O R I G I N A L A R T I C L E
Rare Causes of Primary Adrenal Insufficiency: Genetic
and Clinical Characterization of a Large Nationwide
Cohort
Tulay Guran, Federica Buonocore, Nurcin Saka, Mehmet Nuri Ozbek,
Zehra Aycan, Abdullah Bereket, Firdevs Bas, Sukran Darcan, Aysun Bideci,
Ayla Guven, Korcan Demir, Aysehan Akinci, Muammer Buyukinan,
Banu Kucukemre Aydin, Serap Turan, Sebahat Yilmaz Agladioglu, Zeynep Atay,
Zehra Yavas Abali, Omer Tarim, Gonul Catli, Bilgin Yuksel, Teoman Akcay,
Metin Yildiz, Samim Ozen, Esra Doger, Huseyin Demirbilek, Ahmet Ucar,
Emregul Isik, Bayram Ozhan, Semih Bolu, Ilker Tolga Ozgen,
Jenifer P. Suntharalingham, and John C. Achermann*
Context: Primary adrenal insufficiency (PAI) is a life-threatening condition that is often due to
monogenic causes in children. Although congenital adrenal hyperplasia occurs commonly, several
other important molecular causes have been reported, often with overlapping clinical and bio-
chemical features. The relative prevalence of these conditions is not known, but making a specific
diagnosis can have important implications for management.
Objective: The objective of the study was to investigate the clinical and molecular genetic char-
acteristics of a nationwide cohort of children with PAI of unknown etiology.
Design: A structured questionnaire was used to evaluate clinical, biochemical, and imaging data.
Genetic analysis was performed using Haloplex capture and next-generation sequencing. Patients
with congenital adrenal hyperplasia, adrenoleukodystrophy, autoimmune adrenal insufficiency,
or obvious syndromic PAI were excluded.
Setting: The study was conducted in 19 tertiary pediatric endocrinology clinics.
Patients: Ninety-five children (48 females, aged 0–18 y, eight familial) with PAI of unknown eti-
ology participated in the study.
Results: A genetic diagnosis was obtained in 77 patients (81%). The range of etiologies was as
follows: MC2R (n  25), NR0B1 (n  12), STAR (n  11), CYP11A1 (n  9), MRAP (n  9), NNT (n 
7), ABCD1 (n  2), NR5A1 (n  1), and AAAS (n  1). Recurrent mutations occurred in several genes,
such as c.560delT in MC2R, p.R451W in CYP11A1, and c.IVS3ds1delG in MRAP. Several important
clinical and molecular insights emerged.
Conclusion: This is the largest nationwide study of the molecular genetics of childhood PAI un-
dertaken. Achieving a molecular diagnosis in more than 80% of children has important transla-
tional impact for counseling families, presymptomatic diagnosis, personalized treatment (eg, min-
eralocorticoid replacement), predicting comorbidities (eg, neurological, puberty/fertility), and
targeting clinical genetic testing in the future. (J Clin Endocrinol Metab 101: 284–292, 2016)
ISSN Print 0021-972X ISSN Online 1945-7197 *Author Affiliations are shown at the bottom of the next page.
Printed in USA Abbreviations: AHC, adrenal hypoplasia congenita; DAX-1, dosage-sensitive sex reversal,
This article has been published under the terms of the Creative Commons Attribution adrenal hypoplasia congenita critical region, on the X chromosome, gene 1; DSD, disorders
License (CC-BY; https://creativecommons.org/licenses/by/4.0/), which permits unrestricted of sex development; FGD, familial glucocorticoid deficiency; NGS, next-generation se-
use, distribution, and reproduction in any medium, provided the original author and source quencing; NNT, nicotinamide nucleotide transhydrogenase; PAI, primary adrenal insuffi-
are credited. Copyright for this article is retained by the author(s). ciency; P450scc, P450 side chain cleavage.
Received August 23, 2015. Accepted October 28, 2015.
