European Journal of Endocrinology (2010) 162 1021–1025 ISSN 0804-4643

CLINICAL STUDY ANE syndrome caused by mutated RBM28 gene: a novel etiology of combined pituitary hormone deficiency Ronen Spiegel1,2,3, Stavit A Shalev2,3, Amin Adawi4, Eli Sprecher3,5 and Yardena Tenenbaum-Rakover3,6 1Pediatric Department A and 2Genetic Institute, Ha’Emek Medical Center, 18101, , 3Bruce and Ruth Rappaport School of Medicine, Technion, , 31096 Israel, 4Pediatric and Neonatal Department, E.M.M.S, Hospital, 50294 Israel, 5Department of Dermatology, Sourasky Medical Center, Tel Aviv, 64239 Israel and 6Pediatric Endocrine Unit, Ha’Emek Medical Center, Afula, 18101 Israel (Correspondence should be addressed to R Spiegel at Pediatric Department A, Ha’Emek Medical Center; Email: [email protected])

Abstract Objective and design: A homozygous loss-of-function mutation in the gene RBM28 was recently reported to underlie alopecia, neurological defects, and endocrinopathy (ANE) syndrome. The aim of the present study was to characterize the endocrine phenotype of ANE syndrome and to delineate its pathogenesis. Methods: Detailed neuroendocrine assessment was performed in five affected male siblings harboring the homozygous p.L351P mutation in RBM28. Results: All five affected patients, aged 20–39 years, displayed absent puberty, hypogonadism, and variable degrees of short stature. Low IGF1 concentration and a lack of GH response to provocative tests in all siblings were consistent with GH deficiency. Low testosterone and gonadotropin levels with absence or low response to GnRH stimulation indicated hypogonadotropic hypogonadism. ACTH deficiency evolved over time, and glucocorticoid replacement therapy was initiated in four patients. Thyroid analysis showed variable abnormal TSH response to TRH stimulation, suggesting hypothalamic compensated hypothyroidism in four subjects and laboratory hypothyroidism (low free thyroxine) in one patient. Low prolactin levels were shown in one case. Conclusions: The endocrine defects characteristic of ANE syndrome are compatible with variable combined anterior pituitary hormone deficiency (CPHD), which evolves gradually over the years, indicating long-term hormonal monitoring. We propose that defects in the cellular Wnt/b-catenin signaling pathway underlie this endocrinopathy. RBM28 gene defects should be added to the growing list of gene defects associated with syndromic CPHD.

European Journal of Endocrinology 162 1021–1025

Introduction Arab–Muslim origin. In a previous study, we showed the absence of mature hair follicles and the presence of Alopecia, neurological defects, and endocrinopathy dermal cysts in histopathology of a skin biopsy (ANE) syndrome is a new autosomal recessive syndrome reminiscent of the features seen in mice lacking recently reported by Nousbeck et al. (1). The syndrome b-catenin epidermal expression, suggesting a role for is caused by decreased expression of the nucleolar b-catenin in skin involvement of patients affected with protein RBM28, known to be associated with ribosome ANE syndrome (1). In the current study, we examined biogenesis (2), and is characterized by a distinctive the biochemical and dynamic hormonal tests in these phenotype. Skin involvement includes alopecia of five affected siblings with variable pituitary hormone variable severity, resulting from abnormal hair follicle deficiencies. We have delineated the pathophysiology of differentiation, multiple facial nevi, and flexural ANE syndrome’s endocrine manifestations and propose hyperpigmentation (1). Neurological defects comprising a novel etiology for hypopituitarism resulting from moderate to severe mental retardation and progressive interruption of the Wnt/b-catenin pathway. motor decline typically begin during the second decade of life, presenting with combined upper and lower motor dysfunction. Additional features of the syndrome Patients and methods include gynecomastia and dental anomalies (1). Patients Endocrine deficiency is a major component of the syndrome. A homozygous p.L351P mutation in the Five affected male siblings of Arab–Muslim descent from RBM28 gene was identified in five affected siblings with a consanguineous family aged 20–39 years and ANE syndrome born to consanguineous parents of presenting with ANE syndrome were enrolled in the

q 2010 European Society of Endocrinology DOI: 10.1530/EJE-10-0077 Online version via www.eje-online.org

