Aromatase Research Articles

W142 Gene chimeras involving CYP21A2 and TNXB genes in Spanish patients with congenital adrenal hyperplasia (CAH)

W142 Gene chimeras involving CYP21A2 and TNXB genes in Spanish patients with congenital adrenal hyperplasia (CAH)

Design, Synthesis, and Molecular Modeling Studies of a Novel Benzimidazole as an Aromatase Inhibitor.

In this study, a series of novel 1,3,4-oxadiazole-benzimidazole derivatives were designed and synthesized. Their cytotoxic activities against five cancer cell lines, including A549, MCF-7, C6, HepG2, and HeLa, were evaluated by the MTT assay. The compounds 5b,c showed satisfactory potencies with much higher anticancer activity in comparison to the reference drug doxorubicin against the studied cancer cell lines. In vitro, enzymatic inhibition assays of aromatase (ARO) enzymes were performed. Molecular docking, molecular dynamics simulations, and binding free energy analyses were used to better understand the structure–activity connections and mechanism of action of the aromatase inhibitors. Two types of satisfactory 3D-QSAR (CoMFA and CoMSIA) models were generated, to predict the inhibitory activities of the novel inhibitors. Molecular docking studies were also carried out to find their binding sites and types of their interactions with the aromatase enzyme. Additionally, molecular dynamics simulations were performed to explore the most likely binding modes of compounds 5b,c with CYP19A1.

CYP21A2 Gene Expression in a Humanized 21-Hydroxylase Mouse Model Does Not Affect Adrenocortical Morphology and Function.

Steroid 21-hydroxylase is an enzyme of the steroid pathway that is involved in the biosynthesis of cortisol and aldosterone by hydroxylation of 17α-hydroxyprogesterone and progesterone at the C21 position. Mutations in CYP21A2, the gene encoding 21-hydroxylase, cause the most frequent form of the autosomal recessive disorder congenital adrenal hyperplasia (CAH). In this study, we generated a humanized 21-hydroxylase mouse model as the first step to the generation of mutant mice with different CAH-causing mutations. We replaced the mouse Cyp21a1 gene with the human CYP21A2 gene using homologous recombination in combination with CRISPR/Cas9 technique. The aim of this study was to characterize the new humanized mouse model. All results described are related to the homozygous animals in comparison with wild-type mice. We show analogous expression patterns of human 21-hydroxylase by the murine promoter and regulatory elements in comparison to murine 21-hydroxylase in wild-type animals. As expected, no Cyp21a1 transcript was detected in homozygous CYP21A2 adrenal glands. Alterations in adrenal gene expression were observed for Cyp11a1, Star, and Cyb11b1. These differences, however, were not pathological. Outward appearance, viability, growth, and fertility were not affected in the humanized CYP21A2 mice. Plasma steroid levels of corticosterone and aldosterone showed no pathological reduction. In addition, adrenal gland morphology and zonation were similar in both the humanized and the wild-type mice. In conclusion, humanized homozygous CYP21A2 mice developed normally and showed no differences in histological analyses, no reduction in adrenal and gonadal gene expression, or in plasma steroids in comparison with wild-type littermates.

Long-read Amplicon Sequencing of the CYP21A2 in 48 Thai Patients With Steroid 21-Hydroxylase Deficiency

Congenital adrenal hyperplasia is most commonly caused by 21-hydroxylase deficiency (21-OHD), an autosomal recessive disorder resulting from biallelic pathogenic variants (PVs) in CYP21A2. With a highly homologous pseudogene and various types of single nucleotide and complex structural variants, identification of PVs in CYP21A2 has been challenging. To leverage long-read next-generation sequencing combined with locus-specific polymerase chain reaction (PCR) to detect PVs in CYP21A2 and to determine its diagnostic yield in patients with 21-OHD. Forty-eight Thai patients with 21-OHD comprising 38 sporadic cases and 5 pairs of siblings were enrolled. Two previously described locus-specific PCR methods were performed. Amplicons were subject to long-read sequencing. Ninety-six PVs in CYP21A2 in the 48 patients were successfully identified. The combined techniques were able to detect 26 structural chimeric variants (27%; 26/96) in 22 patients with 18 having monoallelic and 4 having biallelic chimeras. The remaining PVs were pseudogene-derived mutations (63%; 60/96), entire gene deletions (2%; 2/96), missense variants (3%; 3/96), a splice-site variant (2%; 2/96), frameshift variants (2%; 2/96), and a nonsense variant (1%; 1/96). Notably, a splice-site variant, IVS7 + 1G > T, which was identified in a pair of siblings, has not previously been reported. Our approach exploiting locus-specific PCR and long-read DNA sequencing has a 100% diagnostic yield for our cohort of 48 patients with 21-OHD.

