The diagnosis of nonclassic congenital adrenal hyperplasia due to 21-hydroxylase deficiency, based on serum basal or post-ACTH stimulation 17-hydroxyprogesterone, can lead to false-positive diagnosis.

OBJECTIVE: As nonclassic congenital adrenal hyperplasia (NCCAH) needs to be taken into account in women with hyperandrogenism, we aimed to assess whether the recommended level of poststimulated 17OHP ≥30 nmol/l confirms NCCAH. PATIENTS AND METHODS: Forty, consecutive women with biochemical and/or clinical hyperandrogenism (aged 25·4, 18-38) suspected of having NCCAH were recruited to the study. In patients with 17OHP level between 5·1 and 29·9 nmol/l an ACTH stimulation test was performed. In patients with basal or poststimulated 17OHP ≥30 nmol/l, twenty-four-hour urinary steroid profile (USP) analysis was performed and CYP21A2 mutation was assessed. In selected patients with poststimulated 17OHP <30 nmol/l USP was also performed. RESULTS: The group was divided into two subgroups with basal or poststimulated 17OHP ≥30 nmol/l (group A) and with poststimulated 17OHP <30 nmol/l (group B). Among 40 patients, basal or poststimulated 17OHP ≥30 nmol/l was found in 21, but NCCAH was confirmed by USP followed by genetic testing only in 5 (24%). Four patients were diagnosed as heterozygotes, and in twelve, no CYP21A2 mutation was detected. CONCLUSION: The diagnosis of NCCAH based only on serum 17OHP measurements (basal or poststimulated) may lead to false-positive diagnosis when performed by immunoassay with a cut-off value of ≥30 nmol/l. The definitive diagnosis can be established based on USP and/or genetic testing.

LC-MS/MS improves screening towards 21-hydroxylase deficiency.

Basal serum 17OHP measurement remains the first screening step for nonclassic congenital adrenal hyperplasia (NCCAH) and the accuracy of the test is of high value. The aim of this study was to compare the accuracy of immunoassays to LC-MS/MS in the assessment of serum 17OHP and androgens concentration in women with hyperandrogenism and controls. 17OHP, total testosterone, androstendione and DHEA-S were measured in 39 women with clinically and/or biochemically evident hyperandrogenism and in 29 age-matched controls without clinical hyperandrogenism. 17OHP and androgens were measured by immunoassays and by LC-MS/MS. In patients group median 17OHP level measured by immunoassays was significantly higher compared to LC-MS/MS (5.49 nmol/l-ELISA NovaTec® and 3.57 nmol/l-ELISA DRG® versus 1.56 nmol/l-LC-MS/MS p < 0.0001) as well as in the control group (2.58 nmol/l-ELISA DRG® versus 1.14 nmol/l-LC-MS/MS p < 0.0001). Additional, unnecessary diagnostic procedures explaining elevated 17OHP level were undertaken in 85% of patients when NovaTec® test was used, in 50% when ELISA DRG® and in none when LC-MS/MS method was applied. Total testosterone, androstendione and DHEA-S concentrations in the patients and the controls assessed by the immunoassays were also significantly higher compared to LC-MS/MS. LC-MS/MS is more reliable diagnostic tool in the measurement of serum 17OHP and androgens concentrations compared to immunoassays in women with hyperandrogenism.

Trends in prenatal diagnosis of non-specific multiple malformations disorders with reference to the own experience and research study on Smith-Lemli-Opitz syndrome.

