosterone (DHEA), whereas the missense mutation p.P341L showed a residual DHEA conversion of 6% of wild-type activity. Additional analysis of p.P341L, including three-dimensional protein modeling, revealed that the mutant's inactivity predominantly originates from a putative structural alteration of the 3beta-HSD protein and is further aggravated by increased protein degradation. The stop mutations cause truncated proteins missing the final G-helix that abolishes enzymatic activity irrespective of an augmented protein degradation. Genital appearance did not correlate with the mutants' residual in vitro activity. CONCLUSIONS: Three novel C-terminal mutants of the HSD3B2 gene are responsible for classical 3beta-HSD deficiency. The C terminus is essential for the enzymatic activity. However, more studies are needed to clarify the exact function of this part of the protein. Our results indicate that the genital phenotype in 3beta-HSD deficiency cannot be predicted from in vitro 3beta-HSD function alone.

Functional characterization of naturally occurring NR3C2 gene mutations in Italian patients suffering from pseudohypoaldosteronism type 1.

OBJECTIVE: The renal form of pseudohypoaldosteronism type 1 (PHA1) is a rare disease caused by mutations in the human mineralocorticoid receptor gene (NR3C2). DESIGN: Aim of the study was to analyze the NR3C2 gene in three Italian patients with clinical signs of renal PHA1 and to evaluate the distribution of the -2G > C, c.538A > G, and c.722C > T single nucleotide polymorphism (SNP) pattern in the PHA1 patients and in 90 controls of the same ethnic origin. METHODS: Analysis of the NR3C2 gene sequence and of the polymorphic SNP markers. Functional characterization of the detected novel NR3C2 mutations utilizing aldosterone-binding assays and reporter gene transactivation assays. RESULTS: One novel nonsense (Y134X) and one novel frameshift (2125delA) mutation were detected. They exhibited no aldosterone binding and no transactivation abilities. No mutation was detected in the third patient. Haploinsufficiency of NR3C2 was ruled out by microsatellite analysis in this patient. The c.722T SNP was detected in 97% of alleles in the Italian population which is significantly different from the general German or US population. CONCLUSIONS: Molecular analysis of the NR3C2 gene in PHA1 patients is warranted to detect novel mutations in order to clarify the underlying genetic cause, which may extend the insight into relevant functional regions of the hMR protein. The effect the different distribution of the c.722T SNP is not clear to date. Further studies are necessary to provide evidence as to a possible advantage of a less sensitive hMR in southern countries.

Exclusion of serum- and glucocorticoid-induced kinase 1 (SGK1) as a candidate gene for genetically heterogeneous renal pseudohypoaldosteronism type I in eight families.

OBJECTIVE: Autosomal-dominant pseudohypoaldosteronism type 1 (PHA1) is mostly caused by mutations in the mineralocorticoid receptor (NR3C2) gene. However, several kindreds with clinical signs of PHA1 but without NR3C2 gene mutations or deletions are reported. Serum- and glucocorticoid-induced kinase 1 (Sgk1) is involved in epithelial sodium reabsorption by facilitating the accumulation of the epithelial sodium channel in the plasma membrane. Therefore variations in the SGK1 gene may aggravate renal salt loss or cause PHA1. METHODS: The SGK1 genewas sequenced in 10 patients with the typical clinical signs of PHA1 but without NR3C2 or SCNN1A, SCNN1B and SCNN1C gene mutation. RESULTS: No disease-causing SGK1 gene mutation was detected in the studied PHA1 patient group. Two novel intronic SNPs which were also present in the normal population were detected in 2 patients. CONCLUSION: Our data do not support that SGK1 gene variations are disease-causing factors in genetically unexplained PHA1. Therefore, systematic investigation of other factors downstream of the MR involved in epithelial sodium reabsorption is warranted in patients with autosomal-dominant PHA1.

Recurrence of the R947X mutation in unrelated families with autosomal dominant pseudohypoaldosteronism type 1: evidence for a mutational hot spot in the mineralocorticoid receptor gene.