First Published Online November 2, 2015
284 press.endocrine.org/journal/jcem J Clin Endocrinol Metab, January 2016, 101(1):284–292 doi: 10.1210/jc.2015-3250
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doi: 10.1210/jc.2015-3250 press.endocrine.org/journal/jcem 285
Primary adrenal insufficiency (PAI) is a potentially life- the condition on to their children (1, 3, 20, 21). Knowingthreatening condition that requires accurate diagno- the genetic etiology can also help to modify treatments,
sis and urgent treatment with glucocorticoid and often such as the need for long-term mineralocorticoid replace-
mineralocorticoid replacement. Because the symptoms ment, and can predict potential comorbidities, such as im-
and signs of adrenal insufficiency are often nonspecific, paired puberty or fertility and neurological dysfunction.
the diagnosis may be easily overlooked (1). Next-generation sequencing (NGS) approaches are
In contrast to the predominance in autoimmune etiol- revolutionizing our ability to sequence large numbers of
ogies in adults, most causes of PAI in childhood have an genes quickly and cost effectively. In this study, a custom
inherited, monogenic origin (1–3). Genetic causes of pe- panel-based NGS approach has been used to sequence all
diatric PAI can be classified into four major groups known PAI-associated genes in a national cohort of 95
according to the underlying pathogenesis; 1) impaired ste- children with PAI of unknown etiology.
roidogenesis, 2) adrenal hypoplasia, 3) familial glucocor-
ticoid deficiency (FGD) and FGD-like disorders, and 4)
adrenal destruction. Patients and Methods
Congenital adrenal hyperplasia (CYP21A2, CYP11B1,
HSD3B2, CYP17A1, POR deficiencies) constitutes the Patients
largest subgroup of impaired steroidogenesis and is the A pediatric cohort study was performed with PAI patients
recruited from 19 pediatric endocrinology clinics in Turkey. In-
most common cause of PAI in children (1, 2, 4). In con- clusion criteria of a PAI phenotype was defined as the presence
trast, there are other individually rare causes of PAI. of signs and symptoms of adrenal insufficiency together with
Several genetic causes of adrenal hypoplasia (NR0B1/dos- high plasma ACTH and low serum cortisol and intermediary
age-sensitive sex reversal, adrenal hypoplasia congenita glucocorticoid metabolites at initial presentation. Exclusion cri-
critical region, on the X chromosome, gene 1 [DAX-1], teria were as follows: 1) congenital adrenal hyperplasia (21-
hydroxylase, 11-hydroxylase, 3-hydroxysteroid dehydroge-
NR5A1/SF-1, CDKN1C gene defects), congenital lipoid nase 2, 17-hydroxylase, or cytochrome P450 reductase
adrenal hyperplasia (CYP11A1, STAR gene defects), fa- deficiencies) diagnosed by a distinctive serum steroid hormone
milial glucocorticoid deficiency (FGD) and FGD-like con- profiles; 2) X-linked adrenoleukodystrophy in boys with neuro-
ditions (MC2R [FGD1], MRAP [FGD2], STAR, MCM4, logical findings and elevated very long-chain fatty acids, or a
NNT, TXNRD2 gene defects) and adrenal destruction family history of affected males with adrenoleukodystrophy; 3)
clinical and biochemical evidence of autoimmune adrenal fail-
(AIRE, ABCD1, PEX1, LIPA gene defects) are now well ure; and 4) known syndromic causes of PAI (specifically, classic
established (5–16). However, it is also emerging that there Triple A syndrome or Xp deletion syndrome involving NR0B1/
is considerable overlap in the clinical and biochemical pre- DAX-1 with Duchenne muscular dystrophy) (Figure 1).
sentation of these conditions. For example, FGD/FGD- All patients were assessed by a pediatric endocrinologist. A
like conditions (MC2R, MRAP, NNT gene defects) can structured questionnaire was used to systematically evaluate all
present with salt loss suggestive of adrenal hypoplasia, and clinical, biochemical, and imaging data related to the diagnosis
and treatment of PAI and all other relevant medical and family
alterations in STAR and CYP11A1 resulting in partial loss history. Studies were performed with the approval of the Ethics
of protein function may have a predominant FGD-like Committee of the Marmara University Faculty of Medicine
phenotype (17–20). (Istanbul, Turkey; B.30.2.MAR.0.01.02/AEK/108). Patients
Establishing a specific genetic diagnosis of PAI is ex- and/or parents provided written informed consent, and all stud-
tremely valuable for identifying presymptomatic children ies were conducted in accordance with the principles of the Dec-
laration of Helsinki.