AIM OF THE STUDY: Biochemical diagnosis of fetuses with multiple malformations--an attempt to determine the frequency of prenatal Smith-Lemli-Opitz syndrome. Discussion on trends in prenatal diagnosis of non-specific multiple malformations disorders. MATERIAL AND METHODS: A total of 117 fetal samples were obtained. They were analyzed with gas chromatography/mass spectrometry (GC/MS) method to assess the concentration of 7-dehydrocholesterol (7DHC) and 8-dehydrocholesterol (8DHC) in amniotic fluid samples and (or) to establish 7-dehydroestriol/estriol and 8-dehydropregnanetriol/pregnanetrio ratios in maternal urine. RESULTS: In 4 cases Smith-Lemll-Opitz syndrome was confirmed. CONCLUSIONS: Biochemical GC/MS sterol analyses of amniotic fluid or maternal urinary metabolites toward Smith- Lemli-Opitz syndrome, as cheap tests, should be performed in all pregnancies with suggestive ultrasound features (holoprosencephaly and(or) atrioventricular canal and(or) genital anomalies), especially when nuchal translucency is increased >3 mm, and after exclusion of chromosomal aberration in routine karyotyping or even arrayCGH.

Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS.

A case of recurrent labial adhesions in a 15-month-old child with asymptomatic non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Labial adhesions (synechia vulvae) are a relatively common disorder of the external genitalia in prepubertal girls. They usually occur between 3 months and 6 years of age, with a peak between 13 and 23 months of life. In the majority of cases, labial adhesions are asymptomatic and noticed by the parents or a physician during a routine physical examination. Frequently, they cause recurrent urinary tract infections. This report presents the case of a 15-month-old girl with recurrent labial adhesions and urinary tract infections. She was followed-up by a urologist from the age of 3 months, treated topically (estriol cream 1 mg/g and emollients) and surgically. No signs of androgenization were seen apart from advanced bone age. Urinary steroid profile and molecular analysis confirmed the diagnosis of non-classic congenital adrenal hyperplasia (NCCAH) due to 21-hydroxylase deficiency. In the described case, a coincidence of NCCAH and labial adhesion cannot be excluded particularly in view of the resolving of urinary tract infections at the same time as resolving of labial adhesions. However, it seems that in cases of recurrent labial adhesion/synechia that require repeated surgical interventions in view of ineffectiveness of conservative treatment - as it was observed in the presented case - one should consider searching for NCCAH. This may allow not only for the causal treatment of labial adhesions but, above all, NCCAH and, in consequence, the prevention of intensification of virilization, hirsutism, menstrual cycle, and fertility disturbances that are observed starting from puberty.

Five novel mutations in CYP11B2 gene detected in patients with aldosterone synthase deficiency type I: Functional characterization and structural analyses.

CONTEXT: Aldosterone synthase deficiency (ASD) is an important differential diagnosis of diseases associated with salt wasting in early infancy. OBJECTIVE: The objective of this study was to investigate the molecular basis for the disorder by (1) molecular genetic analysis in the CYP11B2 from patients suffering from ASD type I. (2) Functional characterization of the missense mutant gene products. (3) Structural simulation of the missense mutations. RESULTS: Patient 1 was a homozygous carrier of a novel mutation located in exon 4 causing a premature stop codon (p.W260X). Patient 2 was analyzed to be compound heterozygous for two novel mutations: The first was an insertion mutation (p.G206WfsX51), and the second was a deletion mutation (p.L496SfsX169). Two siblings (patients 3 and 4) were compound heterozygous carriers of two novel missense mutations (p.S315R, p.R374W). The expression studies of the mutant proteins in COS-1 cells showed a complete absence of CYP11B2 activity of p.S315R and p.R374W mutants for the conversion of 11-deoxycorticosterone to aldosterone. A 3-D model of CYP11B2 p.S315R and p.R374W indicated a change of the hydrogen bond network which might explain the cause of the dysfunction. CONCLUSION: We have identified the first CYP11B2 gene defects in two Polish families associated with phenotypes of ASD type I. Analysis of the enzymatic function as a complementary procedure to genotyping revealed data for understanding the clinical phenotype of ASD. Molecular modeling of the mutated enzyme provided a rational basis for understanding the changed activities of the mutant proteins.

Differences between predicted and established diagnoses of Smith-Lemli-Opitz syndrome in the Polish population: underdiagnosis or loss of affected fetuses?

