0021-972X/05/$15.00/0 Printed in U.S.A.

The Journal of Clinical Endocrinology & Metabolism 90(6):32433250 Copyright 2005 by The Endocrine Society doi: 10.1210/jc.2004-0016

Primary Adrenal Insufficiency in Children: Twenty Years Experience at the Sainte-Justine Hospital, Montreal
Rebecca Perry, Oufae Kecha, Jean Paquette, Celine Huot, Guy Van Vliet, and Cheri Deal
Endocrinology Service and Research Center, Sainte-Justine Hospital, and Department of Pediatrics, University of Montreal, Quebec, Canada H3T 1C5
Primary adrenal insufficiency (PAI) in the pediatric population (0 18 yr) is most commonly attributed to congenital adrenal hyperplasia (CAH), which occurs in about 1 in 15,000 births, followed by Addisons disease, with an assumed autoimmune etiology. However, molecular advances have increased the number of possible diagnoses. The objective of this study was to determine the incidence and etiologies of PAI in our pediatric population. All patients with a diagnosis of PAI followed by the Endocrinology Service at our institution between September 1981 and September 2001 were studied. One hundred three patients (48 boys) were identified, primarily by the Endocrinology Clinic case registry. CAH was the most frequent etiology (71.8%). However, non-CAH etiologies accounted for 28.2%, of which 55% were nonautoimmune in etiology. Importantly, the CAH sex ratio was 1:1, despite the absence of biochemical screening for this condition in Quebec newborns. Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dysplasia (APECED) developed adrenal insufficiency 4 yr earlier than those with non-autoimmune disease. Finally, we review the rare etiologies of PAI and propose an algorithm to aid in targeted genetic testing. (J Clin Endocrinol Metab 90: 32433250, 2005)

RIMARY ADRENAL INSUFFICIENCY (PAI) in children is an uncommon but potentially lethal condition. It comprises a heterogeneous group of both congenital and acquired disorders. Because PAI from infectious causes has declined in developed countries, physicians have more recently diagnosed PAI, except when associated with congenital adrenal hyperplasia (CAH), as idiopathic or as Addison disease, with the underlying etiology thought to be autoimmune in nature (1). Mutations in genes such as ACTHR (2 4), NROB1 (DAX1) (5), AIRE (6), and AAAS (7, 8) and autoantibodies to gland-specific target antigens such as 21-hydroxylase have been described in the past decade, but the frequency of their occurrence in children with PAI has not been evaluated. The aim of this study was therefore to determine the incidence and molecular etiology of PAI in a large contemporary cohort of children.
Patients and Methods

Patients
All patients followed by the Endocrinology Service at the SainteJustine Hospital between September 1981 and September 2001 for PAI were included. The patients were identified primarily by the outpatient case registry of the Endocrinology Service and secondarily through archival retrieval to identify all hospitalized patients with PAI who might have been lost to follow-up. The diagnostic categories retained First Published Online April 5, 2005 Abbreviations: AADC, Aromatic l-amino acid decarboxylase; ALD, adrenoleukodystrophy; APECED, autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy syndrome; CAH, congenital adrenal hyperplasia; CT, computed tomography; nt, nucleotides; 17OH, 17-hydroxylase; 17OHP, 17-hydroxyprogesterone; 21OH, 21-hydroxylase; PAI, primary adrenal insufficiency; SCC, side-chain cleavage enzyme; SV, simple-virilizing form; SW, salt-wasting form. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

