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

BackgroundCongenital 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.AimThe aim of the study was to study the functional consequences of three novelCYP11B1gene mutations (p.His125Thrfs*8, p.Leu463_Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate this data with the clinical phenotype.MethodsFunctional analyses were done by using a HEK293 cellin vitroexpression system comparing WT with mutant P450c11 activity. Mutant proteins were examinedin silicoto study their effect on the three-dimensional structure of the protein.ResultsTwo mutations (p.His125Thrfs*8 and p.Leu463_Leu464dup) detected in patients with classic 11OHD showed a complete loss of P450c11 activity. The mutation (p.Ser150Leu) detected in a patient with non-classic 11OHD showed partial functional impairment with 19% of WT activity.ConclusionFunctional mutation analysis enables the correlation of novelCYP11B1mutations 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.

Download Full-text

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

Endocrinology ◽

10.1210/endo.151.2.9999 ◽

2010 ◽

Vol 151 (2) ◽

pp. 839-839

Author(s):

Silvia Parajes ◽

Lourdes Loidi ◽

Nicole Reisch ◽

Vivek Dhir ◽

Ian T. Rose ◽

...

Keyword(s):

Expression System ◽

Phenotypic Expression ◽

Three Dimensional ◽

Compound Heterozygous ◽

Three Dimensional Model ◽

Hydroxylase Deficiency ◽

Functional Consequences ◽

Heterozygous Carriers ◽

21 Hydroxylase Deficiency

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

Download Full-text

Congenital Adrenal Hyperplasia: The Molecular Basis of 21-Hydroxylase Deficiency in H-2aw18 Mice

Endocrinology ◽

10.1210/en.2004-1563 ◽

2005 ◽

Vol 146 (6) ◽

pp. 2563-2574 ◽

Cited By ~ 13

Author(s):

Felix G. Riepe ◽

Stephan Tatzel ◽

Wolfgang G. Sippell ◽

Jürgen Pleiss ◽

Nils Krone

Keyword(s):

Enzyme Activity ◽

Congenital Adrenal Hyperplasia ◽

Genetic Model ◽

Three Dimensional ◽

Dimensional Structure ◽

Adrenal Hyperplasia ◽

Hydroxylase Deficiency ◽

P Gene ◽

21 Hydroxylase Deficiency

Abstract The mouse strain H-2aw18 shows typical characteristics of 21-hydroxylase deficiency (21-OHD). A deletion of the active Cyp21a1 gene has been postulated; however, the changes on the nucleotide level are still unknown. To investigate whether this animal model, the only one available, is suitable for studying congenital adrenal hyperplasia in man, a detailed analysis of the Cyp21 locus has been performed to ascertain the genetic cause of 21-OHD in H-2aw18 mice. We demonstrate that 21-OHD is caused by unequal crossing over between the active Cyp21a1 gene and the pseudogene resulting in a hybrid Cyp21a1-Cyp21a2-p gene including a partial deletion of Cyp21a1. Next to several pseudogene-specific point mutations, various novel missense mutations and a nonsense mutation are present. Enzyme activity for each point mutation has been determined in vitro and the structure-function relationship has been studied by sequence conservation analysis and a three-dimensional murine 21-hydroxylase protein (Cyp21) structure model. The mutations are classified in three classes: I, no or minor decrease in enzyme activity: R238Q, P465L, R361K, A362V, P458L; II, loss of enzyme activity caused by inefficient electron flux: R346H, R400C; III, loss of activity due to deficient substrate binding: I462F, L464F. The combination of in vitro protein expression and three-dimensional structure modeling provides a valuable tool to understand the role of the different mutations and polymorphisms on the resulting enzyme activity. The underlying genetic mechanisms are also known to be responsible for 21-OHD in humans, so rodent 21-OHD turns out to be an excellent genetic model for studying the human disease.

Download Full-text

Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

Endocrine Connections ◽

10.1530/ec-21-0262 ◽

2021 ◽

Author(s):

Lanping Jiang ◽

Xiaoyan Peng ◽

Bingbin Zhao ◽

Lei Zhang ◽

Lubin Xu ◽

...

Keyword(s):

Three Dimensional ◽

Gene Mutations ◽

Dimensional Structure ◽

Novel Mutations ◽

Three Dimensional Structure ◽

Slc12a3 Gene ◽

Uptake Experiment ◽

Frequent Mutations ◽

22Na Uptake

Purposes: This study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict three-dimensional structure change of human Na-Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo. Methods: SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, CNKI and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevis oocyte expression system. 35 GS patients and 20 healthy volunteers underwent the thiazide test. Results: T60M, T163M，D486N, R913Q, R928C and R959 frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all 6 mutations: T60M 22±9.2%, R928C 29±12%, L215F 38±14%, N534K 41±15.5%, Q617R 63±22.1% and D486N 77±20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations. Conclusions: Frequent mutations (T60M, D486N, R928C) and novel mutations (L215F, N534K and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on patients.