who could benefit from treatment before the onset of po- A total of 95 PAI patients (48 females and 47 males) from 85
tentially life-threatening symptoms and for counseling families and their unaffected siblings and parents were included
family members appropriately about the risk of passing (Figure 1). The most common presenting features were hyper-
Department of Pediatric Endocrinology and Diabetes (T.G., Z.A., A.B., S.T.), Marmara University, Istanbul 34899, Turkey; Institute of Metabolism and Systems Research (T.G.), University of
Birmingham, Birmingham B15 2TT, United Kingdom; Department of Genetics and Genomic Medicine (F.B., J.S., J.C.A.), University College London Institute of Child Health, University College
London, London WC1N 1EH, United Kingdom; Department of Pediatric Endocrinology and Diabetes (N.S., F.B., B.K.A., Z.Y.A.), Istanbul Faculty of Medicine, Istanbul University, 34452 Istanbul,
Turkey; Clinics of Pediatric Endocrinology (M.N.O., H.D.), Diyarbakir Children’s Hospital, 21100 Diyarbakir, Turkey; Clinics of Pediatric Endocrinology (Z.A., S.Y.A.), Dr Sami Ulus Obstetrics and
Gynecology, Children‘s Health and Disease Training and Research Hospital, 06100 Ankara, Turkey; Department of Pediatric Endocrinology and Diabetes (S.D., S.O.), Ege University, 35040 Izmir,
Turkey; Department of Pediatric Endocrinology and Diabetes (A.B., E.D.), Gazi University, 06550 Ankara, Turkey; Pediatric Endocrinology Clinic (A.G., M.Y.), Goztepe Educational and Research
Hospital,34810 Istanbul, Turkey;DepartmentofPediatrics (A.G.),AmasyaUniversityMedical Faculty,05189Amasya,Turkey;PediatricEndocrinologyClinic (K.D.),DrBehçetUzChildren’sHospital,
Izmir, Turkey;Departmentof Pediatric EndocrinologyandDiabetes (A.A.), InonuUniversity,Malatya, Turkey;Clinicsof Pediatric Endocrinology (M.B.), KonyaTrainingandResearchHospital, 42100
Konya, Turkey; Department of Pediatric Endocrinology and Diabetes (O.T.), Uludag University, 16059 Bursa, Turkey; Department of Pediatric Endocrinology and Diabetes (G.C.), Eylul University,
35210 Izmir, Turkey; Department of Pediatric Endocrinology and Diabetes (B.Y.), Cukurova University, 01330 Adana, Turkey; Clinics of Pediatric Endocrinology (T.A.), Kanuni Sultan Suleyman
EducationandResearchHospital,34303 Istanbul,Turkey;PediatricEndocrinologyClinic (A.U.), SanliurfaChildren’sHospital,63300Sanliurfa,Turkey;PediatricEndocrinologyClinic (E.I.),Gaziantep
Children’s Hospital, 27010 Gaziantep, Turkey; Department of Pediatric Endocrinology and Diabetes (B.O.), Faculty of Medicine, Pamukkale University, 20160 Denizli, Turkey; Department of
Pediatric EndocrinologyandDiabetes (S.B.), FacultyofMedicine,DuzceUniversity, 81620Duzce, Turkey;DepartmentofPediatric EndocrinologyandDiabetes (I.T.O.), Bezm-iAlemVakifUniversity,
34093 Istanbul, Turkey
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286 Guran et al Primary Adrenal Insufficiency in Children J Clin Endocrinol Metab, January 2016, 101(1):284–292
Figure 1. Overview of the study design, recruitment, and outcome of genetic analysis. ALD, adrenoleukodystrophy.
pigmentation (94%), salt-wasting crisis/electrolyte imbalance genes potentially causing PAI, congenital adrenal hyperplasia-
(51%), hypoglycemia with/without convulsions (47%), vomit- related genes, potential syndrome-related genes and candidate
ing/abdominal pain (26%), prolonged jaundice (24%), fatigue genes based on data from biochemical/biological pathways,
(18%), neonatal respiratory distress (17%), frequent infections mouse models of adrenal dysfunction, and gene expression (Sup-
(11%), and failure to thrive or weight loss (7%). Seven patients plemental Methods).
had 46,XY disorders of sex development (DSD). Detailed clinical
findings are provided in Supplemental Table 1. Sequence capture and NGS
Parents of patients were consanguineous in 63 (74%) fami- Sequence capture was performed according to the HaloPlex
lies, whereas 22 families did not report consanguinity. A total of
Target Enrichment Protocol version D.5 (Agilent Technologies
51 patients were on hydrocortisone treatment alone, whereas 44
were also commenced on mineralocorticoid replacement due to Inc) for Illumina sequencing (Supplemental Methods). Patient
salt wasting, high plasma renin activity, or low aldosterone. genomic DNA aliquots (225 ng) were processed in batches of 24
Eight families had multiple affected siblings (six pairs, two trios, samples at a time with an enrichment control DNA sample as a
n  18). positive control. Sequencing was performed on a MiSeq next-
generation sequencer (Illumina Inc).
