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Pathophysiology, Genetics, and Treatment of Congenital Adrenal Hyperplasia

• Deborah P. Merke, MD, MS, Adjunct Investigator, NICHD & Chief, Section of Congenital Disorders, CC
• Ashwini Mallappa, MD, Associate Research Physician
• Vipula Kolli, PhD, Staff Scientist
• Qizong Lao, PhD, Staff Scientist
• Elizabeth Joyal, MSN, Nurse Practitioner
• Lee Ann Keener, MSN, Nurse Practitioner
• Jay Desai, BA, Postbaccalaureate Intramural Research Training Award Fellow
• Emily Frucci, BS, Postbaccalaureate Intramural Research Training Award Fellow
• Megan Parker, BS, Postbaccalaureate Intramural Research Training Award Fellow
• Breesa Bennett, BS, Pathways Intern

In its most severe classic form, congenital adrenal hyperplasia (CAH) is a life-threatening, rare orphan disease that is part of the neonatal screen performed in all 50 U.S. states [Reference 1]. In its mildest non-classical form, CAH is one of the most common autosomal recessive diseases and may be a common cause of female infertility. Our intramural NIH research program strives to elucidate the pathophysiology and genetics of CAH, thus facilitating the development of new approaches to the diagnosis, evaluation, and treatment of the disease. We are conducting the largest ever Natural History Study of CAH, with over 450 patients enrolled. We were the first to identify adrenaline deficiency as a new hormonal imbalance in CAH and the first to report in CAH smaller-than-normal amygdala, the emotion regulator of the brain, providing insight into hormonal effects on the brain. We found that approximately 10 to 15 percent of patients with CAH owing to 21-hydroxylase deficiency have a contiguous gene deletion syndrome resulting in connective tissue dysplasia, hypermobility-type Ehlers-Danlos syndrome, which represents a novel phenotype named CAH-X. Central to our work is the study of new treatments, including a long-term trial testing sex hormone blockade in children, and novel ways of replacing cortisol, aimed at mimicking the normal circadian rhythm of cortisol secretion. The NIH Clinical Center is the ideal venue in which to carry out such studies and is one of the few places in the world that facilitates the conduct of long-term studies of rare diseases.

Adrenal crisis prevention

Patients with adrenal insufficiency are at risk for life-threatening, salt-wasting adrenal crises. Management of illness episodes aims to prevent adrenal crises. We evaluated rates of illnesses and associated factors in our large cohort of patients with adrenal insufficiency attributable to congenital adrenal hyperplasia, who were followed prospectively at the NIH Clinical Center and received repeated glucocorticoid stress-dosing education. We performed longitudinal analysis of over 2,200 visits from 156 CAH patients over 23 years [Reference 2]. During childhood, there were more illness episodes and stress dosing than during adulthood; however, more emergency room visits and hospitalizations occurred during adulthood. The most robust predictors of stress dosing were young age, low hydrocortisone dose, and high fludrocortisone dose during childhood, and, during adulthood, female sex. Gastrointestinal and upper respiratory-tract infections were the two most common precipitating events for adrenal crises and hospitalizations across all ages. Life-threatening adrenal crisis with hypoglycemia occurred in 11 pediatric patients (ages 1.1–11.3 years). Undetectable epinephrine was associated with emergency room visits during childhood and illness episodes during adulthood.

This longitudinal assessment of illnesses, glucocorticoid stress-dosing practices, and illness sequelae in patients with adrenal insufficiency from CAH resulted in recommendations to revise age-appropriate glucocorticoid stress-dosing guidelines to include more frequent glucocorticoid dosing and frequent intake of simple and complex carbohydrates. Our new age-appropriate guidelines aim to reduce adrenal crises and prevent hypoglycemia, particularly in children. Our suggestions were incorporated into the Endocrine Society Clinical Practice Guideline for Congenital Adrenal Hyperplasia.

Genotype-phenotype studies of CAH-X

CAH is most commonly caused by 21-hydroxylase deficiency. The gene encoding 21-hydroxylase, CYP21A2, and a highly homologous pseudogene, CYP21A1P, map to the short arm of chromosome 6 within the human leukocyte antigen histocompatibility complex. The deleterious sequence in the CYP21A1P pseudogene can be transferred to the CYP21A2 functional gene by homologous recombination, and such events produce common mutations that account for approximately 95% of all CYP21A2 disease–causing mutations. Of the common mutations, approximately 30% are large deletions. The TNXB gene, encoding tenascin-X, an extracellular matrix protein that is highly expressed in connective tissue, and a highly homologous pseudogene, TNXA, flank CYP21A2 and CYP21A1P, respectively. Autosomal recessive tenascin X deficiency was described as a cause of Ehlers-Danlos syndrome in 2001. We hypothesized that deletions of CYP21A2 might commonly extend into the TNXB gene, and we have been studying this phenomenon in our Natural History Study.

