Steroid 11β-hydroxylase Deficiency Research Articles

Molecular analysis of 12 Chinese patients with 11β-hydroxylase deficiency and in vitro functional study of 20 CYP11B1 missense variants.

Steroid 11β-hydroxylase deficiency (11β-OHD) is a rare autosomal recessive disorder caused by pathogenic variants of CYP11B1 gene. This study aimed to perform molecular analysis of a Chinese 11β-OHD series and in vitro functional study of twenty CYP11B1 missense variants. Twelve Chinese patients with clinical diagnosis of 11β-OHD were included in the study to analyze their molecular etiology. Genomic DNA of patients was extracted to be sequenced all coding exons and intronic flanking sequences of CYP11B1. Fourteen missense variants found in 12 patients mentioned above along with 6 missense variants previously reported by our team were evaluated functionally. Amino acid substitutions were analyzed with computational program to determine their effects on the three-dimensional structure of CYP11B1 protein. Clinical characteristics and hormone levels at baseline of the 18 patients carrying 18 missense variants aforementioned were recorded to perform genotype-phenotype correlation. A total of 21 rare variants including 9 novel and 12 recurrent ones were identified in 12 patients, out of which 17 were missense, 2 were nonsense, 1 was a splice site variant, and 1 was a deletion-insertion variant. Results of in vitro functional study revealed that 3 out of 20 missense mutants (p.Leu3Pro, p.Gly267Ser, and p.Ala367Ser) had partial enzyme activity and the other 17 had little enzymatic activity. The impairment degree of enzymatic activity in vitro functional study was also reflected in the severity degree of interaction change between the wild-type/mutant-type amino acid and its adjacent amino acids in three-dimensional model. In conclusion, the addition of 9 novel variants expands the spectrum of CYP11B1 pathogenic variants. Our results demonstrate that twenty CYP11B1 variants lead to impaired 11β-hydroxylase activity in vitro. Visualizing these variants in the three-dimensional model structure of CYP11B1 protein can provide a plausible explanation for the results measured in vitro.

Compound heterozygosity of a novel Q73X mutation and a known R141X mutation in CYP11B1 resulting in 11β-hydroxylase deficiency in a Chinese boy with congenital adrenal hyperplasia

Steroid 11β-hydroxylase deficiency (11β−OHD), which is caused by mutations of the CYP11B1 gene, is the second leading cause of congenital adrenal hyperplasia (CAH), an autosomal recessive inherited disorder. Here, we report a case of classic 11β−OHD in a Chinese boy characterized by hypertension, penile enlargement, skin pigmentation, and acne. Molecular analysis of CYP11B1 revealed that the patient was compound heterozygous for a c.217C > T (p.Q73X) mutation in exon 1 and a c.421C > T (p.R141X) mutation in exon 3. His parents carried the novel c.217C > T (p.Q73X) mutation and the prevalent c.421C > T (p.R141X) mutation. Furthermore, we identified a novel 217-bp substitution mutation (Q73X) in CYP11B1 that generates a truncated protein without biological activity, which is likely to be pathogenic. Pursuant to the phenotype of the proband and his family, the Q73X mutation is inferred to exacerbate the disease burden of the R141X mutation, a known pathogenic variant. To further explore this possibility, selecting the x-ray structure of human CYP11B2 as a template, we built three-dimensional homologous models of the normal and mutant proteins. In the mutant model, a change from a helix to terminal structure in amino acids 73 and 141 occurred that affected the binding capacity of CYP11B1 with heme and impaired 11β-hydroxylase activity. Taken together, our findings expand the spectrum of known mutations leading to 11β−OHD and provide evidence to study genotype-phenotype concordance, confirm early diagnosis and treatment of 11β−OHD, and prevent most complications.

