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Disorders of sex development (DSD) are a group of rare conditions characterized by discrepancy between chromosomal sex, gonads and external genitalia. Congenital abnormalities of the kidney and urinary tract are often associated with DSD, mostly in multiple malformation syndromes. We describe the case of an 11-year-old Caucasian boy, with right kidney hypoplasia and hypospadias. Genome-wide copy number variation (CNV) analysis revealed a unique duplication of about 550 kb on chromosome Xq27, and a 46,XX karyotype, consistent with a sex reversal phenotype. This region includes multiple genes, and, among these, SOX3 emerged as the main phenotypic driver. This is the fifth case reporting a genomic imbalance involving the SOX3 gene in a 46,XX SRY-negative male, and the first with associated renal malformations. Our data provide plausible links between SOX3 gene dosage and kidney malformations. It is noteworthy that the current and reported SOX3 gene duplications are below the detection threshold of standard karyotypes and were found only by analyzing CNVs using DNA microarrays. Therefore, all 46,XX SRY-negative males should be screened for SOX3 gene duplications with DNA microarrays.

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., (© 2018 S. Karger AG, Basel.)

Objective: Heterozygosity in 21-hydroxylase deficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry., Design: Eleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared., Method: Hormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 58 carriers (35 males, 23 females, age range 6-78 years) and 44 random controls (25 males, 19 females, age range 8-58 years)., Results: Heterozygotes could be identified best applying the 17-hydroxyprogesterone+21-deoxycortisol/cortisol×1000 ((17OHP+21S)/F×1000) equation 30  min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-off is 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%., Conclusion: Considering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHP+21S)/F×1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC-MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-off and to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHP+21S)/F×1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism., (© 2015 European Society of Endocrinology.)

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

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.

Background: 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., Aim: The aim of the study was to study the functional consequences of three novel CYP11B1 gene mutations (p.His125Thrfs*8, p.Leu463&#95;Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate this data with the clinical phenotype., Methods: 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., Results: Two mutations (p.His125Thrfs*8 and p.Leu463&#95;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., Conclusion: 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.

In this study, we present a Sudanese 46,XY patient raised as a female and diagnosed at the age of 20 years with having 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency. She presented with primary amenorrhea, undeveloped breasts and a male pattern of secondary sexual characteristics. Examination of her external genitalia showed type IV genital circumcision. Steroid measurements both in urine and serum pointed to 17β-HSD3 deficiency. A novel homozygous splice-site mutation [c.524 + 2T>A] was detected in intron 7 of the HSD17B3 gene. In this patient, steroid concentration clearly supported both the clinical diagnosis of 17β-HSD3 deficiency and the functional relevance of the mutation. Interestingly, despite of the type IV genital circumcision, the patient expressed her interest in reassigning her sex from female to male., (© 2014 S. Karger AG, Basel.)

Background: Mutations of the CYP17A1 gene cause 17α-hydroxylase deficiency (17OHD) resulting in 46,XY disorder of sex development, hypertension, hypokalemia and absent pubertal development. It is a rare, autosomal recessive form of congenital adrenal hyperplasia (CAH)., Patient: We report on a neonate with prenatally determined 46,XY karyotype. At 20 weeks of gestation, lack of development of male external genitalia was noticed. A phenotypically female child was born at 41 weeks of gestation., Results: Postnatal ultrasound revealed testes in both labia majora, an absence of uterus and normal adrenal glands. Steroid hormone analysis in serum revealed low basal levels of cortisol, testosterone and androstenedione in the presence of massively elevated corticosterone at the age of 2 weeks. The urinary steroid profile from spot urine showed excessive excretion of 17-desoxysteroids, decreased glucocorticoid metabolites and absent C19 steroids, thus proving 17OHD. Molecular analysis identified a novel mutation of the CYP17A1 gene: c.896T>A (p.I299N) in exon 5. Substitution with hydrocortisone was started. The child is raised as a girl and is developing well so far., Conclusion: Herein, we report the unusually early diagnosis of a newborn with the rare CAH form of 17OHD allowing an early start of treatment., (© 2014 S. Karger AG, Basel.)

Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS) due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

Various genes located at imprinted loci and regulated by epigenetic mechanisms are involved in the control of growth and differentiation. The broad phenotypic variability of imprinting disorders suggests that individuals with inborn errors of imprinting might remain undetected among patients born small for gestational age (SGA). We evaluated quantitative DNA methylation analysis at differentially methylated regions (DMRs) of 10 imprinted loci (PLAGL1, IGF2R DMR2, GRB10, H19 DMR, IGF2, MEG3, NDN, SNRPN, NESP, NESPAS) by bisulphite pyrosequencing in 98 patients born SGA and 50 controls. For IGF2R DMR2, methylation patterns of additional 47 parent pairs and one mother (95 individuals) of patients included in the SGA cohort were analyzed. In six out of 98 patients born SGA, we detected DNA methylation changes at single loci. In one child, the diagnosis of upd(14)mat syndrome owing to an epimutation of the MEG3 locus in 14q32 could be established. The remaining five patients showed hypomethylation at GRB10 (n=2), hypomethylation at the H19 3CTCF-binding site (n=1), hypermethylation at NDN (n=1) and hypermethylation at IGF2 (n=1). IGF2R DMR2 hypermethylation was detected in five patients, six parents of patients in the SGA cohort and two controls. We conclude that aberrant methylation at imprinted loci in children born SGA exists but seems to be rare if known imprinting syndromes are excluded. Further investigations on the physiological variations and the functional consequences of the detected aberrant methylation are necessary before final conclusions on the clinical impact can be drawn.

Background: Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disease caused by mutations in the genes encoding the human mineralocorticoid receptor (MR) or the α (SCNN1A), β (SCNN1B) or γ (SCNN1G) subunit of the epithelial Na(+) channel (ENaC). While autosomal dominant mutation of the MR cause renal PHA1, autosomal recessive mutations of the ENaC lead to systemic PHA1. In the latter, affected children suffer from neonatal onset of multi-organ salt loss and often exhibit cystic fibrosis-like pulmonary symptoms., Objective: We searched for underlying mutations in seven unrelated children with systemic PHA1, all offsprings of healthy consanguineous parents., Methods and Results: Amplification of the SCNN1A gene and sequencing of all 13 coding exons unraveled mutations in all of our patients. We found five novel homozygous mutations (c.587&#95;588insC in two patients, c.1342&#95;1343insTACA, c.742delG, c.189C>A, c.1361-2A>G) and one known mutation (c.1474C>T) leading to truncation of the αENaC protein. All parents were asymptomatic heterozygous carriers of the respective mutations, confirming the autosomal recessive mode of inheritance. Five out of seven patients exhibited pulmonary symptoms in the neonatal period., Conclusion: The α subunit is essential for ENaC function and mutations truncating the pore-forming part of the protein leading to systemic PHA1. Based on current knowledge, the pulmonary phenotype cannot be satisfactorily predicted.

Background: Sensitive and accurate determination of steroids is essential for diagnosing congenital and acquired adrenal diseases. Since plasma concentrations change during childhood, age-specific reference ranges are the prerequisite for clinical interpretation. The objectives of this study were to develop a sensitive and reliable method for simultaneous detection and quantification of progesterone, 17-hydroxyprogesterone, deoxycorticosterone (DOC), 11-deoxycortisol, 21-deoxycortisol, corticosterone, cortisol (F) and cortisone (E) by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and to establish age- and sex-specific reference ranges from birth to adulthood., Methods: All eight steroids were measured simultaneously in 0.1 ml plasma by UPLC-MS/MS. Samples of 905 children were measured and grouped in five age groups., Results: The assay was linear up to 70 ng/ml (700 ng/ml for F; r(2) > 0.992). The limit of detection ranged between 0.01 ng/ml for DOC and 0.07 ng/ml for E. Correlations with radioimmunoassays yielded a coefficient of determination between 0.82 and 0.99. Reference data are reported as a function of age and sex., Conclusions: The UPLC-MS/MS method presented here for the simultaneous detection of eight C-21 adrenal hormones together with the detailed reference ranges for children provides a valuable methodology for assessing adrenal steroids in clinical routine and research., (Copyright © 2013 S. Karger AG, Basel.)