Download Full-text

Phenotype-Genotype Correlations of 13 Rare CYP21A2 Mutations Detected in 46 Patients Affected with 21-Hydroxylase Deficiency and in One Carrier

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2009-1202 ◽

2010 ◽

Vol 95 (3) ◽

pp. 1288-1300 ◽

Cited By ~ 42

Author(s):

V. Tardy ◽

R. Menassa ◽

V. Sulmont ◽

A. Lienhardt-Roussie ◽

C. Lecointre ◽

...

Keyword(s):

Three Dimensional ◽

Intermediate Form ◽

Severe Phenotype ◽

Three Dimensional Model ◽

Hydroxylase Deficiency ◽

Cyp21a2 Gene ◽

Functional Studies ◽

In Silico Studies ◽

21 Hydroxylase Deficiency

Abstract Context: Steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia with genotype/phenotype relationships for common mutations. Novel mutations of the CYP21A2 gene must be well studied to propose right genetic counseling for patients. Objective: Thirteen CYP21 mutations have been studied. A detailed description of phenotype was performed for all mutations (p.I77T, p.L167P, p.I230T, p.R233K, p.G291S, p.G292D, p.E320K, p.R341P, p.R354H, p.R369W, p.R408C, p.G424S, and p.R426H). In vitro and in silico studies were performed only for those not previously described (p.L167P, p.I230T, p.R233K, p.G292D, p.E320K, and p.R369W). Results: Regarding phenotype, patients with 10 of these mutations had a classical form. A patient with isolated p.I230T presented with nonclassical form and a patient with the association p.I230T + p.V281L in cis presented with a more severe phenotype. The p.R233K mutation was detected in a carrier partner. A patient with p.R369W presented with an intermediate form. Functional studies showed that all mutations except p.I230T and p.R369W decreased enzyme activity more than p.P30L: severity of p.R369W was intermediate between p.P30L and p.V281L, and finally p.I230T was less severe than p.V281L. Mutation analysis in a three-dimensional model structure of the CYP21 protein explained the observed in vitro effects, severe mutations being implicated in important functional domains of the protein. Conclusion: According to phenotype and functional studies, 11 of the mutations described, except the isolated p.R369W and p.I230T, may be responsible for a severe phenotype underlying the necessity to manage children having them. The p.I230T is a nonclassical mutation, and for the p.R369W, we need more cases to precise its severity.

Download Full-text

Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst

Polymers ◽

10.3390/polym13142273 ◽

2021 ◽

Vol 13 (14) ◽

pp. 2273

Author(s):

Wan-Ying Huang ◽

Norichika Hashimoto ◽

Ryuhei Kitai ◽

Shin-ichiro Suye ◽

Satoshi Fujita

Keyword(s):

Three Dimensional ◽

Hollow Structure ◽

Dimensional Structure ◽

The Novel ◽

Fibrous Scaffold ◽

Hydrogel Beads ◽

Inner Surface ◽

Intracranial Epidermoid ◽

Hollow Ball

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the “nanofiber-mâché ball.” This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

Download Full-text

Protein Folding: Search for Basic Physical Models

The Scientific World JOURNAL ◽

10.1100/tsw.2003.50 ◽

2003 ◽

Vol 3 ◽

pp. 623-635 ◽

Cited By ~ 3

Author(s):

Ivan Y. Torshin ◽

Robert W. Harrison

Keyword(s):

Protein Folding ◽

Tertiary Structure ◽

Three Dimensional ◽

Physical Models ◽

Dimensional Structure ◽

Computational Techniques ◽

Linear Sequence ◽

Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

Download Full-text

Putative sequences on Ro60 three-dimensional structure accessible for 4-hydroxy-2-nonenal (HNE) modification compared to in vitro HNE modification of Ro60 sequences

Molecular Immunology ◽

10.1016/j.molimm.2011.12.010 ◽

2012 ◽

Vol 50 (4) ◽

pp. 185-192 ◽

Cited By ~ 3

Author(s):

Biji T. Kurien ◽

Anil D'Souza ◽

Simon Terzyan ◽

R. Hal Scofield

Keyword(s):

Three Dimensional ◽

Dimensional Structure ◽

Three Dimensional Structure

Download Full-text

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

Journal of Bacteriology ◽

10.1128/jb.01094-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 2056-2064 ◽

Cited By ~ 17

Author(s):

Jonathan E. Ulmer ◽

Yap Boum ◽

Christopher D. Thouvenel ◽

Hannu Myllykallio ◽

Carol Hopkins Sibley

Keyword(s):

Mycobacterium Tuberculosis ◽

Thymidylate Synthase ◽

Three Dimensional ◽

Pyrimidine Ring ◽

Dimensional Structure ◽

Directed Mutagenesis ◽

Catalytic Residue ◽

Amino Acid Residues ◽

Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

Download Full-text

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Journal of Virology ◽

10.1128/jvi.77.6.3669-3679.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3669-3679 ◽

Cited By ~ 85

Author(s):

Caterina Trozzi ◽

Linda Bartholomew ◽

Alessandra Ceccacci ◽

Gabriella Biasiol ◽

Laura Pacini ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Serine Protease ◽

In Vitro Selection ◽

Three Dimensional ◽

Host Cells ◽

Dimensional Structure ◽

In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Download Full-text