Molecular Diagnosis of Steroid 21-Hydroxylase Deficiency: A Practical Approach.

Adrenal insufficiency in paediatric patients is mostly due to congenital adrenal hyperplasia (CAH), a severe monogenic disease caused by steroid 21-hydroxylase deficiency (21-OHD, encoded by the CYP21A2 gene) in 95% of cases. CYP21A2 genotyping requires careful analyses that guaranty gene-specific PCR, accurate definition of pseudogene-gene chimeras, gene duplications and allele dropout avoidance. A small panel of well-established disease-causing alterations enables a high diagnostic yield in confirming/discarding the disorder not only in symptomatic patients but also in those asymptomatic with borderline/positive results of 17-hydroxyprogesterone. Unfortunately, the complexity of this locus makes it today reluctant to high throughput techniques of massive sequencing. The strong relationship existing between the molecular alterations and the degree of enzymatic deficiency has allowed genetic studies to demonstrate its usefulness in predicting/classifying the clinical form of the disease. Other aspects of interest regarding molecular studies include its independence of physiological variations and analytical interferences, its usefulness in the diagnosis of simple virilizing forms in males and its inherent contribution to the genetic counseling, an aspect of great importance taking into account the high carrier frequency of CAH in the general population. Genetic testing of CYP21A2 constitutes an irreplaceable tool to detect severe alleles not just in family members of classical forms but also in mild late-onset forms of the disease and couples. It is also helpful in areas such as assisted reproduction and preimplantation diagnosis. Molecular diagnosis of 21-OHD under expert knowledge definitely contributes to a better management of the disease in every step of the clinical course.

Identification of a fungal cytochrome P450 with steroid two-step ordered selective hydroxylation characteristics in Colletotrichum lini

Microbial hydroxylation reaction has greatly enriched the number of steroids and created many meaningful new compounds. The dihydroxylation of dehydroepiandrosterone (DHEA) by filamentous fungi produces an important product 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA), which can be used as a key intermediate for the synthesis of contraceptive drospirenone. The introduction of microbial hydroxylation reaction reduces the traditional chemical synthesis process by 4 steps and greatly improves the productivity and economic efficiency. Colletotrichum lini is an industrial strain producing 7α,15α-diOH-DHEA, but the related cytochrome P450 that plays hydroxylation effect has not yet been discovered. In this work, a combination of quantitative proteomics, qRT-PCR, and functional expression in Pichia pastoris was used to identify highly induced steroid hydroxylase from Colletotrichum lini ST-1. A novel fungal cytochrome P450 monooxygenase CYP68JX was identified. The biotransformation in recombinant yeast confirmed that the cytochrome P450 has steroid C7α and C15α hydroxylase activities. The hydroxylation of DHEA by CYP68JX is an ordered reaction, proceeding from the C7 to the C15 site of the steroidal nucleus. The cloning and identification of the CYP68JX gene provide useful information for deepening the understanding regarding the structural basis of its regional and stereoselectivity.

Congenital adrenal hyperplasia with homozygous and heterozygous mutations: a rare family case report

BackgroundCongenital adrenal hyperplasia (CAH), characterized by defective adrenal steroidogenesis, is transmitted in an autosomal recessive manner. Mutations in the steroid 21-hydroxylase gene CYP21A2 causing steroid 21-hydroxylase deficiency account for most cases of CAH. The c.145l-1452delGGinsC gene mutation is rare, and only one case has been reported, but the form of gene mutation is different from this case, resulting in different clinical phenotype. The most common pathogenic genotype of CAH is a homozygous or compound heterozygous mutation, but CAH patients homozygous for the p.I173N mutation and heterozygous for the c.1451-1452delGGinsC mutation have not been reported previously. We report herein a familial case of CAH, in which both siblings carry the rare homozygous p.I173N mutation and heterozygous c.1451-1452delGGinsC mutation.Case presentationThe proband showed amenorrhea, infertility, polycystic ovaries, and increased levels of androgen, rather than the typical clinical manifestations of CAH such as an adrenal crisis or masculine vulva, so was misdiagnosed with polycystic ovary syndrome for many years. Following a correct diagnosis of CAH, she was given glucocorticoid treatment, her menstruation became more regular, and she became pregnant and delivered a healthy baby girl.ConclusionsThe genotypes may be p.I173N homozygous or p.I173N/c.1451-1452delGGinsC heterozygous, both mutations could be pathogenic. This complex combination of mutations has not been reported or studied before. Through the report and analysis of this genotype, the content of CAH gene bank is enriched and the misdiagnosis rate of CAH is reduced.