17-Alpha-hydroxylase/17,20-lyase deficiency (17OHD) is a rare autosomal recessive disorder resulting from mutations in the CYP17A1 gene, leading to impaired adrenal and gonadal steroidogenesis. We report for the first time a patient with a missense mutation at codon 96 (R96Q) of the CYP17A1 gene causing a 46,XY disorder of sexual development (DSD) that additionally showed lack of breast development despite highly dosed estradiol replacement treatment. This phenomenon could be attributed to irreversible breast tissue alterations following high serum progesterone levels., (Copyright © 2013 S. Karger AG, Basel.)

Pseudohypoaldosteronism (PHA) is a rare syndrome of mineralocorticoid resistance. PHA type 1 (PHA1) can be divided into two different forms, showing either a systemic or a renal form of mineralocorticoid resistance. The first is caused by mutations of the genes coding the epithelial sodium channel, the latter is caused by mutations in the mineralocorticoid receptor coding gene NR3C2. The clinical manifestation of systemic PHA1 is overt dehydration and hyponatremia due to systemic salt loss and severe hyperkalemia. The leading clinical sign of the less severe renal PHA1 is insufficient weight gain due to chronic dehydration. Hyperkalemia is generally mild. The patients manifest clinical signs mainly in early infancy. In both entities, plasma renin and aldosterone concentrations are highly elevated, reflecting a resistance of the kidney and other tissues to mineralocorticoids. PHA2 is characterized by hyperkalemia and hypertension. It has been described by Gordon's group as a syndrome with highly variable plasma aldosterone concentrations, suppressed plasma renin activity, various degrees of hyperchloremia and metabolic acidosis. PHA3 comprises transient and secondary forms of salt-losing states caused by various pathologies. Urinary tract infections and obstructive uropathies are the most frequent cause. Contrary to PHA1 and PHA2, the glomerular filtration rate is decreased in PHA3., (Copyright © 2013 S. Karger AG, Basel.)

Background: The steroidogenic acute regulatory protein (StAR) is essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause lipoid congenital adrenal hyperplasia., Objective and Methods: To identify causative mutations in a patient presenting with adrenal failure during early infancy. The objective was to study the functional and structural consequences of the novel StAR mutation p.Trp147Arg in a Turkish patient detected in compound heterozygosity with the p.Glu169Lys mutation., Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.Trp147Arg mutant. The previously described p.Glu169Lys mutant led to significantly lower cholesterol conversion than wild-type StAR protein. As derived from three-dimensional protein modeling, the residue W147 is stabilizing the C-terminal helix in a closed conformation hereby acting as gatekeeper of the ligand cavity of StAR., Conclusions: The novel mutation p.Trp147Arg causes primary adrenal insufficiency and complete sex reversal in the 46,XY patient. Clinical disease, in vitro studies and three-dimensional protein modeling of the mutation p.Trp147Arg underscore the relevance of this highly conserved residue for StAR protein function., (Copyright © 2013 S. Karger AG, Basel.)

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of steroid biosynthesis in humans. More than 90% of all CAH cases are caused by mutations of the 21-hydroxylase gene (CYP21A2), and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. In this study, the CYP21A2 gene was genotyped in 50 patients in Tunisia with the clinical diagnosis of 21-hydroxylase deficiency. CYP21A2 mutations were identified in 87% of the alleles. The most common point mutation in our population was the pseudogene specific variant p.Q318X (26%). Three novel single nucleotide polymorphism (SNP) loci were identified in the CYP21A2 gene which seems to be specific for the Tunisian population. The overall concordance between genotype and phenotype was 98%. With this study the molecular basis of CAH has been characterized, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aldosterone synthase deficiency (ASD) type II was diagnosed in a 3 week old boy with severe dehydration. Elevated plasma renin activity, low-normal aldosterone, increased levels for 18-OH corticosterone (18-OHB) and 18-OH-deoxycorticosterone were measured. Sequencing revealed a homozygous mutation for c554C > T in exon 3 (p.T185I) (CYP11B2). Hypospadias has so far not been reported in ASD.

