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PubMed2006

A leuC mutation leading to increased L-lysine production and rel-independent global expression changes in Corynebacterium glutamicum.

We previously found by transcriptome analysis that global induction of amino acid biosynthetic genes occurs in a classically derived industrial L-lysine producer, Corynebacterium glutamicum B-6. Based on this stringent-like transcriptional profile in strain B-6, we analyzed the relevant mutations from among those identified in the genome of the strain, with special attention to the genes that are involved in amino acid biosynthesis and metabolism. Among these mutations, a Gly-456-->Asp mutation in the 3-isopropylmalate dehydratase large subunit gene (leuC) was defined as a useful mutation. Introduction of the leuC mutation into a defined L-lysine producer, AHD-2 (hom59 and lysC311), by allelic replacement led to the phenotype of a partial requirement for L-leucine and approximately 14% increased L-lysine production. Transcriptome analysis revealed that many amino acid biosynthetic genes, including lysC-asd operon, were significantly upregulated in the leuC mutant in a rel-independent manner.

Corynebacterium glutamicum↗

PubMed2005

Comparative genomic hybridization profiles in human BRCA1 and BRCA2 breast tumors highlight differential sets of genomic aberrations.

BRCA1 or BRCA2 germline mutations cause approximately 30% of breast cancers within high-risk families. This represents 5% of total breast cancer incidence. Although BRCA1 and BRCA2 are both implicated in DNA repair and genome stability, it is unknown whether BRCA1 and BRCA2 are associated with similar or distinct diseases. In a previous study we reported that BRCA1-related breast carcinomas show a distinct genomic profile as determined by comparative genomic hybridization (CGH). We now hypothesize that, if functionally equivalent, mutations in BRCA1 and BRCA2 would result in similar genomic profiles in tumors. Here we report the chromosomal gains and losses as measured by CGH in 25 BRCA2-associated breast tumors and compared them with our existing 36 BRCA1 and 30 control profiles. We compared all chromosomal regions and determined the regions of differential gain or loss between tumor classes and controls. BRCA2 and control tumors have very similar genomic profiles. As a consequence, and in contrast to BRCA1-associated tumors, CGH profiles from BRCA2-associated tumors could not be distinguished from control tumors using the classification methodology as we have developed before. The largest number of significant differences existed between BRCA1 and controls, followed by BRCA1 compared with BRCA2, suggesting different tumor development pathways for BRCA1 and BRCA2.

Breast Neoplasms↗

PubMed2003

Fluoroquinolones: structure and target sites.

The quinolones are a potent group of drugs that target the essential bacterial enzymes DNA gyrase and topoisomerase IV. DNA gyrase is the primary target of Gram negative organisms however, it is topoisomerase IV that is the primary target of Gram positive organisms. Within these enzymes is a highly conserved region centered round the active site where resistance mutations occur. These mutations are almost always identical, irrespective of organism. In spite of the homology of this region, amino acid sequence analysis shows that there are defined differences between the Gram groups, particularly in topoisomerase IV, and it is speculated that herein lies the origin of target preference. Since the first quinolone nalidixic acid was developed, the quinolones have undergone structural modifications, in particular the addition of a fluorine at position 6, to produce the fluoroquinolones. This has seen their potency and pharmakokinetic profile greatly increase. In vitro selection of resistance mutations has allowed the observation of how resistance is acquired and some of the modifications in newer fluoroquinolones have resulted in the shift of primary target from topoisomerase IV to gyrase with Gram positives. Curiously, purified topoisomerase IV is still more sensitive even if gyrase is the primary target. Gyrase remains the primary target for Gram negatives.

Amino Acid Sequence↗

PubMed2026

Real-World Actionability Analysis of Comprehensive Genomic Profiling Versus Single/Small-Gene Panels.