Copy Number Variations in Genetic Diagnosis of Congenital Adrenal Hyperplasia Children.

Background: Congenital adrenal hyperplasia (CAH) is a monogenic disorder caused by genetic diversity in the CYP21A2 gene, with 21-hydroxylase deficiency (21-OHD) as the most common type. Early sex assignment and early diagnosis of different genetic variations with a proper technique are important to reduce mortality and morbidity. Proper early sex identification reduces emotional, social, and psychological stress. Aim: Detection of a spectrum of aberrations in the CYP21A2 gene, including copy number variations, gene conversion, chimeric genes, and point variations. Methods: The CYP21A2 gene was screened using MLPA assay in 112 unrelated Egyptian children with 21-OHD CAH (33 males and 79 females). Results: In the studied group, 79.5% were diagnosed within the first month of life. 46.8% of the genetic females were misdiagnosed as males. Among the copy number variation results, large deletions in 15.4% and three types of chimeric genes in 9% (CH-1, CH-7, and CAH-X CH-1) were detected. Regarding gene dosage, one copy of CYP21A2 was found in 5 cases (4.5%), three copies were detected in 7 cases (6.3%), and one case (0.9%) showed four copies. Eight common genetic variants were identified, I2G, large deletions, large gene conversion (LGC), I172N, F306 + T, -113 SNP, 8bp Del, and exon 6 cluster (V237E and M239K) with an allelic frequency of 32.62%, 15.45%, 7.30%, 3.00%, 2.58%, 2.15%, 0.86%, and 0.86%, respectively. Conclusion: High prevalence of copy number variations highlights the added value of using MLPA in routine laboratory diagnosis of CAH patients.

Abstract 109: Long-range PCRs and MLPA for molecular analysis of large rearrangements in 21 hydroxylase deficiency

Background: Genetics of 21 hydroxylase deficiency is complex with pseudogene derived point mutations and rearrangements in the CYP21A2 gene. 20-30% of these mutations are due to large 30kb deletion forming chimeric genes and large gene conversion. In general, MLPA is the choice of technique in molecular analysis of large deletions and duplications.Aim and Objectives: To evaluate long-range PCRs, restriction digestion, ASPCR and MLPA to identify various chimeric forms of CYP21A2 gene.Results: In this study, 24.2% (n=16/66) were identified with large rearrangements based on long-range PCR and restriction digestion results. Thirteen subjects were positive for chimeric genes described in 21-hydroxylase deficiency. Junction site analysis with both MLPA and ASPCR results revealed that ASPCR could detect the actual extent of rearrangement in all the chimeric genes while MLPA could miss two of the chimeric genes in four subjects.Conclusion: In this study, MLPA as a direct stand-alone technique has shown to be inadequate in identifying the extent of some rearrangements in the CYP21A2 gene. Since, it is crucial to differentiate various chimeric forms of this gene; we recommend a multistep approach including long-range PCR, restriction digestion and ASPCR/MLPA to interpret and understand the associated phenotypic severity in 21-hydroxylase deficiency.

Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome.

Congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency is an autosomal recessive disorder. The 21-hydroxylase enzyme P450c21 is encoded by the CYP21A2 gene located on chromosome 6p21.33 within the HLA major histocompatibility complex. This locus also contains the CYP21A1P, a non-functional pseudogene, that is highly homologous to the CYP21A2 gene. Other duplicated genes are C4A and C4B, that encode two isoforms of complement factor C4, the RP1 gene that encodes a serine/threonine protein kinase, and the TNXB gene that, encodes the extracellular matrix glycoprotein tenascin-X (TNX). TNX plays a role in collagen deposition by dermal fibroblasts and is expressed in the dermis of the skin and the connective tissue of the heart and skeletal muscle. During meiosis, misalignment may occur producing large gene deletions or gene conversion events resulting in chimeric genes. Chimeric recombination may occur between TNXB and TNXA. Three TNXA/TNXB chimeras have been described that differ in the junction site (CH1 to CH3) and result in a contiguous CYP21A2 and TNXB gene deletion, causing CAH-X syndrome. TNXB deficiency is associated with Ehlers Danlos syndrome (EDS). EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders. As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB-gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

