Aromatase Research Articles

Congenital Adrenal Hyperplasia: A Clinical Review

CYP21A2 genes code for 21-hydroxylase (21OH), an enzyme required for the production of cortisol and aldosterone by the adrenal cortex. Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that occurs when both copies of a patient’s CYP21A2 genes are mutated and their ability to produce the adrenal cortical hormones cortisol, and also aldosterone in severe cases, is compromised but their production of adrenal androgens is increased. Neonates with severe CAH will experience life-threatening acute adrenal crisis unless promptly diagnosed and appropriately managed. CAH-induced adrenal crisis presents most critically with hypotension, hyponatremia, hypoglycemia, and hyperkalemia, as well as with less specific symptoms (fatigue, nausea, and vomiting). CAH is also the most common cause of androgenized genitalia in 46XX newborns. Patients with severe CAH will require lifelong hormone replacement and “sick day” dosing in response to physiologic stressors. Adults with CAH face lifelong health challenges, many associated with reproduction. Thus, a practitioner including an advanced practice nurse could encounter a patient with CAH in the emergency department or primary care office. This clinical review discusses the pathophysiology, presentation, diagnosis, and management of CAH. Its goal is to prepare advanced practice nurses and other practitioners to recognize CAH and contribute to the care of patients with CAH across the lifespan, with the goal of reducing complications.

Chimeric CYP21A1P/CYP21A2 Genes in 21-Hydroxylase Deficiency Detected by Long-Read Sequencing and Phenotypes Correlation.

21-Hydroxylase deficiency (21-OHD) is caused by pathogenic variants in CYP21A2. High homology between CYP21A2 and its pseudogene CYP21A1P causes mismatches, leading to deletions and CYP21A1P/CYP21A2 chimeras. To detect chimeric CYP21A1P/CYP21A2 in 21-OHD patients using long-read sequencing (LRS) and analyze genotype-phenotype correlations. From 2015 to 2023, 869 21-OHD patients were enrolled at Peking Union Medical College Hospital, with 113 identified harboring CYP21A2 large deletion. Long-range PCR and LRS were used to identify the types of CYP21A1P/CYP21A2 chimeric. Haplotype analysis explored founder effects, and in vitro assays assessed the functional impact of novel mutations. Clinical data were retrospectively collected and patients were classified into 4 groups based on genotypes and residual enzyme activity to study genotype-phenotype correlations. Ten types of chimeric CYP21A1P/CYP21A2 genes were identified across 119 alleles, including a novel type, CH-10. The most common, CH-1, accounted for 50.4% of all types. Haplotype analysis of 24 SNPs within CYP21A1P/CYP21A2 CH-1 revealed 25 haplotypes, with haplotype 11 being the most prevalent. Variants p.L100P and p.L301V of CYP21A2 showed enzyme activities of 1.36 ± 0.44% or 1.63 ± 0.19% for 17-hydroxyprogesterone to 11-deoxycortisol, and 1.36 ± 0.58% or 3.99 ± 1.09% for progesterone to 11-deoxycorticosterone, respectively, linked to the simple virilizing type. Genotype-phenotype consistency rates were 78.6% to 84% across the 4 groups. LRS is a comprehensive genetic testing method for 21-OHD patients, effectively detecting both CYP21A2 gene variants and CYP21A1P/CYP21A2 chimeric gene types. This study expands the CYP21A2 variant spectrum by identifying a novel chimera. Haplotype analysis revealed diverse haplotypes for each chimeric gene type, suggesting the absence of a common founder effect. The strong genotype-phenotype correlation aids genetic counseling and supports personalized treatment.

Genetics in Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency and Clinical Implications.

Of all congenital adrenal hyperplasia (CAH), 95% to 99% is 21-hydroxylase deficiency (21OHD), an autosomal recessive disease. 21OHD is due to an insufficiency of 21-hydroxylase enzyme, which is encoded by the CYP21A2 gene and involved in cortisol and aldosterone production. The clinical presentation differs widely from severe classic to mild nonclassic CAH. 21OHD represents one of the most complex and at the same time intriguing topics in human genetics and its molecular diagnosis involves ongoing challenges. To provide a meticulous presentation of the topic, we searched the past and present literature, including original articles and reviews from PubMed, ScienceDirect, Web of Science, Embase, and Scopus, using search terms for genetics of 21OHD, 21OHD variants, molecular diagnosis of 21OHD, and 21OHD genetic testing. We offer a comprehensive review focusing on recent developments, new concepts, and conclusions.

