Simple Virilizing Research Articles

SAT275 Generation And Characterization Of A Humanized CYP21A2 Mouse Model For Congenital Adrenal Hyperplasia

Abstract Disclosure: A. Huebner: None. S. Thirumalasetty: None. T. Schubert: None. R. Naumann: None. I. Reichardt: None. M. Rohm: None. D. Landgraf: None. F. Gembardt: None. M.F. Hartmann: None. S.A. Wudy: None. M. Peitzsch: None. N. Reisch: None. K. Koehler, PhD: None. 21-hydroxylase deficiency (21OHD) is the most common form of congenital adrenal hyperplasia (CAH) and is caused by mutations in the CYP21A2 gene. 21OHD causes a wide array of clinical symptoms that result from gluco- and mineralocorticoid deficiency and adrenal androgen excess. Treatment firstly aims to substitute lacking steroid hormones, and secondly to restore negative feedback towards CRH and pituitary ACTH secretion to diminish adrenal androgen overproduction. In most cases supra-physiological glucocorticoid doses are necessary which may cause short stature, obesity, hypertension, and cardiovascular and metabolic co-morbidity with reduced quality of life. Hence, current steroid substitution regimens have significant limitations, so novel therapeutic strategies are required. In recent years new therapeutic approaches have emerged including new non-glucocorticoid substances interfering with the HPA axis to minimize adrenal androgen production and to lower external glucocorticoid substitution to physiological levels. However, valuable in-vivo models for pre-clinical testing of such drugs are lacking. Here we present the first viable and humanized mouse model in which the mouse gene Cyp21a1 is replaced by the human orthologue CYP21A2 in which the human point mutation R484Q is integrated. Twenty-weeks-old homozygous mice show marked adrenal hyperplasia, enhanced expression of the CYP21A2 gene and a weak increase of Cyp11a1 and Cyp11b2 gene expression. Tandem mass spectrometry measurements of the mice plasma at 20 weeks show decreased corticosterone and 11-deoxycorticosterone levels in the presence of increased ACTH levels in both male and female homozygous animals. Progesterone levels in homozygous mice are significantly higher (p<0.01) than in wildtype mice. We also observed increased aldosterone levels in female mutants whereas blood pressure does not differ between wildtype and mutant mice strains. Histologically, mutants exhibit adrenocortical hyperplasia. While mutant male mice are fertile with normal appearing testes, females are infertile, remain in the diestrus phase and present with a reduced number of ovarian follicles. In parallel, a second mouse strain bearing the I173N mutation was developed. This mutation is frequent in human patients causing simple virilizing or rarely salt wasting CAH. Homozygous mice require dexamethasone treatment during pregnancy and until weaning but are viable without treatment afterwards. Preliminary results show adrenal hyperplasia and alteration in steroidogenic gene expression and steroid profiles. In conclusion, we demonstrate that the humanized mutant CYP21A2 mice may represent an excellent animal CAH model to test novel treatment strategies for CAH patients. We believe that this model(s) will facilitate the transition from basic research into clinical application. Presentation: Saturday, June 17, 2023

Genotyping in patients with congenital adrenal hyperplasia by sequencing of newborn bloodspot samples.

Genotype-phenotype correlation in congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency ranges from 45 to 97 %. We performed massively parallel sequencing of CYP21A2 on stored newborn bloodspot samples to catalogue the genotypes present in our patients with CAH and enable genotype-phenotype comparison. Participants≤15 years old with clinically diagnosed CAH were recruited from The Sydney Children's Hospitals Network. Phenotype was classified from clinical and biochemical details in the medical record as salt wasting (SW), simple virilising (SV), non-classic (NC) or an intermediate phenotype (SW/SV; SV/NC). Amplicon-based sequencing for CYP21A2 was performed on stored newborn bloodspot samples by the New South Wales Newborn Bloodspot Screening Laboratory on MiSeq™Dx (Illumina, California). Available genetic test results were also obtained from the medical records. Samples from 67 participants (43 % female, age 0.3-15 years) were sequenced, including 9 sibships. SW phenotype was present in 33/67 participants (49 %), SV in 9 (13 %) and NC in 16 (24 %). Intermediate phenotypes included SW/SV in seven participants (10 %) and SV/NC in two (3 %). Variants were identified in 90/116 alleles (78 %). A complete genotype was available in 47/67 participants (70 %). The most common genotype was homozygous c.293-13A/C>G (I2G) in 7/47 participants (15 %). Genotype correlated with the mostcommonly reported phenotype in 36/44 cases (82 %). Correlation was higher in SW and NC phenotypes. This study uses genetic testing of newborn bloodspots to identify and characterise the genotypes present in an ethnically diverse Australian population with CAH. It further strengthens our knowledge of genotype-phenotype correlations in CAH.