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 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 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&#95;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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3) deficiency is a rare cause of 46,XY disorders of sex development (DSD). At puberty, these patients experience a surge of androstenedione and also testosterone, leading to substantial virilization. The origin of testosterone synthesis in these patients remains elusive. We investigated the expression of the isoenzyme AKR1C3 (17β-HSD 5) in the testis and patient-derived genital skin fibroblasts (GSF) as well as the ability of GSF to synthesize testosterone. Supernatants of GSF cultures and serum samples of one patient before and after gonadectomy were analyzed by liquid and gas chromatography/mass spectrometry. The androgenic potential of GSF-derived supernatants was also assessed by androgen receptor-mediated transactivation of a reporter gene in transiently transfected Chinese hamster ovary cells. Although AKR1C3 is expressed both in the testes and in GSF, androstenedione is rapidly metabolized and is not synthesized to testosterone. The transactivation potential of GSF supernatants towards the androgen receptor is declining within 48 h. However, under testis-equivalent androstenedione concentration, testosterone can be synthesized in 17β-HSD 3-negative GSF. After gonadectomy, both androstenedione and testosterone decline rapidly in vivo. In 17β-HSD 3 deficiency, relevant amounts of testosterone are synthesized most probably through AKR1C3 in the testis and not peripherally in GSF., (Copyright © 2012 S. Karger AG, Basel.)

Background: 21-Hydroxylase deficiency (21-OHD) is the target disease of newborn screening for congenital adrenal hyperplasia (CAH). We describe the additional detection of patients suffering from 11β-hydroxylase deficiency (11-OHD) by second-tier testing., Method: Over a period of 5 years, screening for CAH was done in a total of 986,098 newborns by time-resolved immunoassay (DELFIA®) for 17α-hydroxyprogesterone (17-OHP). Positive samples were subsequently analyzed in an LC-MS/MS second-tier test including 17-OHP, cortisol, 11-deoxycortisol, 4-androstenedione and 21-deoxycortisol., Results: In addition to 78 cases of 21-OHD, 5 patients with 11-OHD were identified. Diagnostic parameters were a markedly elevated concentration of 11-deoxycortisol in the presence of a low level of cortisol. Androstenedione was also increased. In contrast to 21-OHD, concentrations of 21-deoxycortisol were normal., Conclusion: Steroid profiling in newborn blood samples showing positive results in immunoassays for 17-OHP allows for differentiating 21-OHD from 11-OHD. This procedure may not detect all cases of 11-OHD in the newborn population because there may be samples of affected newborns with negative results for 17-OHP in the immunoassay., (Copyright © 2012 S. Karger AG, Basel.)

Background/aim: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder characterized by primary adrenal insufficiency and hypogonadic hypogonadism. It is caused by deletions or point mutations of the NR0B1 gene, on Xp21. AHC can be associated with glycerol kinase deficiency, Duchenne muscular dystrophy and mental retardation (MR), as part of a contiguous gene deletion syndrome. A synthetic probe set for multiplex ligation-dependent probe amplification analysis was developed to confirm and characterize NR0B1 deletions in patients with AHC and to correlate their genotypes with their divergent phenotypes., Results: In 2 patients, isolated AHC was confirmed, while a patient at risk for metabolic crisis was revealed as the deletion extends to the GK gene. A deletion extending to IL1RAPL1 was confirmed in both patients showing MR. Thus, a good genotype-phenotype correlation was confirmed., Conclusions: Multiplex ligation-dependent probe amplification analysis is a valuable tool to detect NR0B1 and contiguous gene deletions in patients with AHC. It is especially helpful for IL1RAPL1 deletion detection as no clinical markers for MR are available. Furthermore, multiplex ligation-dependent probe amplification has the advantage to identify female carriers that, depending on the deletion extension, have a high risk of giving birth to children with MR, AHC, glycerol kinase deficiency and Duchenne muscular dystrophy., (Copyright © 2012 S. Karger AG, Basel.)

