Challenging Molecular Diagnosis of Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency: Case Series and Novel Variants of CYP21A2 Gene.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive genetic defects in cortisol synthesis and shows elevated ACTH concentrations, which in turn has downstream effects. The most common variant of CAH, 21-hydroxylase deficiency (21OHD), is the result of pathogenic variants in the CYP21A2 gene and is one of the most common monogenic disorders. However, the genetics of 21OHD is complex and challenging. The CYP21A2 gene is located in the RCCX copy number variation (CNV), a complex, multiallelic, and tandem CNV in the major histocompatibility complex (MHC) class III region on chromosome 6 (band 6p21.3). Here, CYP21A2 and its pseudogene CYP21A1P are located 30 kb apart and share a high nucleotide homology of approximately 98% and 96% in exons and introns, respectively. This high-sequence homology facilitates large structural rearrangements, copy number changes, and gene conversion through intergenic recombination. There is a good genotype-phenotype correlation in 21OHD, and genotyping can be performed to confirm the clinical diagnosis, predict long-term outcomes, and determine genetic counseling. Thus, genotyping in CAH is clinically relevant but the interpretations can be challenging for non-initiated clinicians. Here, there are some concrete examples of how molecular diagnosis can sometimes require the use of multiple molecular strategies.

Increased risk of nephrolithiasis: an emerging issue in children with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

PURPOSE: To investigate the incidence of nephrolithiasis in a cohort of children with congenital adrenal hyperplasia (CAH), and to study if there is an association with the metabolic control of the disease. METHODS: This study was designed as a multicenter 1 year-prospective study involving 52 subjects (35 males) with confirmed molecular diagnosis of CAH due to 21-hydroxylase deficiency (21-OHD). Each patient was evaluated at three different time-points: T0, T1 (+6 months of follow-up), T2 (+12 months of follow up). At each follow up visit, auxological data were collected, and adrenocorticotrophic hormone (ACTH), 17-hydroxyprogesterone (17-OHP), Δ4-androstenedione, dehydroepiandrosterone sulfate (DHEAS) serum levels, and urinary excretion of creatinine, calcium, oxalate and citrate were assayed. Moreover, a renal ultrasound was performed. RESULTS: The incidence of nephrolithiasis, assessed by ultrasound was 17.3% at T0, 13.5% at T1 and 11.5% at T2. At T0, one subject showed nephrocalcinosis. In the study population, a statistically significant difference was found for 17-OHP [T0: 11.1 (3.0-25.1) ng/mL; T1: 7.1 (1.8-19.9) ng/mL; T2: 5.9 (2.0-20.0) ng/mL, p < 0.005], and Δ4-androstenedione [T0: 0.9 (0.3-2.5) ng/mL; T1: 0.3 (0.3-1.1) ng/mL; T2: 0.5 (0.3-1.5) ng/mL, p < 0.005] which both decreased over the follow up time. No statistically significant difference among metabolic markers was found in the group of the subjects with nephrolithiasis, even if 17-OHP, DHEAS and Δ4-androstenedione levels showed a tendency towards a reduction from T0 to T2. Principal component analysis (PCA) was performed to study possible hidden patterns of associations/correlations between variables, and to assess the trend of them during the time. PCA revealed a decrease in the amount of the variables 17-OHP, Δ4-androstenedione, and ACTH that occurred during follow-up, which was also observed in subjects showing nephrolithiasis. CONCLUSIONS: our data demonstrated that children affected with 21-OHD can be at risk of developing nephrolithiasis. Additional studies are needed to clarify the pathogenesis and other possible risk factors for this condition, and to establish if regular screening of kidney ultrasound in these patients can be indicated.

A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency.

CONTEXT: Recently developed long-read sequencing (LRS) technology has been considered an option for CYP21A2 analysis. However, the clinical use of LRS for CYP21A2 analysis is limited. OBJECTIVE: This study's objective is to develop an efficient and low-cost LRS system for CYP21A2 screening. METHODS: A DNA fragment library was prepared in a single polymerase chain reaction (PCR) that covers the entire CYP21A2 gene and all known junctions caused by TNXB gene structural rearrangements, yielding a single 8-kb product of CYP21A2 or CYP21A1P/CYP21A2 chimera. After barcoding, the PCR products were sequenced on a MinION-based platform with Flongle Flow Cell R9.4.1 and R10.4.1. RESULTS: The reference genotypes of 55 patients with 21-hydroxylase deficiency (21OHD) were established using the conventional method with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS using Flongle Flow Cell R9.4.1 yielded consistent results. Additionally, the recently updated LRS "duplex" analysis with Flongle flow cell R10.4.1 was tested to reveal an advantage of accurately sequencing a variant located on the homopolymer region. By introducing a barcode system, the cost was reduced to be comparable to that of conventional analysis. A novel single-nucleotide variation was discovered at the acceptor site of intron 7, c.940-1G > C. We also identified a subtype of the classical chimeric junction CH2, "CH2a," in the region from the latter part of intron 5 to exon 6. CONCLUSION: We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.