Genetic Characterization of a Cohort of Italian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

Defects in the steroid 21-hydroxylase gene (CYP21A2) cause 21-hydroxylase deficiency (21OHD), the main cause of congenital adrenal hyperplasia (CAH). The disease shows a broad spectrum of clinical forms, ranging from severe or classical (salt wasting, SW, and simple virilizing, SV), to mild late onset or nonclassical (NC). 21OHD affects 1 in 15,000 in its severe classic form and 1 in 200-1000 in its mild NC form. There are many studies reporting the frequency of CYP21A2 pathogenic variants in different populations; however, few of them provide comprehensive information about Italian patients. Here, we present genetic results from a cohort of 245 unrelated Italian individuals with clinical diagnosis of CAH due to 21OHD. A specific polymerase chain reaction (PCR) protocol combined with Sanger sequencing was used for CYP21A2 analysis. The multiplex ligation-dependent probe amplification (MLPA) assay was employed for copy number variation (CNV) determination. One hundred fourteen (46.5%) of the index cases had the NC form, 57 (23.3%) had the SV form, and 74 (30.2%) presented the SW form of the disease. The most prevalent variant found in NC patients was the p.Val282Leu (51.3%), while the most frequent variants in the classical form were p.Ile173Asn (8.6%) and c.293-13C>G (26.0%). In our study, the frequency of large rearrangements was 15.3%, with CAH-X alleles representing 40% of all DEL/CONV. In addition, 12 alleles carried rare variants, and 1 had a novel variant p.(Arg342Gln). We observed phenotype-genotype correlation in 94.7% of cases. A complete concordance was observed in Groups 0 (enzyme activity completely impaired) where all patients had the SW form as expected. In Group A (0-1% residual enzyme activity), 78.4% of patients had the anticipated SW form while 21.6% were diagnosed with the SV form. Within Group B (~2% residual enzyme activity), 93.4% of patients exhibited SV form and 6.5% SW disease. Finally, 92.6% and 7.4% of patients belonging to Group C (enzyme partially impaired to ~20-60% residual activity) exhibited NC and SV phenotypes, respectively. This work, representing a comprehensive genetic study, expanded the CYP21A2 variants spectrum of Italian patients with 21OHD and could be helpful in prenatal diagnosis and genetic counseling.

Assessment of the Degree of Clinical Suspicion of 21-Hydroxylase Deficiency Prior to the Newborn Screening Result.

The aim of the study was to analyze the clinical suspicion and where patients were when they received the positive result of the neonatal screening for CAH 21OHD. The present data derived from a retrospective analysis of a relatively large group of patients with classical CAH 21OHD patients nosed by newborn screening in Madrid, Spain. During the period from 1990 to 2015 of this study 46 children were diagnosed with classical 21OHD [36 with the salt-wasting (SW) form and 10 with simple virilizing (SV)]. In 38 patients, the disease had not been suspected before the neonatal screening result (30 SW and 8 SV). Thirty patients (79%) were at home without suspicion of any disease, as healthy children, 3 patients (8%) were at home pending completion of the study due to clinical suspicion of any disease (ambiguous genitalia, cryptorchidism) and 5 patients (13%) were admitted to the hospital for reasons unrelated to CAH (sepsis, jaundice, hypoglycemia). It is relevant to note that 69.4% of patients (25/36) with SW form were at home with potential risk of adrenal crisis. Six females had been incorrectly labeled as male at birth. The most frequent reason for clinical suspicion was genital ambiguity in women followed by family history of the disease. Neonatal screening provided better results than clinical suspicion. In the majority of patients with 21OHD the diagnosis by screening was anticipated to the clinical suspicion of the disease even in female patients with ambiguous genitalia.

Epidemiology and diagnostic trends of congenital adrenal hyperplasia in Denmark: a retrospective, population-based study.