Hormone concentrations decline with aging. Up to now it was not clear, whether the decrease of hormone concentrations in blood samples are also present in cutaneous suction blister fluids, and whether skin from different anatomical sites shows different hormone concentrations.Analysis of suction blister fluids and paired blood samples from young (mean 27.8 y) and old (mean 62.6 y) male subjects by UPLC-MS/MS showed that DHEA concentration in blood samples was age-dependently significantly reduced, but increased in suction blister fluids, while androstenedione behaved in an opposite manner to DHEA. Testosterone decreased age-dependently in blood samples and in suction blister fluids. Regarding skin sites, DHEA was lower in samples from upper back compared with samples from the forearm. In contrast, the concentrations of androstenedione and testosterone were higher in samples from upper back.In vitro analyses showed that SZ95 sebocytes, but neither primary fibroblasts nor keratinocytes, were able to use DHEA as precursor for testosterone biosynthesis, which was confirmed by expression analysis of 3β-hydroxysteroiddehydrogenase in skin biopsies.In conclusion, we show an inverse pattern of DHEA and androstenedione concentrations in blood vs. suction blister fluids, highlighting age-dependent changes of dermal testosterone biosynthesis, and a stronger metabolism in young skin. Furthermore, sebocytes play a central role in cutaneous androgen metabolism.

Background: There are very few laboratory markers which reflect the biological sensitivity of children to recombinant human growth hormone (rhGH) treatment. Genome-wide transcriptional changes in peripheral blood mononuclear cells (PBMC) have been widely used as functional readout for different pharmacological stimuli., Objective: To characterize transcriptional changes in PBMC induced by rhGH during a routine short-term IGF-I generation test (IGFGT) in children with growth disorders., Materials and Methods: Blood was obtained for IGF-I determination and RNA-preparation from PBMC of 12 children before and after 4days treatment with 30μgrhGH/kg body weight/day s.c. Transcriptional changes were assessed by cDNA-microarrays in the first six children. Selected genes were validated in all 12 cases by RT-qPCR., Results: Serum IGF-I rose in all patients except one (p<0.0001), confirming biological response to rhGH. Unsupervised microarray data analysis in the first six children revealed 313 transcripts with abundant transcriptional changes but considerable inter-individual variability of response patterns. Many patients showed a large cluster of up-regulated genes, including EGR1, EGR2, FOS and to a lesser extent STAT2 and 5b. Exemplarily, EGR1, EGR2 and FOS data were independently reproduced by RT-qPCR. Gene ontology analysis revealed that pathways involved in cell proliferation and immune functions were significantly over represented., Conclusion: The IGFGT is a suitable method for measuring reproducible and biologically conclusive transcriptional changes in PBMC. As our unsupervised data analysis strategy exposed a considerable inter-individual variability of response profiles a search for molecules of diagnostic and even prognostic value needs to be based on large long-term studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Liquid-chromatography - tandem mass spectrometry (LC-MS/MS) is becoming the method of choice for clinical steroid analysis. In most instances, it has the advantage of higher sensitivity, better reproducibility and greater specificity than commercial immunoassay techniques. The method requires only minimal sample preparation and a small sample volume. Furthermore, it has the potential to analyze multiple steroids simultaneously. Modern instruments guarantee high throughput, allowing an affordable price for the individual assay. All this makes LC-MS/MS an attractive method for use in a clinical setting. Reliable reference ranges for the detected analytes are the pre-requisite for their clinical use. If these are available, LC-MS/MS can find application in congenital disorders of steroid metabolism, such as congenital adrenal hyperplasia, disorders of sex development and disorders of salt homeostasis, as well as in acquired disorders of steroid metabolism, such as primary aldosteronism, Cushing's disease, Addison's disease, and hyperandrogenemia, as well as in psychiatric disease states such as depression or anxiety disorders. The principles of LC-MS/MS for steroid measurement, the pros and cons of LC-MS/MS compared with conventional immunoassays and the possible applications in clinical routine, with a special focus on pediatric endocrinology needs, are discussed here.