The clinical and biochemical significance of 11-oxygenated androgens in human health and disease.

For many decades, the prevailing paradigm in endocrinology was that testosterone and 5α-dihydrotestosterone are the only potent androgens in the context of human physiology. The more recent identification of adrenal derived 11-oxygenated androgens and particularly 11-ketotestosterone have challenged these established norms, prompting a revaluation of the androgen pool, particularly in women. Since being recognized as bone fide androgens in humans, numerous studies have focused their attention on understanding the role of 11-oxygenated androgens in human health and disease and have implicated them as role players in conditions such as castration resistant prostate cancer, congenital adrenal hyperplasia, polycystic ovary syndrome, Cushing's syndrome, and premature adrenarche. This review therefore provides an overview of our current knowledge on the biosynthesis and activity of 11-oxygenated androgens with a focus on their role in disease states. We also highlight important analytical considerations for measuring this unique class of steroid hormone.

Challenges in treatment of patients with non-classic congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) due to 21α-hydroxylase deficiency (21OHD) or 11ß-hydroxylase deficiency (11OHD) are congenital conditions with affected adrenal steroidogenesis. Patients with classic 21OHD and 11OHD have a (nearly) complete enzyme deficiency resulting in impaired cortisol synthesis. Elevated precursor steroids are shunted into the unaffected adrenal androgen synthesis pathway leading to elevated adrenal androgen concentrations in these patients. Classic patients are treated with glucocorticoid substitution to compensate for the low cortisol levels and to decrease elevated adrenal androgens levels via negative feedback on the pituitary gland. On the contrary, non-classic CAH (NCCAH) patients have more residual enzymatic activity and do generally not suffer from clinically relevant glucocorticoid deficiency. However, these patients may develop symptoms due to elevated adrenal androgen levels, which are most often less elevated compared to classic patients. Although glucocorticoid treatment can lower adrenal androgen production, the supraphysiological dosages also may have a negative impact on the cardiovascular system and bone health. Therefore, the benefit of glucocorticoid treatment is questionable. An individualized treatment plan is desirable as patients can present with various symptoms or may be asymptomatic. In this review, we discuss the advantages and disadvantages of different treatment options used in patients with NCCAH due to 21OHD and 11OHD.

The High Relevance of 21-Deoxycortisol, (Androstenedione + 17α-Hydroxyprogesterone)/Cortisol, and 11-Deoxycortisol/17α-Hydroxyprogesterone for Newborn Screening of 21-Hydroxylase Deficiency.

CONTEXT: There are limited reports on the detailed examination of steroid profiles for setting algorithms for 21-hydroxylase deficiency (21OHD) screening by liquid chromatography-tandem mass spectrometry (LC-MS/MS). OBJECTIVE: We aimed to define an algorithm for newborn screening of 21OHD by LC-MS/MS, measuring a total of 2077 dried blood spot samples in Tokyo. METHODS: Five steroids (17α-hydroxyprogesterone [17αOHP], 21-deoxycortisol [21DOF], 11-deoxycortisol [11DOF], androstenedione [4AD], and cortisol [F]) were included in the panel of LC-MS/MS. Samples from 2 cohorts were assayed: Cohort A, 63 "screening positive" neonates who were referred to an endocrinologist (n = 26 with 21OHD; n = 37 false-positive; obtained from 2015 to 2020); and Cohort B, samples (n = 2014) with 17αOHP values in the 97th percentile or above, in the first-tier test with 17αOHP ELISA from 2020 to 2021. RESULTS: Analysis of Cohort A revealed that the 3 indexes 21DOF, 11DOF/17αOHP, and (4AD + 17αOHP)/F had higher area under the curve (AUC) values (0.999, 0.997, 0.989, respectively), while the 17αOHP AUC was lower (0.970). Accordingly, in addition to 17αOHP, the 3 markers were included for defining the screening algorithm. The assay of Cohort B revealed that the new algorithm gave 92% of predicted positive predictive value without false-negative cases. We also determined the reference values for the 5 steroids at 4 to 7 days after birth, according to sex and gestational age (GA), revealing extremely low levels of 21DOF at any GA irrespective of sex differences. CONCLUSION: Our study demonstrated the high relevance of 21DOF, (4AD + 17αOHP)/F, and 11DOF/17αOHP, rather than 17αOHP, for 21OHD screening.