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic disorder in which an error in cholesterol biosynthesis results in congenital anomalies/mental deficits. The results of our previous newborn screening, based on the carrier frequency of the two most common SLOS-causing mutations in Poland (p.W151X and p.V326L), would make SLOS one of the most frequent recessive disorders in our country (with an incidence of 1:2,300 - 1:3,937). This prompted us to carry out a 3-year (2006-2008) national surveillance program in which about 2,000 physicians were asked to identify potential SLOS patients pre- and postnatally based on clinical identification forms. The incidence of SLOS in Poland was estimated to be from 1:60,941 to 1:105,395 (1: 83,168 ± 22,227) live births, and its 3-year prevalence 1:866,273 ± 16,242. The mean carrier frequency was calculated to be from 1:123 to 1:165. The notable discrepancy between our previous carrier newborn screening and these prospective data may result from reduced fertility in SLOS carriers, intrauterine death of affected fetuses, or underdiagnosis in postnatal life. Since we did not notice significant data supporting the first two aspects, our study may support the suggestion that screening for the most frequent DHCR7 alleles does not reflect the true disease rates in the Polish population. Hence, further studies in which maternal urinary steroids (7-dehydroestriol/estriol and 8-dehydropregnanetriol/pregnanetriol ratios) would serve as screening markers in early pregnancies may be justified.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency - management in adults.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive hereditary diseases. The impairment of cortisol synthesis leads to excessive stimulation of the adrenal glands by adrenocorticotropic hormone (ACTH), adrenal hyperplasia, and excessive androgen synthesis. The syndrome is characterised by a considerable correlation between the genotype and the phenotype with the type of CYP21A2 gene mutation affecting the severity of 21-hydroxylase deficiency. The clinical manifestations of CAH in adults result from adrenocortical and adrenomedullary insufficiency, hyperandrogenism, and the adverse effects of glucocorticosteroids used for the treatment of the condition. Non-classic CAH may sometimes be asymptomatic. In patients with classic CAH obesity, hyperinsulinaemia, insulin resistance, and hyperleptinaemia are more often seen than in the general population. These abnormalities promote the development of metabolic syndrome and its sequelae, including endothelial dysfunction, and cardiovascular disease. Long-term glucocorticosteroid treatment is also a known risk factor for osteoporosis. Patients with CAH require constant monitoring of biochemical parameters (17a-hydroxyprogesterone [17-OHP] and androstenedione), clinical parameters (body mass, waist circumference, blood pressure, glucose, and lipids), and bone mineral density by densitometry. The principal goal of treatment in adults with CAH is to improve quality of life, ensure that they remain fertile, reduce the manifestations of hyperandrogenisation in females, and minimise the adverse effects of glucocorticosteroid treatment. Patients with classic CAH require treatment with glucocorticosteroids and, in cases of salt wasting, also with a mineralocorticosteroid. Radical measures, such as bilateral adrenalectomy, are very rarely needed. Asymptomatic patients with non-classic CAH require monitoring: treatment is not always necessary. Medical care for patients with CAH should be provided by reference centres, as the management of such patients requires collaboration between an endocrinologist, diabetologist, gynaecologist, andrologist, urologist, and psychologist.

Steroid biomarkers and genetic studies reveal inactivating mutations in hexose-6-phosphate dehydrogenase in patients with cortisone reductase deficiency.