Pseudohypoaldosteronism type 1 (PHA-1, MIM #264350) is caused by defective transepithelial sodium transport. Affected patients develop life-threatening neonatal-onset salt loss, hyperkalemia, acidosis, and elevated aldosterone levels due to end-organ resistance to aldosterone. In this report, we present a patient diagnosed as PHA-1 who had clinical and laboratory findings compatible with the diagnosis and had genetically proven autosomal recessive PHA-1. The patient received high doses of sodium supplementation and potassium-lowering therapies; however, several difficulties were encountered in the management of this case. The aim of this presentation was to point out the potential pitfalls in the treatment of such patients in the clinical practice and to recommend solutions., (©Journal of Clinical Research in Pediatric Endocrinology, Published by Galenos Publishing.)

Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level., (2011 S. Karger AG, Basel.)

17α-Hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

Context: Current immunoassays for analysis of plasma androgens in children have several limitations due to antibody-specific variations of data and normal ranges. Mass spectrometry-based methods are available for individual steroids but need complex sample preparation and report only fragmentary reference data for the pediatric population., Objective: Our objective was to develop a state of the art sensitive and specific tandem mass spectrometry method for high-throughput simultaneous determination of plasma concentrations of androstenedione (A), testosterone (T), and dihydrotestosterone (DHT) and to report age-, sex-, and pubertal stage-specific reference levels for these steroids in children aged 0-18 yr., Subjects and Methods: Plasma (100 microl) was mixed with internal standard and extracted by solid-phase extraction. Androgens were measured by ultrapressure liquid chromatography tandem mass spectrometry. Samples of 138 boys and 131 girls with neither signs of endocrine nor systemic disease were considered for the generation of reference data. The following age groups were used: less than 1 wk, 2 wk to 2 months, 3-5 months, 6-11 months, 1-3 yr, 4-6 yr, 7-9 yr, 10-12 yr, 13-15 yr, and over 16 yr., Results: Lower quantification limit was 2.9 ng/dl (0.1 nmol/liter) for A, T, and DHT. No relevant interference with other steroids was detected. Reference data for A, T, and DHT are reported as functions of age, sex, pubertal maturation, and testicular volume., Conclusion: Simplicity, velocity, sensitivity, specificity, and the availability of pediatric reference data allow application of our new method in clinical routine as well as in research settings.

Context: In 21-hydroxylase (CYP21A2) deficiency (21OHD), the level of in vitro enzymatic function allows for classification of mutation groups (null, A, B, C) and prediction of disease severity. However, genital virilization in affected females correlates only weakly with CYP21A2 mutation groups, suggesting the influence of genetic modifiers., Objective: The objective of the study was to investigate the influence of the polymorphic CAG and GGn repeats of the androgen receptor (AR) gene on the degree of genital virilization in 21OHD females., Design and Patients: Design of the study was the determination of CYP21A2 genotype, degree of genital virilization (Prader stage), and X-weighted biallelic mean of AR CAG and GGn repeat length in 205 females with 21OHD., Outcome Measurements: Correlation of AR CAG and GGn repeat lengths with Prader stages using nested stepwise logistic regression analysis was measured., Results: CYP21A2 mutation groups null and A showed significantly higher levels of genital virilization than groups B and C (P < 0.01). However, Prader stages varied considerably within mutation groups: null, Prader I-V (median IV); A, Prader I-V (median IV); B, Prader I-V (median III); C, 0-III (median I). Mean GGn repeat length of patients was not significantly associated with Prader stages, classified as low (0-I), intermediate (II-III), or severe (IV-V) (odds ratio per repeat: 0.98, 95% confidence interval 0.71-1.35). In contrast, patients with Prader 0-I showed a trend toward longer CAG repeats without reaching statistical significance (P = 0.07, odds ratio per repeat: 0.82, 95% confidence interval 0.65-1.02)., Conclusion: Neither CAG nor GGn repeat lengths are statistically significant modifiers of genital virilization in females with 21OHD.

