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Pathophysiology, Genetics, and Treatment of Congenital Adrenal Hyperplasia

• Deborah P. Merke, MD, MS, Adjunct Investigator, NICHD, and Chief, Section of Congenital Disorders, Clinical Center
• Rida Javaid, MD, Staff Clinician
• Vipula Kolli, PhD, Staff Scientist
• Qizong Lao, PhD, Staff Scientist
• Elizabeth Joyal, MSN, Nurse Practitioner
• Lee Ann Keener, MSN, Nurse Practitioner
• Daniella Bick, BS, Postbaccalaureate Intramural Research Training Award Fellow
• Annie Schulman, BS, Postbaccalaureate Intramural Research Training Award Fellow
• Charles Sukin, BS, Postbaccalaureate Intramural Research Training Award Fellow

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. In its mildest non-classic form, CAH is one of the most common autosomal recessive diseases and may be a common cause of female infertility. Our 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 smaller-than-normal amygdala, the emotion regulator of the brain, in CAH, providing insight into hormonal effects on the brain. We also found that approximately 15 percent of patients with CAH owing to 21-hydroxylase deficiency have a contiguous gene-deletion syndrome resulting in connective tissue dysplasia and a hypermobility-type Ehlers-Danlos syndrome, which represents a novel phenotype and which we named CAH-X. Our studies over the years have had a major impact on the management of patients with CAH, including changing the recommended stress-dosing practices in the Endocrine Society Clinical Practice Guidelines and, more recently, establishing recommendations for the management of women with CAH during pre-conception, pregnancy, and postpartum. Central to our work is the study of new treatments, including a long-term trial testing sex hormone blockade in children, novel ways of replacing cortisol aimed at mimicking the normal circadian rhythm of cortisol secretion, and antagonists of the CRH (corticotropin-releasing hormone) type 1 receptor to suppress the drivers of excess androgen production to allow for lower-dose glucocorticoid therapy. 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. Through the study of a large cohort of these unique patients, there is a great potential to expand our understanding of hormonal effects on normal development and physiology, provide insights into gene regulation and expression, and develop and test new treatment approaches.

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, which 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 TNXA, a highly homologous pseudogene, 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 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 in 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 lab continues to investigate how TNXB contributes to the phenotype of CAH patients, and to identify novel chimeric genes.

To date, we have described 24 patients (19 families) with monoallelic CAH-X and three patients with biallelic CAH-X. It is now estimated that approximately 15% of patients with CAH resulting from 21-hydroxylase deficiency are affected by CAH-X. Overall, CAH-X patients have generalized joint hypermobility, sub-luxations, 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. The assay had 100% sensitivity and 99.2% specificity and was recognized as an important additional molecular genetic testing platform by the European Molecular Genetics Quality Network, an international group of genetic methodology experts. We continue to refine and improve genetic testing methodology. This year we reported that pseudogene TNXA variants may interfere with the genetic testing of CAH-X.

The study of the 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.

Longitudinal assessments of cardiovascular disease risk and metabolic morbidity

Our long-term natural history study has been critical for understanding disease-specific outcomes, and the long duration of our study has enabled us to analyze longitudinal data and disease trajectory. Longitudinal assessment of risk factors for cardiovascular disease (CVD) revealed higher prevalence of metabolic morbidity in patients with CAH than in the general population (National Health and Nutrition Examination Survey–NHANES) across the lifespan. Associations with treatment- and disease-related factors differed during childhood and adulthood.

Prior studies of risk factors for cardiovascular disease and metabolic morbidity in CAH had been reported in cross-sectional studies with varied results. In this first longitudinal assessment of CVD risk factors, we identified the prevalence and age of onset of risk factors for CVD and metabolic morbidity, described risk factors over time, and evaluated these outcomes in relation to treatment- and disease-related characteristics in our large cohort of patients followed during childhood and into adulthood for an average of 18 years. We found that children with classic CAH have metabolic risk (obesity, hypertension, fasting hyperglycemia, and hypertriglyceridemia) starting at a young age (prior to 10), and that risks are distinctly different during adulthood. In particular, hypertension during childhood, but not adulthood, was associated with mineralocorticoid dosing. The study provided guidance regarding treatment practices in relation to metabolic and cardiovascular-disease risk.

The effect of GnRHa on bone health in CAH

Our study was the first clinical study to report the effect of GnRHa (gonadotropin-releasing hormone analog) on bone health in the CAH population. Reduced bone mineral density (BMD) is a known possible risk of delaying puberty, and our longitudinal data were especially valuable for evaluating the long-term effects of BMD into adulthood. Childhood GnRHa therapy to delay central puberty for an average of 4.5 years in patients with CAH did not affect BMD at attainment of adult height or during early adulthood when peak BMD occurs in healthy individuals. However, an overall reduction in BMD occurred during the second and third decades of life in our CAH cohort, regardless of GnRHa therapy, a possible effect of chronic supra-physiologic glucocorticoids. Our study demonstrated that children with CAH who experience early puberty benefit from GnRHa treatment, as evidenced by a positive effect on adult height. The height gain for treated patients was 0.8 SD (approximately 2.5 inches) from predicted height at the start of puberty, which is clinically meaningful.

We found that GnRHa therapy can be used safely to delay central puberty in children with CAH and has a positive effect on adult height. Importantly, we also found that the CAH population is at risk for early-onset low bone mass, given the decline in z-scores observed over time and in early adulthood.