Molecular genetic diagnosis and surgical management in a cohort of children with 46,XY disorders/differences of sex development

ObjectiveA firm diagnosis revealing the etiology of disorders/differences of sex development (DSD) is most helpful in guiding clinical management. The aim of this study is to investigate molecular genetic diagnoses and surgical treatment in a cohort of children with 46,XY DSD.MethodsA retrospective study was conducted on children with 46,XY DSD. They were referred to a tertiary surgical center during the period between 2011 and 2022 and were found to have genetic alterations, which were considered etiologies for their DSD. Data on clinical presentations, sex of rearing, genetic findings, surgical treatment, and comorbidities were collected and reviewed.ResultsA total of 21 patients were included in the study: 11 and 10 were reared as male and female, respectively. Genetic alterations were found as the causes for androgen insensitivity syndrome (n = 4), 5-alpha reductase type II deficiency (n = 5), 17-beta hydroxysteroid dehydrogenase III deficiency (n = 1), 17-alpha hydroxylase deficiency (n = 1), and gonadal dysgenesis (n = 10). Of those with gonadal dysgenesis, the genetic alterations were NR5A1 mutation/deletion (n = 3), DMRT1 deletion (n = 4), WT1 mutation (n = 2), and DAX1 duplication (n = 1). A total of 20/21 patients underwent one or more surgical procedures including hypospadias repair (n = 10), gonadectomy (n = 11), gonadal biopsy (n = 4), hernia repair (n = 4), orchidopexy (n = 1), and feminizing genitoplasty (n = 1). A total of 5/21 had germ cell neoplasms in one or both gonads. A total of 8/10 patients with gonadal dysgenesis had comorbidities involving other systems. Of the whole group, seven patients were found to inherit genetic alterations from their parents.ConclusionsMolecular genetic diagnosis enhances the understanding of etiology, improves diagnostic accuracy, and provides precise guidance in the counseling and surgical management of children with 46,XY DSD.

O-16 A CASE OF VITAMIN D-DEPENDENT RICKETS DIAGNOSED IN ADULTHOOD FOLLOWING PREGNANCY

Abstract Discussion Vitamin-D (Vit-D)-dependent rickets are the result of a deficiency in 1-alpha hydroxylase. It is a rare condition that is typically diagnosed in childhood. We present a case of Vit-D-dependent rickets that was detected in adulthood after pregnancy. Clinical Case A 25-year-old female patient has been sent from the neurosurgical clinic to the endocrinology department for evaluation of Vit-D deficiency, which is believed to be causing muscle weakness. She experienced chronic lower back discomfort that began two years ago. She experienced challenges when going down stairs and when bending her body forward. Rest alleviated the lower back pain, whereas physical activity exacerbated it, and it did not interfere with her sleep. The weakness in the legs was there with it. There was no previous fractures or other form of physical injury. The woman's issues became evident after her pregnancy. She consulted a physician nine months postpartum, following the birth of a robust infant. The patient's personal and family history was unremarkable. She had no smoking or drinking habits. During the physical examination, the patient stood 150cm tall and weighed 60kg. Except for left lower extremity muscle strength, which was 3-4/5 on the neurological assessment, everything else was normal. In her laboratory results; calcium 8 mg/dl (8.8-10.6), ionized calcium 3.61mg/dL (4.6-5.0), albumin 4.7 g/dl (3.5-5.2), phosphorus 2.1 mg/dl (2.5-4.5), parathormone 640 ng/L (12-88), creatinine kinase 50 U/L (0-145), alkaline phosphatase 345 U/L (33-98), 25-OH Vit-D 24.2 ug/L, leukocyte: 7100/uL, hemoglobin 11.6 gr/dl, thrombocyte 336000/uL, ALT: 6.6 U/L, osteocalsin: 65 ng/mL (11-48), 1,25-OH Vit-D level: 43.1 pg/mL (15-90). Computed CT revealed type H vertebrae in the thoracic vertebrae, with no disease found in the lumbar, cervical, pelvic, or femur. (Figure 1). Bone density (BD): L1-L4 Z score: -2.7 BMD: 0.858; Femoral neck Z score: -2.7 BMD: 0.644. The EMG results were consistent with moderate myopathy. After one year of complaints, she became wheelchair-dependent. At that point, a neurosurgical doctor prescribed Vit-D and calcium tablets. She was able to walk again in two months and referred to our clinic two years after the initial issue. Genetic testing revealed that CYP27b1>homozygous positive was compatible with Vit-D dependent Rickets Type 1. During the two-year diagnostic period, the patient's height dropped from 153 cm to 150 cm. Calcitriol 0.25 mcg was administered every other day. Her calcium level: 9.4 mg/dL and phosphorus level: 2.2 mg/dL following treatment. This type of rickets is caused by a lack of the 1-alpha hydroxylase enzyme, which is essential for Vit-D production in the kidney. Untreated patients may have muscular and bone abnormalities. It is a sickness that responds exceptionally well to treatment.Figure 1:Vertebral lateral X -ray revealed H shaped vertebrae