Back where it belongs: 11ß-hydroxyandrostenedione compels the re-assessment of C11-oxy androgens in steroidogenesis.

Adrenal steroidogenesis has, for decades, been depicted as three biosynthesis pathways -the mineralocorticoid, glucocorticoid and androgen pathways with aldosterone, cortisol and androstenedione as the respective end products. 11ß-hydroxyandrostenedione was not included as an adrenal steroid despite the adrenal output of this steroid being twice that of androstenedione. While it is the end of the line for aldosterone and cortisol, as it is in these forms that they exhibit their most potent receptor activities prior to inactivation and conjugation, 11ß-hydroxyandrostenedione is another matter entirely. The steroid, which is weakly androgenic, has its own designated pathway yielding 11-ketoandrostenedione, 11ß-hydroxytestosterone and the potent androgens, 11-ketotestosterone and 11-ketodihydrotestosterone, primarily in the periphery. Over the last decade, these C11-oxy C19 steroids have once again come to the fore with the rising number of studies contradicting the generally accepted notion that testosterone and it's 5α-reduced product, dihydrotestosterone, are the principal potent androgens in humans. These C11-oxy androgens have been shown to contribute to the androgen milieu in adrenal disorders associated with androgen excess and in androgen dependant disease progression. In this review, we will highlight these overlooked C11-oxy C19 steroids as well as the C11-oxy C21 steroids and their contribution to congenital adrenal hyperplasia, polycystic ovarian syndrome and prostate cancer. The focus is on new findings over the past decade which are slowly but surely reshaping our current outlook on human sex steroid biology.

11α-Hydroxyprogesterone, a potent 11ß-hydroxysteroid dehydrogenase inhibitor, is metabolised by steroid-5α-reductase and cytochrome P450 17α-hydroxylase/17,20-lyase to produce C11α-derivatives of 21-deoxycortisol and 11-hydroxyandrostenedione in vitro.

11α-Hydroxyprogesterone (11αOHP4) and 11ß-hydroxyprogesterone (11ßOHP4) have been reported to be inhibitors of 11ß-hydroxysteroid dehydrogenase (11ßHSD) type 2, together with 11ß-hydroxytestosterone and 11ß-hydroxyandrostenedione, and their C11-keto derivatives being inhibitors of 11ßHSD1. Our in vitro assays in transiently transfected HEK293 cells, however, show that 11αOHP4 is a potent inhibitor of 11ßHSD2 and while this steroid does not serve as a substrate for the enzyme, the aforementioned C11-oxy steroids are indeed substrates for both 11ßHSD isozymes. 11ßOHP4 is metabolised by 11ßHSD2 yielding 11-ketoprogesterone with 11ßHSD1 catalysing the reverse reaction, similar to the reduction of the other C11-oxy steroids. In the same model system, novel 11αOHP4 metabolites were detected in its conversion by steroid-5α-reductase (SRD5A) types 1 and 2 yielding 11α-hydroxydihydroprogesterone and its conversion by cytochrome P450 17A1 (CYP17A1) yielding the hydroxylase product, 11α,17α-dihydroxyprogesterone, and the 17,20 lyase product, 11α-hydroxyandrostenedione. We also detected both 11αOHP4 and 11ßOHP4 in prostate cancer tissue- ∼23 and ∼32 ng/g respectively with 11KP4 levels >300 ng/g. In vitro assays in PC3 and LNCaP prostate cancer cell models, showed that the metabolism of 11αOHP4 and 11ßOHP4 was comparable. In LNCaP cells expressing CYP17A1, 11αOHP4 and 11ßOHP4 were metabolised with negligible substrate, 4%, remaining after 48 h, while the steroid substrate 11ß,17α-dihydroxyprogesterone (21dF) was metabolised to C11-keto C19 steroids yielding 11-ketotestosterone. Despite the fact that 11αOHP4 is not metabolised by 11ßHSD2, it is a substrate for SRD5A and CYP17A1, yielding C11α-hydroxy C19 steroids as well as the C11α-hydroxy derivative of 21dF-the latter associated with clinical conditions characterised by androgen excess. With our data showing that 11αOHP4 is present at high levels in prostate cancer tissue, the steroid may serve as a precursor to unique C11α-hydroxy C19 steroids. The potential impact of 11αOHP4 and its metabolites on human pathophysiology can however only be fully assessed once C11α-hydroxyl metabolite levels are comprehensively analysed.