Sexual dimorphism in Odontobutis sinensis brain-pituitary-gonad axis and liver highlighted by histological and transcriptomic approach

In this study, sexual dimorphism in Chinese dark sleeper (Odontobutis sinensis) brain-pituitary-gonad axis and liver was highlighted by histological and transcriptomic approach. The results showed that there were two significant differences between males and females. Firstly, males grew larger and faster than females. Transcriptomic analysis and qPCR validation indicated that two key growth genes, insulin-like growth factor (igf) and 25-hydroxyvitamin D-1 alpha hydroxylase (cyp27b), were more highly detected in male liver than that in female liver. Secondly, histological analysis displayed that the liver in males showed an obvious ivory fatty phenomenon with more fat vacuoles and lipid droplet aggregation compared to that in females. Transcriptomic analysis indicated that the transcript level of vitellogenin (vtg) in male liver were significantly lower than that in female liver. After 17β-estradiol (E2) treatment of primary cultured hepatocytes, the vtg mRNA expression was induced significantly, while dihydrotestosterone (DHT) treatment had little effect on it. Generally, this study will provide some ideas for further exploring the mechanism of sexual dimorphism in Odontobutis sinensis.

Characterization of the CYP21A2 Gene Mutations in Children with Classic Congenital Adrenal Hyperplasia.

To characterize the CYP21A2 gene mutations in children with classic congenital adrenal hyperplasia (CAH). A prospective, cross-sectional study was conducted on 24 children with classic CAH. Molecular characterization of the CYP21A2 gene was carried out by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), or clinical exome sequencing. Another 21 previously mutation-proven CAH patients were also included and a combined result was drawn. Out of 45 children, pathogenic variants in the CYP21A2 gene were identified in 43 patients (95.5%). Homozygous, probable compound heterozygous, and heterozygous variants were seen in 69%, 22%, and 18% of patients, respectively. The most common variant was c.293-13C/A>G(33%), followed by deletion/duplication (24%), and c.955C>T (p.Gln319Ter) (21%), similar to previous Indian studies. Allelic frequencies of c.332_339del and c.518T>A(p.Ile173Asn) were 9% and 4%, respectively. Less common variants were c.923dupT(p.Leu308PhefsTer6), c.92C>T(p.Pro31Leu), c.1069C>T(p.Arg357Trp), c.1267G>C(p.Gly423Arg), and c.710_719delins(p.Ile237_Met240delinsAsnGluGluLys). A good genotype-phenotype correlation was observed; only p.Pro31Leu and p.Ile173Asn variants showed discordance. The diagnostic yield of Sanger sequencing alone, Sanger sequencing with MLPA, and clinical exome alone was 85%, 100%, and 100%, respectively. All children, except two, diagnosed clinically as classic CAH, showed pathogenic variants in the CYP21A2 gene; the most common variant was c.293-13 C/A>G. The results suggest a broad mutation spectrum in the authors' single-center cohort of children with CAH. Clinical exome sequencing is the preferred stand-alone method for molecular diagnosis of CAH.

Molecular analysis and genotype-phenotype correlations in patients with classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency from southern Poland — experience of a clinical center

PurposeThe prevalence of CYP21A2 gene variants and genotype-phenotype correlations are variable among populations. The aim of this study was to characterize CYP21A2 gene variants in adult patients with classical congenital adrenal hyperplasia (CCAH) from southern Poland and to analyze genotype-phenotype correlations.Materials/MethodsA total of 48 patients (30 women and 18 men) with CCAH were included in the study. Patients were divided into two clinical subgroups, namely, salt-wasting (SW) — 38 patients and simple virilizing (SV) — 10 patients. A genetic analysis MLPA (multiplex ligation-dependent probe amplification) was performed in all of them. In dubious cases, the analysis was complemented by Sanger sequencing. Genotypes were classified into five groups (depending on the residual in vitro enzymatic activity), namely, null, A, B, C, and D, and correlated with the clinical picture.ResultsMolecular defects were investigated and identified in 48 patients. The most common variant in the studied group was I2G, followed by whole or partial gene copy deletion, and I172N. One novel variant c.[878G>T] (p.Gly293Val) was found. In nine patients, a non-concordance between genotype and phenotype was observed. Genotype-phenotype correlations measured by positive predictive value (PPV) were as follows: 100% in group null, 90.5% in group A, and 66.7% in group B.ConclusionsCYP21A2 variants in the studied cohort were similar to values previously reported in other countries of the region. There was a good correlation between genotype and phenotype in the null and A groups, the correlation being considerably lower in group B.