The prevalence of newborns with congenital adrenal hyperplasia (CAH) detected by neonatal screening is well-described, but data including patients diagnosed later in life are extremely limited. This study aimed to describe diagnostic trends for all patients with CAH in Denmark. A nationwide population-based registry study including medical record review. We identified 462 patients (290 females) with any form of CAH. The prevalence of CAH combined was 15.1 (95% confidence interval [CI]: 12.3-16.1) and 9.0 (CI: 7.6-10.4) per 100,000 newborn females and males. There was a prevalence of salt-wasting (SW), simple-virilizing (SV), and non-classic (NC) CAH due to 21-hydroxylase deficiency of: SW-CAH: 6.4 (CI: 5.3-7.6) and 5.6 (CI: 4.6-6.8); SV-CAH: 2.0 (CI: 1.4-2.8) and 1.6 (CI: 1.0-2.7); and NC-CAH: 5.5 (CI: 4.4-6.9) and 2.5 (CI: 1.7-3.7) per 100,000 newborn females and males, respectively. Diagnosis of NC-CAH increased significantly during the course of the study. There was a female preponderance for SV-CAH (ratio: 1.8) and NC-CAH (ratio: 3.2). Median age at diagnosis, females and males respectively: SW-CAH: 4 (interquartile range [IQR]: 0-11) and 14 (IQR: 8-24) days, SV-CAH: 3.1 (IQR: 1.2-6.6) and 4.8 (IQR: 3.2-6.9) years, and NC-CAH: 15.5 (IQR: 7.9-22.5) and 9.4 (IQR: 7.2-23.2) years. The combined prevalence of CAH was 15.1 and 9.0 per 100,000 newborn females and males, respectively. The female preponderance was primarily due to diagnosis of more females than males with NC-CAH. International Fund of Congenital Adrenal Hyperplasia, Health Research Fund of Central Denmark Region, Aase and Einar Danielsen Fund, and "Fonden til Lægevidenskabens Fremme".

ODP028 Association Between Serum Steroid Profile and Metabolic Risks in Adults with Classic Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency

Abstract Objective In adult patients with congenital adrenal hyperplasia (CAH), long-term health outcomes, including metabolic and cardiovascular health, are a priority for the management of CAH. The enzyme 21-hydroxylase deficiency, which accounts for a majority of CAH, leads to a large perturbation in serum steroid profile under glucocorticoid replacement therapy. Serum levels of several adrenal steroids, including 17-hydroxyprogesterone and androstenedione, are associated with disease severity and sexual dysfunction of CAH patients. However, the association between the serum steroid profiles with metabolic risk in CAH patients is not elucidated yet. We aimed to investigate the serum steroid profile of adult CAH patients using liquid chromatography-mass spectrometry (LC-MS) and their association with metabolic risk. Methods Adult patients with classic CAH due to 21-hydroxylase deficiency were included in this study (36 females and 27 males). A multi-steroid panel composed of 25 steroids and their metabolites was applied to morning serum samples of study subjects using LC-MS. An unsupervised clustering algorithm was applied to the serum steroid panel to discover any unique pattern which divides CAH subjects into clusters. The association between serum steroid profiles and clinical characteristics, including age, sex, body mass index (BMI), subtypes (simple virilizing and salt wasting), glucocorticoid dose, and the presence of hypertension, diabetes mellitus (DM), and metabolic syndrome (MetS) was analyzed. The discriminatory power of each steroid or a combination of steroids was estimated using the area under the curve of receiver operating characteristics (AUROC) for metabolic diseases. Results Fifteen steroids including pregnenolone sulfate, 17α-hydroxyprogesterone, cortisol, cortisone, 21-deoxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, α-cortolone, β-cortolone, tetrahydrocortisone, dehydroepiandrosterone sulfate, testosterone, androstenedione, 11β-hydroxytestosterone, and 11β-hydroxyandrostenedione were successfully quantified in all subjects. The median age of subjects was 28 years (interquartile range: 25-35). The prevalence of DM, hypertension, and MetS was 3.2% (2/63), 15.8% (10/63), and 28.6% (18/63), respectively. The subjects were divided into two clusters based on the pattern of serum steroid profile using unsupervised hierarchical clustering. The prevalence of MetS was significantly different between the two clusters (cluster 1, 37.8% [17/45] vs. cluster 2, 5.6% [1/18], P = 0. 011). The prevalence of HTN was numerically higher in cluster 1 than cluster 2 (20. 0% [9/45] vs. 5.6% [1/18], P = 0.257). Other clinical characteristics, including age, sex, BMI, subtypes, and glucocorticoid dose, were not different between the two clusters. Among fifteen steroids, the level of tetrahydrocortisone showed the highest discriminatory power for MetS (AUROC 0.795, 95% confidence interval: 0.675-0.914). The multivariate logistic regression model of all 15 steroids showed AUROC of 0.832 (95% CI: 0.732-0.933) for MetS. Conclusion The serum steroid profiles of CAH patients were significantly associated with the presence of MetS. This suggests that serum steroid signatures can guide the optimal management of adult CAH patients to minimize the risk of MetS. Presentation: No date and time listed