Nationwide carrier screening for congenital adrenal hyperplasia: integrated approach of CYP21A2 pathogenic variant genotyping and comprehensive large gene deletion analysis

BackgroundCongenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD CAH) is an autosomal recessive disorder resulting from pathogenic variants in the CYP21A2 gene. The disorder exhibits variable clinical severity, with the classical form manifesting as salt-wasting crisis in neonates, while inducing ambiguous genitalia in females and precocious puberty in males through simple virilization. Identifying at-risk couples during the preconception stage holds significance for optimizing reproductive choices. Methods: This study included 204 unrelated preconception individuals undergoing carrier screening. A robust molecular approach was devised for rapid detection of nine prevalent CYP21A2 pathogenic variants, utilizing Amplification-Refractory Mutation System (ARMS) PCR and mass spectrometry (MS) genotyping. Complementary quantitative real-time PCR (qPCR) and PCR-based Restriction Fragment Length Polymorphism (PCR-based RFLP) assays were employed for comprehensive gene deletion analysis. The concordance of pathogenic variant detection between ARMS-PCR and MS, as well as the consistency observed in molecular insights from qPCR and PCR-based RFLP, fortified the accuracy of our methodologies. Results: Our combined method could detect common pathogenic variants and large gene deletions with high concordance between ARMS-PCR, MS genotyping, qPCR, and PCR-based RFLP assays. Remarkably, two carriers exhibited significant large-scale deletions, while another manifested a carrier state due to minor-scale gene conversion. The estimated carrier frequency in our cohort using these methods was approximately 1 in 65 individuals. Conclusions: The methods used for 21-OHD CAH carrier screening offer a reliable, swift, and cost-effective approach for detecting common pathogenic variants and large deletions. Despite some limitations, such as the inability to detect all rare mutations, the techniques provide a practical solution for carrier screening, with an estimated carrier frequency of 1 in 65 in our study population. These findings support the potential adoption of these methods in national carrier screening programs, offering a practical balance between efficiency and affordability.

Altered expression of aromatase and estrogen receptors in adipose tissue from men with obesity or type 2 diabetes.

Obesity and insulin resistance in men are linked to decreased testosterone and increased estradiol (E2) levels. Aromatase (ARO) converts testosterone into E2, and this occurs mainly in adipose tissue in men. E2 acts through estrogen receptors ESR1 and ESR2, and they potentially affect development of type 2 diabetes (T2D). This study explored alterations in ARO, ESR1 and ESR2 in men with obesity or T2D. Subcutaneous adipose tissue (SAT) from men with or without obesity or T2D was analyzed for ARO, ESR1, and ESR2 gene and protein expression. Data were compared across groups and correlated with markers of obesity, glycaemia, insulin resistance, and sex hormones. Moreover, SAT was incubated with E2 or testosterone for ex vivo glucose uptake measurements. Aromatase ARO levels were higher in SAT from men with obesity compared to non-obese men, and gene expression correlated positively with adiposity, hyperglycaemia and insulin resistance. No association was found between ARO and circulating E2. Men with obesity had lower levels of ESR1 and ESR1:ESR2 ratio, but not ESR2. ESR1 gene expression in SAT correlated negatively with adiposity and insulin resistance markers as well as with ARO expression, and tended to be lower in men with T2D. E2 reduced insulin-stimulated glucose uptake, while testosterone increased basal glucose uptake in adipocytes. Elevated ARO in SAT was found in obese men, and this was linked to insulin resistance and glycaemia, supporting that local estrogen production contributes to metabolic dysregulation. ESR1 was reduced in men with T2D and was linked to adiposity and insulin resistance. Taken together, high ARO and low ESR1 expression in SAT in obese men may contribute to insulin resistance and T2D development.