were as follows: primary adrenal failure, CAH, Addison disease, polyglandular autoimmune syndrome, triple A syndrome/Allgrove syndrome, Wolman disease, and adrenoleukodystrophy (ALD)/ Schilder syndrome. The following was recorded for all patients: date of birth, gestational age, age at diagnosis, and clinical presentation. For patients with a diagnosis of CAH, diagnostic test results retained included electrolytes, blood glucose, capillary blood gases, adrenal steroidogenic profile, renin, aldosterone, adrenal ultrasound, and genitogram. We also reviewed all notes pertaining to neurological development and school progress. For patients with non-CAH PAI, we recorded the evidence supporting mineralocorticoid and glucocorticoid deficiency, initial ACTH level, autoantibody screen, very-long-chain fatty acids, and adrenal imaging. Transient cases of adrenal insufficiency, with or without stress-related elevations of 17-hydroxyprogesterone (17OHP) levels (9), were excluded. Non-CAH primary adrenal insufficiency was defined as insufficient cortisol production with elevated ACTH and/or evidence of adrenal disease. We also included adrenal disorders diagnosed at autopsy that will eventually lead to adrenal insufficiency, e.g. Wolman disease. Classic CAH due to 21-hydroxylase (21OH) deficiency was subdivided as defined previously (10) into the salt-wasting (SW) form and the simplevirilizing (SV) form; nonclassic 21OH CAH was diagnosed when signs of androgen excess in childhood/adolescence were accompanied by an ACTH-stimulated 17OHP level greater than 45.5 nmol/liter (10). For children with classic 21OH CAH, we used the year 1990 as an arbitrary cutoff to see whether there was any difference between the two decades included in the study in terms of age at diagnosis of CAH (particularly of boys). We defined severe metabolic compromise as more than 10% dehydration with electrolyte abnormalities, namely hyponatremia of no more than 125 mmol/liter and hyperkalemia of no less than 7.5 mmol/liter.

Assay of antibodies
The presence of antiadrenal antibodies in serum was evaluated by indirect immunofluorescence using monkey adrenal tissue (Inova Diagnostic, Inc., San Diego, CA). Antibodies against aromatic l-amino acid decarboxylase (AADC), 21OH, 17-hydroxylase (17OH), and side-chain cleavage enzyme (SCC) were kindly determined by Dr. O. Ka mpe (Uppsala, Sweden). The method used is based on the in vitro transcribed and translated protein, as described by Ekwall et al. (11). The upper normal limit of each antibody index was the mean value of negative controls

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plus three sd values; values above this cutoff were taken to indicate the presence of autoantibodies.

Classic 21OH CAH (Table 2)

DNA isolation
For all antibody-negative patients without an obvious etiology to their adrenal failure, DNA was isolated from peripheral blood leukocytes after informed consent by standard methodology for sequencing of appropriate candidate genes, including ACTHR, NROB1 (DAX1), and AAAS. DNA was also obtained after informed consent from patients with positive antiadrenal antibodies and at least two components of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED).

Fifty-nine patients (29 boys) from 53 families had classic 21OH CAH. Fifty-three patients had the SW form, and six had the SV form. Thirteen patients had nonclassic CAH. However, there may be some overlap between these three categories. SW form. All of these infants were born at 36 wk or greater, with a median of 40 wk, except for two monozygotic twin girls concordant for CAH who were delivered electively at 35 wk due to intrauterine growth retardation of one of the twins. There were no reported deaths of siblings. The median age at diagnosis before 1990 was similar for both boys and girls at 24 and 21 d, respectively. However, after 1990, girls were diagnosed significantly earlier than their male counterparts, with a median age of 2.5 and 23 days (P 0.005), respectively. All girls had sexual ambiguity or virilization, although this was not recognized in the delivery room in all. Four girls were declared male at birth in peripheral hospitals; in three, sex assignment was reversed before discharge and, in one, after a salt-losing crisis led to the diagnosis at 10 d of age. In contrast, boys presented most commonly with severe metabolic compromise (20 of 26 vs. 7 of 27 in girls; P 0.0002). Three boys had a cardiorespiratory arrest at presentation; all were diagnosed before 1990 at 14 16 d of age. Three boys were diagnosed later due to signs of precocious sexual development along with evidence of aldosterone deficiency. One of these boys only came to medical attention after his infant sister was diagnosed with CAH at 8 months when she presented with a salt-losing episode and her mild virilization was recognized. School performance was tracked in 36 patients who were older than 6 yr. Six of 17 boys (35%) have repeated grades, whereas four of 19 girls (21%) have been held back. In the general population in Quebec, the corresponding percentages at 12 yr of age are 25 and 17.8% (14). Two unrelated children died during the study period; both deaths were

Mutational analysis of the AIRE gene

In four patients with features suggestive of APECED, AIRE was sequenced in our laboratory as described previously (6).