Congenital adrenal hyperplasia. Role of dentist in early diagnosis.

Congenital adrenal hyperplasia (CAH) is a genetic disorder characterized by an impairment of steroid synthesis due to an altered production of 21-hydroxylase enzyme. Corticoid hormones are involved in the development and functioning of many organs. The aim of the present study was to review the international literature to collect data regarding oral manifestations of CAH. A review of the literature describing oral features of patients affected by CAH was performed using electronic databases (PubMed and Scopus). The data about number of patients, form of CAH, and oral findings were extracted and analyzed. Seven studies were included in the final analysis. The principal findings reported regarded an advanced dental development observed in patients with CAH. One paper reported amelogenesis imperfecta and periodontal issues. The dentist could be the first specialist involved in the CAH syndrome diagnosis, identifying the characteristic features described above, especially for the classical simple virilizing and non-classical form.

Various Clinical Manifestations of Congenital Adrenal Hyperplasia in Children

The study's objectives was to determine the frequency of various clinical manifestations of congenital adrenal hyperplasia in children. Materials & Methods: The research methodology is descriptive and cross sectional. The event was held at the Department of Pediatric Medicine at DHQ Hospital in Faisalabad. The study will last from September 29th to March 28th, 2019. A total of 90 children with congenital adrenal hyperplasia of age 0-10 years of either gender were included. After taking informed written consent from all children’s parents, different clinical presentations (ambiguous genitalia, simple virilising and salt wasting) were noted in each patient. Results: Mean age of children was 4.84 ± 1.78 years. Out of the 90 patients, 62 (68.89%) were male and 28 (31.11%) were females with male to female ratio of 2.2:1. Mean duration of symptoms was 5.79 ± 2.59 days. In this study, I have found the ambiguous genitalia in 27 (30.0%), simple virilising in 17 (18.88%) and salt wasting in 59 (65.56%) children with congenital adrenal hyperplasia. Conclusion: This study has shown that the salt wasting is the most common clinical presentations of congenital adrenal hyperplasia in children followed by ambiguous genitalia and simple virilising. Keywords: Congenital adrenal hyperplasia, ambiguous genitalia, salt wasting.

Characterization of a cohort of pediatric patients with Congenital Adrenal Hyperplasia

Observational descriptive longitudinal study. Medical records from the pediatric endocrinology outpatients from four institutions in Medellín, Colombia were reviewed. Sociodemographic, clinical (type of CAH, salt-wasting crisis, associated endocrinopathies), labora tory (17-hydroxyprogesterone, testosterone, dehydroepiandrosterone sulfate, androstenedione, cor tisol, and adrenocorticotropic hormone) variables were analyzed. A descriptive statistical analysis was carried out. 132 patients (65% female) were included. The median age at diagnosis was 2 months, 44.7% neonatal diagnosis. Seventy-nine children with classical salt-wasting CAH, 31 with simple virilizing, and 22 with non-classical form were documented. Median 17-OHP at diagnosis was 4820 ng/dl. Sexual differentiation disorder was presented in 47% of patients and 48% presented with adrenal crisis (AC) at diagnosis; the median age of the first AC was 15 days. Ninety-three patients required fludrocortisone and 32 patients presented AC after diagnosis and treatment. Median height/ age (last appointment): -0.49 SD, difference between bone and chronological age: 26 months. More than 60% of patients had elevated androstenedione and/or testosterone at the last appointment. Sociodemographic and clinical characteristics are similar to those reported in the literature. In 48% of patients, AC was the initial manifestation, making neonatal screening important, as it would allow an early diagnosis. We found virilization in 71% of women in our study. A CAH should be suspected in a newborn with different genitalia.