Downloaded from Bioscientifica.com at 09/24/2021 03:19:16AM via free access 1022 R Spiegel and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 162 study. They were previously reported to carry the 30, 60, 90, and 120 min following i.v. injection of p.L351P mutation in RBM28. The pedigree and 100 mgTRH. detailed clinical phenotype have been reported pre- GH and IGF1, ACTH, and cortisol were measured by viously (1). The present study was approved by the Immulite 2000 DPC (Los Angeles, CA, USA). TSH, FT4, Ha’Emek Medical Center ethics committee. FT3, testosterone, PRL, LH, and FSH serum levels were All patients were clinically examined by the same measured by direct automated chemiluminescence endocrinologist (Y T-R). Patients’ height, Tanner assays using an ADVIA Centaur (Bayer Corporation). pubertal stages, penile length, and testicular volume were recorded. Results Hormonal assays Clinical features and hormonal studies are summarized in Table 1. All patients were short compared to target Basal levels of TSH, free thyroxine (FT ), free tri- 4 height (mean K2.2G1.5, range K0.86 to K4.0 S.D., iodothyronine (FT ), insulin-like growth factor 1 (IGF1), 3 target height 179.5 cm (0.72 S.D)). Bone age assess- GH, ACTH, prolactin (PRL), LH, FSH, and testosterone ments confirmed that they completed their growth. were measured at fasting at 0800 h. GH excretion They had very low serum IGF1 levels (mean 4.5G1.6, was evaluated by clonidine provocative test (samples range 3.3–7.2 nmol/l) and lacked a GH response to were taken at intervals of 0, 30, 60, 90, and 120 min). provocative test, consistent with the diagnosis of GH The hypothalamic–pituitary–gonadal axis was studied deficiency (GHD; Table 1). All five male patients by GnRH test. LH and FSH levels at basal, 30, 60, 90, displayed absent or markedly delayed pubertal develop- and 120 min were measured following i.v. injection ment. All patients except patient 5 had variable degrees of 100 mg GnRH. Hypothalamic–pituitary–adrenal of bilateral gynecomastia. Hormonal studies revealed axis was evaluated by ACTH stimulation test (cortisol very low basal levels of serum testosterone and was measured at 0 and 60 min following i.v. injection gonadotropins combined with either blunted or no of 0.25 mg synacthen). Thyroid function was increase in LH and FSH response to GnRH stimulation, assessed by sequential sampling of serum TSH at basal, consistent with hypogonadotropic hypogonadism.

Table 1 Clinical and hormonal characteristics of affected family members.