Novel treatment approaches: circadian cortisol replacement

Humans have biological clocks with characteristic patterns of hormone secretion. Cortisol has a circadian rhythm, with levels low at sleep onset, rising between 0200hr and 0400hr, peaking in the early morning, and then declining throughout the day. Existing glucocorticoid replacement is non-physiologic, and the lack of diurnal rhythm may contribute to the many adverse outcomes observed in patients with adrenal insufficiency. In CAH, physiologic cortisol replacement might improve control of adrenal androgens at lower glucocorticoid doses, thus improving patient outcome. A promising treatment approach we have therefore been studying is circadian cortisol replacement in patients with CAH.

In 2016, we successfully replaced cortisol in a physiologic manner through the use of a pump usually used to deliver insulin. A programmed 24-hour infusion of hydrocortisone was delivered subcutaneously for six months to eight patients with adrenal insufficiency resulting from 21-hydroxylase deficiency and with multiple comorbidities. Following six months of pump therapy, patients experienced significant improvement in disease control at similar or lower daily doses of glucocorticoid, and significant improvement in their quality of life and fatigue compared with oral conventional therapy. The improvements achieved in androgen control, lean body mass, and health-related quality of life after six months of pump therapy were maintained at eighteen months.

Our group was the first to study circadian cortisol replacement in CAH patients with the use of a modified-release formulation of hydrocortisone, (MR-HC, Chronocort®, CRADA #02800). We successfully completed a phase 2, open-label trial of 16 adults with classic CAH. Compared with various forms of conventional therapy prior to entry, six months of twice daily MR-HC yielded improved disease control throughout the day, using a lower hydrocortisone dose equivalent. Successful completion of this phase 2 study, carried out at the NIH Clinical Center, resulted in a multicenter international phase 3, parallel arm, randomized, open-label study to determine whether this new modified-release preparation of hydrocortisone improves short-term clinical outcome; 122 Adults with classic CAH completed the phase 3 study in 2021. The primary endpoint, 17α-hydroxyprogesterone 24-hour area under the curve standard deviation, did not differ between the two groups; however, improved biochemical control of CAH was observed in the morning and early afternoon in those receiving the MR-HC compared with standard treatments. Sustained benefits with decreased dosage were observed in 18 months extension. Based on these data, MR-HC is now licensed in the UK and Europe. Our patients who were enrolled in the phase 3 study are continuing MR-HC therapy in a long-term follow-up study, and future US studies are under way.

Studies of circadian cortisol replacement provide insight into the role that circadian rhythm plays in the development of the comorbidities associated with adrenal insufficiency. Physiologic cortisol replacement represents a novel treatment approach that promises to improve treatment outcome for patients with CAH, as well as other forms of adrenal insufficiency.

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 on the effect of an antiandrogen (flutamide) and an aromatase inhibitor (letrozole), as well as reduced hydrocortisone dose vs. conventional treatment, on adult height is near completion; 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.

Since the inception of our study on 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 17α-hydroxylase, a key enzyme required for testosterone synthesis, in a multicenter Phase 1/2 study in prepubescent children (NCT02574910).

Novel treatment approaches: hypothalamic-pituitary-adrenal axis suppression

Potential strategies to address the drivers of excess androgen production in CAH include suppression of the hypothalamic-pituitary-adrenal axis. Newly developed small-molecule antagonists of the corticotropin-releasing factor type 1 (CRF1) receptor antagonist are being studied. Successful phase 2 studies completed in 2021 led to multicenter, randomized, double-blind, placebo-controlled, long-term (52 weeks) clinical trials in adults with CAH (NCT04490915) to study the effect of CRF-1 antagonists on outcomes such as improved adrenal androgen control, as well as the potential of reducing glucocorticoid dose as an add-on therapy.

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
• Cooperative Research and Development (CRADA) for a Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Safety and Efficacy of Crinecerfont in Adult Subjects with CAH, Followed by Open-Label Treatment

Publications

1. Mallappa A, Merke DP. Management challenges and therapeutic advances in congenital adrenal hyperplasia. Nat Rev Endocrinol 2022 18(6):337–352.
2. Maher JY, Gomez-Lobo V, Merke DP. The management of congenital adrenal hyperplasia during preconception, pregnancy, and postpartum. Rev Endocr Metab Disord 2022 24(1):71–83.
3. Lao Q, Zhou K, Parker M, Faucz FR, Merke DP. Pseudogene TNXA variants may interfere with the genetic testing of CAH-X. Genes 2023 14(2):265.
4. Torky A, Sinaii N, Jha S, El-Maouche, Desai J, El-Maouche D, Mallappa A, Merke DP. Cardiovascular disease risk factors and metabolic morbidity in a longitudinal study of congenital adrenal hyperplasia. J Clin Endocrinol Metab 2021 106(12):e5247–5257.
5. Flokas ME, Wakim P, Kollender S, Sinai N, Merke DP. Gonadotropin-releasing hormone agonist therapy and longitudinal bone mineral density in congenital adrenal hyperplasia. J Clin Endocrinol Metab 2023 dgad514.

Collaborators

• Veronica Gomez-Lobo, MD, Pediatric and Adolescent Gynecology, NICHD, Bethesda, MD
• James Marko, MD, Radiology and Imaging Sciences, NIH Clinical Center, Bethesda, MD
• Martha Quezado, MD, Laboratory of Pathology, Center for Cancer Research, 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
• 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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