Mutational analysis of ACTHR (MC2R), AAAS, NROB1 (DAX1), and HSD3B2

In four patients with isolated glucocorticoid deficiency, ACTHR (MC2R) was sequenced by Dr A. J. Clark (London, UK). The coding exon (exon 2), short intronic flanking sequences in both directions, and several overlapping fragments were sequenced. Based on clinical evidence, Dr. A. Huebner (Dresden, Germany) sequenced AAAS in one patient, Dr. E. McCabe (Los Angeles, CA) sequenced NROB1 (DAX1) in another, and Dr. J. Simard (Quebec City, Canada) sequenced HSD3B2 in two others. The sequencing methods for all of these genes have been reported previously (4, 8, 12, 13).

Statistical analyses
Continuous quantitative variables were expressed as median and range and were compared by the Mann-Whitney U test. Categorical data were compared by the 2 test. The level of significance was set at 0.05 (two-tailed).

Results

21OH CAH (of which 79% were classic and 21% nonclassic) accounted for 71.8% of the total PAI cases (Table 1).
TABLE 1. Etiologies of PAI
Etiology Females (n 55)

Males (n 48)

% of total (n 103)

Age at diagnosis of PAI

21OH CAH Classic SW form SV form Nonclassic 3--hydroxysteroid dehydrogenase deficiency CAH Autoimmune APECED Non-APECED Adrenoleukodystrophy Syndromes Wolman disease Triple A Zellweger disease X-linked AHC Unexplained isolated glucocorticoid deficiency Unexplained glucocorticoid and mineralocorticoid deficiencies

30 27 3 11 1 3 5 0 1 0 0 0 2 2

29 26 3 2 1 2 3 4 2 1 1 1 1 1

57.3 51.5 5.8 12.6 1.9 4.9 7.8 3.9 2.9 1.0 1.0 1.0 2.9 2.9

16 (12281) da 5.8 (3.77) yr 7.4 (510.3) yr 17.5 (14 21) d 10.7 (5.4 13.6) yr 14.6 (7.8 16.3) yr 10.5 (4.314.3) yr 26 (25 49) db 10.3 yr 8.2 yr 4.6 yr 42 (6 122) d 2.3 (1.2 4.3) yr

a If the boy with a late diagnosis made after identification of his virilized infant sister is excluded, the median is unchanged, but the range becomes 11125 d. b Age at presentation, not diagnosis (which was made postmortem; see Results).

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TABLE 2. Patients with classic 21OH CAH

No. of patients Salt wasting Female Male Female Simple virilizing Male

No. of patients Age at diagnosis [median (range)] (d) Before 1990 After 1990 Presentation [n (total no.)] Metabolic compromise Before 1990 After 1990 Ambiguity Prader 2 Prader 3 SW (no shock) Pseudo-precocious puberty Basal 17OHP [median (range)] (nmol/liter) Bone age SD values [median (range)]

27 21 (2243) (n 13) 2.5 (118) (n 14)a 7 (27)b 5 (13) 2 (14) 5 (27) 22 (27) 7 0 905 (1132446)

26 24 (22281) (n 17) 23 (11912) (n 9)a 20 (26)b 14 (17) 6 (9)

3 6.2 (3.77) yr

3 5.8 (4.35.8) yr

2 3 1901 (79 7370)

3 0 0 0 294 (95 827) 6.4 (5.19.8)

0 3 34.4 (39.7 45) 8.2 (8 11.5)

Normal values: basal 17 OHP: age-dependent, refer to Ref. 45. Conversion factor: 17 OHP nmol/liter 31.7 ng/dl. Difference between males and females: a P 0.005; b P 0.0002.

non-CAH related: one was accidental, and the other was due to a codiagnosis of cystinosis. SV form. Six children (three boys) had the SV form. The median age at diagnosis was 5.8 yr. Boys presented with early pubarche, acceleration of linear growth and bone maturation, acne, and marked penile development contrasting with a prepubertal testicular volume. The girls also presented with early pubarche and growth acceleration; all three had clitoromegaly, which was associated with posterior labial fusion in one.
Nonclassic 21OH CAH (Table 3)