Case 1 Case 2 Case 3 Case 4 Case 5 MeanGS.D. (range) Normal range

Gender M M M M M 5M Age (years) 39 36 30 27 20 30.4G7.5 (20–39) Height (cm) 150 166 167 169 148 160G10 (148–169) Target height, 179.5 Height (S.D.) K3.7 K1.3 K1.16 K0.86 K4.0 K2.2G1.5 (K4.0 to K0.86) 0.72 Tanner stage (T,P,Pu) 1,1,1 1,3,3 1,1,1 1,3,2 1,1,1 Normal puberty 5,5,5 Penile length (cm) 4 8 5 9.5 4 Testicular volume (ml) 2.5 2 2 3 2 15–20 Prolactin (mU/l) 48.0 380.9 194.6 107.6 108.4 168G130 (48–380) 58–489 Testosterone (nmol/l) 0.7 4.2 0.8 4.2 0.7 2.1G1.9 (0.7–4.2) 8.3–28.6 IGF1 (nmol/l) 4.89 3.67 3.25 3.63 7.19 4.5G1.6 (3.3–7.2) 15.5–62 FT4 (pmol/l) 16.7 13.1 8.5 14.8 15.4 13.7G3.2 (8.5–16.7) 9.9–22.8 ACTH (pg/ml) 13.3 10.8 10.7 15.1 19.3 13.8G3.6 (10.7–19.3) 0–46 Clonidine test Basal GH (mIU/l) 1.3 1.3 1.3 1.3 1.3 0–13 Peak GH (mIU/l) 1.3 1.3 1.3 1.3 1.56 O26 ACTH test Basal cortisol (mmol/l) 249 420 39 54 97 172G162 (39–420) O276 Peak cortisol (mmol/l) 497 662 186 262 409 303G189 (186–662) O552 TRH test Basal TSH (mIU/ml) 4.0 3.1 9.5 7.5 5.0 5.8G2.6 (3.1–9.5) 0.35–5.4 Peak TSH (mIU/ml) 11.0 12.8 28.3 28.8 16.0 19.4G8.6 (11–28.8) 5–20 GnRH test Basal LH (mIU/ml) 0.5 1.7 1.8 1.8 0.5 1.3G0.7 (0.5–1.8) 1.1–7.4a Peak LH (mIU/ml) 1.2 3.8 3.1 5.8 0.7 1.9G2.0 (0.3–5.8) 10.4–34.4a Basal FSH (mIU/ml) 0.81 5.0 2.24 1.17 0.30 1.9G1.9 (0.3–5.0) 0.3–4.8a Peak FSH (mIU/ml) 1.3 5.8 3.0 2.73 0.3 2.6G2.1 (0.3–5.8) 12.2–19.9a

M, male; T, testes; P, penis; Pu, pubic hair. aAdult male.