INTRODUCTION: Comprehensive genomic profiling (CGP) enables identification of patients eligible for targeted treatments, making it essential in the management of advanced cancer. This retrospective real-world study compared actionable mutations in CGP-tested patients with advanced/metastatic solid tumors to those who received single-gene/small-panel (SP) tests. METHODS: Patients aged > 18 years with advanced/metastatic solid tumors (including non-small cell lung cancer (NSCLC), colorectal cancer, prostate cancer, breast cancer, or melanoma) with a CGP or SP test reported between 1 January 2018, and 31 December 2022, were included. OncoKB-derived actionability was compared between the two cohorts. Inverse probability of treatment weighting (IPTW) was used to adjust for baseline characteristics. A weighted generalized linear model with log link was used to report actionability ratio (AR) and 95% confidence intervals (CI). RESULTS: Among 406 patients (CGP-tested cohort: 202, SP-tested cohort: 204), approximately half were diagnosed with NSCLC. After adjusting for baseline characteristics, CGP testing detected more actionable alterations than SP: OncoKB level 1 (50.0% versus 31.4%; AR 1.76 [95% CI: 1.24, 2.51]; p = 0.002), OncoKB level 2 (22.8% versus 10.3%; AR 2.53 [95% CI: 1.17, 5.48]; p = 0.018), and OncoKB level 1 or level 2 or level R1 (55.9% versus 41.2%; AR 1.5 [95% CI: 1.11, 2.01]; p = 0.008). CONCLUSIONS: CGP testing identified more actionable genetic alterations compared with SP testing methods for patients with advanced/metastatic NSCLC, colorectal cancer, prostate cancer, breast cancer, or melanoma. Expanding reimbursement and coverage for CGP testing as well as expanding CGP use can facilitate equitable treatment access for patients with advanced/metastatic cancer.

Actionable mutations↗

PubMed2006

Mutation screening of mismatch repair gene Mlh3 in familial esophageal cancer.

AIM: To shed light on the possible role of mismatch repair gene Mlh3 in familial esophageal cancer (FEC). METHODS: A total of 66 members from 10 families suggestive of a genetic predisposition to hereditary esophageal cancer were screened for germline mutations in Mlh3 with denaturing high performance liquid chromatography (DHPLC), a newly developed method of comparative sequencing based on heteroduplex detection. For all samples exhibiting abnormal DHPLC profiles, sequence changes were evaluated by cycle sequencing. For any mutation in family members, we conducted a segregation study to compare its prevalence in sporadic esophageal cancer patients and normal controls. RESULTS: Exons of Mlh3 in all samples were successfully examined. Overall, 4 missense mutations and 3 polymorphisms were identified in 4 families. Mlh3 missense mutations in families 9 and 10 might be pathogenic, but had a reduced penetrance. While in families 1 and 7, there was no sufficient evidence supporting the monogenic explanations of esophageal cancers in families. The mutations were found in 33% of high-risk families and 50% of low-risk families. CONCLUSION: Mlh3 is a high risk gene with a reduced penetrance in some families. However, it acts as a low risk gene for esophageal cancer in most families. Mutations of Mlh3 may work together with other genes in an accumulated manner and result in an increased risk of esophageal tumor. DHPLC is a robust and sensitive technique for screening gene mutations.

Carrier Proteins↗

PubMed2023

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

src-Family Kinases↗

PubMed2004

Characterization of structural determinants of granzyme B reveals potent mediators of extended substrate specificity.

Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile99, Tyr174, Arg192, and Asn218. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile99 completely defines and predicts P2 specificity, primarily through the binding constant Km. Asn218 variants have minor effects alone but in combination with mutations at Arg192 and Ile99 alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn218 variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn218 not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members.

Alanine↗

PubMed1996

Mutagenesis of transmembrane domain 11 of P-glycoprotein by alanine scanning.

The biochemical and genetic analyses of P-glycoprotein (P-gp) have indicated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport. Predicted transmembrane domain 11 (TM11) maps near a major drug binding site revealed by photoaffinity labeling, and mutations in this domain alter the substrate specificity of P-gp. To investigate further the role of TM11 in P-gp function in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of endogenous alanines. After transfection and overexpression in Chinese hamster ovary cells, pools of stable transfectants were analyzed for qualitative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by the wild-type protein. While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of activity against one of the four drugs tested. Although the phenotype of individual mutants was varied, replacements at most mutation-sensitive positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp. When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutral mutations was not random but segregated with a more hydrophobic (mutation-insensitive) face and a more hydrophilic (mutation-sensitive) face of a putative amphipathic helix. The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more hydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp. Finally, the conservation of the two residues most sensitive to mutations (Y949 and Y953) in TM11, and in the homologous TM5, of all mammalian P-gps and also in other ABC transporters, suggests that these residues and domains may play an important role in structural as well as mechanistic aspects common to this family of proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Modulation of polyglutamine-induced cell death by genes identified by expression profiling.

