177:2 AUTHOR COPY ONLY N Amano and others Pediatric-onset adrenal 177:2 187–194 Clinical Study insufficiency Genetic defects in pediatric-onset adrenal insufficiency in Japan Naoko Amano1,2, Satoshi Narumi1,3, Mie Hayashi1, Masaki Takagi1,4, Kazuhide Imai5, Toshiro Nakamura6, Rumi Hachiya1,4, Goro Sasaki1,7, Keiko Homma8, Tomohiro Ishii1 and Tomonobu Hasegawa1 1Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan, 2Department of Pediatrics, Tokyo Saiseikai Central Hospital, Tokyo, Japan, 3Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan, 4Department of Endocrinology and Metabolism, Tokyo Metropolitan Children’s Medical Center, Tokyo, Japan, 5Department of Pediatrics, Nishibeppu National Hospital, Oita, Japan, Correspondence 6Department of Pediatrics, Kumamoto Chuo Hospital, Kumamoto, Japan, 7Department of Pediatrics, should be addressed Tokyo Dental College Ichikawa General Hospital, Chiba, Japan, and 8Clinical Laboratory, to T Hasegawa Keio University Hospital, Tokyo, Japan Email [email protected] Abstract Context: Most patients with pediatric-onset primary adrenal insufficiency (PAI), such as 21-hydroxylase deficiency, can be diagnosed by measuring the urine or serum levels of steroid metabolites. However, the etiology is often difficult to determine in a subset of patients lacking characteristic biochemical findings. Objective: To assess the frequency of genetic defects in Japanese children with biochemically uncharacterized PAI and characterize the phenotypes of mutation-carrying patients. Methods: We enrolled 63 Japanese children (59 families) with biochemically uncharacterized PAI, and sequenced 12 PAI-associated genes. The pathogenicities of rare variants were assessed based on in silico analyses and structural modeling. We calculated the proportion of mutation-carrying patients according to demographic characteristics. Results: We identified genetic defects in 50 (85%) families:STAR in 19, NR0B1 in 18, SAMD9 in seven, AAAS in two, NNT in two, MC2R in one and CDKN1C in one. NR0B1 defects were identified in 78% of the male patients that European Journal European of Endocrinology received both glucocorticoid and mineralocorticoid replacement therapy and had normal male external genitalia. STAR defects were identified in 67% of female and 9% of male patients. Seven of the 19 patients with STAR defects developed PAI at age two or older, out of whom, five did not have mineralocorticoid deficiency. Conclusions: Molecular testing elucidated the etiologies of most biochemically uncharacterized PAI patients. Genetic defects such as NR0B1 defects are presumed based on phenotypes, while others with broad phenotypic variability, such as STAR defects, are difficult to diagnose. Molecular testing is a rational approach to diagnosis in biochemically uncharacterized PAI patients. European Journal of Endocrinology (2017) 177, 187–194 Introduction Primary adrenal insufficiency (PAI) is a rare life-threatening (1, 2). These enzymatic defects usually yield specific condition caused by disruption of cortisol synthesis findings in the urine and serum steroid metabolites such in the adrenal cortex despite high ACTH levels. A large as high serum 17α-hydroxyprogesterone levels and can be proportion of patients with PAI have congenital adrenal diagnosed biochemically (3). hyperplasia (CAH), most of which is caused by enzymatic However, a minor subset of patients with PAI lacks defects in cortisol biosynthesis, such as deficiencies in these biochemical features, and it is often difficult to 21-hydroxylase and 3β-hydroxysteroid dehydrogenase determine the etiology based on the clinical findings alone. www.eje-www.eje-online.orgonline.org © 2017 European Society of Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/EJE-17-0027 Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 09:04:02PM via free access 10.1530/EJE-17-0027 AUTHOR COPY ONLY Clinical Study N Amano and others Pediatric-onset adrenal 177:2 188 insufficiency Most of these biochemically uncharacterized PAI patients In the present study, we conducted genetic analyses would be classified into four groups based on International in 63 nonconsanguineous Japanese children with Classification of Pediatric Endocrine Diagnosis (ICPED, biochemically uncharacterized PAI. We estimated www.icped.org). The first group is CAH, which is caused the frequency of genetic defects in our cohort and by STAR or CYP11A1 defects (4, 5). The patients belonging characterized the phenotypes of the mutation- to this group usually have PAI with complete female carrying patients. phenotype due to destruction of the initial step of steroid biosynthesis in adrenal and gonads. The second group Subjects and methods is congenital adrenal hypoplasia, which