Autoimmune adrenal failure (Table 4)

The two boys presented with premature pubarche, which was associated with accelerated growth velocity in one and with acne in the other. In girls, 10 presented with premature pubarche, of which three had an increased growth velocity, four had acne, and one presented with clitoromegaly alone.
3--hydroxysteroid dehydrogenase deficiency

One boy and one girl were both homozygous for the A10E mutation of HSD3B2. Their clinical presentation and molecular diagnosis have been published previously (13).
TABLE 3. Patients with nonclassic 21OH CAH
Females Males

APECED. Five patients (three girls; four families) with adrenal failure due to APECED were included in our series based on a positive history for candidiasis and/or hypoparathyroidism. Median age at presentation was 10.7 yr. In the family of two female affected patients with compound heterozygosity at the AIRE locus [exon 2, missense L93R; and exon 8, deletion nucleotides (nt) 10851097], which we reported previously (6), symptomatic glucocorticoid deficiency developed at 10.7 and 13.6 yr in the younger and older sister, respectively, followed by salt loss. The third unrelated female, with a history of hypoparathyroidism from age 4 yr and of juvenile idiopathic arthritis, developed glucocorticoid deficiency at the age of 12.4 yr and does not yet require mineralocorticoid replacement; her parents are first cousins, and she is homozygous for the R257X mutation in exon 6 of AIRE. The two unrelated boys presented at 5.4 and 5.7 yr, respectively, with shock secondary to a saltlosing crisis. Both patients had a history of chronic mucocutaneous candidiasis, and one of them also had previously undiagnosed hypoparathyroidism and hypoplasia of dental enamel. AIRE mutational analysis was only possible for one of the two boys; compound heterozygosity was detected for an exon 8 mutation (deletion nt 10851097) and an exon 14 mutation (P539L). Non-APECED. Eight patients (five girls) had a diagnosis of autoimmune adrenal insufficiency (non-APECED, based on clinical history). Median age at presentation was 14.6 yr, older than in APECED patients (P 0.005). All had evidence of positive antiadrenal antibodies (positive IF and/or anti-21OH antibodies). Four patients (three girls) also had thyroid dysfunction, suggesting a diagnosis of autoimmune polyglandular syndrome type 2. The remaining four patients had isolated PAI, although three had a family history of thyroid dysfunction. Seven patients showed evidence of both glucocorticoid and mineralocorticoid deficiencies at diagnosis, and the eighth presented initially with only glucocorticoid deficiency but subsequently developed mineralocorticoid deficiency within the first 6 months.

No. of patients Age at diagnosis (yr) Cortisola Basal Peakb 17OHPa Basal Peakb Bone age SD values Treated with glucocorticoids

11 7.3 (5 8.8) 275 (113 469) 431 (265 669) 31 (5164) 216 (59 409) 2.2 (0 5.1) 2

2 9.4 (8.6 10.3) 217 (164 270) 287 (280 293) 59 (26 92) 415 (352 478) 2.95 (2.4 3.5) 0

Normal values, refer to Ref. 10. Conversion factors: cortisol, nmol/ liter 0.036 g/dl; 17OHP, nmol/liter 31.7 ng/dl. a Median (range) in nmol/liter. b After 0.25 mg Cortrosyn, iv.

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TABLE 4. PAI patients who had specific target antigen panels performeda
Index cutoff 3 SD values 5.4 yr M 12.4 yr F 13.6 yr F 11 yr M 4.3 yr F

Antiadrenal antibodies Immunofluorescence 21OH SCC 17--OH AADC Diagnosis APECED Non-APECED Unexplained
a

24 18 30 17

1/5 46 94 16 15

1/20 67 1 4 7

1/40 43 96 192 188

1/80 81 17 11 8

ve 12 5 4 17

M, Male; F, female. All of these patients had AIRE mutational analysis.