Genotype-phenotype correlation study and mutational and hormonal analysis in a Chinese cohort with 21-hydroxylase deficiency.

BACKGROUND: Steroid 21-hydroxylase deficiency (21OHD) is the most common enzymatic defect, but the genotype-phenotype associations have not been well established in Chinese patients. Here, a Chinese 21OHD cohort was enrolled to investigate the clinical, biochemical, and genetic characteristics of this disorder. METHODS: Mutation analysis of CYP21A2 gene, 21-hydroxylase activity assays and in silico predictions of protein structure were performed. Genotype-phenotype associations were analyzed in both the cohort and 487 Chinese CAH patients ever reported. RESULTS: Among the total cohort (72 patients), 47 patients (65.3%) were diagnosed as salt-wasting (SW) phenotype, 11 (15.3%) were simple virilizing (SV) type, and 14 (19.4%) were nonclassic (NC) type. The value of FSH and LH for prediction of the SW phenotype was up to 0.862 and 0.669, respectively. Overall, the detection rate of CYP21A2 mutation was 97.9%, which revealed 25 mutations and 36 genotypes. Four novel mutations (p.L199X, p.E321del, p.H393Q, and p.L459-P464del) were detected and induced a significantly reduced 21-hydroxylase activity. Generally, disease severity can be predicted with the genotypes. The most common genotypes in Chinese population were I2G/I2G (12.5%), I2G/Large lesion (12.1%), I173N/I2G (10.3%), and I173N/Large lesion (9.2%). The SW form of CAH is prominent in deletion or intronic splice mutations, namely I2G/I2G (18.6%), I2G/Large lesion (17.2%) and Large lesion/Large lesion (8.6%). CONCLUSION: Four novel mutations were identified and a high consistency of genotype-phenotype association was found in SW CAH. Moreover, FSH and LH levels were proved to be a promising marker for predicting the severity of the disease.

The 11ß-hydroxysteroid dehydrogenase isoforms: pivotal catalytic activities yield potent C11-oxy C19 steroids with 11ßHSD2 favouring 11-ketotestosterone, 11-ketoandrostenedione and 11-ketoprogesterone biosynthesis.

The 11ß-hydroxysteroid dehydrogenase (11ßHSD) types 1 and 2 are primarily associated with glucocorticoid inactivation and reactivation. Several adrenal C11-oxy C19 and C11-oxy C21 steroids, which have been identified in prostate cancer, 21-hydroxylase deficiency and polycystic ovary syndrome, are substrates for these isozymes. This study describes the kinetic parameters of 11ßHSD1 and 11ßHSD2 towards the C11-keto and C11-hydroxy derivatives of the C19 and C21 steroids. The apparent Km and Vmax values indicate the more prominent 11ßHSD2 activity towards 11ß-hydroxy androstenedione, 11ß-hydroxytestosterone and 11ß-hydroxyprogesterone in contrast to the 11ßHSD1 reduction of the C11-keto steroids, as was demonstrated in the LNCaP cell model in the production of 11-ketotestosterone and 11-ketodihydrotestosterone. Data highlighted the role of 11ßHSD2 and cytochrome P450 17A1 in the contribution of C11-oxy C21 steroids to the C11-oxy C19 steroid pool in the C11-oxy backdoor pathway. In addition, 11ßHSD2 activity, catalysing 11-ketotestosterone biosynthesis, was shown to be key in the production of prostate specific antigen and in the progression of prostate cancer to castration resistant prostate cancer. The study at hand thus provides evidence that 11ßHSD isozymes play key roles in pathophysiological states, more so than was previously put forward.

Clinical presentation and mutational spectrum in a series of 166 patients with classical 21-hydroxylase deficiency from South China.