Molecular analyses
Variant analysis
DNA samples Sequence alignment and variant calling were performed using
Genomic DNA was extracted from whole blood of patients, SureCall (version 2.0) software (Agilent Technologies Inc). All
parents, and available unaffected siblings using a QIAamp DNA potential disease causing variants were confirmed by PCR and
blood maxikit (QIAGEN Inc). Sanger sequencing. Variants in known disease genes were con-
sidered highly likely to be pathogenic if they segregated with the
Design of targeted gene panel phenotype with an appropriate inheritance pattern within fam-
A custom HaloPlex DNA target enrichment panel (Agilent ilies, were determined damaging or likely damaging by several
Technologies Inc) was designed (SureDesign) to capture 160 bioinformatic prediction models (Ensembl Variant Effector Pre-
known and candidate genes involved in adrenal development dictor; SIFT; PolyPhen2; and Mutation Taster) and if they had
and function. All coding exons and 100 base pairs of intronic been reported previously. In addition, novel missense changes
flanking sequence were included. The panel covered known were absent in at least 200 Turkish control samples and had a
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doi: 10.1210/jc.2015-3250 press.endocrine.org/journal/jcem 287
Table 1. Sequence Variations Detected in Our Cohort of 95 Children With Primary Adrenal Insufficiency
Gene Familial, Sporadic, Total, Mineralocorticoid
(Chromosome) n n n Variants n Consanguinity Treatment
MC2R (18p11.21) 4 21 25 22/25 (88%) 2/25 (8%)
p.D103N 1
p.G116V 2
p.R137W 1
p.V142L 1
p.T143S 1
p.L225R 1
p.G226R 1
p.A233P 2
p.C251W 2
c.560delT (p.V187Afs*29) 10
Deletion 3
NR0B1 (Xp21.2)a 6b 6 12 3/12 (25%) 12/12 (100%)
p.W235* 3
p.W236* 1
p.E256* 3
p.W291C 1
p.L299R 1
p.Y378* 1
p.C396* 1
p.V269del 1
STAR (8p11.23) 2 9 11 8/11 (72%) 11/11 (100%)
p.S13P 3
p.W96C 2
p.L157P 1
p.E169K 1
p.R182H 1
p.W250*/p.I166M 1
p.S12Afs*9 1
p.K159del 1
CYP11A1 (15q24.1) 2 7 9 8/9 (89%) 6/9 (67%)
p.R451W 9
MRAP (21q22.11) 2 7 9 5/9 (56%) 2/9 (22%)
p.L53P 1
c.IVS3ds  1insT 1
c.IVS3ds  1delG 5
p.K30del 2
NNT (5p12) 2 5 7 7/7 (100%) 2/7 (29%)
p.D178G 1
p.H370R 1
c.1769dupA (p.D590Efs*29) 1
c.2396delC (p.P799Qfs*22) 1
c.127_128delTG (p.W43Vfs*2) 2
Deletion (exon 2–3) 1
ABCD1 (Xq28)a 0 2 2 0/2 1/2
p.G512S 1
p.Y547C 1
NR5A1 (9q33.1) 0 1 1 0/1 1/1
p.R92Q 1
AAAS (12q13.13) 0 1 1 1/1 0/1
p.R445* 1
Total 18 59 77 54/77 (70%) 37/77 (48%)
Novel variants are marked in bold. All mutations are homozygous except for hemizygous mutations in X-linked genes and p.W250*/p.I166M in
STAR which was compound heterozygous.
a Hemizygous mutations in X-linked genes.
b Familial cases included sibling pairs except for NR0B1 in which two sibling trios were identified. Nucleotide position of variants is shown in
Supplemental Table 1.
minor allele frequency less than 1:100 000 in the Exome Aggre- Results
gation Consortium (ExAC) browser (ExAC; Cambridge, MA,
http://exac.broadinstitute.org; accessed July 2015). A molecular genetic diagnosis was obtained in 81% chil-
More detailed description of methods, workflows, coverage, dren with PAI (77 of 95) using this targeted NGS ap-
and quality control are provided in Supplemental Methods. proach. A total of 43 different deleterious nonsynony-
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288 Guran et al Primary Adrenal Insufficiency in Children J Clin Endocrinol Metab, January 2016, 101(1):284–292
(Supplemental Table 1), several notable findings have
emerged. For example, most patients with MC2R, MRAP,
and STAR mutations presented in the first weeks or
months of life, whereas children with the p.R451W mu-
tation in CYP11A1 presented in early childhood (1–6 y)
(Figure 4). Children with nicotinamide nucleotide trans-
hydrogenase (NNT) changes presented at different ages in
the first 2 years, whereas boys with NR0B1 (DAX-1) mu-
tations had a bimodal pattern, presenting either in the first
month of life or else after 18 months.