Genetics and Pathophysiology of Classic Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease that manifests clinically in varying forms depending on the degree of enzyme deficiency. CAH is most commonly caused by 21-hydroxylase deficiency (21OHD) due to mutations in the CYP21A2 gene. Whereas there is a spectrum of disease severity, 21OHD is generally categorized into 3 forms. The classic form encompasses salt-wasting and simple virilizing CAH and the least affected form is termed nonclassic CAH. The classic form of 21OHD occurs in ∼1 in 16 000 births with the most severe salt-wasting cases presenting in the neonatal period with cortisol and aldosterone deficiencies and virilization of external female genitalia. Cortisol deficiency removes normal feedback on the hypothalamic-pituitary-adrenal axis leading to elevations in ACTH and adrenal androgen levels, which often accelerate skeletal maturation, leading to premature epiphyseal growth plate closure. Additionally, supraphysiologic doses of glucocorticoids are necessary to suppress androgen levels, adversely affecting final adult height. This paper highlights a brief history of 21OHD and provides an overview of the genetic basis and pathophysiology of 21OHD.

High clinical utility of long-read sequencing for precise diagnosis of congenital adrenal hyperplasia in 322 probands

BackgroundThe molecular genetic diagnosis of congenital adrenal hyperplasia (CAH) is very challenging due to the high homology between the CYP21A2 gene and its pseudogene CYP21A1P.MethodologyThis study aims to assess the clinical efficacy of targeted long-read sequencing (T-LRS) by comparing it with a control method based on the combined assay (NGS, Multiplex ligation-dependent probe amplification and Sanger sequencing) and to introduce T-LRS as a first-tier diagnostic test for suspected CAH patients to improve the precise diagnosis of CAH.ResultsA large cohort of 562 participants including 322 probands and 240 family members was enrolled for the perspective (96 probands) and prospective study (226 probands). The comparison analysis of T-LRS and control method have been performed. In the perspective study, 96 probands were identified using both the control method and T-LRS. Concordant results were detected in 85.42% (82/96) of probands. T-LRS performed more precise diagnosis in 14.58% (14/96) of probands. Among these, a novel 4141 kb deletion involving CYP21A2 and TNXB was established. A new diagnosis was improved by T-LRS. The duplications were also precisely identified to clarify the misdiagnosis by MLPA. In the prospective study, Variants were identified not only in CYP21A2 but also in HSD3B2 and CYP11B1 in 226 probands. Expand to 322 probands, the actual frequency of duplication haplotype (1.55%) could be calculated due to the accurate genotyping. Moreover, 75.47% of alleles with SNVs/indels, 22.20% of alleles with deletion chimeras.ConclusionT-LRS has higher resolution and reduced cost than control method with accurate diagnosis. The clinical utility of L-LRS could help to provide precision therapy to CAH patients, advance the life-long management of this complex disease and promote our understanding of CAH.

Identification of deleterious variants associated with male infertility genes in a cohort of idiopathic hypospermatogenesis patients.

Hypospermatogenesis is a common histopathological subtype of non-obstructive azoospermia and is characterized by a decrease in the total number of germ cells within the seminiferous tubule as a result of spermatogenic failure. Determination of genetic factors before intracytoplasmic sperm injection can prevent the inheritance of these factors, as hypospermatogenesis patients gives high successful sperm retrieval rate. This study aimed to identify the structural variants associated with idiopathic hypospermatogenesis (iHS) by analyzing patient cohorts diagnosed with azoospermia using whole exome sequencing. It is a hospital-based observational study in which patients reporting with azoospermia due to spermatogenic failure were recruited prospectively. Comprehensive clinical history, blood samples, semen analysis parameters, and reproductive endocrine evaluation reports of 51 hypospermatogenesis patients were collected. The known genetic causes were investigated using XY fluorescent in situ hybridization and Yq microdeletion for exclusion. Whole exome sequencing was performed, and the data of 42 iHS patients was analyzed to identify single nucleotide variants associated with diagnostically important male infertility genes. Genomic analysis of SNVs identified rare deleterious candidate variants in CFTR (c.1265C>T; p.Ser422Phe), CYP21A2 (c.955C>T; p.Gln319Glu), SRD5A2 (c.737G>A; p.Arg245Gln), LHCGR (c.378A>C; p.Lys126Asn) and AR (c.2179C>A; p.Arg727Ser) genes associated with 7/42 idiopathic hypospermatogenesis patients. In silico analysis of variants shows deleterious and probably damaging effects on canonical transcripts of the genes. This exploratory genomic analysis conducted on idiopathic hypospermatogenesis patients shows prevalence of rare deleterious candidate variants in genes associated with human male infertility. The candidate variants in idiopathic hypospermatogenesis patients are heterozygous and genotypically associated with syndromic male infertility. The symptomatic heterozygosity leading to mild spermatogenic failure resulting in hypospermatogenesis points towards a multifactorial etiology of the disease. This study justifies the importance of genetic screening of idiopathic hypospermatogenesis patients for the presence of structural variants in known human male infertility genes.

