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  • Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency Ahmed Khattaba,b,1,2, Shozeb Haiderc,1, Ameet Kumara,b,1, Samarth Dhawana,b, Dauood Alama,b, Raquel Romeroc, James Burnsc, Di Lic, Jessica Estaticoa,b, Simran Rahia,b, Saleel Fatimaa,b, Ali Alzahranid, Mona Hafeze, Noha Musae, Maryam Razzghy Azarf, Najoua Khaloulg, Moez Gribaag, Ali Saadg, Ilhem Ben Charfeddineg, Berenice Bilharinho de Mendonçah, Alicia Belgoroskyi, Katja Dumicj, Miroslav Dumicj, Javier Aisenbergk, Nurgun Kandemirl, Ayfer Alikasifoglul, Alev Ozonl, Nazli Goncl, Tina Chenga,b, Ursula Kuhnle-Krahlm, Marco Cappan, Paul-Martin Holterhuso, Munier A. Nourp, Daniele Pacaudq, Assaf Holtzmana,b, Sun Lia,b, Mone Zaidia,b,2, Tony Yuena,b,2, and Maria I. Newa,b,2,3

    aDivision of Adrenal Steroid Disorders, Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029; bDepartment of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029; cSchool of Pharmacy, University College London, London WC1N 4AX, United Kingdom; dKing Faisal Specialist Hospital and Research Centre, Riyadh 12713, Saudi Arabia; eDiabetes, Endocrine, and Metabolism Pediatrics Unit, Department of Pediatrics, Cairo University, 11617 Cairo, Egypt; fAli Asghar Children’s Hospital, Iran University of Medical Sciences, 10000 Tehran, Iran; gLaboratory of Human Cytogenetic Molecular Genetics and Biology of Reproduction, Farhat Hached University Hospital, Sousse, Tunisia; hDevelopmental Endocrinology Unit, Hormone and Molecular Genetics Laboratory, University of São Paulo, Sao Paulo 05508, Brazil; iGarrahan Pediatric Hospital, 1881 Buenos Aires, Argentina; jUniversity Hospital Centre Zagreb, 10000 Zagreb, Croatia; kPediatric Endocrinology and Diabetes Medicine, Hackensack University Medical Center, Hackensack, NJ 07601; lFaculty of Medicine, Hacettepe University, 06100 Ankara, Turkey; mChildrens Hospital, University of Munich, 80539 Munich, Germany; nBambino Gesù Hospital, 00146 Roma, Italy; oDepartment of Pediatrics, University Hospital of Schleswig-Holstein, 24105 Kiel, Germany; pDepartment of Pediatrics, University of Saskatchewan College of Medicine, Saskatoon, SK, Canada S7N 0W8; and qDepartment of Pediatrics, University of Calgary, Calgary, AB, Canada T3B 6A8

    Contributed by Maria I. New, January 3, 2017 (sent for review October 26, 2016; reviewed by Vidya Darbari, Joe Leigh Simpson, and Rajesh V. Thakker)

    Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1, a gene encoding 11β-hydroxylase, represents a rare auto- somal recessive Mendelian disorder of aberrant sex steroid produc- tion. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where con- sanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not fre- quently measured, is the most robust biochemical marker for diag- nosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifica- tions in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, ge- netic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β- hydroxylase deficiency CAH.

    steroid hormones | missense mutations | classic CAH | ambiguous genitalia

    Congenital adrenal hyperplasia (CAH) is a Mendelian disor-der transmitted as an autosomal recessive trait. The most prevalent form of CAH arises from steroid 21-hydroxylase en- zyme deficiency, accounting for ∼90–95% of all cases (1, 2). In contrast, CAH caused by steroid 11β-hydroxylase deficiency is considerably rare, with a prevalence of 5–8% (3), from which we estimate an overall frequency of 1 in 100,000 live births. Two homologous enzymes, 11β-hydroxylase and aldosterone syn-