Long-Term Results of Surgical Treatment and Patient-Reported Outcomes in Congenital Adrenal Hyperplasia-A Multicenter European Registry Study.

Representatives for congenital adrenal hyperplasia (CAH) continue to desire early feminizing surgery in girls with 46,XX-CAH. The aim of this analysis, which included 174 46,XX- individuals with salt-wasting (SW) or simple-virilizing (SV) CAH, a female gender identity, and an age > 16 years participating in a multicenter cross-sectional clinical evaluation study (dsd-LIFE), was to evaluate the long-term results of surgery and patient-reported outcomes (PRO). The gynecological examination (n = 84) revealed some shortcomings concerning surgical feminization. A clitoris was absent in 9.5% of cases, while a clitoral hood was missing in 36.7% of cases. Though all women had large labia, they didn’t look normal in 22.6% of cases. Small labia were absent in 23.8% of cases. There was no introitus vaginae, and the urethra and vagina had no separate opening in 5.1% of cases. A mucosal lining was missing in 15.4% of cases. Furthermore, 86.2% of the women had scars at the region of their external genitalia. A vaginal stenosis was described in 16.5% of cases, and a meatal stenosis was described in 2.6% of cases. Additionally, PRO data showed a very-/high satisfaction rate of 21.3%/40.2% with cosmesis and 23.8%/38.1% with functionality, while 3.3%/10.7% showed a very-/low satisfaction with cosmesis as well as 5.6%/10.3% with functionality. The remaining women—24.6% and 23.8%—were indifferent. Satisfaction concerning sex life was very-/high in 9.6%/27.7%. In 12.0%/16.9% it was very-/low. Furthermore, 33.7% had no opinion. Furthermore, 27.0%/31.6% of the women reported that clitoriplasty, but not clitoridectomy, had a very-/positive influence on their lives, while 1.3%/8.9% felt it to be very-/negative, and 28.4% were indifferent. Vaginoplasty had a very-/positive influence in 25.7%/33.8% and a very-/negative effect in 3.6%/6.8%. 29.7% had no opinion. Additionally, 75.7% of the women preferred feminizing surgery during infancy/childhood, especially concerning clitoreduction. In conclusion, though the majority of the participants (76%) preferred early feminizing surgery and 60% described a positive effect on their lives, about 10% felt it to have been negative. About 15% of the women suffered from insufficient cosmesis and functionality after surgery. Sex life was even described as poor in nearly 30%. Therefore, the decision about early genital surgery in 46,XX-CAH girls should be considered carefully. Parents should get detailed information about possible complications of surgery and should receive support to understand that postponing surgery does not inevitably cause harm for their child. Importantly, genital surgery when performed in children should only be performed in expert centers with a specialized team including surgeons who are trained in feminizing surgery.

Genetic analysis and novel variation identification in Chinese patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency is an autosomal-recessive disorder caused by mutations in the CYP21A2 gene. The aim of the study was to analyze the molecular data of 155 21-OHD patients and retrospectively investigated the common allelic mutations of CYP21A2 in 1442 Chinese 21-OHD patients. Clinical features and mutations of CYP21A2 gene in 155 unrelated 21-OHD patients were examined. Of the 155 patients, 103 cases were salt-wasting (SW) forms, 38 were simple virilizing (SV) forms and 14 were non-classical (NC) forms. In general, two types of mutations including common allelic mutations (281/310, 90.6%) and rare mutations (29/310, 9.4%) were detected, among them four novel variants c.835G>T, c.1081C>T, c.1423C>T and c.651 + 2 T > G were identified. In 1442 Chinese 21-OHD patients, the most frequently mutations were I2G (36.2%), large deletion/conversion (20.7%) and p.I173N (17.8%), while p.V282L has the lowest frequency. In this study, we provided detailed clinical data and mutation spectrum in Chinese 21-OHD patients. Moreover, four novel CYP21A2 variants (c.835G>T, c.1081C>T, c.1423C>T and c.651 +2 T > G) were identified and computational structural modeling indicated that these novel variations probably affect structural stability. Our findings improve the understanding of CYP21A2 mutational spectrum and contribute to the precise diagnosis and prenatal counseling.