The majority of triplet-repeat diseases are caused by mutated genes with an extended polyglutamine tract, exemplified by Huntington's disease (HD). In order to model HD pathogenesis in a controlled system, we developed stable PC12 cell lines that express exon 1 fragments of the huntingtin gene with 23 or 74 polyglutamines driven by an inducible doxycycline (dox)-sensitive promoter (HD-23Q or HD-74Q). We aimed to identify early perturbations induced by the mutation by studying expression levels of 1824 genes at 0, 5, 10 and 18 hours after induction, using adaptor-tagged competitive PCR (ATAC-PCR). At these time points, the cells show no appreciable death or mitochondrial impairment and very low inclusion levels. A total of 126 genes, including 69 known genes, exhibited statistically significant alterations in the HD-74Q cell lines but no changes in the HD-23Q lines. We tested 11 of these genes for their abilities to modulate polyglutamine-induced cell death in transiently transfected cell models. Five genes [glucose transporter 1 (Glut1), phosphofructokinase muscle isozyme (Pfkm), prostate glutathione-S -transferase 2 (Gstm2), RNA-binding motif protein 3 (Rbm3) and KRAB-A interacting protein 1 (Krip-1)] significantly suppressed cell death in both neuronal precursor and non-neuronal cell lines, suggesting that these transcriptional changes were relevant to polyglutamine pathology. The efficient recovery of functionally relevant genes supports the utility of gene expression profiling for discovering pathways related to pathogenesis, and the importance of analyzing molecular events in the early stages of disease.

Animals↗

PubMed2006

Cognitive deficits in familial Alzheimer's disease associated with M239V mutation of presenilin 2.

The neuropsychological assessment of non-demented subjects with gene mutation of familial Alzheimer's disease (AD) provides a model for exploring the early cognitive features of the disease. We evaluated 1 patient and 6 non-demented subjects belonging to a family with AD with M239V mutation of the presenilin 2 gene, aiming to verify the contribution of specific cognitive patterns to the characterization of familial AD. One patient, 3 non-demented subjects with M239V mutation and 3 subjects without mutation from the same family underwent neuropsychological testing. The patient's cognitive profile was characterized by anosognosia, visuospatial agnosia, apraxia and fluent aphasia. Of the 3 non-demented subjects with mutation, 1 showed no deficits, another constructive apraxia and the third spatial perception and memory deficits. The 3 subjects without mutation showed normal abilities. The cognitive deficits of the non-demented subjects with mutations indicate focal dysfunction of the posterior cortical areas, resembling the more extended parieto-occipito-temporal dysfunction of the demented patient. Such grading of visuospatial, praxis, and language impairments highlights a distinctive pattern related to the M239V mutation of the presenilin 2 gene.

Aged↗

PubMed2006

Global transcriptome analysis of the responses of a fluoroquinolone-resistant Streptococcus pneumoniae mutant and its parent to ciprofloxacin.

Streptococcus pneumoniae M22 is a multidrug-resistant mutant selected after exposure of capsulated wild-type S. pneumoniae NCTC 7465 (strain M4) to ciprofloxacin. DNA microarray analysis comparing the gene expression profiles of strain M22 with those of strain M4 showed that strain M22 constitutively expressed 22 genes at levels higher than those observed in strain M4 under all conditions studied. These included the genes encoding the enzymes involved in branched-chain amino acid biosynthesis and two genes (patA and patB) with sequences suggestive of ABC transporter proteins. Expression of the patA and patB genes was induced by ciprofloxacin in both strains, but in strain M4 it only reached the levels observed in strain M22 after long incubation with high concentrations of ciprofloxacin. The altered expression profile observed with strain M22 suggested that the mutation or mutations acquired during resistance selection bring the cell into a state in which the expression of critical genes is preemptively altered to correct for the potential effects of ciprofloxacin on gene expression in the parent strain.

Ciprofloxacin↗

PubMed2006

A role for immunoglobulin G in donor-specific Streptococcus sanguis-induced platelet aggregation.

There is increasing evidence for a relationship between bacterial infections and several cardiovascular disorders. Although the precise mechanism(s) underlying this association is unknown, the direct activation of platelets by bacteria is one possibility. Individual strains of S. sanguis activate platelets in a non-uniform, donor-dependent manner. In the current study, platelet aggregation profiles were obtained for fourteen donors in response to four strains of S. sanguis (2017-78, 133-79, SK112, SK108a) and one of S. gordonii (SK8) . The platelets from all donors responded to strains 2017-78 and 133-79, whereas strains SK112, SK8 and SK108a caused aggregation in one, five and twelve donors, respectively. Immunoglobulin G (IgG) binding to strains 2017-78, 133-79 and SK108a were significantly greater than to strains SK112 and SK8. Absorption of IgG by strain 2017-78 caused significant decreases in IgG binding, and platelet aggregation in response, to all strains. Single-strand conformational polymorphisms were observed in the Fcgamma RIIA gene from four donors. Sequencing revealed two known and two novel point mutations, none of which correlated with the aggregation profile. Thus, platelet activation to the various strains depends on a common IgG and, while in most cases the level of IgG binding to S. sanguis determines platelet responsiveness, neither the levels of IgG nor FcgammaRIIA polymorphisms can fully account for donor variability.