SUN-389 Consequences of Late Diagnosis of 11β-Hydroxylase Deficiency

Background: Steroid 11β-hydroxylase (CYP11B1) deficiency (11βOHD) is the second most common form of congenital adrenal hyperplasia (CAH). CAH is more likely to be missed in boys, particularly in patients with 11βOHD. Here, we report two boys with 11βOHD who presented late and the consequences of late diagnosis in them. Case presentation: Case 1: A-4-year-old boy presented with acne, appearance of axillary and pubic hair since the age of 3 years. Examination revealed tall stature (height z score: 4.6; predicted adult height <143 cm), hypertension (BP = 130/80 mmHg), P4, G3 (stretched phallic length: 8.5 cm) and nodular testes measuring 2 cc on right and 3 cc on left. His bone age was 13 years and had mild hypokalemia (serum potassium: 3.3 mEq/L). Serum total testosterone was elevated (263.13 ng/dl) with suppressed LH (0.04 mIU/ml). Serum 8:00 am cortisol was low (0.71 µg/dl) with markedly elevated corticosterone levels (>5000 ng/dl) and deoxycortisol (>1000 ng/dl). 17OHP was slightly elevated (334.73 ng/dl). Ultrasound scrotum revealed the presence of testicular adrenal rests (TARTs) in both testes. He had grade 1 hypertensive retinal changes and concentric left ventricular hypertrophy. Case 2: A-12-year-old presented with hypertensive encephalopathy. On examination he had normal stature for age (height: 138 cm; ≈predicted adult height =short), P4, G4 (stretched phallic length: 9 cm) and nodular testes measuring 4 cc on right and 6 cc on left. His bone age was 18 years and serum potassium was 2.67 mEq/L. Serum total testosterone was 229.77 ng/dl but LH was suppressed (0.03 mIU/ml). Serum 8:00 am cortisol level was low (0.39 µg/dl) with markedly elevated corticosterone levels (>5000 ng/dl) and deoxycortisol (>2000 ng/dl). 17OHP was slightly elevated (239.3 ng/dl). Ultrasound scrotum revealed bilateral TARTs. He had grade 1 hypertensive retinal changes and mild concentric left ventricular hypertrophy. Conclusion: Late diagnosis is common in boys with classic 11βOHD. It leads to disastrous complications such as hypertensive encephalopathy and/or hypertensive end organ damage, significantly compromises adult height potential and may have adverse impact on future fertility. Hence, early diagnosis of 11βOHD is essential.

Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1, a gene encoding 11β-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β-hydroxylase deficiency CAH.

Phenotypic, metabolic, and molecular genetic characterization of six patients with congenital adrenal hyperplasia caused by novel mutations in the CYP11B1 gene

Congenital adrenal hyperplasia (CAH) is an autosomal recessive inherited disorder of steroidogenesis. Steroid 11β-hydroxylase deficiency (11β-OHD) due to mutations in the CYP11B1 gene is the second most common form of CAH.In this study, 6 patients suffering from CAH were diagnosed with 11β-OHD using urinary GC–MS steroid metabolomics analysis. The molecular basis of the disorder was investigated by molecular genetic analysis of the CYP11B1 gene, functional characterization of splicing and missense mutations, and analysis of the missense mutations in a computer model of CYP11B1.All patients presented with abnormal clinical signs of hyperandrogenism. Their urinary steroid metabolomes were characterized by excessive excretion rates of metabolites of 11-deoxycortisol as well as metabolites of 11-deoxycorticosterone, and allowed definite diagnosis. Patient 1 carries compound heterozygous mutations consisting of a novel nonsense mutation p.Q102X (c.304C>T) in exon 2 and the known missense mutation p.T318R (c.953C>G) in exon 5. Two siblings (patient 2 and 3) were compound heterozygous carriers of a known splicing mutation c.1200+1G>A in intron 7 and a known missense mutation p.R448H (c.1343G>A) in exon 8. Minigene experiments demonstrated that the c.1200+1G>A mutation caused abnormal pre-mRNA splicing (intron retention). Two further siblings (patient 4 and 5) were compound heterozygous carriers of a novel missense mutation p.R332G (c.994C>G) in exon 6 and the known missense mutation p.R448H (c.1343G>A) in exon 8. A CYP11B1 activity study in COS-1 cells showed that only 11% of the enzyme activity remained in the variant p.R332G. Patient 6 carried a so far not described homozygous deletion g.2470_5320del of 2850bp corresponding to a loss of the CYP11B1 exons 3–8. The breakpoints of the deletion are embedded into two typical 6 base pair repeats (GCTTCT) upstream and downstream of the gene.Experiments analyzing the influence of mutations on splicing and on enzyme function were applied as complementary procedures to genotyping and provided a rational basis for understanding the clinical phenotype of CAH.