is caused by mutations in NR0B1 encoding DAX1 or NR5A1 encoding Subjects SF-1 (6, 7). The second group includes the syndromic PAI, such as IMAGe syndrome (caused by CDKN1C mutations) We enrolled 63 patients belonging to 59 families with and SERKAL syndrome (caused by WNT4 mutations) (8, biochemically uncharacterized PAI in Japan from 2000 9). The recently reported MIRAGE syndrome (caused by to 2015. The inclusion criteria were as follows: (i) age at SAMD9 mutations) might also belong to the second group diagnosis less than 15 years; (ii) clinical diagnosis of PAI (10). IMAGe syndrome comprises intrauterine growth based on symptoms (e.g. fatigue, hyperpigmentation, restriction, metaphyseal dysplasia, adrenal hypoplasia and vomiting) and laboratory examinations (i.e., low congenital and genital anomalies; SERKAL syndrome or normal serum cortisol levels with high plasma ACTH comprises 46,XX sex reversal with dysgenesis of the levels). We excluded the patients with any specific kidneys, adrenals and lungs; and MIRAGE syndrome findings of urine or serum steroid metabolites suggesting comprises myelodysplasia, infection, restriction of growth, enzymatic defects (i.e. 21-hydroxylase deficiency and adrenal hypoplasia, genital phenotypes and enteropathy. 3β-hydroxysteroid dehydrogenase deficiency), and two The third group is familial glucocorticoid deficiency patients with adrenoleukodystrophy. (FGD), which is predominantly isolated glucocorticoid We divided the subjects into two groups according to insufficiency. This group is mainly composed of patients the onset age (infancy, <1 year; childhood, 1–15 years). with ACTH resistance caused by MC2R and MRAP defects Moreover, we also classified male patients into two groups (11, 12). NNT, TXNRD2 and MCM4 have been reported according to the phenotypes of their external genitalia as additional causative genes for this third group (13, 14, (normal or underdeveloped). 15). Triple A syndrome (caused by AAAS defects), which We obtained written informed consent for study European Journal European of Endocrinology comprises achalasia, addisonianism (adrenal insufficiency) participation from each patient or his/her parents. The and alacrima, also belongs to the third group (16). The Ethics Committee of Keio University School of Medicine fourth group is adrenal destruction due to metabolic and approved the study. In the 63 patients, one patient autoimmune causes (AIRE, ABCD1, PEX and LIPA defects) with IMAGe syndrome and eight patients with MIRAGE (17, 18, 19, 20). Some recently identified novel causative syndrome were reported previously (10, 23). genes and atypical phenotypes of these genetic defects have complicated the clinical approaches used to treat Molecular analysis biochemically uncharacterized PAI patients. There are few studies on the frequency of genetic We extracted genomic DNA from peripheral lymphocytes defects in biochemically uncharacterized PAI patients. In of the study subjects using a standard technique. 2015, Guran et al. conducted a genetic screening study We selected the following 12 genes associated with targeting 95 non-syndromic pediatric patients having biochemically uncharacterized PAI: CYP11A1, STAR biochemically uncharacterized PAI in Turkey and revealed (associated with CAH); CDKN1C, NR0B1, NR5A1 and that 81% of the study subjects had genetic defects (21). SAMD9 (associated with congenital adrenal hypoplasia); On the other hand, Chan et al. also conducted genetic AAAS, MCM4, MC2R, MRAP, NNT and TXNRD2 analyses targeting more than 300 FGD patients in the (associated with isolated or FGD). All coding exons and United Kingdom and diagnosed about 60% of them flanking introns of these 12 genes were analyzed using genetically (22). However, it is not clear whether these standard PCR-based sequencing, targeted next-generation mutation frequencies can be generalized to other ethnic sequencing or whole exome sequencing (Supplementary groups including Japanese. Table 1, see section on supplementary data given at the www.eje-online.org Downloaded from Bioscientifica.com at 09/30/2021 09:04:02PM via free access AUTHOR COPY ONLY Clinical Study N Amano and others Pediatric-onset adrenal 177:2 189 insufficiency end of this article). Regarding the PCR-based sequencing, Table 1 Subject characteristics. the primer sequences and PCR conditions are available Variable upon request. In 20 of 63 patients, we performed targeted Number of the probands 59 next-generation sequencing using the panel of congenital Legal sex; male/female 35/24 endocrine disorders (160 genes), which was included Family history 10 all coding exons and flanking introns of the following Potential inheritance; X-linkeda 6 Othersb 4 13 PAI-associated