Detection and characterization of the types of CYP21A1P/CYP21A2 and TNXA/TNXB fused genes by long-read sequencing among children with Steroid 21-hydroxylase deficiency

To assess the diagnostic efficiency of long-read sequencing (LRS) for the determination of CYP21A1P/CYP21A2 and TNXA/TNXB fusion genotypes among children with 21-hydroxylase deficiency (21-OHD) and explore their clinical characteristics. LRS sequencing was carried out on 30 children diagnosed with 21-OHD at the Department of Endocrinology, Fujian Children's Hospital between November 2022 and September 2023 by clinical symptoms or conventional Sanger sequencing combined with multiple ligation-dependent probe amplification (MLPA). The results of the two methods were compared. Clinical data of the children were collected and analyzed. This study has been approved by the Medical Ethics Committee of the Fujian Children's Hospital (Ethic No. 2022ETKLR10024). Of the 30 children with 21-OHD, 11 (36.7%) were found to carry CYP21A1P/CYP21A2 and TNXA/TNXB fusion genes by LRS. The most common type of fused CYP21A1P/CYP21A2 gene was CH-1 (72.7%), and 1 (3.3%) was found to harbor TNXA/TNXB CH-1. Eleven cases (36.7%) were found to carry large deletions by Sanger sequencing combined with MLPA, with the most common one being CYP21A2 exons 1-3 del (72.7%), which was followed by CYP21A2 exons 1-7 del (18.2%). Follow up of 11 patients carrying a fusion gene revealed that 6 were sale wasting (SW) types, 5 were simple virilizing (SV) types, whilst no non-classical (NC) type was found. Four girls had presented with central precocious puberty (CPP). One child carrying TNXA/TNXB CH-1 had presented with CAH-X syndrome. Compared with Sanger sequencing combined with MLPA detection method, LRS sequencing was able to differentiate the subtypes of CYP21A1P/CYP21A2 and TNXA/TNXB fusion genes, pinpoint the breakpoints of the deletions, and directly determine the cis-trans position without the need to analyze the genotype of the pedigree members, which has provided a reliable method for the typing of 21-OHD. As some fusion genes may retain 21-hydroxylase activity, female carriers may have a higher incidence of CPP.

Novel rapid molecular diagnosis methods for comprehensive genetic analysis of 21-hydroxylase deficiency

BackgroundMolecular analysis of the CYP21A2 gene is highly important for understanding the aetiology of 21-hydroxylase deficiency (21-OHD). The aim of this study was to use a novel approach named CNVplex, together with the SNaPshot assay and direct sequencing, to identify CYP21A2 mutations efficiently and comprehensively. Targeted CYP21A2 mutation analysis was performed in 113 patients and 226 parents. Large rearrangements of CYP21A2 were characterized by CNVplex; twenty prevalent mutations, including nine common micro-conversions and eleven high-frequency mutations reported in the literature, were detected by SNaPshot; and rare mutations were investigated by direct sequencing.ResultsAmong the 113 21-OHD patients, 95.6% of the affected alleles were detected accurately by SNaPshot and CNVplex. Prevalent mutations were detected in 69.5% of the alleles; 62.4% of alleles contained pseudogene-derived micro-conversions, 1.8% contained nonpseudogene-derived mutations, and 5.3% contained complex variations resulting from multiple recombinations between CYP21A2 and CYP21A1P. Large rearrangements were identified in 27.0% of the alleles, including five types (CH-1, CH-3, CH-4, CH-5 and CH-8) of chimeric CYP21A1P/CYP21A2 genes. Two novel CYP21A2 haplotypes and four de novo CYP21A2 mutations were characterized. A rare haplotype with a c.955 C > T mutation in the duplicated CYP21A2 gene was found in 0.9% of the probands and 33.3% of the parents. In addition, four parents were also diagnosed with 21-OHD.ConclusionCNVplex and SNaPshot appear to be highly efficient and reliable techniques for use in a molecular diagnosis laboratory, and combined with direct sequencing based on locus-specific PCR, they might constitute a definitive way to detect almost all common and rare 21-OHD-related alleles.