Adrenoleukodystrophy (Table 5)

Triple A syndrome

Four boys presented at a median age of 10.5 yr. Adrenal failure was the first clinical sign of ALD in three of the four patients; only one of the three had a known affected halfbrother and had had yearly ACTH testing, although in the other two, retrospective review of family histories included maternal male relatives with neurological disease. The fourth patient presented with transient blindness, which progressed relatively rapidly to spasticity and epilepsy. He was the only patient without a family history suggestive of ALD. Currently, only one patient has mineralocorticoid deficiency.
Wolman disease

A 10-yr-old boy presented with glucocorticoid deficiency and later developed mineralocorticoid deficiency. His parents had also noted longstanding dysphagia for solids and hypolacrimia. Bilateral lacrimal gland aplasia was demonstrated on orbital computed tomography (CT) scan, multiple small diverticulae were observed on a barium meal, and manometry showed primary dysmotility. The clinical suspicion of triple A syndrome was confirmed by mutational analysis of the AAAS gene [compound heterozygosity: R286X, 1332-2AG (splice mutation)].
Zellweger disease

Three patients (two families) had Wolman disease. The first two are brother and sister who presented at 49 and 26 d, respectively, with hepatosplenomegaly, anemia, and elevated transaminases and triglycerides. They were both treated for a presumptive diagnosis of familial erythrophagocytic lymphohistiocytosis with chemotherapy and an allogenic bone marrow transplant, respectively. The diagnosis of Wolman disease was only made retrospectively at autopsy of the second infant, in which marked accumulation of cholesterol crystals and lipids was observed in several organs (liver, intestines, spleen, lymph nodes, and adrenals). There was adrenal hypertrophy (weight of 19.1 g; normal is 4.6 g) with microscopic areas of calcification. On revising the autopsy of the first infant, enlarged adrenals with calcifications were noted. The diagnosis was confirmed by the finding of low acid lipase activity on liver biopsy. The third patient, a boy, presented at 25 d with hepatosplenomegaly, abdominal distension, elevated transaminases, and adrenal calcification noted on ultrasound. In both families, there was a history of consanguinity. Death occurred early, with a median age of 67 d (range of 60 72 d).
TABLE 5. Patients with X-linked ALD
Age (yr) at diagnosis; sex Heralding feature of ALD Type

One boy with Zellweger disease developed glucocorticoid and mineralocorticoid deficiencies at the age of 8 yr. He had been diagnosed at 11 months with Zellweger syndrome based on the clinical findings of global developmental delay and hypotonia, dysmorphic facial features, neurosensory deafness, and optic atrophy. The diagnosis was confirmed by alterations in the very-long-chain fatty acids: presence of C26:1, increased levels of C26:0, and increased ratios C24:C22 and C26:C22. Due to his longevity, he has been reclassified as having Zellweger disease rather than syndrome, because classically, Zellweger syndrome patients have a considerably shorter lifespan, with most affected infants succumbing during the first 2 yr of life (15).
Congenital adrenal hypoplasia

This boy initially presented at 4.6 yr with adrenal insufficiency with salt wasting. A maternal uncle had died at 3 wk of age after protracted vomiting. At 15 yr, a diagnosis of hypogonadotrophic hypogonadism was made based on lack of response of gonadotrophins after stimulation with LHRH and

Nervous system

Family history

Treatment used for ALD

10.8; M 4.3; M 10.2; M 14.3; M

Adrenal failure Adrenal failure Transient blindness Adrenal failure

Adolescent cerebral Addison only Childhood cerebral Addison only

Hyperactivity, deafness, strabismus ADHD, normal CNS Hyperactivity, epilepsy, spasticity Normal CNS

Maternal uncles Brother, mother (AMN) None ? Maternal grandfather Lorenzos oil, Lovastatin Bone marrow transplant, Lovastatin, Lorenzos oil

M, Male; F, female; ADHD, attention deficit hyperactivity disorder; CNS, central nervous system; AMN, adrenomyeloneuropathy.