Classical 21-hydroxylase deficiency (21-OHD) due to mutations in the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene is the most common type of congenital adrenal hyperplasia (CAH). In this study, we analyzed clinical and molecular data of 166 patients with classical CAH in South China. Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) method were used to detect mutations in these 99 salt wasting (SW) patients and 67 simple virilizing (SV) patients. Micro-conversion mutation IVS2-13A/C > G (I2G) was the most frequent mutation in both SW form (42.9%) and SV form (41.8%) in our large cohort, and large gene deletion or large gene conversion also commonly resulted in classical CAH. Rare mutations only account for 8.4% of all alleles, among them four novel variants p.S126X, p.C429X, c.1209_1210insT and c.840delG were responsible for the clinical presentations. CYP21A2 gene duplications linked to the mutation Q319X were found in our cohort, though these cases were rather rare. In this study, we provided detailed clinical data and mutation spectrum to confirm the common mutations in Chinese populations, especially in South China,which will contribute to further genetic consultation and prenatal diagnosis. Sanger sequencing combined with MLPA method could detect most mutation types in the CYP21A2 gene effectively.

The in vitro metabolism of 11ß-hydroxyprogesterone and 11-ketoprogesterone to 11-ketodihydrotestosterone in the backdoor pathway.

Increased circulating 11ß-hydroxyprogesterone (11OHP4), biosynthesised in the human adrenal, is associated with 21-hydroxylase deficiency in congenital adrenal hyperplasia. 17α-hydroxyprogesterone levels are also increased, with the steroid's metabolism to dihydrotestosterone in the backdoor pathway contributing to hyperandrogenic clinical conditions. In this study we investigated the in vitro biosynthesis and downstream metabolism of 11OHP4. Both cytochrome P450 11ß-hydroxylase and aldosterone synthase catalyse the biosynthesis of 11OHP4 from progesterone (P4) which is converted to 11-ketoprogesterone (11KP4) by 11ß-hydroxysteroid dehydrogenase type 2, while type 1 readily catalysed the reverse reaction. We showed in HEK-293 cells that these C11-oxy C21 steroids were metabolised by steroidogenic enzymes in the backdoor pathway-5α-reductase (SRD5A) and 3α-hydroxysteroid type 3 (AKR1C2) converted 11OHP4 to 5α-pregnan-11ß-ol,3,20-dione and 5α-pregnan-3α,11ß-diol-20-one, while 11KP4 was converted to 5α-pregnan-3,11,20-trione and 5α-pregnan-3α-ol-11,20-dione (alfaxalone), respectively. Cytochrome P450 17α-hydroxylase/17,20-lyase catalysed the hydroxylase and lyase reaction to produce the C11-oxy C19 steroids demonstrated in the conversion of alfaxalone to 11-oxy steroids demonstrated in the conversion of alfaxalone to 11ketoandrosterone. In LNCaP cells, a prostate cancer cell model endogenously expressing the relevant enzymes, 11OHP4 and 11KP4 were metabolised to the potent androgen, 11-ketodihydrotestosterone (11KDHT), thus suggesting the C11-oxy C21 steroids contribute to the pool of validating the in vitro biosynthesis of C11-oxy C19 steroids from C11-oxy C21 steroids. The in vitro reduction of 11KP4 at C3 and C5 by AKR1C2 and SRD5A has confirmed the metabolic route of the urinary metabolite, 3α,20α-dihydroxy-5ß-pregnan-11-one. Although our assays have demonstrated the conversion of 11OHP4 and 11KP4 by steroidogenic enzymes in the backdoor pathway yielding 11KDHT, thus suggesting the C11-oxy C21 steroids contribute to the pool of potent androgens, the in vivo confirmation of this metabolic route remains challenging.

Adrenal C11-oxy C21 steroids contribute to the C11-oxy C19 steroid pool via the backdoor pathway in the biosynthesis and metabolism of 21-deoxycortisol and 21-deoxycortisone.

21-Hydroxylase deficiency presents with increased levels of cytochrome P450 21-hydroxylase substrates, progesterone and 17α-hydroxyprogesterone, which have been implicated in the production of androgens via the backdoor pathway. This study shows the biosynthesis of C11-oxy C21 steroids, 21-deoxycortisol and 21-deoxycortisone, and their metabolism by steroidogenic enzymes in the backdoor pathway yielding novel steroid metabolites: 5α-pregnan-11ß,17α-diol-3,20-dione; 5α-pregnan-17α-ol-3,11,20-trione; 5α-pregnan-3α,11ß,17α-triol-20-one and 5α-pregnan-3α,17α-diol-11,20-dione. The metabolism of 21-deoxycortisol was validated in LNCaP cells expressing the relevant steroidogenic enzymes showing for the first time that the steroid, produced at high levels in 21OHD, is metabolised via the C11-oxy derivatives of 5α-pregnan-17α-ol-3,20-dione and 5α-pregnan-3α,17α-diol-20-one to substrates for the lyase activity of CYP17A1, leading to the production of C11-oxy C19 steroids. 21-Deoxycortisol thus contributes to the pool of potent androgens in 21OHD, with novel steroid metabolites also presenting possible biomarkers in disease identification.