Characterization of the molecular genetic pathology in patients with 11β‐hydroxylase deficiency

Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia. Nonclassic or mild 11OHD appears to be a rare condition. Our study assessed the residual CYP11B1 function of detected mutations, adding to the spectrum of mild 11OHD, and illustrates the variability of the clinical presentation of 11OHD. Five patients presented with mild to moderate 11OHD. Two women presented with mild hirsutism and in one case with secondary amenorrhoea. Two men presented with precocious pseudopuberty, gynaecomastia and elevated blood pressure. One 46,XX female patient was diagnosed with virilization of the external genitalia 2 years after birth. Direct DNA sequencing was carried out to perform CYP11B1 mutation analysis. The CYP11B1 mutations were functionally characterized using an in vitro expression system. CYP11B1-inactivating mutations were detected in all patients. Two novel missense mutations (p.P42L and p.A297V) and the previously characterized p.R143W mutation had residual CYP11B1 activities between 10% and 27%. A novel p.L382R and the previously uncharacterized p.G444D mutation both caused complete loss of CYP11B1 enzymatic activity. Mutations causing partial impairment of 11β-hydroxylase activity (residual activity of 10% or above) are associated with a less severe clinical presentation of 11OHD, which can be classified as a nonclassic form. Our data demonstrate that patients with nonclassic 11OHD can present with androgen excess, precocious pseudopuberty and increased blood pressure. Timely diagnosis of nonclassic 11OHD and consequently initiation of personalized treatment is essential to prevent co-morbidities caused by androgen excess and hypertension.

Phenotype of patients with congenital adrenal hyperplasia due to 11β-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is one of the most common metabolic diseases. It is caused by a severe or partial impairment of adrenal steroidogenesis affecting cortisol biosynthesis. Approximately 5–8% of all cases are due to steroid 11β-hydroxylase deficiency (11OHD; OMIM +202010), which occurs in approximately 1:100,000 to 1:200,000 live births in non consanguineous populations. Mutations in the CYP11B1 gene, causing 11b-hydroxylase deficiency in the zona fasciculate in the adrenal cortex, have been identified. Our aim is to describe clinical and biochemical features in patients with CAH due to 11β-hydroxylase deficiency. The case series report included 9 patients (6 male and 3 female) from 7 unrelated families who was identified novel and/or reported homozygous or compound heterozygous mutations in CYP11B1 gene. Diagnosed age was from 2 to 11 years old. All three female cases presented with ambiguous genitalia at birth. Other clinical features were hypertension (6/7 cases); hyperpigmentation (5/7 cases); pseudo-precocious puberty (male) (5/5 cases). Hypokalemia was noted in 3/7 cases. Three cases need antihypertensive drug associated with hydrocortisone replacement therapy. In conclusions, the clinical hallmark of 11β hydroxylase deficiency is variable and virilization and hypertension are the prominent clinical features of 11b hydroxylase deficiency. Biochemical identification of elevated precursor metabolites is not usually available and mutation analysis of CYP11B1 will held confirmation of diagnosis.

Identification of seven novel CYP11B1 gene mutations in Chinese patients with 11β-hydroxylase deficiency

Steroid 11β-hydroxylase deficiency (11β-OHD), one of common cause of congenital adrenal hyperplasia (CAH), is an autosomal recessive disorder characterized by virilization, precocious pseudo-puberty, and hypertension. It is caused by CYP11B1 gene mutation. We performed molecular genetic analysis of the CYP11B1 gene in six patients with preliminary clinical diagnosis of 11β-OHD and four patients identified as potential 11β-OHD from a CAH cohort in which CYP21A2 gene mutations consecutively screened. Seven novel CYP11B1 mutations, including p.R454H, p.Q472P, p.Q155X, p.K173X, IVS2-1G>A, R454A fs 573X, and g.2704_g.3154del, and six previously described mutations (p.P94L, p.G267S, p.G379V, p.R448H, p.R454C and p.R141X) were identified. These mutations mainly clustered in exons 3 and 8. Eight of twenty alleles carried mutations occurring at the Arg454 position, which is a mutational hot spot for Han Chinese. The pathogenic nature of novel p.R454H mutation was predicted by protein sequence alignment and in silico analysis. All the identified mutations were responsible for the clinical features observed in these ten unrelated Chinese patients. This study expands the CYP11B1 mutation spectrum and provides evidence for prenatal diagnosis and genetic counseling. Genetic analysis is an alternative approach to help clinicians confirm uncertain 11β-OHD diagnosis, facilitating reasonable steroid replacement.