CONTEXT: Cortisone reductase deficiency (CRD) is characterized by a failure to regenerate cortisol from cortisone via 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), resulting in increased cortisol clearance, activation of the hypothalamic-pituitary-axis (HPA) and ACTH-mediated adrenal androgen excess. 11beta-HSD1 oxoreductase activity requires the reduced nicotinamide adenine dinucleotide phosphate-generating enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the endoplasmic reticulum. CRD manifests with hyperandrogenism resulting in hirsutism, oligo-amenorrhea, and infertility in females and premature pseudopuberty in males. Recent association studies have failed to corroborate findings that polymorphisms in the genes encoding H6PDH (R453Q) and 11beta-HSD1 (Intron 3 inserted adenine) interact to cause CRD. OBJECTIVE: Our objective was to reevaluate the genetics and steroid biochemistry of patients with CRD. DESIGN: We analyzed 24-h urine collection for steroid biomarkers by gas chromatography/mass spectrometry and sequenced the HSD11B1 and H6PD genes in our CRD cohort. PATIENTS: Patients included four cases presenting with hyperandrogenism and biochemical features clearly indicative of CRD. RESULTS: Gas chromatography/mass spectrometry identified steroid biomarkers that correlated with CRD in each case. Three cases were identified as homozygous (R109AfsX3, Y316X, and G359D) and one case identified as compound heterozygous (c.960G-->A and D620fsX3) for mutations in H6PD. No mutations affecting enzyme activity were identified in the HSD11B1 gene. Expression and activity assays demonstrate loss of function for all reported H6PDH mutations. CONCLUSIONS: CRD is caused by inactivating mutations in the H6PD gene, rendering the 11beta-HSD1 enzyme unable to operate as an oxoreductase, preventing local glucocorticoid regeneration. These data highlight the importance of the redox control of cortisol metabolism and the 11beta-HSD1-H6PDH pathway in regulating hypothalamic-pituitary-adrenal axis activity.

[The Smith-Lemli-Opitz syndrome--case report, early diagnosis]. / Zespól Smitha, Lemlego i Opitza--opis przypadku, wczesna diagnostyka.

Smith-Lemli-Opitz syndrome (SLOS) is one of the most frequent metabolic disorders in Poland and manifests itself with multiple congenital anomalies, psychomotor delay and intellectual disability. It is caused by mutations in DHCR7 gene, which codes one of the cholesterol biosynthesis enzymes. Clinical diagnosis of the syndrome is difficult due to lack of pathognomonic features and their variable expression. Despite high carrier frequency in Poland, SLO syndrome is rarely suspected and recognized. Its early diagnosis is essential, mainly because of high genetic risk (25%) as well as possibilities of treatment. Authors present a female-newborn, diagnosed with SLOS during the neonatal period. The diagnosis was based on biochemical and molecular tests. Mutations in DHCR7 gene have been found in both, the child and the parents.

X-Linked Adrenal Hypoplasia Congenita in a Boy due to a Novel Deletion of the Entire NR0B1 (DAX1) and MAGEB1-4 Genes.

X-linked Adrenal Hypoplasia Congenita (AHC) is caused by deletions or point mutations in the NR0B1 (DAX1) gene. We present a boy with AHC who came at the age of 25 days in a severe state due to prolonged vomiting and progressive dehydration. Laboratory studies showed prominent hyponatremia and hyperkaliemia but not hypoglycemia. Primary adrenal insufficiency was confirmed with low serum cortisol levels and high plasma ACTH levels. Hydrocortisone therapy combined with saline and glucose infusions was started immediately after blood collection. Two exons of the NR0B1 (DAX1) gene were impossible to amplify using the standard PCR method. Array CGH was used to confirm the putative copy-number variation of NR0B1 (DAX1) revealing a novel hemizygous deletion encompassing the entire NR0B1 (DAX1) gene together with the MAGEB genes. This genetic defect was also present in heterozygosity in the patient's mother. We show that NR0B1 (DAX1) gene analysis is important for confirmation of AHC diagnosis and highlights the role of genetic counseling in families with AHC patients, particularly those with X chromosome microdeletions, covering more than NR0B1 (DAX1) alone. We hope that further clinical follow-up of this patient and his family will shed a new light on the role of MAGEB genes.

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 11ß-hydroxylase deficiency (11ß-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 11ß-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 2850 bp 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.

Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study.