Abstract 64: Clinical and hormonal profile of congenital adrenal hyperplasia from a tertiary centre in India

Background: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders due to defect in enzymes involved in adrenal steroidogenesis with varied clinical presentations.Aims and Objectives: To understand the clinical and hormonal profile of individuals with CAH.Results: Twenty nine patients were included in this cross sectional study ( males (n) =15 (51.7%)) and females (n=14 (48.3%)) (adults=11) (born out of consanguineous marriage = 22 and 3 pairs of siblings). Thirteen patients had salt wasting (SW) phenotype, 14 had simple virilizing (SV) and 2 had non classical CAH (NCCAH). The presentation of SW phenotype was salt wasting crisis in males and ambiguous genitalia in females. The presentation of SV phenotype was clitoromegaly in females; precocious puberty and premature adrenarche in males. All the patients with NCCAH were females with virilization and primary amenorrhea. Majority of patients are on hydrocortisone (82.7%), the rest on prednisolone (13.7%) and dexamethasone (3.4%). Adult females with SV and NCCAH phenotypes had higher 17OHP levels at follow up. Mean adult height of patients with NCCAH was better than SV patients (154.5 cms vs 145.2 cms)Conclusions: The presentation of CAH may be varied but a screening at birth will help us to detect it early.

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.

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.

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.

Prevalence of testicular adrenal rest tumor and factors associated with its development in 6 month to 18 years-old patients with congenital adrenal hyperplasia

Introduction: Congenital adrenal hyperplasia (CAH) is a potentially life-threatening form of primary adrenal insufficiency characterized by cortisol, aldosterone, and epinephrine deficiencies, as well as overproduction of androgen. Infertility is one of the most important complications in male patients with CAH, and testicular adrenal rest tumors (TARTs) are known to be the most important caause of infertility in these patients. In the present study, the prevalence of TART and factors associated with its development were evaluated in patients with CAH. Methods & Materials: This is a descriptive cross-sectional study evaluating 30 males (6 month -18 years) with the classical 21-hydroxylase deficiency (21-OHD) through testicular ultrasonography. Data including age, bone age, puberty, 21-OHD phenotype (salt wasting (SW) or simple virilizing (SV)) and serum levels of 17- hydroxyprogesterone (17-OHP), androstenedione and adrenocorticotropic hormone (ACTH) were records. Results: The prevalence of TART was determined 56.7%, which increased with age with higher prevalence in children >12 (52.9%) year old. The mean age in patients with TART was 12.4 ± 4.18 years. No association was found between TART and 21-OHD phenotype, androstenedione, or 17OHP levels, but an association was found between TART and elevated levels of ACTH (p= 0.049), advanced bone age (p= 0.030) and puberty (p= 0.003). Conclusion: According to the results, TART is very common and can occur in pre-pubertal and young patients, and the disease control could be a factor associated with its development. Therefore, it is suggested to investigate the TART development early in childhood, mainly in poorly controlled 21-OHD patients.

Impact of Newborn Screening on Clinical Presentation of Congenital Adrenal Hyperplasia.

Background and Objectives: The main reason for Newborn screening (NBS) for congenital adrenal hyperplasia (CAH) is to prevent adrenal insufficiency that can lead to life-threatening conditions. On the other hand, screening programs are not always sensitive and effective enough to detect the disease. We aimed to evaluate impact of the national NBS on the clinical presentation of patients with CAH in Lithuania. Materials and Methods: A retrospective study was performed on data of 88 patients with CAH from 1989 to 2020. Patients with confirmed CAH were divided into two groups: (1) 75 patients diagnosed before NBS: 52 cases with salt-wasting (SW), 21 with simple virilising (SV) and two with non-classical (NC) form; (2) 13 patients diagnosed with NBS: 12 cases with SW and 1 case with SV form. For the evaluation of NBS effectiveness, data of only male infants with salt-wasting CAH were analysed (n = 36, 25 unscreened and nine screened). Data on gestational age, birth weight, weight, symptoms, and laboratory tests (serum potassium and sodium levels) on the day of diagnosis, were analysed. Results: A total of 158,486 neonates were screened for CAH from 2015 to 2020 in Lithuania and CAH was confirmed in 13 patients (12 SW, one–SV form), no false negative cases were found. The sensitivity and specificity of NBS program for classical CAH forms were 100%; however, positive predictive value was only 4%. There were no significant differences between unscreened and screened male infant groups in terms of age at diagnosis, serum potassium, and serum sodium levels. Significant differences were found in weight at diagnosis between the groups (−1.67 ± 1.12 SDS versus 0.046 ± 1.01 SDS of unscreened and screened patients respectively, p = 0.001). Conclusions: The sensitivity and specificity of NBS for CAH program were 100%, but positive predictive value—only 4%. Weight loss was significantly lower and the weight SDS at diagnosis was significantly higher in the group of screened patients.