A Novel Mutation in theCYP11B1Gene Causes Steroid 11β-Hydroxylase Deficient Congenital Adrenal Hyperplasia with Reversible Cardiomyopathy

Congenital adrenal hyperplasia (CAH) due to steroid 11β-hydroxylase deficiency is the second most common form of CAH, resulting from a mutation in the CYP11B1 gene. Steroid 11β-hydroxylase deficiency results in excessive mineralcorticoids and androgen production leading to hypertension, precocious puberty with acne, enlarged penis, and hyperpigmentation of scrotum of genetically male infants. In the present study, we reported 3 male cases from a Saudi family who presented with penile enlargement, progressive darkness of skin, hypertension, and cardiomyopathy. The elder patient died due to heart failure and his younger brothers were treated with hydrocortisone and antihypertensive medications. Six months following treatment, cardiomyopathy disappeared with normal blood pressure and improvement in the skin pigmentation. The underlying molecular defect was investigated by PCR-sequencing analysis of all coding exons and intron-exon boundary of the CYP11B1 gene. A novel biallelic mutation c.780 G>A in exon 4 of the CYP11B1 gene was found in the patients. The mutation created a premature stop codon at amino acid 260 (p.W260∗), resulting in a truncated protein devoid of 11β-hydroxylase activity. Interestingly, a somatic mutation at the same codon (c.779 G>A, p.W260∗) was reported in a patient with papillary thyroid cancer (COSMIC database). In conclusion, we have identified a novel nonsense mutation in the CYP11B1 gene that causes classic steroid 11β-hydroxylase deficient CAH. Cardiomyopathy and cardiac failure can be reversed by early diagnosis and treatment.

Characterisation of three novel CYP11B1 mutations in classic and non-classic 11β-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine diseases. Steroid 11β-hydroxylase (P450c11) deficiency (11OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel CYP11B1 gene mutations (p.His125Thrfs*8, p.Leu463_Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate this data with the clinical phenotype. Functional analyses were done by using a HEK293 cell in vitro expression system comparing WT with mutant P450c11 activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.His125Thrfs*8 and p.Leu463_Leu464dup) detected in patients with classic 11OHD showed a complete loss of P450c11 activity. The mutation (p.Ser150Leu) detected in a patient with non-classic 11OHD showed partial functional impairment with 19% of WT activity. Functional mutation analysis enables the correlation of novel CYP11B1 mutations to the classic and non-classic 11OHD phenotype respectively. Mutations causing a non-classic phenotype show typically partial impairment due to reduced maximum reaction velocity comparable with non-classic mutations in 21-hydroxylase deficiency. The increasing number of mutations associated with non-classic 11OHD illustrate that this disease should be considered as diagnosis in patients with otherwise unexplained hyperandrogenism.

Uniparental disomy of chromosome 8 leading to homozygosity of a &lt;i&gt;CYP11B1&lt;/i&gt; mutation in a patient with congenital adrenal hyperplasia: Implication for a rare etiology of an autosomal recessive disorder