Characteristics of In2G Variant in Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

Substantial research has been performed during the last decades on the clinical and genetic variability of congenital adrenal hyperplasia (CAH) and its most common form, 21-hydroxylase deficiency (21OHD). CAH is one of the most prevalent autosomal recessive diseases in humans, and it can be divided into classic—further subdivided into salt wasting (SW) and simple virilizing (SV)—and non-classic (NC) forms. Pathogenic variants of CYP21A2 gene, encoding the 21-hydroxylase enzyme, have been reported with variable prevalence in different populations. NM_000500.9:c.293-13C/A>G (In2G) variant represents the most common CYP21A2 gene changes related to the classic 21OHD form. However, the phenotype of In2G carriers is variable depending on the variant homozygous/heterozygous status and combination with other CYP21A2 pathogenic variants. In addition, identical genotypes, harboring the homozygous In2G variant, can present with variable phenotypes including the SW and SV or rarely NC form of the disease. Here, we analyze and present the clinical aspects, genotype/phenotype correlations, and other characteristics related to the CYP21A2 In2G variant.

Allele-specific and multiplex PCR based tools for cost-effective and comprehensive genetic testing in Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder due to enzyme defects in adrenal steroidogenesis. Several genes code for these enzymes, out of which mutations in the CYP21A2 gene resulting in 21 hydroxylase deficiency, contribute to the most common form of CAH. However, pseudogene imposed challenges complicate genotyping CYP21A2 gene, and there is also a lack of comprehensive molecular investigations in other genetic forms of CAH in India. Here, we describe a cost-effective, highly specific, and sensitive Allele Specific PCR (ASPCR) assay designed and optimized in-house to screen eight common pathogenic mutations in the CYP21A2 gene. We have also established and utilized a multiplex PCR assay for target enrichment and Next-generation sequencing (NGS) of CYP11B1, CYP17A1, POR, and CYP19A1 genes. Following preliminary amplification of the functional gene CYP21A2, ASPCR based genotyping of eight common mutations - P30L, I2G, 8BPdel, I172N, E6CLUS (I235N, V236E, M238K) V281L, Q318X, and R356W was carried out. These results were further validated using Sanger and Next-generation sequencing. Once optimized to be specific and sensitive, the advantage of ASPCR in CYP21A2 genotyping extends to provide genetic screening for both adult and paediatric subjects and carrier testing at a low cost and less time. Furthermore, multiplex PCR coupled NGS has shown to be cost-effective and robust for parallel multigene sequencing in CAH.

Long-term morbidity in congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive disorders and is characterized by cortisol deficiency. The most common cause of CAH is amutation in the CYP21A2 gene, resulting in 21-hydroxylase deficiency in the adrenal cortex. The lack of cortisol causes an increase in adrenocorticotropic hormone (ACTH), which in turn results in an excess of adrenal androgens. Aldosterone synthesis may also be impaired. The clinical manifestation of CAH depends on the residual activity of 21-hydroxylase and the subsequent lack of cortisol and adrenal androgen excess. While classic CAH is apotentially life-threatening condition, non-classic CAH is mild to asymptomatic. Therapy of classic CAH consists of glucocorticoid and mineralocorticoid substitution. Despite optimization of therapy, CAH still leads to increased morbidity and mortality in patients. The clinical consequences of androgen excess in affected women range from intrauterine virilisation of external genitalia in classic CAH patients to mild symptoms of hyperandrogenism in non-classic forms. Increased demand for cortisol during illness or physical and psychological stress situations can trigger life-threatening adrenal crises. As current glucocorticoid therapy cannot mimic the physiological circadian rhythm and is usually supraphysiological in dose to control androgen excess, therapy-associated long-term consequences such as decreased bone health and an increased cardiometabolic risk profile are common. The burden of the disease may also lead to impaired quality of life and mental health. For this reason, regular screening and follow-up of patients with CAH should be performed in specialized centers to detect and treat possible comorbidities at an early stage.