Functional characterization and unraveling the structural determinants of novel steroid hydroxylase CYP154C7 from Streptomyces sp. PAMC26508

This study characterized cytochrome P450 enzyme CYP154C7 from Streptomyces sp. PAMC26508, emphasizing its capability to hydroxylate steroids, especially at the 16α-position. The enzymatic assay of CYP154C7 demonstrated effective conversion across a pH range of 7.2-7.6, with optimal activity at 30°C in the Pdx/PdR plus NADH system. Kinetic analysis on most converted steroids (androstenedione and adrenosterone) was performed which shows a greater affinity for androstenedione (K m , 11.06±1.903μM; V max, 0.0062±0.0002 sec-1) compared to adrenosterone (K m , 34.50±6.2μM; V max, 0.0119±0.0007 sec-1). A whole-cell system in Escherichia coli, overexpressing recombinant CYP154C7, achieved substantial conversion for steroids, indicating that CYP154C7 can also be used as a potential whole-cell biocatalyst. To gain structural insights, homology models of CYP154C7 and its homologs were constructed using CYP154C5 (PDB ID: 6TO2), refined, validated, and used for docking studies. Comparative docking analysis suggests that lysine (K236) in the active site and tyrosine (Y197) in the substrate access channel of CYP154C7 are crucial for substrate selectivity and catalytic efficiency. This study suggests that CYP154C7 could be a promising candidate for developing modified steroids, providing valuable insights for protein engineering to design commercially useful CYP steroid hydroxylases with diverse substrate specificities.

Expanding families: a pilot study on preconception expanded carrier screening in Bahrain

BackgroundPreconception expanded carrier screening (ECS) is a genetic test that enables the identification of at-risk carriers of recessive disorders by screening for up to hundreds of genes. Next-generation sequencing (NGS) development has paved the way for its integration into ECS. This study aims to identify the carrier genetic status of couples experiencing or anticipating conception challenges through NGS-based ECS and to gain an overview of the rare genetic disorders in a population with increased consanguinity.MethodsThirty couples who presented to the Genetic Disease Clinic between 2015 and 2024 with failed reproductive outcomes or with a positive personal or family history of genetic disorders and underwent ECS were included and retrospectively analyzed.ResultsFifty-four individuals (90.00%) were found to carry at least one variant of 95 identified genes, totaling 174 variants. Six individuals (10.00%) tested negative for any variant. Seven individuals had one variant (11.67%), 13 had two variants (21.67%), and 34 had 3 or more variants (56.67%). The most common variants identified were of HBA, HBB, CYP21A2, and G6PD genes. Most of the detected variants were unknown or unexpected (n = 143, 82.18%). Eight couples carried two or more variants in common. Consanguinity was reported in 14 couples (46.67%).ConclusionsPreconception ECS is crucial for reproductive planning, permitting couples to evaluate their combined genetic risks and make informed decisions, reducing the chance of having children with genetic disorders.

Synchronously Mature Intersex Japanese Flounder (Paralichthys olivaceus): A Rare Case.

Japanese flounder is usually gonochoristic, with gonads that are either testes or ovaries. Here, we report an unusual case of hermaphroditism in Japanese flounder captured from the Bohai Sea. In the intersex flounder, the membrane of the upper ovary was closely connected to the abdominal muscles and internal organs, and the eggs filled the entire abdomen. The lower ovary was small and closely connected to the testes. The testes contained few fully mature sperm. Both eggs and sperm were capable of fertilization. The levels of several reproduction-related hormones (17β-estradiol, 11-ketotestosterone, 17α, 20β-dihydroxyprogesterone, luteinizing hormone, follicle-stimulating hormone, and testosterone) in the intersex flounder were intermediate, between those in females and males. The results showed that the heterozygosity of the intersex flounder was 0.632, and there were 28 single-nucleotide polymorphisms in the cyp21a gene. Compared with that of wild flounder, the activity of 21-hydroxylase was reduced by approximately 20.0%, and expressions of cyp19a, amh, and dmrt1 differed. We present the first report of its kind, detailing the anatomy, hormonal endocrinology, molecular biology, and physiology of the intersex Japanese flounder.