BACKGROUND: The renal form of pseudohypoaldosteronism type 1 (PHA1) is a rare disease characterized by congenital mineralocorticoid resistance of the kidney. Twenty-two different loss-of-function mutations in the mineralocorticoid receptor gene have been described in families with PHA1. These mutations were not recurrent and resulted in a large phenotypic variability. OBJECTIVE: The objective of this study is to analyze the recurrence of an inactivating mutation in the mineralocorticoid receptor gene in unrelated families with autosomal dominant PHA1. PATIENTS: Seventeen members from three unrelated families with autosomal dominant PHA1 were studied, including 11 affected patients with variable clinical manifestations. Fifty healthy subjects were used as controls. METHODS: Genomic DNA was extracted, and the entire coding region of the mineralocorticoid receptor gene was submitted to automatic sequencing. Four dinucleotide microsatellite markers spanning a region of 3.2 cM in the human mineralocorticoid receptor gene locus, and two intragenic polymorphisms were used for haplotype analysis. RESULTS: A heterozygous point mutation at codon 947 (c.2839C>T) changing arginine to stop codon (R947X) was found in the three families. Different haplotypes segregated with the R947X mutation in each family, demonstrating the absence of a founder effect for this mutation. CONCLUSION: Codon 947 of the mineralocorticoid receptor is the first mutational hot spot for autosomal dominant PHA1.

Four novel missense mutations in the CYP21A2 gene detected in Russian patients suffering from the classical form of congenital adrenal hyperplasia: identification, functional characterization, and structural analysis.

CONTEXT: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The most frequent cause is the deficiency of steroid 21-hydroxylase (CYP21) due to mutations in the CYP21A2 gene. OBJECTIVE: We analyzed the functional and structural consequences of the four CYP21A2 missense mutations (C169R, G178R, W302R, and R426C) to prove their clinical relevance and study their impact on CYP21 function. RESULTS: Analyzing the mutations in vitro revealed an almost absent or negligible CYP21 activity for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. Protein translation and intracellular localization were not affected by the mutants, as could be demonstrated by Western blotting and immunofluorescence studies. Analysis of these mutants in a three-dimensional model structure of the CYP21 protein explained the observed in vitro effects because all the mutations severely interfere either directly or indirectly with important structures of the 21-hydroxylase protein. CONCLUSION: The in vitro expression analysis of residual enzyme function is a complementary method to genotyping and an important tool for improving the understanding of the clinical phenotype of 21-hydroxylase deficiency. This forms the foundation for accurate clinical and genetic counseling and for prenatal diagnosis and treatment. Moreover, this report demonstrates that the combination of in vitro enzyme analysis and molecular modeling can yield novel insights into CYP450 structure-functional relationships.

Elucidating the underlying molecular pathogenesis of NR3C2 mutants causing autosomal dominant pseudohypoaldosteronism type 1.

CONTEXT: Pseudohypoaldosteronism type 1 (PHA1) is a rare salt-wasting syndrome. Mutations in the NR3C2 gene coding for the mineralocorticoid receptor (MR) cause autosomal dominant PHA1. OBJECTIVE: Our objective was to reveal the cause of renal salt loss in six PHA1 patients and analyze the mutants' functional impact on MR function. DESIGN: Our study included the following: clinical and hormonal characterization of the patients' phenotype, analysis of the NR3C2 gene, determination of receptor affinities to aldosterone and the transcriptional activation abilities of the MR mutants, investigation of subcellular translocation using fluorescence-labeled MR, and studying changes in mutant receptor conformation with proteolysis experiments and three-dimensional modeling. RESULTS: Six heterozygous NR3C2 mutations were detected. One frameshift mutation (c.1131dupT) has been reported previously. The second frameshift mutation (c.2871dupC), which has only recently been reported by our group, showed no aldosterone binding and no transactivation because of a major change in receptor conformation. Two novel nonsense mutations generate a truncated receptor protein. Two missense mutations differently affect MR function. S818L was reported recently without complete in vitro data. S818L does not bind aldosterone or activate transcription or translocate into the nucleus. A major displacement of several residues involved in aldosterone binding was PHA1 causing. The novel E972G mutation showed a significantly lower ligand-binding affinity and only 9% of wild-type transcriptional activity caused by major changes in receptor conformation. CONCLUSIONS: Our data on six mutations extend the spectrum of PHA1-causing NR3C2 gene mutations. Studying naturally occurring mutants helps to clarify their pathogenicity and to identify crucial residues for MR structure and function.