Context: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia., Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient)., Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the alpha4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein., Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient's phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

Background: Relative adrenocortical insufficiency is often seen in sick premature newborns. As the human fetal adrenal cortex does not express the 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme before about 23 weeks of gestation, we hypothesized that this enzymatic step may be rate limiting in cortisol synthesis in premature infants of less than 28 weeks postmenstrual age at birth., Methods: We measured cord, first day (D0) and median fourth day (D4) serum 17-OH-pregnenolone (17-OHPreg), 17-OH-progesterone (17-OHProg), 11-deoxycortisol, cortisol (F) and dehydroepiandrosterone sulphate concentrations and calculated the substrate/product ratios in 67 infants with gestational age 23.6-33.1 weeks., Results: The mean 17-OHPreg/17-OHProg ratio as a marker of 3β-HSD activity did not differ between the gestational age groups (gestational age <28 vs. ≥28 weeks: 0.40 vs. 0.48, p = 0.52 for cord, 3.1 vs. 2.4, p = 0.25 for D0, and 1.6 vs. 1.9, p = 0.62 for D4). In addition, the 17-OHPreg/17-OHProg ratio did not differ between the infants in the lowest F tertile compared to those in the highest F tertile group, and the serum 17-OHPreg and 17-OHProg concentrations were parallel with the respective F concentrations., Conclusion: We did not find evidence of significant immaturity in adrenal 3β-HSD activity in preterm infants between 24 and 28 weeks of gestation., (Copyright © 2010 S. Karger AG, Basel.)

Patients with rare and complex diseases such as congenital adrenal hyperplasia (CAH) often receive fragmented and inadequate care unless efforts are coordinated among providers. Translating the concepts of the medical home and comprehensive health care for individuals with CAH offers many benefits for the affected individuals and their families. This manuscript represents the recommendations of a 1.5 day meeting held in September 2009 to discuss the ideal goals for comprehensive care centers for newborns, infants, children, adolescents, and adults with CAH. Participants included pediatric endocrinologists, internal medicine and reproductive endocrinologists, pediatric urologists, pediatric surgeons, psychologists, and pediatric endocrine nurse educators. One unique aspect of this meeting was the active participation of individuals personally affected by CAH as patients or parents of patients. Representatives of Health Research and Services Administration (HRSA), New York-Mid-Atlantic Consortium for Genetics and Newborn Screening Services (NYMAC), and National Newborn Screening and Genetics Resource Center (NNSGRC) also participated. Thus, this document should serve as a "roadmap" for the development phases of comprehensive care centers (CCC) for individuals and families affected by CAH.

Hashimoto encephalopathy is characterized by severe neuropsychiatric findings, including psychosis, confusion, seizures, stupor, stroke-like episodes, tremor, and myoclonus. The combination of findings is variable. Hashimoto encephalopathy constitutes an important differential diagnosis in patients with encephalopathy. The triad of encephalopathy, corresponding electroencephalographic slowing, and increased protein content in cerebrospinal fluid should prompt testing of anti-thyroid antibodies in blood and cerebrospinal fluid. Elevated antibody levels support the diagnosis. We describe a 15-year-old girl with a fluctuating course of Hashimoto encephalopathy. Electroencephalograms revealed no specific alterations, but widespread slowing of the background activity occurred during two episodes of fluctuating encephalopathy. Cortical edema was indicated by cranial magnetic resonance imaging during the first episode of encephalopathy, in the context of cerebral seizures. Laboratory findings were in accordance with Hashimoto encephalopathy, which was steroid-responsive.