Most children had extremely high ACTH levels at di-
Figure 2. Pie chart showing the percentage of mutations in each
gene in this cohort of children with PAI. agnosis and almost all children, even babies, were clini-
cally hyperpigmented. Hypoglycemia was a frequent find-
ing, and hypoglycemic convulsions at presentation were
mous variations were detected in nine different genes
(Table 1). These changes were all confirmed by Sanger more common in children with MC2R (22 of 25, 88%)
sequencing and included missense mutations (n  24), and MRAP (five of nine, 56%) mutations than in children
nonsense mutations (n 7), frameshift mutations (n 5), with an alternative diagnosis (5 of 43, 12%) (P  .0001). 
in-frame single codon deletions (n 3), splice site disrup- Salt-wasting states requiring mineralocorticoid replace-
tions (n  2), and whole gene/exon deletions (n  2). Of ment occurred in all children with NR0B1 (DAX-1) and
these variations, 22 (51%) had not been reported previ- STAR mutations and in most children with the CYP11A1
ously but were considered to be causative because they p.R451W change (six of nine, 66%) (Table 1). Fewer chil-
segregated with the phenotype in the family, they were dren with NNT (two of seven, 28%), MRAP (two of nine,
classed as damaging or probably damaging by several 22%), and MC2R (2 of 25, 8%) mutations required min-
bioinformatic predictions, and they were not found in the eralocorticoid replacement, although four additional
control samples or databases (Supplemental Table 2). The children with MC2R defects had transient hyponatremia
remaining 21 mutations have been reported (Supplemen- (sodium 117–133 mmol/L) that resolved without fludro-
tal Table 1). A molecular diagnosis was reached in all eight cortisone treatment (Supplemental Table 1). Adrenal im-
families with multiple affected siblings, including novel aging was generally uninformative, showing normal sized
changes in five families (Table 1). or hypoplastic glands for most of these diagnoses, includ-
The range of genetic etiologies found in this cohort were ing many children with STAR mutations (congenital li-
as follows: MC2R (n  25), NR0B1 (n  12), STAR (n  poid adrenal hyperplasia) in which enlarged adrenals are
11), CYP11A1 (n  9), MRAP (n  9), NNT (n  7), reported (22).
ABCD1 (n  2), NR5A1 (n  1), and AAAS (n  1) Additional clinical features were seen in many children
(Figure 2). Most patients were homozygous for recessive in this cohort, such as altered growth, neuromotor delay,
changes (62 of 77, 80%), one patient carried compound learning difficulties, and cardiac defects (Supplemental
heterozygous changes (1 of 77, 1.3%), and 14 patients had Table 1). Abnormalities in thyroid function such as sub-
hemizygous mutations in X-linked genes (NR0B1, clinical hypothyroidism were common (n  20, 26%),
ABCD1) (14 of 77, 18%). As expected, consanguinity whereas thyroglossal cysts (n  2), primary hypothyroid-
rates were much higher in families of patients harboring ism (n  2), and transient hypothyroidism (n  1) were
mutations in recessive genes (51 of 63, 81%) compared also found. DSD occurred in all six 46,XY infants with
with X-linked genes (3 of 14, 21%; P  .0001) (Table 1). STAR mutations. Five of these children were phenotypic
Recurrent mutations were detected in several genes, such females who presented with a salt-losing crisis, and in two
as c.560delT in MC2R (10 patients from nine unrelated cases a karyotype was not available prior to this genetic
families), p.R451W in CYP11A1 (nine patients from eight analysis. The one boy with severe hypospadias due to
unrelated families), c.IVS3ds1delG in MRAP (five pa- STAR deficiency developed hypergonadotropic hypogo-
tients from five unrelated families), and p.S13P in STAR nadism in puberty and needed T replacement. One of the
(three patients from two unrelated families). Geographical four boys with the p.R451W mutation in CYP11A1 had a
hot spots were found for the p.R451W CYP11A1 muta- small penis and cryptorchidism. One boy with a NR0B1
tion in eastern Turkey and for the c.IVS3ds1delG MRAP mutationandX-linkedadrenalhypoplasia congenita (AHC)
mutation in the west (Figure 3, A and B). had macrophallia and another one had cryptorchidism. Co-
Although there was considerable overlap in the clinical incidental hypospadias and unilateral cryptorchidism was
and biochemical features of children within this cohort found in one boy with disruption of MC2R.