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absence of testicular enlargement. NR0B1 sequencing revealed a 343delG mutation (frame shift; patient included in Ref. 12).
PAI with no molecular cause identified (Table 6)

Isolated glucocorticoid deficiency. Three patients (two girls) were identified with isolated, early-onset glucocorticoid deficiency. The three patients were born at 40 42 wk with an appropriate birth weight and normal genitalia and were diagnosed at 6, 42, and 122 d. All presented with persistent hypoglycemia, normal 17OHP levels and very-long-chain fatty acids, and negative antiadrenal antibodies. None showed unexplained tall stature. The girl with the earliest presentation also had unexplained developmental delay, an atrial septal defect, and dysmorphic facial features (hypertelorism and anteverted nares) reminiscent of Aarskog syndrome. The second girl had a stormy neonatal period. She was diagnosed at 6 wk with streptococcal osteomyelitis and adrenal insufficiency; she subsequently developed iatrogenic cardiac insufficiency. The dilated cardiomyopathy, which was probably secondary to fluid overload in the context of high-dose corticosteroid therapy, completely resolved. The boys clinical course was complicated by acute lymphoblastic leukemia, marked obesity, hepatitis secondary to varicella, hypercholesterolemia, and hypertriglyceridemia. ACTHR sequencing was normal in all three cases, and no specific underlying cause has been identified. Combined glucocorticoid and mineralocorticoid deficiencies. All three patients have normal very-long-chain fatty acids and 17OHP levels and negative antiadrenal antibodies. The first patient was diagnosed at 4.3 yr with isolated glucocorticoid deficiency (but ACTHR sequencing was normal), and she later developed mineralocorticoid deficiency. This patients antibody panel is interesting (Table 4): antiadrenal antibodies by immunofluorescence and anti-21OH antibodies are negative, but anti-AADC antibodies are positive (3 sd values), which is highly suggestive of APECED (16). However, she has no other features suggestive of APECED, such as mucocutaneous candidiasis or hypoparathyroidism, which usually predate the adrenal failure seen with APECED. She also has a normal AIRE sequence (all 14 exons). The second girl was diagnosed at 2.3 yr after an acute, probably septic, illness (bacterial tracheitis), which led to a cardiorespiratory arrest. Her parents describe symptoms, including growth delay and poor weight gain, which predate the acute episode by 1 yr. Episodic vomiting the week before admission had
TABLE 6. Patients with unexplained PAI
Age/sex at presentation Serum cortisol (nmol/liter) Basal Peak
a

been attributed to a minor head injury with no loss of consciousness. Upon her initial presentation, she was not hyperpigmented but was hyponatremic (119 mmol/liter), hyperkalemic (6.8 mmol/liter), and hypoglycemic (0.4 mmol/ liter). Her adrenal ultrasound and CT scan at this time was normal. At the age of 4 yr, an adrenal CT scan revealed punctiform calcifications. Adrenal insufficiency could be explained by bilateral adrenal hemorrhage, but because there was no evidence of this on CT at presentation, this is unlikely. Two ACTH stimulation tests performed after her initial presentation confirmed adrenal failure; her dehydroepiandrosterone sulfate at the age of 13.5 yr was undetectable, although thelarche had begun at age 13 yr and puberty appeared to be progressing normally. The boy presented at 1.2 yr with hypoglycemic convulsions and salt loss. His screening investigations were all normal, and, now aged 21 yr, he has not developed any other endocrinopathies.
Discussion

ACTH (pg/ml) (n 130)

6 d/F 42 d/F 122 d/M 4.3 yr/Fb 2.3 yr/Fb 1.2 yr/Mb

214.4 15 19 37 143 19.3

270.3 15 44 43 143 27.6

N.A. 288 195.3 1721 481 500

Normal values: cortisol, 193 690 nmol/liter (basal); 550 nmol/liter (peak). a After 0.25 mg Cortrosyn or on a critical sample when glucose 2.2 mmol/liter. b Combined gluco- and mineralocorticoid deficiencies.