BACKGROUND: Congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency is associated with accumulation of steroid metabolites, indicating impaired activity of 17alpha-hydroxylase and 21-hydroxylase. However, no mutations have been reported in the CYP17 and CYP21 genes, which encode these P450 enzymes. Affected girls are born with ambiguous genitalia, but their circulating androgens are low, and virilisation does not progress. We aimed to investigate the underlying molecular basis of congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency in affected children. METHODS: We did sequence analysis of the human gene encoding P450 oxidoreductase, an enzyme that is important in electron transfer from NADPH to P450C17 and P450C21. We studied two unrelated families with a total of three affected children and 100 healthy controls. Wild-type and mutant P450 oxidoreductase proteins were bacterially expressed, purified, and assayed for cytochrome c reductase activity. FINDINGS: We identified four mutations encoding single aminoacid changes in P450 oxidoreductase. All patients were compound heterozygotes, whereas their parents and an unaffected sibling harboured a mutation in only one allele. By contrast, no mutations were noted in the controls. Bacterial expression of recombinant mutant proteins revealed deficient or reduced enzyme activity. INTERPRETATION: Molecular pathogenesis of this form of congenital adrenal hyperplasia is caused by mutations in the gene encoding P450 oxidoreductase. Deficiency of this enzyme could suggest an alternative pathway in human androgen synthesis, present only in fetal life, which explains the combination of antenatal androgen excess and postnatal androgen deficiency.

The diagnosis of congenital adrenal hyperplasia in the newborn by gas chromatography/mass spectrometry analysis of random urine specimens.

Definitive neonatal diagnosis of congenital adrenal hyperplasia (CAH) is frequently complicated by normal 17-hydroxyprogesterone levels in 21-hydroxylase-deficient patients, residual maternal steroids, and other interfering substances in neonatal blood. In an effort to improve the diagnosis, we developed a gas chromatography/mass spectrometry method for simultaneous measurement of 15 urinary steroid metabolites as early as the first day of life. Furthermore, we developed 11 precursor/product ratios that diagnose and clearly differentiate the four enzymatic deficiencies that cause CAH. Random urine samples from 31 neonatal 21-hydroxylase-deficient patients and 59 age-matched normal newborns were used in the development. Additionally, samples from two 11 beta-hydroxylase-deficient patients and one patient each for 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase deficiencies were used. The throughput for one bench-top gas chromatography/mass spectrometry instrument is 20 samples per day. Thus, this method affords an accurate, rapid, noninvasive means for the differential diagnosis of CAH in the newborn period without the need for invasive testing and ACTH stimulation.

Apparent pregnene hydroxylation deficiency (APHD): seeking the parentage of an orphan metabolome.

This paper collates and reviews a number of clinical cases published over the last 20 years that we believe describe a novel steroid disorder associated with genital ambiguity. The authors of the original papers were unable to diagnose their patients conclusively. Females with the disorder are frequently masculinized and males feminized. The central feature of steroid biosynthesis is overproduction of pregnenolone and progesterone, which results in elevated concentrations of these steroids in plasma and exaggerated response to ACTH. Equivalent urinary data show elevated excretion of 5-pregnene-3beta,20alpha-diol (the major pregnenolone metabolite) and pregnanediol. Another notable feature of the metabolome is the relatively high plasma concentrations of the analytes for 21-hydroxylase deficiency (17-hydroxyprogesterone) and 17-hydroxylase deficiency (corticosterone). Correspondingly, metabolites of these steroids are prominent in urine. Circulating cortisol was generally normal but had blunted response to ACTH. Taken together these features pointed to an apparent relative deficiency of 21- and 17-hydroxylation, but no mutations have been found in the enzymes responsible for these transformations in cases where it has been investigated. The simultaneous presence of mutated genes on two chromosomes is also extremely unlikely. Until more is known of this interesting condition, and the genetic cause (presumed) is known, we propose naming it apparent pregnene hydroxylation deficiency (APHD). The metabolome is similar to that of isolated 17,20-lyase deficiency with which it has been confused. In fact, diminished (but not isolated) lyase activity is probably a significant factor in some patients. We believe that the attenuated steroid hydroxylation could be associated with deficiency of the required co-factors and modulators of hydroxylation such as P450 reductase and cytochrome b5. Post-translational modifications of the hydroxylases such as phosphorylation/dephosphorylation have also been suggested as influential in directionally regulating steroid synthesis. Some patients with the disorder have no dysmorphology apart from genital malformations while others have skeletal abnormalities attributed to Antley-Bixler syndrome. Whether we are looking at different conditions, or a severity continuum is not known. It is possible that the presence of this metabolome may become a required feature for diagnosis of Antley-Bixler syndrome. A combination of intersex phenotype and dysmorphology suggests that an error in a transcription factor may be an alternative to hydroxylation redox partner deficit as causative of the condition.The virilization of female patients with APHD probably results from a transient hyperandrogenism prior to birth.

Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

CONTEXT: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. OBJECTIVE: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. DESIGN: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. RESULTS: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. CONCLUSIONS: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

Pubertal presentation in seven patients with congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

CONTEXT: P450 oxidoreductase (POR) is a crucial electron donor to all microsomal P450 cytochrome (CYP) enzymes including 17α-hydroxylase (CYP17A1), 21-hydroxylase (CYP21A2) and P450 aromatase. Mutant POR causes congenital adrenal hyperplasia with combined glucocorticoid and sex steroid deficiency. P450 oxidoreductase deficiency (ORD) commonly presents neonatally, with disordered sex development in both sexes, skeletal malformations, and glucocorticoid deficiency. OBJECTIVE: The aim of the study was to describe the clinical and biochemical characteristics of ORD during puberty. DESIGN: Clinical, biochemical, and genetic assessment of seven ORD patients (five females, two males) presenting during puberty was conducted. RESULTS: Predominant findings in females were incomplete pubertal development (four of five) and large ovarian cysts (five of five) prone to spontaneous rupture, in some only resolving after combined treatment with estrogen/progestin, GnRH superagonists, and glucocorticoids. Pubertal development in the two boys was more mildly affected, with some spontaneous progression. Urinary steroid profiling revealed combined CYP17A1 and CYP21A2 deficiencies indicative of ORD in all patients; all but one failed to mount an appropriate cortisol response to ACTH stimulation indicative of adrenal insufficiency. Diagnosis of ORD was confirmed by direct sequencing, demonstrating disease-causing POR mutations. CONCLUSION: Delayed and disordered puberty can be the first sign leading to a diagnosis of ORD. Appropriate testosterone production during puberty in affected boys but manifest primary hypogonadism in girls with ORD may indicate that testicular steroidogenesis is less dependent on POR than adrenal and ovarian steroidogenesis. Ovarian cysts in pubertal girls may be driven not only by high gonadotropins but possibly also by impaired CYP51A1-mediated production of meiosis-activating sterols due to mutant POR.