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that usually results from paternally and maternally transmitted mutations in genes for steroidogenic enzymes. Recent studies on steroid 21-hydroxylase deficiency, the most common form of CAH, have revealed that a small percentage of patients have a non-carrier parent; uniparental disomy (UPD) and de novo mutations were reported as disease-causing mechanisms in these patients. However, it remains unknown whether UPD and de novo mutations underlie other forms of CAH. Here, we report a male patient with steroid 11β-hydroxylase deficiency (11OHD) born to a non-carrier mother. The patient was identified by an elevated 17-hydroxyprogesterone level at a neonatal mass-screening test. His clinical features were comparable to those of previously reported patients with 11OHD. Direct sequencing of CYP11B1 identified a homozygous IVS7+1G>A mutation in the patient, which was not shared by his mother. Comparative genomic hybridization of the patient detected UPD of chromosome 8 [UPD(8)]. Microsatellite analysis indicated non-maternal origin of the UPD(8) and confirmed parentage of other chromosomes. This study shows for the first time that 11OHD can be caused by UPD in the presence of a non-carrier parent. Awareness of such rare cases should improve the accuracy of genetic counseling for families with CAH. Our data support the importance of UPD as an underlying mechanism of autosomal recessive disorders.

Two Novel CYP11B1 Gene Mutations in Patients from Two Croatian Families with 11 β -Hydroxylase Deficiency.

Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common cause of congenital adrenal hyperplasia. Mutations in the CYP11B1 gene, which encodes steroid 11β-hydroxylase, are responsible for this autosomal recessive disorder. Here, we describe the molecular genetics of two previously reported male siblings in whom diagnosis of 11β-OHD has been established based on their hormonal profiles displaying high levels of 11-deoxycortisol and hyperandrogenism. Both patients are compound heterozygous for a novel p.E67fs (c.199delG) mutation in exon 1 and a p.R448H (c.1343G>A) mutation in exon 8. We also report the biochemical and molecular genetics data of one new 11β-OHD patient. Sequencing of the CYP11B1 gene reveals that this patient is compound heterozygous for a novel, previously undescribed p.R141Q (c.422G>A) mutation in exon 3 and a p.T318R (c.953C>G) mutation in exon 5. All three patients are of Croatian (Slavic) origin and there is no self-reported consanguinity in these two families. Results of our investigation confirm that most of the CYP11B1 mutations are private. In order to elucidate the molecular basis for 11β-OHD in the Croatian/Slavic population, it is imperative to perform CYP11B1 genetic analysis in more patients from this region, since so far only four patients from three unrelated Croatian families have been analyzed.

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

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.

A Diagnosis Not to Be Missed: Nonclassic Steroid 11β-Hydroxylase Deficiency Presenting With Premature Adrenarche and Hirsutism

Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia. Milder nonclassic forms are rare and at risk to be missed. The objective of the study was to demonstrate the challenges in diagnosing nonclassic 11OHD. Patient 1, a 10-year-old boy, presented with high-normal blood pressure and previously unexplained exaggerated adrenarche from age 4 years. Previous tests at the age of 8 years showed normal 17-hydroxyprogesterone concentrations with increased androgens. Patient 2, a 14-year-old female, presented with facial hirsutism, primary amenorrhea, and high-normal blood pressure. Novel CYP11B1 mutations were functionally analyzed in transiently transfected COS7 cells measuring the conversion of 11-deoxycortisol to cortisol by liquid chromatography-tandem mass spectrometry. Biochemical findings including urinary steroid metabolite analysis by gas chromatography-mass spectrometry were suggestive of 11OHD in all patients. CYP11B1 mutation analysis revealed compound heterozygosity in patient 1 (g.235T>A, p.F79I/g.2608C>T, p.R138C) and a homozygous mutation in patient 2 and two siblings (g.2623C>T, p.R143W). Functional in vitro analysis demonstrated partially impaired CYP11B1 activity compared with wild-type (p.F79I: 8.8% ± 0.8% (SEM); p.R138C: 9.8% ± 0.8%; p.R143W: 10.6% ± 1.2%). In addition to nonclassic 21-hydroxylase deficiency and steroid-secreting tumors, nonclassic 11OHD should be considered as an important differential diagnosis in patients with unexplained hyperandrogenism without 46,XX disorder of sex development. Nonclassic 11OHD is likely to be missed when relying on measuring standard steroid hormone panels. This diagnosis needs to be established early in life to avoid long-term health problems such as short stature, hyperandrogenism-related metabolic complications, potentially severe arterial hypertension, and cardiovascular consequences.

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

Steroid 11β hydroxylase deficiency (11β-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 40 kb 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 11β-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 11β hydroxylase deficiency is variable, and biochemical identification of elevated precursor metabolites is not usually available. In order to clarify the underlying mechanism causing 11β-OHD, we performed the molecular genetic analysis of the CYP11B1 gene in a female patient diagnosed as classical 11β-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 11β hydroxylase deficiency.

Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V

Steroid 11β-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11β-hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11β-hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population.

Three Novel CYP11B1 Mutations in Congenital Adrenal Hyperplasia due to Steroid 11Beta-Hydroxylase Deficiency in a Moroccan Population

Background/Aims: Steroid 11β-hydroxylase deficiency (11OHD), the second cause of congenital adrenal hyperplasia (CAH), accounts only for 5% of all CAH. To date, only 51 different mutations have been reported with poor clinical and biological data. Most of them could be considered as private mutations except one, p.R448H, identified especially in Moroccan Jews but also in Caucasian patients. As two other CYP11B1 mutations have a high incidence in Tunisian patients, we report from another Maghreb population the clinical, follow-up and molecular genetics of 5 Moroccan patients with classical 11OHD. Methods: Patients belonging to 3 families were recruited on clinical data. The diagnosis was confirmed by 11-deoxycortisol determination. Sequencing of the CYP11B1 gene and molecular modeling were performed. Results: Clinical, hormonal and follow-up data were consistent with a severe form of 11OHD. Gender reassignment and evolution of hypertension were discussed. Three novel mutations, p.Ala259Asp, p.Gly446Val and IVS5+2T>G were identified. As each patient was homozygous for one mutation, we could deduce from their phenotype and our modeling studies that the p.Gly446Val mutation was more severe than p.Ala259Asp. Conclusion: This study shows a good correlation between phenotype and genotype. Each CYP11B1 mutation is new and private, contrasting with the high incidence of two Tunisian mutations.

Functional Consequences of Seven Novel Mutations in the CYP11B1 Gene: Four Mutations Associated with Nonclassic and Three Mutations Causing Classic 11β-Hydroxylase Deficiency

ABSTRACT Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD.

Mutations in CYP11B1 and Congenital Adrenal Hyperplasia in Moroccan Jews

In Jews of Moroccan descent (MJ), the prevalence of steroid 11beta-hydroxylase deficiency (11-OHD) is relatively high, with a carrier rate estimated as approximately one in 40. A single mutation in the CYP11B1 gene (encoding 11beta-hydroxylase), R448H, was suggested to account for the disease alleles in this population. We screened 236 healthy MJ for R448H. Only two of the subjects screened were found to be carriers, suggesting that the R448H allele frequency is lower than was assumed previously. An R448H/R448C compound heterozygote patient, diagnosed with 11-OHD, was identified. However, a subsequent screen of MJ subjects for R448C failed to detect any carriers, suggesting that this was a private mutation of this family. The high incidence of 11-OHD in MJ, therefore, is only partially explained by the presence of R448H as a founder mutation.

Deletion Hybrid Genes, due to Unequal Crossing Over betweenCYP11B1(11β-Hydroxylase) andCYP11B2(Aldosterone Synthase) Cause Steroid 11β-Hydroxylase Deficiency and Congenital Adrenal Hyperplasia1

Chromosomal rearrangements are natural experiments that can provide unique insights into in vivo regulation of genes and physiological systems. We have studied a patient with congenital adrenal hyperplasia and steroid 11beta-hydroxylase deficiency who was homozygous for a deletion of the CYP11B1 and CYP11B2 genes normally required for cortisol and aldosterone synthesis, respectively. The genes were deleted by unequal recombination between the tandemly arranged CYP11B genes during a previous meiosis, leaving a single hybrid gene consisting of the promoter and exons 1-6 of CYP11B2 and exons 7-9 of CYP11B1. The hybrid gene also carried an I339T mutation formed by intracodon recombination at the chromosomal breakpoint. The mutant complementary DNA corresponding to this gene was expressed in COS-1 cells and was found to have relatively unimpaired 11beta-hydroxylase and aldosterone synthase activities. Apparently the 11beta-hydroxylase deficiency and the adrenal hyperplasia are due to the lack of expression of this gene in the adrenal zona fasciculata/reticularis resulting from replacement of the CYP11B1 promoter and regulatory sequences by those of CYP11B2.