    thase, are encoded by the CYP11B1 andCYP11B2 genes, respectively. The two genes are 40-kb apart, each comprising nine exons and mapped to chromosome 8q21-22 (3, 4) (Fig. 1A). In contrast to CYP21A2 and its CYP21A1P pseudogene, CYP11B1 and CYP11B2 are both active and do not have a pseudogene. The two encoded homologs, however, have distinct functions in cortisol and aldosterone synthesis, respectively (3). In the zona fasciculata, 11β-hydroxylase converts 11-deoxycortisol and 11-deoxycorticosterone to cortisol and corticosterone, respectively, and is regulated by adrenocorticotro- pic hormone secreted by the pituitary. In contrast, in the zona glomerulosa aldosterone synthase converts corticosterone to aldoste- rone with the intermediate production of 18-hydroxycorticosterone. These latter conversions are controlled mainly by the renin angio- tensin II system and serum potassium concentration (3).

    Deficiency of 11β-hydroxylase prevents the conversion of 11- deoxycortisol to cortisol and 11-deoxycorticosterone to corticoste- rone. This results in high levels of 11-deoxycortisol and 11-deoxy- corticosterone, respectively, which are shunted into the androgen synthesis pathway, resulting in high levels of the androgenic steroid, androstenedione. Female newborns are thus profoundly virilized and exhibit significant masculinization of the external genitalia (5). Precocious puberty, rapid somatic growth, and rapid skeletal matura- tion because of hyperandrogenemia occur in both genders. Accumu- lation of the potent mineralocorticoid 11-deoxycorticosterone also leads to hypertension, which is not seen with 21-hydroxylase deficiency. We recently published evidence for genotype–phenotype concor-

    dance in 1,507 families with 21-hydroxylase deficiency; however, 7% of the patients demonstrated nonconcordance (6). In a separate study, we used computational modeling to define in silico the structural


    Congenital adrenal hyperplasia resulting from mutations in the CYP11B1 gene, which encodes a steroidogenic enzyme 11β- hydroxylase, is a rare inherited disorder associated with hyperandrogenemia, short stature, hypertension, and viriliza- tion of female newborns. We present a comprehensive clinical, genetic, and hormonal characterization for 68 of 108 patients with a genotype from an International Consortium on Rare Steroid Disorders. We also use computational modeling to de- fine the effect of each of the missense mutations on the struc- ture of 11β-hydroxylase, information that can be used to predict clinical severity prenatally in high-risk mothers.

    Author contributions: M.Z., T.Y., andM.I.N. designed research; S.H., A. Kumar, S.D., D.A., R.R., J.B., D.L., J.E., A. Alzahrani, and N.M. performed research; A. Khattab, S.H., A. Kumar, R.R., J.B., D.L., J.E., S.R., S.F., A. Alzahrani, M.H., M.R.A., N. Khaloul, M.G., A.S., I.B.C., B.B.d.M., A.B., K.D., M.D., J.A., N. Kandemir, A. Alikasifoglu, A.O., N.G., T.C., U.K.-K., M.C., P.-M.H., M.A.N., D.P., A.H., S.L., M.Z., T.Y., and M.I.N. analyzed data; and A. Khattab, S.H., M.Z., T.Y., and M.I.N. wrote the paper.

    Reviewers: V.D., Queen Mary University of London; J.L.S., March of Dimes Foundation; and R.V.T., University of Oxford.

    The authors declare no conflict of interest. 1A. Khattab, S.H., and A. Kumar contributed equally to this work. 2A. Khattab, M.Z., T.Y., and M.I.N. contributed equally to this work. 3To whom correspondence should be addressed. Email:

    This article contains supporting information online at 1073/pnas.1621082114/-/DCSupplemental. PNAS | Published online February 22, 2017 | E1933–E1940

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  • changes that each mutation in the CYP21A2 gene induces in the 21- hydroxylase enzyme, and by correlating these changes with phenotype,

    defined structural derangements underpinning the clinical variants of CAH, namely salt-wasting, simple virilizing, and nonclassic CAH (7).








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