Context: Steroid 17alpha-hydroxylase (CYP17A1, alias P450c17) deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The CYP17A1 enzyme catalyzes two distinct reactions, 17alpha-hydroxylase and 17,20-lyase activities., Objective: The aim of the study was to analyze the structural and functional consequences of three novel (A174E, V178D, and L465P) and one previously reported (R440C) CYP17A1 mutation found in three patients clinically and biochemically presenting with 17OHD., Patients and Methods: Two patients suffering from 46,XY disordered sex development presented at ages 5.5 and 8.8 yr, respectively, with tall stature and hypertension. Mutation analysis revealed compound heterozygous CYP17A1 mutations (A174E/K388X; V178D/R440C). The third patient (46,XX) presented with primary amenorrhea and hypertension at age 15 yr. She was homozygous for the novel L465P mutation. Functional studies employing a yeast microsomal expression system compared wild-type and mutant CYP17A1 both with regard to 17alpha-hydroxylase and 17,20-lyase activity. Mutants were examined in a computational three-dimensional model of the CYP17A1 protein., Results: The activity assays showed that all three mutants retain only 0-7% of both 17alpha-hydroxylase and 17,20-lyase activity relative to CYP17A1 wild-type activity, corresponding to the in vivo situation. Enzyme kinetic studies proved the impairment of both reactions, respectively. Computer-based three-dimensional model analysis of CYP17A1 using CYP2B4 as template showed that three of the mutations had no direct effect on the active center, whereas one affects the heme coordination., Conclusion: The functional studies revealed that the described missense mutations result in severe 17OHD. Our data are important to predict the phenotypic expressions and provide important information for patient management and genetic counseling.

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.

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., ((c) 2008 Wiley-Liss, Inc.)

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&#95;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., (Copyright 2009 S. Karger AG, Basel.)

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., (Copyright 2009 S. Karger AG, Basel.)

Background: Extreme hyponatremia (<105 mmol/l) has rarely been reported in infants. It is potentially life-threatening and requires intensive care treatment., Patient: We report on a male infant with absence of weight gain from birth to day 33 of life despite adequate nutrition. On admission serum sodium and potassium were 104 and 5.9 mmol/L respectively. The infant's physical status revealed dehydration, but normal activity with no apparent neurological, circulatory or respiratory impairment., Main Results: Global adrenocortical insufficiency was diagnosed and treated with hormonal substitution in addition to intravenous application of fluid, glucose and electrolytes. The rise of serum sodium was carefully monitored and adjusted to a target rate of 0.5 mmol/L/h. X-linked adrenal hypoplasia congenita (X-AHC) was confirmed by the identification of a novel nonsense NR0B1 (DAX-1) mutation (W236X)., Conclusions: X-AHC in infants may present as failure to thrive despite adequate nutrition. Extreme hyponatremia may be associated with little symptoms if developing slowly. Rehydration and slow correction of serum sodium with solutions containing less sodium than normal saline is essential.

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.

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.

Background/aim: 21-Hydroxylase deficiency congenital adrenal hyperplasia (CAH) is one of the target diseases in many newborn screening programs. 11beta-Hydroxylase defiency is less frequent and does not cause salt-losing crisis. Thus, it is not a target disease for newborn screening. However, affected newborns might show slightly elevated levels of 17-OH-progesterone (17-OHP) in the standard immunoassay screening test. The objective is to show that the diagnosis of 11beta-hydroxylase deficiency can be done using a dried blood spot from newborn screening., Case Report: A male newborn was born at term. Blood sample for newborn screening was taken 36 h after birth. 17-OHP was slightly elevated using time-resolved fluorescence immunoassay (72.8 nmol/l; cut-off <60 nmol/l)., Results: We performed second-tier LC-MS/MS from the same blood sample and found elevated levels of 11-deoxycortisol and androstenedione and low cortisol. The family history was positive with an affected older sister born with ambiguous genitalia. Confirmation of diagnosis was done by hormonal analysis and molecular genetic testing of the CYP11B1 gene. A known CYP11B1 gene mutation W116C was identified in this family., Conclusions: The diagnosis of 11beta-hydroxylase deficiency can be made by second-tier LC-MS/MS from dried blood spots. This method is very helpful in the work-up of elevated immunoassay 17-OHP., ((c) 2007 S. Karger AG, Basel)