The first evaluation of the potential clinical implications of TNXB heterozygosity in CAH patients was performed in our Natural History Study of CAH (www.ClinicalTrials.gov Identifier No. NCT00250159) at the NIH Clinical Center. In 2013, we prospectively studied 193 consecutive unrelated patients with CAH with clinical evaluations for manifestations of Ehlers-Danlos syndrome and genetic evaluations for TNXB mutations. Heterozygosity for a TNXB deletion was present in 7% of CAH patients; such CAH patients were more likely than age-and sex-matched CAH patients with normal TNXB to have joint hypermobility, chronic joint pain, multiple joint dislocations, and a structural cardiac valve abnormality detected by echocardiography. Six of 13 probands had a cardiac abnormality, including the rare quadricuspid aortic valve, a left ventricular diverticulum, and an elongated anterior mitral valve leaflet. As a result of the study, the term CAH-X was coined to describe the subset of CAH patients who display an Ehlers-Danlos syndrome phenotype resulting from to the monoallelic presence of a CYP21A2 deletion extending into the TNXB gene.

The study of CAH-X has provided insight into the recombination events that occur in the class III region of the major histocompatibility complex (MHC) locus, a region of the genome that is predisposed to genetic recombination and misalignment during meiosis. The majority of deletions generate chimeric CYP21A1P/CYP21A2 genes. Chimeric recombination between TNXB and TNXA also occurs (Figure 1). The recombination event deletes CYP21A2 and therefore represents a CAH disease–causing allele. We described three unique types of TNXA/TNXB chimera (CH): CAH-X CH-1 renders the gene nonfunctional, resulting in reduced dermal and serum TNX expression; CAH-X CH-2 alters protein structure; and CAH-X CH-3 is predicted to reduce protein folding energy. Our laboratory continues to investigate how TNXB contributes to the phenotype of CAH patients.

To date, we have described 24 patients (19 families) with monoallelic CAH-X and three patients with biallelic CAH-X. Approximately 10 to 15 percent of patients with CAH resulting from 21-hydroxylase deficiency are now estimated to be affected by CAH-X. Overall, CAH-X patients have generalized joint hypermobility, subluxations, and chronic arthralgia, and about 25% have cardiac structural abnormalities. Patients with biallelic CAH-X show severe skin hyperextensibility with delayed wound healing and significant joint hypermobility. Other connective-tissue disease manifestations in CAH-X patients include chronic tendonitis and/or bursitis, rectal prolapse, severe gastroesophageal reflux, and cardiac abnormalities. Genetic testing for CAH-X is complex and complicated by pseudogene interference and the large, 70kb size of the TNXB gene. In 2019, we developed a PCR–based, high-throughput, cost-effective assay that accurately identifies CAH-X [Reference 3]. The assay had 100% sensitivity and 99.2% specificity.

The study of CAH-X syndrome provides insight into the complex clinical and genetic characteristics associated with CAH and promises to improve patient outcome through the development of focused medical management aimed at preventing long-term consequences.

New and improved biomarkers of CAH

The diagnosis and management of CAH has been limited by inadequate biomarkers. Several pitfalls have been identified in the use of 17-hydroxyprogesterone, the most commonly used biomarker, for both diagnosis and management. The development of liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels of adrenal steroids has expanded the repertoire of potential new and improved steroid biomarkers. We found that steroids synthesized with the participation of 11beta-hydroxylase (11-oxygenated C19 steroids) are abundant in patients with CAH resulting from 21-hydroxylase deficiency (Figure 2). With our collaborators Richard Auchus and Adina Turcu, we compared traditional and 11-oxygenated androgens in patients with non-classic (mild) CAH resulting from 21-hydroxylase deficiency and patients with symptoms of hyperandrogenism from other causes. Patients with non-classic CAH present with clinical manifestations of hyperandrogenism, features that are shared with other disorders of androgen excess. In particular, the clinical phenotype of women with non-classic CAH is similar to the more common polycystic ovarian syndrome. The diagnosis of non-classic CAH is based on serum 17-hydroxyprogesterone and usually requires dynamic testing with synthetic ACTH (cosyntropin) testing. We found that 11-oxygenated C19 steroids are disproportionately elevated compared with conventional androgens in non-classic CAH, and steroid panels can accurately diagnose non-classic CAH in unstimulated blood tests [Reference 4]. We continue to explore the utility of these newly described steroids in the diagnosis and management of CAH.