Antigens, CD↗

PubMed2006

Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk.

AIMS: To determine the relative frequency of mutations in three different genes (low-density lipoprotein receptor (LDLR), APOB, PCSK9), and to examine their effect in development of coronary heart disease (CHD) in patients with clinically defined definite familial hypercholesterolaemia in UK. PATIENTS AND METHODS: 409 patients with familial hypercholesterolaemia patients (158 with CHD) were studied. The LDLR was partially screened by single-strand conformational polymorphism (SSCP) (exons 3, 4, 6-10 and 14) and by using a commercial kit for gross deletions or rearrangements. APOB (p.R3500Q) and PCSK9 (p.D374Y) were detected by specific assays. Coding exons of PCSK9 were screened by SSCP. RESULTS: Mutations were detected in 253 (61.9%) PATIENTS: 236 (57.7%) carried LDLR, 10 (2.4%) carried APOB p.Q3500 and 7 (1.7%) PCSK9 p.Y374. No additional mutations were identified in PCSK9. After adjusting for age, sex, smoking and systolic blood pressure, compared to those with no detectable mutation, the odds ratio of having CHD in those with an LDLR mutation was 1.84 (95% CI 1.10 to 3.06), for APOB 3.40 (0.71 to 16.36), and for PCSK9 19.96 (1.88 to 211.5; p = 0.001 overall). The high risk in patients carrying LDLR and PCSK9 p.Y374 was partly explained by their higher pretreatment cholesterol levels (LDLR, PCSK9 and no mutation, 10.29 (1.85), 13.12 and 9.85 (1.90) mmol/l, respectively, p = 0.001). The post-statin treatment lipid profile in PCSK9 p.Y374 carriers was worse than in patients with no identified mutation (LDL-C, 6.77 (1.82) mmol/l v 4.19 (1.26) mmol/l, p = 0.001, HDL-C 1.09 (0.27) mmol/l v 1.36 (0.36) mmol/l, p = 0.03). CONCLUSIONS: The higher CHD risk in patients carrying PCSK9 p.Y347 or a detected LDLR mutation supports the usefulness of DNA testing in the diagnosis and management of patients with familial hypercholesterolaemia. Mutations in PCSK9 appear uncommon in patients with familial hypercholesterolaemia in UK.

Adult↗

PubMed2006

Differential contributions of Shaker and Shab K+ currents to neuronal firing patterns in Drosophila.

Different K(+) currents participate in generating neuronal firing patterns. The Drosophila embryonic "giant" neuron culture system has facilitated current- and voltage-clamp recordings to correlate distinct excitability patterns with the underlying K(+) currents and to delineate the mutational effects of identified K(+) channels. Mutations of Sh and Shab K(+) channels removed part of inactivating I(A) and sustained I(K), respectively, and the remaining I(A) and I(K) revealed the properties of their counterparts, e.g., Shal and Shaw channels. Neuronal subsets displaying the delayed, tonic, adaptive, and damping spike patterns were characterized by different profiles of K(+) current voltage dependence and kinetics and by differential mutational effects. Shab channels regulated membrane repolarization and repetitive firing over hundreds of milliseconds, and Shab neurons showed a gradual decline in repolarization during current injection and their spike activities became limited to high-frequency, damping firing. In contrast, Sh channels acted on events within tens of milliseconds, and Sh mutations broadened spikes and reduced firing rates without eliminating any categories of firing patterns. However, removing both Sh and Shal I(A) by 4-aminopyridine converted the delayed to damping firing pattern, demonstrating their actions in regulating spike initiation. Specific blockade of Shab I(K) by quinidine mimicked the Shab phenotypes and converted tonic firing to a damping pattern. These conversions suggest a hierarchy of complexity in K(+) current interactions underlying different firing patterns. Different lineage-defined neuronal subsets, identifiable by employing the GAL4-UAS system, displayed different profiles of spike properties and K(+) current compositions, providing opportunities for mutational analysis in functionally specialized neurons.

Action Potentials↗

PubMed2005

ProfDist: a tool for the construction of large phylogenetic trees based on profile distances.