Analyzing the functional and structural consequences of two point mutations (P94L and A368D) in the CYP11B1 gene causing congenital adrenal hyperplasia resulting from 11-hydroxylase deficiency.

CONTEXT: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The deficiency of steroid 11-hydroxylase (CYP11B1) resulting from mutations in the CYP11B1 gene is the second most frequent cause. OBJECTIVE: We studied the functional and structural consequences of two CYP11B1 missense mutations, which were detected in a 1.8-yr-old boy with acne and precocious pseudopuberty, to prove their clinical relevance and study their impact on CYP11B1 function. RESULTS: The in vitro expression studies in COS-7 cells revealed an almost complete absence of CYP11B1 activity for the P94L mutant to 0.05% for the conversion of 11-deoxycortisol to cortisol. The A368D mutant severely reduced the CYP11B1 enzymatic activity to 1.17%. Intracellular localization studies by immunofluorescence revealed that the mutants were correctly localized. Introducing these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the effects measured in vitro. We hypothesize that the A368D mutation interferes with structures important for substrate specificity and heme iron binding, thus explaining its major functional impact. However, according to structural analysis, we would expect only a minor effect of the P94L mutant on 11-hydroxylase activity, which contrasts with the observed major effect of this mutation both in vitro and in vivo. CONCLUSION: Analyzing the in vitro enzyme function is a complementary procedure to genotyping and a valuable tool for understanding the clinical phenotype of 11-hydroxylase deficiency. This is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides valuable insights in cytochrome P450 structural-functional relationships, although one must be aware of the limitations of in silico-based methods.

The residue E351 is essential for the activity of human 21-hydroxylase: evidence from a naturally occurring novel point mutation compared with artificial mutants generated by single amino acid substitutions.

Congenital adrenal hyperplasia (CAH) [OMIM 201910] is a group of autosomal recessive disorders, caused in 90-95% of cases by a deficiency of steroid 21-hydroxylase due to mutations in the CYP21A2 gene. The functional and structural effects of a novel rare missense mutation (E351K) in CYP21A2 found in a male patient with simple virilizing CAH were studied. The novel E351K point mutation is located in the ERR triad of the 21-hydroxylase. The ERR triad is a glutamine-arginine-arginine motif conserved in all cytochrome P450 sequences. The glutamate and first arginine residue are invariant in all P450 cytochrome enzymes, whereas the second arginine residue is present as arginine, histidine, or asparagine. Although the ERR triad is involved in some way to heme binding by the cytochrome P450 monooxygenases, the E351K mutation leads to severe but not complete loss of CYP21 enzyme activity. The functional analysis in COS-7 cells revealed a reduced conversion of 17-hydroxyprogesterone to 11-deoxycortisol of 1.1+/-0.5% (SD) and of progesterone to 11-deoxycorticosterone of 1.2+/-0.3% of wild-type activity. Analyzing the artificial mutants (E351D, E351I) of the E351 residue did not show a restoration of the in vitro 21-hydroxylase activity. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. The combination of in vitro enzyme function and computerized protein analysis of the E351 residue of the CYP21 protein provides experimental evidence for the ERR triad being a fundamental structural element of cytochrome P450 enzymes.

Congenital adrenal hyperplasia: the molecular basis of 21-hydroxylase deficiency in H-2(aw18) mice.

The mouse strain H-2(aw18) shows typical characteristics of 21-hydroxylase deficiency (21-OHD). A deletion of the active Cyp21a1 gene has been postulated; however, the changes on the nucleotide level are still unknown. To investigate whether this animal model, the only one available, is suitable for studying congenital adrenal hyperplasia in man, a detailed analysis of the Cyp21 locus has been performed to ascertain the genetic cause of 21-OHD in H-2(aw18) mice. We demonstrate that 21-OHD is caused by unequal crossing over between the active Cyp21a1 gene and the pseudogene resulting in a hybrid Cyp21a1-Cyp21a2-p gene including a partial deletion of Cyp21a1. Next to several pseudogene-specific point mutations, various novel missense mutations and a nonsense mutation are present. Enzyme activity for each point mutation has been determined in vitro and the structure-function relationship has been studied by sequence conservation analysis and a three-dimensional murine 21-hydroxylase protein (Cyp21) structure model. The mutations are classified in three classes: I, no or minor decrease in enzyme activity: R238Q, P465L, R361K, A362V, P458L; II, loss of enzyme activity caused by inefficient electron flux: R346H, R400C; III, loss of activity due to deficient substrate binding: I462F, L464F. The combination of in vitro protein expression and three-dimensional structure modeling provides a valuable tool to understand the role of the different mutations and polymorphisms on the resulting enzyme activity. The underlying genetic mechanisms are also known to be responsible for 21-OHD in humans, so rodent 21-OHD turns out to be an excellent genetic model for studying the human disease.