[Congenital adrenal hyperplasia due to 21-hydroxylase deficiency--management in adults]. / Wrodzony przerost nadnerczy z niedoboru 21-hydroksylazy--problemy i postepowanie u doroslych.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive hereditary diseases. The impairment of cortisol synthesis leads to excessive stimulation of the adrenal glands by adrenocorticotropic hormone (ACTH), adrenal hyperplasia, and excessive androgen synthesis. The syndrome is characterised by a considerable correlation between the genotype and the phenotype with the type of CYP21A2 gene mutation affecting the severity of 21-hydroxylase deficiency. The clinical manifestations of CAH in adults result from adrenocortical and adrenomedullary insufficiency, hyperandrogenism, and the adverse effects of glucocorticosteroids used for the treatment of the condition. Non-classic CAH may sometimes be asymptomatic. In patients with classic CAH obesity, hyperinsulinaemia, insulin resistance, and hyperleptinaemia are more often seen than in the general population. These abnormalities promote the development of metabolic syndrome and its sequelae, including endothelial dysfunction, and cardiovascular disease. Long-term glucocorticosteroid treatment is also a known risk factor for osteoporosis. Patients with CAH require constant monitoring of biochemical parameters (17a-hydroxyprogesterone and androstenedione), clinical parameters (body mass, waist circumference, blood pressure, glucose, and lipids), and bone mineral density by densitometry. The principal goal of treatment in adults with CAH is to improve quality of life, ensure that they remain fertile, reduce the manifestations of hyperandrogenisation in females, and minimise the adverse effects of glucocorticosteroid treatment. Patients with classic CAH require treatment with glucocorticosteroids and, in cases of salt wasting, also with a mineralocorticosteroid. Radical measures, such as bilateral adrenalectomy, are very rarely needed. Asymptomatic patients with non-classic CAH require monitoring: treatment is not always necessary. Medical care for patients with CAH should be provided by reference centres, as the management of such patients requires collaboration between an endocrinologist, diabetologist, gynaecologist, andrologist, urologist, and psychologist.

Concomitant mutations in the P450 oxidoreductase and androgen receptor genes presenting with 46,XY disordered sex development and androgenization at adrenarche.

CONTEXT: Undervirilization in males, i.e. 46,XY disordered sex development (46,XY DSD), is commonly caused by either lack of androgen action due to mutant androgen receptor (AR) or deficient androgen synthesis, e.g. due to mutations in 17alpha-hydroxylase (CYP17A1). Like all other microsomal cytochrome P450 (CYP) enzymes, CYP17A1 requires electron transfer from P450 oxidoreductase (POR). OBJECTIVE: The objective of the study was to analyze the clinical and biochemical phenotype in a 46,XY individual carrying concomitant POR and AR mutations and to dissect their impact on phenotypic expression. METHODS: We characterized the clinical and biochemical phenotype, genetic identification, and functional analysis of POR missense mutation by yeast micrososomal coexpression assays for CYP17A1, CYP21A2 and CYP19A1 activities. RESULTS: The patient presented neonatally with 46,XY DSD and was diagnosed as partial androgen insensitivity syndrome carrying a disease causing AR mutation (p.Q798E). She was raised as a girl and gonadectomized at the age of 4 yr. At 9 yr progressive clitoral enlargement prompted reassessment. Urinary steroid analysis was indicative of POR deficiency, but surprisingly androgen production was normal. Genetic analysis identified compound heterozygous POR mutations (p.601fsX12/p.Y607C). In vitro analysis confirmed p.Y607C as a pathogenic mutation with differential inhibition of steroidogenic CYP enzymes. CONCLUSION: Both mutant AR and POR are likely to contribute to the neonatal presentation with 46,XY DSD. Virilization at the time of adrenarche appears to suggest an age-dependent, diminishing disruptive effect of both mutant proteins. This case further highlights the importance to assess both gonadal and adrenal function in patients with 46,XY DSD.

[Pseudohypoaldosteronism in infants with salt wasting syndrome. Two case reports]. / Pseudohipoaldosteronizm u niemowlat jako przyczyna zespolu utraty soli. Opis dwóch przypadków.

The paper discusses two cases of male infants, who developed a markedly elevated salt wasting syndrome in early infancy, resistant to treatment with mineralocorticoids. Steroid urinary profiles excluded congenital adrenal hyperplasia. However, both patients presented with extremely high excretion of aldosterone metabolite THAldo without effects of aldosterone action, what resulted in pseudohypoaldosteronism (PHA) diagnosis. The patients were treated with sodium supplementation, which normalized their clinical state and serum electrolytes. In the first patient the sporadic form of renal PHA1 is suspected. In the second patient congenital urinary tract anomalies and infection resulted in transient PHA1. Pseudohypoaldosteronism should be considered in the differential diagnosis of a salt wasting syndrome in infants, especially when it is accompanied by infections or congenital defects of the urinary tract.