Analysis of the screening results for congenital adrenal hyperplasia involving 7.85 million newborns in China: a systematic review and meta-analysis

Background Congenital adrenal hyperplasia (CAH) is a group of congenital genetic diseases caused by defective steroidogenesis. Our study aims to systematically analyze the screening results for CAH in Chinese newborns. Methods Studies were searched from PubMed, Web of Science, Cochrane library and some Chinese databases up to September, 2020. Meta-analysis was performed after quality assessment and data extraction. Results After a review of 2 694 articles, we included 41 studies enrolling 7 853 756 newborns. In our study, we found that the incidence of CAH in China was 0.43‱ [95% confidence intervals(CI), (0.39‱, 0.48‱)], or 1/23 024 [95%CI, (1/25 757,1/20 815)]. 27 studies were included for analysis of the screening positive rate, which gave a rate of 0.66% [95%CI, (0.54%, 0.78%)]. As for the recall rate of positive cases, 17 studies were included and showed that the recall rate reached 86.17% [95%CI, (82.70%, 89.64%)]. Among the CAH patients, the ratio of males to females was 1.92:1 (119:62), and the ratio of salt wasting (SW) to simple virilization (SV) type was 3.25:1 (104:32). The average 17-hydroxyprogesterone (17-OHP) value of CAH was 393.40 ± 291.85 nmol/L (Range 33-1 300 nmol/L); there was no significant difference between male and female patients (437.17 ± 297.27 nmol/L v.s. 322.25 ± 293.04 nmol/L, P=0.16), but a significant difference was found between SW and SV patients (483.29 ± 330.07 nmol/L v.s. 73.80 ± 7.83nmol/L, P=0.04). Conclusion We systematically analyzed the current situation of neonatal CAH screening in China, which will deepen our understanding for future CAH screening and early diagnosis.

The validity of the Bayley-Pinneau method in predicting final adult height at the onset of puberty in patients with classic congenital adrenal hyperplasia.

The final adult height (FAH) of patients with congenital adrenal hyperplasia (CAH) is often lower than the predicted adult height (PAH) using the Bayley-Pinneau (B&P) method. The aim of the current work was to test the validity of B&P in predicting FAH from a bone age (BA) measurement performed at onset of puberty. This was a retrospective longitudinal observational convenience single-centre study. The study included 54 patients (male and female) with classic CAH, whether salt-wasting (SW) or simple virilising (SV), who had reached FAH. The results of auxological measurements and hormonal data around the time of puberty were retrieved from files. Predicted adult height (PAH) was calculated from a BA taken at onset of puberty and compared with FAH. The median PAH SDS at the onset of puberty (-1.5) was significantly greater than the median FAH SDS (-2.2) (p < 0.001). The median target height SDS (-0.8) was significantly higher than the median FAH SDS (-2.2) (p < 0.001). FAH and FAH SDS were significantly worse in females (150.36 ± 7.23; -2.05 ± 1.13) than in their male counterparts (162.86 ± 3.30; -1.53 ± 0.51) (p value < 0.001; 0.048). In patients with good control, there was no difference between PAH SDS (-1.7) and FAH SDS (-1.5) (p value = 0.37). In patients with poor control (over- or under-treated) FAH SDS (-2.1) was significantly lower than PAH SDS (-1.4) (p value < 0.001). The B&P method was able to accurately predict FAH in children with classic CAH, who were medically well controlled (based on 17-hydroxyprogesterone levels), but overestimated it by a significant 0.7 SD in poorly-controlled patients.