Implications of CYP21A2 gene duplications in carrier screening and prenatal diagnosis of congenital adrenal hyperplasia due to 21 Hydroxylase deficiency

Background: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that presents as salt wasting or simple virilization (SV). It is due to biallelic mutations in the CYP21A2 gene that encodes the 21-hydroxylase enzyme. This gene is susceptible to deletions and duplications due to the presence of a homologous pseudogene and its location in the RCCX module. This complicates the interpretation of molecular analysis of the CYP21A2 gene. Clinical Description: During preconception counseling and subsequent workup of a couple, the wife (who had been diagnosed with simple virilizing CAH at the age of 14 years, based on clinical and metabolic profile) was identified with c.373C >T variant on one and a deletion on the other allele of CYP21A2. Her asymptomatic husband harbored a novel c. 939+5G>A variant in intron 7 of CYP21A2. Prenatal diagnosis by Sanger sequencing revealed the presence of both maternal (c.373C>T) and paternal (c. 939+5G>A) variants in the fetus, indicative of SV form. After genetic counseling, the parents decided to continue with the pregnancy. Management and Outcome: A baby boy was born who underwent investigations according to the standard protocol. However, a diagnosis of CAH could not be established conclusively. The molecular diagnosis of both baby and parents was revisited. It was found that the baby harbored a duplication of CYP21A2 (inherited from his father) along with a novel variant. The duplication neutralized the paternal variant, and thus the baby was not affected, but a carrier. Conclusion: Evaluation of duplication in parents is crucial before prenatal testing, as duplications have important bearing on the carrier status.

Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

An Iranian Congenital Adrenal Hypoplasia Patient with Elevated Testosterone in Infancy due to a Novel Pathogenic Frameshift Variant in NR0B1.

X-linked congenital adrenal hypoplasia due to NR0B1 mutation is characterized by hypogonadotropic hypogonadism (HH) and infertility. Here, we describe a novel pathogenic frameshift variant in NR0B1 associated with congenital adrenal hypoplasia by whole exome sequencing in an Iranian case with high level of testosterone. Clinical evaluations and pedigree drawing were performed. Point mutations, gene conversions, and large deletions of the CYP21A2 gene were checked. WES and segregation analyses were conducted. In silico analysis was also performed for the novel variant. The ACTH, 17-hydroxy progesterone c, and DHEA sulfate values were elevated up to 624.6 pg/mL, 8.6 pmol/L, and 17.8UMOL/L, respectively. No mutation was found in the CYP21A2 gene. WES identified a novel hemizygous frameshift insertion c.218_219insACCA: p.His73GlnfsTer41 variant in the NR0B1 gene with a pathogenic effect according to ACMG criteria. Genetic testing is helpful for differential diagnosis in primary adrenal insufficiency disorders. NR0B1 may be a common cause of congenital adrenal hypoplasia in our population.

Mutations of the CYP21A2 gene: Association of p.V281L mutation with polycystic ovarian syndrome (PCOS)

Purpose: The CYP21A2 gene encodes the enzyme 21–hydroxilase, which is responsible for the production of steroids. These hormones are key mediators of sexual development and conception. Patients with 21–hydroxilase deficiency tend to be affected in different stages of life. The purpose of this study was to compare the genetic profiles of women with unexplained infertility problems with the genetic profiles of healthy controls. Furthermore, were analyzed associations between mutations of the CYP21A2 gene and various clinical and laboratory parameters.
 Methods: We enrolled 300 women, diagnosed with unexplained infertility problems, into this retrospective study. For each subject, we recorded important clinical and laboratory parameters during different phases of the menstrual cycle. In the control group, we enrolled 100 samples. Each subject provided a blood sample, which was used to isolate DNA for subsequent polymerase chain reaction (PCR).
 Results: In total, 11.6% of the test subjects exhibited significant hormonal deviations (HD) (estrogens and/ or progestins and/or total testosterone) and 5.3% were diagnosed with polycystic ovarian syndrome (PCOS). We identified a significant association between the p.V281L mutation and the frequency of test subjects with significant HD (x2=6.99, p=0.01). A similar association was also observed between p.V281L mutation and the frequency of test subjects with PCOS (x2=16.78, p=0.00). However, we did not establish any associations between the frequency of mutations in test subjects when compared with controls. In addition, we did not find any significance in the frequency of CYP21A2 gene mutations and any of the laboratory parameters tested.
 Conclusion: Our results identify a significant association of the p.V281L mutation in the CYP21A2 gene with the frequencies of both PCOS and significant HD.