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ACTH stimulation tests yielded variable responses The spectrum of pituitary hormone deficiencies may ranging from a normal increase in serum cortisol vary in ANE syndrome. It encompasses complete GH (case 2), a partial response (cases 1 and 5), and a and gonadotropin deficiency, variable degrees of ACTH blunted response (cases 3 and 4). Basal ACTH levels deficiency, and mild TSH and PRL abnormalities. were not elevated, indicating central adrenal insuffi- Unfortunately, we were unable to trace any previous ciency. Repeated ACTH stimulating tests over 1 year in records documenting anterior pituitary function before patients 3 and 4 revealed decreased peak cortisol from the age of 20 years in any of our patients. Nevertheless, 453 to 185 and from 323 to 235 mmol/l respectively, we can assume that GHD had a juvenile onset with a thus confirming a progressive course of adrenal impairment. Patient 3 was admitted due to clinical progressive course, allowing three of our five patients to signs of adrenal insufficiency. Replacement therapy with achieve a final height within the low range of the oral hydrocortisone was initiated in patients 1, 3, 4, and normal spectrum, albeit significantly below their target 5 with clinical improvement. height. Testosterone deficiency as seen in our patients Basal TSH levels were moderately elevated in patients may result in low GH peak following provocative tests. Yet, the complete deficiency of GH is in favor of primary 3 and 4. Normal FT4 serum levels were shown in all pituitary cause, rather than a secondary effect of subjects apart from patient 3, who had low FT4 concentrations. TRH stimulation study yielded an testosterone shortage. inappropriate TSH response, with delayed peak response Hypogonadotropic hypogonadism in ANE syndrome (case 4) and TSH levels remaining abnormallyelevated at appears to present before or in the second decade, 90 and 120 min. Mean PRL serum concentrations were resulting in absent puberty. ACTH insufficiency is low, but only below the normal male range in case 1. assumed to evolve during the second or even third Brain magnetic resonance imaging of patient 5 demon- decade of life, resulting in slowly progressive, mildly strated a severely hypoplastic thin anterior pituitary symptomatic adrenal insufficiency. The development of gland at the bottom of the sella turcica and an ectopic adrenal insufficiency of central origin, clearly seen in posterior pituitary located at the proximal level of the repeated ACTH stimulation tests in the same subject pituitary stalk. This finding was previously reported (1). over time, supports the need for long-term monitoring for evolving pituitary hormone deficiencies to prevent clinical adrenal insufficiency. Discussion TSH dysfunction is reflected by mildly elevated basal TSH and sustained TSH response to TRH stimulation, We report on five male siblings with ANE syndrome indicating hypothalamic hypothyroidism. Replacement caused by a p.L351P mutation in the RBM28 gene who therapy with L-T4 was indicated in only one patient. The displayed endocrine features consistent with combined RBM28 protein has recently been shown to be a anterior pituitary hormone deficiency (CPHD). Thus, component of the mammalian spliceosomal small ANE syndrome should be considered in the differential nuclear ribonucleoproteins (2), and to play a major diagnosis of CPHD. role in ribosome assembly (1). It suggests that deficiency CPHD refers to a rare heterogeneous group of of this protein may have pleiotropic effects, as seen in conditions, in which there is a true deficiency of at least ANE syndrome. Moreover, abnormal ribosome assembly two anterior pituitary hormone axes. It may have a and/or defective global splicing alterations may result in purely pituitary phenotype or may be associated with variable expression of the disease phenotypes, as extra pituitary features (syndromic CPHD) (3). Mutations exemplified by the cutaneous (1) and endocrine features in at least ten genes encoding both signaling molecules observed in the kindred reported in this study. and transcription factors have been implicated in various Meticulous examination of skin biopsies from our forms of inherited hypopituitarism. These genes are patients revealed the absence of mature follicles and the HESX1, PROP1, POU1F1, LHX3, LHX4, TBX19, SOX2, presence of dermal cysts (1) reminiscent of the features SOX3, TBCE,andOTX2 (4–6). Non-syndromic CPHD has seen in humans and mice affected by a large spectrum of been found to be caused by mutations in PROP1 and genetic defects, all known or suspected to result in POU1F1 (7–9), while mutations in the other genes defective stabilization of epidermal b-catenin expression typically cause a syndromic subtype of CPHD, featuring (17, 18). Accordingly, b-catenin expression is defective septo-optic dysplasia (HESX1) (10), short stiff neck in the skin of ANE syndrome patients (1). b-catenin is a (LHX3) (11),cerebellaranomalies(LHX4) (12, 13), key protein of the Wnt signaling pathway which plays a mental retardation (SOX3) (14), anophthalmia, esopha- pivotal role in gene expression, cell adhesion, and tissue geal atresia, and genital anomalies (SOX2) (15, 16), development (19). Activation of the Wnt pathway hypoparathyroidism-retardation and dysmorphism results in cytosolic b-catenin accumulation and its (TBCE) (5), and microphthalmia and anophthalmia translocation into the nucleus, where it engages in gene (OTX2) (6). Associated neuroradiological findings, activation and the cell cycle (20). Interestingly, recent including anterior pituitary hypoplasia, absent infundi- data have established a major role for the Wnt/ bulum, and an ectopic posterior pituitary, may present in b-catenin signaling pathway in regulating pituitary 50% of patients with CPHD (4). development and growth (21). These studies

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Downloaded from Bioscientifica.com at 09/24/2021 03:19:16AM via free access 1024 R Spiegel and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 162 demonstrated that b-catenin plays an essential role 8 Hala´sz Z, Toke J, Pato´cs A, Bertalan R, To¨mbo¨l Z, Sallai A, in transducing the GnRH signal by interacting with Hosszu´ E, Muzsnai A, Kova´cs L, So´lyom J, Fekete G & Ra´cz K. High prevalence of PROP1 gene mutations in Hungarian patients with multiple DNA-binding proteins in gonadotropes childhood-onset combined anterior pituitary hormone deficiency. (22–24). In addition, in a mouse model, b-catenin Endocrine 2006 30 255–260. acts as a cofactor for a number of pituitary transcription 9 Wu W, Cogan JD, Pfaffle RW, Dasen JS, Frisch H, O’Connell SM, factors that control pituitary cell specification and exert Flynn SE, Brown MR, Mullis PE, Parks JS, Phillips JA III & both stimulatory (prop1 in regulating expression of Rosenfeld MG. 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