7030 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

9266 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing

Abstract Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome. Presentation: 6/1/2024

8528 An Unusual Case Of Adrenal Hemorrhage As An Initial Manifestation Of Castleman Disease In A 29 yo Male With Unsuspected Late-Onset Congenital Adrenal Hyperplasia

Abstract Disclosure: L.M. Martel: None. F. Mercier: None. S. Parisien-La Salle: None. A. Lacroix: None. A. Liberman: None. N. Bettache: None. H. Olney: None. I. Bourdeau: None. Background: Castleman disease is a lymphoproliferative disorder with no clear etiology. We report the case of a 29 yo man presenting with an adrenal hemorrhage as initial manifestation of Castleman disease and unsuspected late-onset congenital adrenal hyperplasia. Clinical case: A 29-year-old male with no significant past medical history except for unilateral orchiectomy at 1 year-old for a possible testicular mass was referred to our adrenal clinic for a 6,2x7,5 cm left adrenal mass showing at CT scan necrosis and an hemorrhagic component (HU52). The patient presented initially with acute left abdominal pain. The adrenal mass showed at peripheral high uptake at PET-FDG (SUVmax 5,3) with no central uptake. His physical exam was unremarkable except for obesity. The hormonal work-up for the adrenal mass revealed normal metanephrines, and normal 1mg-dexamethasone suppression test with morning cortisol of 18 nmol/L (N<50). Further hormonal investigations based on the high adrenal mass uptake and possible adrenocortical carcinoma included: 17-OH Progesterone: 7.7 nmol/L (N:1.1-7.0), Androstenedione: 14.1 nmol/L (N: 1.5-9.0), Testosterone: 11.3 nmol/L (N: 9.0-28.3) and DHEAS: 3.6 umol/L (2.3-18.7). The ACTH levels were increased at 21.2 pmol/L (N:2-11) with morning cortisol of 220 nmol/L. During the consultation, the patient reported a pleuritic chest pain and underwent an urgent CT pulmonary angiography showing a pulmonary embolism with lung infarction in addition to mediastinal lymphadenopathy (biopsy negative) and a mass in his thymus. He underwent thymectomy and the pathology report described Castleman disease (hyaline vascular type). Taking into account the increased level of 17-OH progesterone, we performed an ACTH stimulation test (250 mcg) that showed an increase of cortisol levels from 333nmol/L to 874nmol/L at 60 minutes and of 17OH Progesterone levels from 5.2nmol/L to 44.2nmo/L excluding adrenal insufficiency, but confirming the diagnosis of late-onset congenital adrenal hyperplasia. Genetic analyses: After written consent, the patient underwent genetic analysis of the CYP21A2 gene by direct sequencing and MLPA. The analyses revealed a heterozygous pathogenic variant in the CYP21A2 gene (c.293-13A/C>G). No other genetic variant was identified in the gene. Follow-up: Subsequent adrenal MRIs showed decreasing size of the left adrenal mass; 3 cm at 3 months, 2 cm at 6 months and no residual mass was seen at 9 months supporting the diagnosis of adrenal hemorrhage. Conclusion: To our knowledge, we report a first case of Castleman disease presenting with adrenal hemorrhage as initial symptom with the association of late-onset congenital adrenal hyperplasia. The possible link between these diseases remains to be explored. Presentation: 6/3/2024