Novel treatment approaches: sex steroid blockade and inhibition

As an alternative approach to the treatment of CAH, the effects of elevated androgen and estrogen could be prevented through the use of sex steroid blockade. Short-term (two-year) administration of an antiandrogen and aromatase inhibitor and reduced hydrocortisone was shown to normalize linear growth rate and bone maturation. A prospective long-term randomized parallel study to adult height of an antiandrogen (flutamide) and an aromatase inhibitor (letrozole), and reduced hydrocortisone dose vs. conventional treatment is near completion. The main outcome is adult height, and we will compare data between the treatment groups. The goal of this novel treatment approach is to normalize the growth and development of children with CAH and, ultimately, to determine whether the treatment regimen is effective in improving the growth of children with CAH. The Clinical Center is the ideal place to carry out such a long-term study of a rare disease.

Since the inception of our study of peripheral blockade of sex hormones using an antiandrogen and aromatase inhibitor, new and improved drugs that block sex steroids have been developed. In collaboration with the group of Perrin White, we are studying abiraterone, an irreversible inhibitor of 17a-hydroxylase, a key enzyme required for testosterone synthesis, in a multicenter Phase 1/2 study in prepubescent children (NCT 02574910).

In 2020, we reported for the first time the use of nevanimibe, an orally administered ACAT1/sterol O-acyltransferase 1 (SOAT1) inhibitor, as adjuvant therapy for CAH [Reference 5]. The enzyme is the gatekeeper for the esterification of cholesterol, a necessary step for adrenocortical steroid biosynthesis. This proof-of-concept study showed that short-term (two-week) use of nevanimibe at various doses reduced 17-hydroxyprogesterone, a biomarker of adrenal androgen production. Larger studies of longer duration are needed, but use of the drug, which selectively inhibits adrenal cortex function, might reduce androgen excess and thus allow for lower glucocorticoid dosing in CAH.

Additional Funding

• Cooperative Research and Development Agreement (CRADA) #02800 for Age-Appropriate Hydrocortisone Formulations for the Treatment of Adrenal Insufficiency including Congenital Adrenal Hyperplasia
• NIH U Grant: Abiraterone Acetate in Children with Classic 21-Hydroxylase Deficiency

Publications

1. Merke DP, Auchus RJ. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. N Engl J Med 2020;83(13):1248-1261.
2. El-Maouche D, Hargreaves CJ, Sinaii N, Mallappa A, Veeraraghavan P, Merke DP. Longitudinal assessment of illnesses, stress dosing and illness sequelae in patients with congenital adrenal hyperplasia. J Clin Endocrinol Metab 2018;103:2336-2345.
3. Lao Q, Brookner B, Merke DP. High throughput screening for CYP21A1P-TNXA/TNXB chimeric genes responsible for Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia. J Mol Diagn 2019;21:924-931.
4. Turcu AF, El-Maouche D, Zhao L, Nanba AT, Gaynor A, Veeraraghavan P, Auchus RJ, Merke DP. Androgen excess and diagnostic steroid biomarkers for nonclassic 21-hydroxylase deficiency without cosyntropin stimulation. Eur J Endocrinol 2020;183:63-71.
5. El-Maouche, D, Merke DP, Vogiatzi MG, Chang AY, Turcu AF, Else T, Joyal EG, Lin VH, Plaunt MR, Mohideen P, Auchus RJ. Phase 2, multicenter study of nevanimibe for the treatment of congenital adrenal hyperplasia. J Clin Endocrinol Metab 2020;105:2771-2778.

Collaborators

• Richard J. Auchus, MD, PhD, University of Michigan, Ann Arbor, MI
• Veronica Gomez-Lobo, MD, Children's National Health System, Washington, DC
• James Marko, MD, Radiology and Imaging Sciences, NIH Clinical Center, Bethesda, MD
• Martha Quezado, MD, Laboratory of Pathology, NCI, Bethesda, MD
• Richard J. Ross, MD, University of Sheffield, Sheffield, United Kingdom
• Ninet Sinaii, PhD, MPH, Biostatistics and Clinical Epidemiology Service, NIH Clinical Center, Bethesda, MD
• Adina Turcu, MD, University of Michigan, Ann Arbor, MI
• Perrin White, MD, University of Texas Southwestern Medical Center, Dallas, TX

Contact

For more information, email dmerke@nih.gov or visit https://irp.nih.gov/pi/deborah-merke.

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