Functional characterization of two novel point mutations in the CYP21 gene causing simple virilizing forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders most often caused by deficiency of steroid 21-hydroxylase due to mutations in the CYP21 gene. We studied the functional and structural consequences of two novel missense mutations in the CYP21 gene, detected in two simple virilizing congenital adrenal hyperplasia patients. Both the male and female patient were compound heterozygous for the novel I77T and A434V point mutations, respectively. The in vitro expression analysis in COS-7 cells revealed a reduced 21-hydroxylase activity in the I77T mutant of 3 +/- 2% (sd) for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and of 5 +/- 3% for the conversion of progesterone to 11-deoxycorticosterone. The A434V mutant had a residual enzyme activity of 14 +/- 2% for 17-hydroxyprogesterone and 12 +/- 6% for progesterone. Substrate affinity was similar in the mutants as in the CYP21 wild-type protein, whereas reaction velocity was markedly decreased in both mutants. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. We hypothesize that the I77T mutation markedly decreases the reaction product release and/or substrate entrance to the enzyme's active site, whereas the A434V mutant reduces both the catalytic capacity and reaction velocity. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 21-hydroxylase deficiency and also provides new insights into cytochrome P450 structure-function relationships.

Congenital adrenal hyperplasia due to 11-hydroxylase deficiency: functional characterization of two novel point mutations and a three-base pair deletion in the CYP11B1 gene.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders second most often caused by deficiency of steroid 11-hydroxylase (CYP11B1) due to mutations in the CYP11B1 gene. We studied the functional and structural consequences of two novel missense mutations (W116C, L299P) and an in-frame 3-bp deletion (DeltaF438) in the CYP11B gene, detected in three unrelated families. All patients are suffering from classical CYP11B1 deficiency. In vitro expression studies in COS-7 cells revealed a decreased CYP11B1 activity in the W116C mutant to 2.9 +/- 0.9% (sd) for the conversion of 11-deoxycortisol to cortisol. The L299P mutant reduced the enzymatic activity to 1.2 +/- 0.9%, whereas the DeltaF438 mutation resulted in no measurable residual CYP11B1 activity. Introduction of these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the in vitro measured effects. We hypothesize that the W116C mutation influences the conformational change of the 11-hydroxylase protein necessary for substrate access and product release. The L299P mutation causes a change in the position of the I helix relative to the heme group, whereas the DeltaF438 mutation results in a steric disarrangement of the heme group relative to the enzyme. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 11-hydroxylase deficiency, which is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides new insights in cytochrome P450 structural-functional relationships.

Homozygous disruption of P450 side-chain cleavage (CYP11A1) is associated with prematurity, complete 46,XY sex reversal, and severe adrenal failure.

Disruption of the P450 side-chain cleavage cytochrome (P450scc) enzyme due to deleterious mutations of the CYP11A1 gene is thought to be incompatible with fetal survival because of impaired progesterone production by the fetoplacental unit. We present a 46,XY patient with a homozygous disruption of CYP11A1. The child was born prematurely with complete sex reversal and severe adrenal insufficiency. Laboratory data showed diminished or absent steroidogenesis in all pathways. Molecular genetic analysis of the CYP11A1 gene revealed a homozygous single nucleotide deletion leading to a premature termination at codon position 288. This mutation will delete highly conserved regions of the P450scc enzyme and thus is predicted to lead to a nonfunctional protein. Both healthy parents were heterozygous for this mutation. Our report demonstrates that severe disruption of P450scc can be compatible with survival in rare instances. Furthermore, defects in this enzyme are inherited in an autosomal-recessive fashion, and heterozygote carriers can be healthy and fertile. The possibility of P450scc-independent pathways of steroid synthesis in addition to the current concept of luteoplacental shift of progesterone synthesis in humans has to be questioned.