Lack of genotypephenotype correlation in congenital adrenal hyperplasia due to a CYP21A2-like gene.

CONTEXT: Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, encoded by CYP21A2 gene, is an autosomal recessive disorder. The CYP21A2 gene, localized in a genetic unit defined RCCX module, is considered one of the most polymorphic of human genes. OBJECTIVES: We considered new evidences about the presence of a RCCX trimodular haplotype with a CYP21A2-like gene to explain the lack of a genotype-phenotype correlation in individuals of two different families. DESIGN AND METHODS: To verify gene duplication we used Multiplex Ligation Probe-Dependent Amplifications (MLPA) and to confirm the presence of a CYP21A2-like gene downstream TNXA gene we used previously described amplification and restriction strategy followed by the sequencing of the CYP21A2 gene downstream TNXB gene. RESULTS: The amplification strategy and restriction analysis of CYP21A1P/CYP21A2-TNXA PCR product in association with MLPA assay and sequencing of CYP21A2 gene downstream TNXB were able to identify the presence of the CYP21A2-like gene in healthy subjects of the two families, wherein the direct sequencing of CYP21A2 gene showed genotypes correlated to pathological phenotypes. CONCLUSIONS: The strategy suggested is useful to facilitate molecular testing in CAH patients, considering the new evidence about possible different haplotypes.

In Vitro Enzyme Assay of CYP21A2 Mutation (R483Q) by A Novel Method Using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS).

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive disorders in humans, and 21-hydroxylase deficiency (21-OHD) accounts for 90 to 95% of all cases of CAH. Approximately 95% mutations are a consequence of recombination between the CYP21A2 and its highly homologous pseudogene CYP21A1P. Recently, other rare mutations have been identified, increasing the number of reported mutations to more than eighty. The in vitro enzyme assay for the detection of mutated 21-hydroxylase is a well-established method. In this study, we report the characterization of the R483Q mutation using a novel in vitro enzyme assay, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). With this system, we evaluated the activity of the R483Q mutation. The enzyme activities of 21-hydroxylase in the convertion of progesterone to deoxycorticosterone (DOC), and 17-hydroxyprogesterone (17-OHP) to 11-deoxycortisol (11-DOF), were measured as 2.00 ± 0.25% and 1.89 ± 0.30% of the wild type, respectively. This result was in agreement with that of a previous report, which measured the activities using the (3)H labeled steroid assay. Our results suggest that the R483Q mutation is compatible with the simple virilizing form of 21-OHD and that the LC-ESI-MS/MS assay using picolinoyl derivatives is an alternative to the existing (3)H-labeled steroid assay for the characterization of the CYP21A2 mutation.

A Case of a Preterm Infant with 21-Hydroxylase Deficiency: Implications of the Biochemical Diagnosis with Urinary Pregnanetriolone by Gas Chromatography/Mass Spectrometry in Selected Ion Monitoring (GCMS-SIM).

The biochemical diagnosis of 21-hydroxylase deficiency (21-OHD) is difficult in preterm infants. To date, no marker for the biochemical diagnosis of 21-OHD has been found. Seventeen α-hydroxyprogesterone (17-OHP), is not useful because of interference by delta 5 steroids from the fetal adrenal cortex. A 5-d-old female infant, born at 31 wk of gestation, was suspected of having 21-OHD based on physical findings (mild clitoromegaly, pigmentation of the tongue and gingiva) as well as laboratory data (17-OHP >93.5 ng/ml by ELISA 7 prime extractive method in filter paper-dried blood spot and 718.3 ng/ml by RIA after high performance liquid chromatography extraction in serum; plasma ACTH 690 pg/ml; and serum testosterone 3,169 ng/dl). We examined her urinary steroid profiles by gas chromatography/mass spectrometry in selected ion monitoring (GCMS-SIM) at 8 d of age. The pregnanetriolone (Ptl) level was noticeably high (0.80 mg/g creatinine), which was strongly suggestive of 21-OHD. Gene analysis of CYP21A2 showed compound heterozygosity, one allele having a cluster mutation in exon 6 and the other having a large deletion including CYP21A2, confirming the diagnosis of 21-OHD. This case suggested that, in preterm infants, urinary Ptl by GCMS-SIM can be useful for the biochemical diagnosis of 21-OHD.