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doi: 10.1210/jc.2015-3250 press.endocrine.org/journal/jcem 289
is unclear how much these genes con-
tribute to pediatric adrenal disease in
theclinicalsettingasmostreportshave
focused on specific categories of adre-
nal disease such as FGD or adrenal hy-
poplasia (6, 9).
In this study, an unbiased nation-
wide cohort of almost 100 children
with PAI was recruited from 19 pedi-
atric endocrinology centers across
Turkey, and a molecular diagnosis
was reached in more than 80% of the
children. This represents the largest
clinical cohort of children with this
rare condition assembled. Because
children could die from convulsions,
respiratory distress, or salt-losing cri-
ses before reaching the hospital or get
misdiagnosed with sepsis, the condi-
tion may be underdiagnosed. In fact, a
history of unexplained death in in-
fancy or childhood in the extended
family was common in many of those
questioned.
Targeted panel-based capture and
high-throughput sequencing proved
very effective in reaching a molecular
Figure 3. Geographical distribution of recurrent mutations identified in this study. A, The MRAP diagnosis in a relatively quick and
c.IVS3ds1delG mutation was identified mainly in patients from west Turkey, whereas the comprehensive manner. A total of 43
CYP11A1 p.R451W mutation was found in patients who originated from east Turkey. The first
report of the CYP11A1 p.R451W mutation was in a family from Germany who were originally known and novel mutations in nine
from Elazig (shown in light pink) (20). B, The MC2R c.560delT mutation showed a wider genes were discovered in 77 patients,
distribution most likely reflecting migration from the east to west of Turkey and has been with 73 of 77 mutations (95%) oc-
described previously in a family from northern Iran (17). curring in six genes (MC2R, NR0B1,
Stature was variable in the 25 children with MC2R STAR, CYP11A1, MRAP, and NNT). Several recurrent
mutations with seven children (28%) having tall stature mutations were discovered, which likely represent
(2 SD score or two percentile lines above parental target founder effects. Some of these are localized to certain geo-
height), four having short stature (2 SD score) and the graphical areas (eg, p.R451W in CYP11A1 in eastern Tur-
rest being within the normal range. There was no history key, c.IVS3ds1delG inMRAP inwesternTurkey),which
of preterm birth associated with CYP11A1 mutations, but could lead to focused cost-effective clinical genetic testing
there were reports of multiple stillbirths in three families for patients and families at risk of adrenal insufficiency in
in which a child had severe disruption of MC2R (deletion these regions (Figure 3A). Other recurrent changes, such
or c.560delT frameshift). Despite the range of neurolog- as the c.560delT in MC2R showed more diverse geograph-
ical and other features seen, the two boys with mutations ical distribution across the country, possibly reflecting mi-
in ABCD1 (causing X-linked adrenoleukodystrophy) and
gration toward the west (Figure 3B). Indeed, this MC2R
one boy with disruption of AAAS (causing Triple A syn-
mutation has been reported previously in the father of two
drome) appeared to have adrenal only phenotypes and
siblings with FGD1 who originated from northern Iran,
would not have been diagnosed without genetic testing.