7534 Characterizing The Presentation Of Non-Classical Congenital Adrenal Hyperplasia

Abstract Disclosure: S. Patel: None. M. Skamagas: None. A.C. Levine: None. M. Yau: None. Introduction: 21-hydroxylase deficiency, due to CYP21A2 gene mutations, is the most common steroidogenic enzymatic defect causing congenital adrenal hyperplasia (CAH). Disease severity is categorized into the classical and milder, non-classical (NCAH) form. Many patients go undiagnosed due to variable presentation and delayed onset. We aim to investigate the specific presenting concerns, age of onset, and genotypes associated with NCAH. Methods: A retrospective chart review of 285 patients with CAH seen over the past 2 years at our academic pediatric and adult endocrine clinics was performed. Records were reviewed for presenting concerns and age of symptom onset. CYP21A2 gene analysis was performed commercially via multiplex ligation-dependent probe amplification or next generation sequencing. Results: Of the 165 patients classified with NCAH, genotypes were available for 124 patients. Of the patients with a V281L allele, 43 were homozygous and 49 were heterozygous. 7 patients had the P30L allele and 7 had other allele combinations. The overall most common presenting concerns were early pubarche (29%), hirsutism (28%), and irregular menses (26%). In V281L homozygotes, family history most commonly led to diagnosis (30%). In patients with V281L heterozygosity or the P30L allele, the predominant concern was early pubarche (37% and 43% respectively). 15% of patients were screened prenatally or only due to family history and were asymptomatic at diagnosis. Patients’ age at diagnosis ranged from 0-40 years, with the mean age at diagnosis for the total group being 13.3 ± 8.9 years. Mean age of onset in symptomatic V281L homozygotes (15.4 ± 7.9 years) trended toward an older age compared to symptomatic V281L heterozygotes (15.1 ± 10.5 years) (P value = 0.90). Those who were heterozygous for V281L/P30L or undetermined whether homozygous for V281L or heterozygous with a deletion were excluded from age of onset comparisons. Discussion: The mean age of symptom onset was in adolescence and did not differ significantly between V281L homozygotes and heterozygotes. Early pubarche is a frequent presenting symptom in children with NCAH, and our data support this finding. P30L has been associated with a more severe phenotype than V281L, and in our cohort, the only patient presenting in adrenal crisis had a genotype with a P30L mutation. As NCAH is one of the most common autosomal recessive disorders in humans and there is an allelic predominance of V281L in NCAH patients, it is not surprising that a large percentage of V281L homozygotes were diagnosed due to family history prior to developing symptoms. Such patients need to be followed clinically and our data describing symptom onset could be useful in counseling these families and those with prenatal diagnoses. Presentation: 6/1/2024

12375 Rhabdomyolysis As A Cause Of PTH-independent Hypocalcemia In A Child With Acute Influenza Infection

Abstract Disclosure: S. Bhattarai: None. K. Halpin: None. Introduction: Hypocalcemia is characterized by low blood calcium level. Normal range for calcium for ages 12- 19-year is 8.5-10.6 mg/dli . Severe hypocalcemia is considered as serum calcium level of <7 mg/dl. Hypoparathyroidism, pseudohypoparathyroidism and Vitamin D deficiency are some of the most common causes encountered by pediatrician endocrinologists. Rhabdomyolysis with hypocalcemia and elevated PTH is a rare presentation that should be considered, particularly for children presenting with acute viral illness. We present a rare case of rhabdomyolysis associated with hypocalcemia not related to hypoparathyroidism. Case: A 12-year-old female presented to the ED with body ache, poor oral intake and vomiting. She was found to be influenza positive. Labs showed hypocalcemia (4.8 mg/dl). She had an undetectable 25-OH vitamin D level (<5 ng/ml) and elevated iPTH level (609 pg/ml). We discussed the possibility of her etiology of hypocalcemia as Vitamin D deficiency although her presentation was atypical at an older age with negative imaging for rickets, hyperphosphatemia (6.0 mg/dl), and normal ALP (334 unit/L). She did not have any phenotypic features of Albright’s hereditary osteodystrophy with normal renal function. She had elevated CK (4829 U/L) supporting rhabdomyolysis secondary to acute influenza as a cause of her hypocalcemia. Rhabdomyolysis, a known complication of influenza infection, causes cell membrane destruction which impairs the normal function of Na-K-ATPase channel leading to increase in intracellular sodium activating Na/Ca exchanger. This in turn causes influx of calcium intracellularly causing hypocalcemia. Additionally, any injury leads to high phosphorus release from cells due to its lysis. High phosphorus is also caused by reduced oxidative metabolism in muscles impairing phosphate use. This excess of phosphate then combines with calcium and causes calcium-phosphate complex in soft tissues. Hyperphosphatemia additionally inhibits 1 alpha hydroxylase limiting formation of calcitriol leading to hypocalcemia. iiConclusion: Our patient had severe hypocalcemia due to influenza-related rhabdomyolysis. Rhabdomyolysis is an important cause of hypocalcemia in children, especially with acute viral illness. Accordingly, it is also important to obtain serum electrolytes in patients presenting with rhabdomyolysis as hypocalcemia may lead to complications like seizures and cardiac arrhythmia. Endnotes:i Roizen, Jeffrey & Shah, Vipul & Levine, Michael & Carlow, Dean. (2013). Determination of Reference Intervals for Serum Total Calcium in the Vitamin D-Replete Pediatric Population. The Journal of clinical endocrinology and metabolism. 98. 10.1210/jc.2013-3105.ii Ding LN, Wang Y, Tian J, Ye LF, Chen S, Wu SM, Shang WB. Primary hypoparathyroidism accompanied by rhabdomyolysis induced by infection: A case report. World J Clin Cases. 2019 Oct 6;7(19) Presentation: 6/2/2024