We were able to determine the etiology of PAI in 94.2% of our patients, including those with biochemically proven CAH due to 21OH deficiency in whom CYP21 sequencing was not routinely performed. Our principal diagnoses fell into three main categories, i.e. impaired steroidogenesis, adrenal destruction, and dysgenesis. CAH was the most frequent cause of PAI, but non-CAH etiologies accounted for 28.2%, of which 55% were non-autoimmune in etiology. Based on our large patient cohort, we propose a new diagnostic tree for genetic testing in children with PAI (Fig. 1). Importantly, the sex ratio of classic 21OH CAH in our large series is 1:1, despite the absence of systematic biochemical screening for CAH in Quebec newborns. This is in contrast to the female preponderance (57 43%), which is still seen in other contemporary series of children with classic 21OH CAH (17). However, boys with the SW form presented later than girls and with more severe metabolic compromise (77 vs. 26%). We do not think that we have underdiagnosed classic CAH: our hospital provides pediatric subspecialty care to 36.4% of the Quebec population (18), yielding a prevalence comparable with that found elsewhere (1 in 16,630) (19, 20). Our proportion of classic (79%) vs. nonclassic (21%) CAH is also similar to that based on neonatal screening results (70% classic, 30% nonclassic) (19). Biochemical screening for CAH was recommended by the Joint LWPES/ESPE CAH Working Group, principally to avoid the death of affected boys (21). Our results suggest that this can be achieved by clinical ascertainment and that severe metabolic compromise at presentation, in both boys and girls, may be decreasing due to greater awareness of CAH among healthcare professionals. Moreover, biochemical screening is plagued by a high rate of false-positive to true-positive results, especially in premature newborns (20). In this respect, it is worth pointing out that, with the exception of the twins, all infants with CAH in our series were born at 36 wk or more and had a median gestational age of 40 wk. This is consistent with the concept that deficient cortisol production by the fetal adrenal may delay the onset of labor (22). In view of a previous report of learning difficulties in CAH (23), we analyzed the percentage of index cases who repeated

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FIG. 1. Diagnostic algorithm in primary adrenal failure. This schematic proposes a diagnostic workup to direct genetic testing to the appropriate gene. Note that less frequent forms of CAH must be considered if steroidogenic profile is suggestive and/or if mutation analysis of CYP21 is negative. Not shown in this algorithm are Smith-Lemli-Opitz syndrome (suggested by microcephaly, micrognathia, syndactyly, sexual ambiguity, and kidney and heart malformations), which should prompt measurement of cholesterol and its metabolites (7 and 8 dehydrocholesterol), as well as the rare entity of 46,XY sex reversal with testicular dysgenesis and adrenal failure due to mutations in steroidogenic factor-1 (38). Finally, the genetics of the IMAGe syndrome and autoimmune polyglandular syndrome type 2 are not yet elucidated. GC, Glucocorticoid; MC, mineralocorticoid; VLCFA, very-long-chain fatty acids; Abs, antibodies; IUGR, intrauterine growth retardation; MCC, mucocutaneous candidiasis; DM, diabetes mellitus; AHC, adrenal hypoplasia congenita; APS-2, autoimmune polyglandular syndrome type 2.

a grade in school. Although this percentage is slightly higher than that found in the general population of Quebec (14), our patient group is relatively small and not matched for age and socioeducational background to a control group. Clearly, the relationship between the number and severity of dehydration episodes and school progress in both boys and girls with CAH deserves additional study. Autoimmune adrenal failure was the second leading cause of PAI in our series, accounting for 12.7% of all cases. Patients with APECED developed PAI 4 yr earlier than non-APECED patients and had a history of chronic candidiasis and/or hypoparathyroidism. All patients with adrenal failure due to APECED had positive antiadrenal antibodies by indirect immunofluorescence. With the identification of specific target antigens in adrenal failure (21OH, SCC, 17OH, and AADC), the specificity of the diagnosis of autoimmune adrenal failure is much improved (24). Indeed, antibodies against 21OH were positive in all four of the patients with APECED investigated, whereas antibodies against SCC and 17OH were only positive in one and two patients, respectively. AntiAADC antibodies, which are thought to be a useful diag-

nostic marker for APECED, were only positive in one of the four patients, although two of the patients tested are still young and may go on to develop antibodies (16). Since the cloning of the AIRE gene (25), more than 45 mutations have been described in APECED patients (26). Genotype-phenotype correlations are not clear because, even in the same pedigree, there is discordance, suggesting the importance of modifying loci or environmental factors (6, 27). Two of the most common mutations in European populations, the exon 6 R257X mutation and the exon 8 13 bp deletion (nt 10851097) were found in our four APECED patients agreeing to mutational analysis. In one patient, an exon 14 mutation was also found. This is an uncommonly affected exon with only two mutations described to date (28, 29). In our series, 25% of boys with non-CAH PAI had X-linked ALD, the most common non-CAH, non-autoimmune cause of adrenal failure for which we were able to identify an etiology. Adrenal failure was the presenting feature in 75%. X-linked ALD is the most common inherited peroxisomal disorder, with an incidence of 1:40,000 to 1:100,000. Although the gene is now known and codes for a peroxisomal integral