close to the Turkish border (17). Sanger sequencing for
just the three changes in MC2R, CYP11A1, and MRAP
Discussion would have diagnosed 22 of 85 different families recruited
in this nationwide cohort (26%), which could represent a
The past 20 years has seen significant progress in our under- cost-effective approach to first-line clinical genetic anal-
standing of the genetic causes of childhood PAI. However, it ysis. Of note, no mutations were found in MCM4 or
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290 Guran et al Primary Adrenal Insufficiency in Children J Clin Endocrinol Metab, January 2016, 101(1):284–292
Hemizygous mutations in NR0B1 (encoding the nu-
clear receptor DAX-1) were found in 12 boys, including in
two families in which three sons were affected. Mutations
in NR0B1 cause X-linked AHC and more than 100 dif-
ferent mutations are reported (5). Most are frameshift or
nonsense mutations that disrupt protein function, with a
clustering of missense changes in three regions of the li-
gand-like binding domain (27). The three missense
changes identified here (p.V269del, p.W291C, p.L299R)
are located in a loop region of helix 3 to helix 5 that in-
teracts with NR5A nuclear receptors (such as steroido-
genic factor-1) (28). The novel p.L299 lies adjacent to the
frequently mutated p.A300 residue, whereas codons
p.V269 and p.W291 have been mutated previously in non-
Turkish pedigree (5). All DAX-1-deficient boys presented
with salt loss and showed a bimodal distribution pattern
Figure 4. Age at presentation of the patients with PAI. of age at presentation as reported previously (Figure 4)
(29). Although currently preadolescent, they are likely to
TXNRD2, which to date have been described only in Irish develop hypogonadotropic hypogonadism and infertility
Traveler and Kashmiri families, respectively (12, 14, 23) as part of their condition, so establishing the diagnosis in
In addition to defining the population genetics of PAI, childhood will help plan endocrine management of pu-
this study has provided some useful clinical and novel mo- berty, counseling, and potential assisted reproduction
lecular insight into several of these specific conditions. (30). Macrophallia in infancy, found in one boy, is emerg-
Mutations in MC2R (encoding the ACTH receptor) are ing as a rare feature of X-linked AHC (31).
well established as the cause of FGD1, and approximately True CYP11A1 deficiency (encoding P450 side chain
40 different missense changes have been reported (9). cleavage [P450scc]) is a relatively recently established
Children typically present with hypoglycemia or hyper- endocrine condition because it was thought that disrup-
pigmentation in early infancy or in childhood, but com- tion of this enzyme, which facilitates the first three steps in
plete lossof functionmutationsare rare (17,24).Although conversion of cholesterol to pregnenolone, would be in-
diverse missense mutations were common in our cohort, compatible with fetal survival in humans (4). However,
severely disruptive changes in MC2R were found in more several children with this condition are now reported (32,
than half of the children (13 of 25), all of whom presented 33). Severe loss of CYP11A1/P450scc function is usually
in the first 6monthsof lifewithhypoglycemic convulsions, associated with severe salt-losing adrenal failure in the
respiratory distress, or both. In addition to the c.560delT neonatal period and a female phenotype in 46,XY children
mutation discussed above, the first complete deletions of (46,XY DSD). Milder changes can present later with ad-
the MC2R locus were found in three patients. Although renal insufficiency and a history of hypospadias in boys
ACTH plays a facilitative role in supporting mineralocor- (34, 35). The p.R451W variant found in all our patients
ticoid release, children with FGD1 do not typically have was described recently in two brothers of Turkish origin
salt loss. However, transient hyponatremia has been re- residing in Germany who had normal genitalia and child-
ported in several children with severe disruption of the hood-onset PAI (20). Their family originated from the
receptor, sometimes leading to a misdiagnosis of adrenal Elazig region of Eastern Anatolia, a province bordering
hypoplasia (17, 25). Similar observations were seen in this two regions where our patients lived (Figure 3A). The
cohort, with 5 of 25 children having evidence of hypona- p.R451W mutation disrupts hydrogen bonds with resi-
tremia (sodium 117–133 mmol/L) but only two of them dues F428 and Y449 in the K-L loop of the enzyme, caus-
receiving long-term fludrocortisone replacement. On the ing partial loss of function consistent with the late pre-
basis of the genetic diagnosis, it is likely the need for this sentation and normal genitalia in three of the four boys
treatment can be reviewed. The significance of recurrent (20). Nevertheless, all children will need careful follow-up
stillbirths in three families with severe loss of MC2R func- through puberty and into adult life to monitor sex hor-
tion is unclear. Although tall stature at diagnosis has been mone production and fertility. These findings confirm that
suggested for MC2R mutations, any effects on growth are mild loss of CYP11A1/P450scc function can present with
difficult to interpret because most of our cohort were di- isolated adrenal insufficiency and normal male genitalia,
agnosed and treated in infancy (26). similar to mild loss of function of STAR (18–20).