SUMMARY: ProfDist is a user-friendly software package using the profile-neighbor-joining method (PNJ) in inferring phylogenies based on profile distances on DNA or RNA sequences. It is a tool for reconstructing and visualizing large phylogenetic trees providing new and standard features with a special focus on time efficency, robustness and accuracy. AVAILABILITY: A Windows version of ProfDist comes with a graphical user interface and is freely available at http://profdist.bioapps.biozentrum.uni-wuerzburg.de

Algorithms↗

PubMed2003

The roles of mutations in gyrA, parC, and ompK35 in fluoroquinolone resistance in Klebsiella pneumoniae.

In a survey of 541 Klebsiella pneumoniae isolates from 44 hospitals in Taiwan, three distinct populations were identified by the disk diffusion method according to the disribution of zone diameters of ciprofloxacin. Isolates with resistant, reduced-susceptible, and susceptible to fluoroquinolone were defined as CIP zone diameters of < or = 15 mm, 16-26 mm, and > or = 27 mm, respectively. Thus, in addition to 38 (7%) resistant isolates, there were 30 (5.5%) reduced-susceptible isolates and 473 (87.5%) susceptible isolates. A total of 34 isolates consisting of nine resistant, 13 reduced-susceptible, and 12 susceptible isolates were assessed for point mutations in gyrA and parC and the outer membrane profiles. The susceptibility to fluoroquinolone of 13 reduced-susceptible isolates was not altered in the presence of carbonyl cyanide m-chlorophenylhydrazone, an efflux inhibitor, showing that efflux is not a major contributor to reduced susceptibility. In addition to single mutation in gyrA, OmpK35 porin loss can also be the first step for developing fluoroquinolone resistance. No strain possesses a parC mutation without the simultaneous presence of a gyrA mutation, suggesting that mutations in parC play a complementary role for higher-level of fluoroquinolone resistance and fluoroquinolone resistance is a multistep process.

Anti-Infective Agents↗

PubMed2002

Missense mutations of the BRAF gene in human lung adenocarcinoma.

Mutations of the BRAF protein serine/threonine kinase gene have recently been identified in a variety of human cancers, most notably melanomas. We sought to determine the frequency of BRAF mutations in human lung cancer pathogenesis. Analysis of BRAF sequence from 127 primary human lung adenocarcinomas revealed mutations in two tumor specimens, one in exon 11 (G465V), and a second in exon 15 (L596R). These specimens belong to the same adenocarcinoma subgroup as defined by clustering of gene expression data. BRAF may provide a target for anticancer chemotherapy in a subset of lung adenocarcinoma patients.

Adenocarcinoma↗

PubMed2003

Limited value of serum steroid measurements in identification of mild form of 21-hydroxylase deficiency.

BACKGROUND: ACTH stimulation test is widely used as a basic diagnostic method for non-classical congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). However, the interpretation of this test has not been definitely established. To determine the cut-off values of basal and post-ACTH serum 17-OHP concentrations, data of patients with suspected 21-OHD has been analysed. PATIENTS AND METHODS: Two hundred and eighty-seven patients with postnatal/peripubertal virilization were investigated. Serum steroid concentrations were measured by RIA, urinary steroid profile was determined by capillary gas chromatography and mutation analysis of CYP21 gene was performed by allele specific PCR. 21-OHD was diagnosed by elevated serum 17-OHP concentrations, high level of the urinary 17-OHP metabolites and/or homozygosity for CYP21 mutations. RESULTS: Twenty-one patients of the total of 287 subjects (7.3 %) were identified as having 21-OHD. The numbers of 21-OHD patients compared to total numbers of patients with different ranges of serum 17-OHP were as follows: basal values below 3.5 ng/ml (mean + 1 SD) 0/225; between 3.5 - 6.6 ng/ml 3/41; above 6.6 ng/ml (mean + 2 SD) 18/21. Post-ACTH values below 6.4 ng/ml (mean + 1 SD) 0/226, between 6.4 - 10.3 ng/ml 0/35, above 10.3 ng/ml (mean + 2 SD) 21/26. CONCLUSION: There are patients with inappropriate peripubertal virilization who have slightly elevated 17-OHP concentrations. In this subgroup of patients more sensitive and specific methods are needed to establish the diagnosis of 21-OHD. Therefore we suggest performing an ACTH stimulation test in patients with a morning 17-OHP level above 3.5 ng/ml. Furthermore, urinary steroid profile and/or CYP21 gene analysis are needed in patients with a stimulated 17-OHP value between 10 and 30 ng/ml. These tests will distinguish between patients with non-classical 21-OHD and patients with other disorders.

Adolescent↗