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doi: 10.1210/jc.2015-3250 press.endocrine.org/journal/jcem 291
Defects in STAR (encoding steroidogenic acute regu- Heterozygous mutations in NR5A1 (encoding the nu-
latory protein) disrupt the transport of cholesterol into clear receptor steroidogenic factor-1) usually cause 46,XY
mitochondria and classically lead to congenital lipoid ad- DSD or primary ovarian insufficiency (5). Adrenal insuf-
renal hyperplasia (4, 22). A range of mutations were ficiency is extremely rare and has been reported only once,
found, including recurrent p.S13P changes in three pa- in a 46,XX girl (39). The homozygous p.R92Q mutation
tients including two siblings. This is by far the most ami- found here in a 46,XX girl with early-onset PAI is fasci-
noterminal STAR mutation reported to date and affects nating because the same homozygous change was reported
the mitochondrial leader that is involved in targeting and in a 46,XY phenotypic female with adrenal failure from
localizing STAR to the outer mitochondrial membrane central Turkey in 2002 (40). This finding reinforces the
(36). Classic congenital lipoid adrenal hyperplasia is as- importance of the A-box motif of steroidogenic factor 1 in
sociated with severe salt-losing adrenal failure and 46,XY monomeric binding to DNA and provides conclusive ev-
DSD due to a block in both adrenal and testicular steroid- idence that severe disruption of steroidogenic factor 1 can
ogenesis. The two-hit hypothesis suggests that after an cause adrenal insufficiency in humans.
initial reduction in steroid production and increased Finally, mutations identified in AAAS (typically caus-
ACTH drive, accumulation of intracellular lipid has a sec- ing Triple A syndrome: achalasia, alacrima, Addison dis-
ondary toxic effect on cells (22). Interestingly, nine of the ease) and ABCD1 (typically causing X-linked adrenoleu-
children presented between 3 weeks and 3 months of age, kodystrophy) in three children without other features
consistent with the two-hit hypothesis, whereas only two shows how genetic screening can identify adrenal-only
presented in the first week (22). Two girls were found to phenotypes in young people who may be at risk of devel-
have 46,XY DSD only after genetic analysis revealed oping other symptoms in later life (11, 15).
STAR deficiency, highlighting the importance of obtain- This nationwide cohort study of high-throughput ge-
ing a karyotype in phenotypic girls with salt-losing adrenal netic screening of children with rare causes of PAI has
failure. As with CYP11A1/P450scc deficiency, these chil- provided many novel and supportive clinical and molec-
dren all need endocrine follow-up for life to monitor pu- ular insights and has significant impact on the manage-
berty and ovarian function in 46,XX girls and for puberty ment of these patients and their families. New genetic tech-
induction and sex steroid replacement in 46,XY DSD. In- nologies are a powerful tool in defining population
deed, the one patient with hypospadias raised male is now genetics of rare conditions and will allow more focused
showing evidence of hypergonadotropic hypogonadism in clinical genetic screening programs to be established.
adolescence.
Mutations in MRAP (encoding melanocortin 2 recep- Acknowledgments
tor-associated protein) and NNT (encoding nicotinamide
nucleotide transhydrogenase) cause adrenal insufficiency We thank the Exome Aggregation Consortium and the groups
without other features and can only be diagnosed by ge- that provided exome variant data for comparison. A full list of
netic analysis, as shown here. MRAP is essential for traf- contributing groups can be found at http://exac.broadinstitute.
ficking the ACTH receptor (melanocortin 2 receptor) to org/about.
the cell membrane, and mutations causing FGD2 were Address all correspondence and requests for reprints to: Tu-
first described in 2005 (9, 10, 24). The intron 3 splice site lay Guran, MD, Department of Pediatric Endocrinology and
is especially vulnerable, often resulting in early-onset ad- Diabetes, Marmara University, Fevzi Cakmak Mh. Mimar Sinan
renal insufficiency. Patients with novel aminoterminal Cd. No 41, 34899 Ustkaynarca/Pendik, Istanbul, Turkey.
point mutations (p.K30del, p.L53P) presented unusually E-mail: tulayguran@yahoo.com.
This work was supported by Turkish Pediatric Endocrinol-
late (3.5–13 y) (Figure 4), consistent with reports of chil- ogy Research Grant UPE-2014-2. J.C.A. is a Wellcome Trust
dren with p.V26A and p.Y59D changes (37). Senior Research Fellow in Clinical Science (Grant 098513/Z/12/
NNT mutations affect cellular oxidation and were Z), with support from the National Institute for Health Research
first decribed in 2012; approximately 20 children have Biomedical Research Centre at Great Ormond Street Hospital
been reported to date (9, 13, 38). The six novel changes for Children NHS Foundation Trust and University College Lon-
don. T.G. is a European Community, Marie-Curie research fel-
found here include familial homozygous deletions of low (Grant PIEF-GA-2012-328959).
exons 2–3 and homozygous missense mutations in the Disclosure Summary: The authors have nothing to disclose.
mitochondrial matrix region (p.D178G, p.H370R).
These findings confirm the importance of NNT for ad-
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