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membrane protein, mutational analysis of the ALD gene is not routinely indicated, because the biochemical finding of elevated very-long-chain fatty acids is accurate in 99.9% of affected males (30). However, 15% of obligate female carriers will have normal results, and thus mutational analysis is required for accurate determination of carrier status with a view to genetic counseling. There is currently no clear correlation between genotype and either neurological or endocrinological phenotype (31). However, if genotype-phenotype correlations become clearer, this will be a compelling reason to routinely perform mutational analysis to provide more useful counseling for affected families. Zellweger syndrome is one of several peroxisomal disorders sharing a common pathogenesis involving mutations in proteins required for peroxisomal biogenesis and proliferation, the so-called PEX proteins (32). Other entities in this diagnostic category that can manifest adrenal failure as part of their clinical presentation are as follows: 1) neonatal, autosomal adrenoleukodystrophy, and 2) Zellweger disease and a milder form of Zellweger syndrome, as demonstrated by our patient. Mutational analysis of the PEX proteins is not routinely performed or available, although there is very active research in this field; in the future, these diseases might be more readily confirmed by mutational analysis. Among the other rare genetic causes of adrenal failure that we encountered, Wolman disease was found in two different consanguineous families. This lethal autosomal recessive storage disease, due to deficiency of lysosomal acid lipase, is rare but should be considered in any neonate with bilateral adrenal calcification, especially if there is a family history of consanguinity or early neonatal deaths. The calcification delineates the outline of both glands and appears to be pathognomonic for Wolman disease and for its milder form, cholesterol ester storage disease (33). It should also be considered if bilateral adrenal calcification is noted on prenatal ultrasound examination. Prenatal diagnosis is possible by measuring acid esterase activity in cultured embryonic cells (34), although mutational analysis of LIPA, the gene coding for lysosomal acid lipase, may provide a more rapid diagnosis (35). Despite the gloomy prognosis, there has been some recent encouraging progress in gene therapy of Wolman disease. After gene transfer of lysosomal acid lipase into affected fibroblasts, there is phenotypic correction of the lipid storage and of the growth arrest (36). It is not yet clear whether patients surviving past the first few months, thanks to earlier diagnosis and better treatment modalities, will require mineralocorticoid and/or glucocorticoid replacement, and therefore adrenal function should be investigated and treatment initiated if basal and/or ACTH-stimulated cortisol is abnormal (33). The gene responsible for the triple A syndrome (AAAS), another rare autosomal recessive cause of adrenal failure, has been discovered recently (7, 8). We describe a patient with compound heterozygosity, including a known substitution mutation (R286X) and a novel splice mutation (1332-2AG). The first mutation has been described previously in a large highly inbred French-Canadian family (8) and probably constitutes a founder mutation, whereas the second allele harbors a novel mutation that is currently being characterized further.

Adrenal hypoplasia congenita is relatively rare (37). It is an X-linked recessive disorder characterized by glucocorticoid and mineralocorticoid deficiency with low levels of adrenal androgens. The molecular etiology involves mutations in NROB1 as found in one of our patients (12). Affected boys present within the first months or years of life and frequently have hypogonadotrophic hypogonadism, as did our patient. Some rare cases develop adrenal failure only in adulthood. Diagnoses not represented in our series include mutations in the gene for steroidogenic factor-1, or SF1 (38), IMAGe syndrome (intrauterine growth retardation/metaphysial dysplasia/adrenal hypoplasia/genital anomalies) (39), SmithLemli-Opitz syndrome (40, 41), and Antley-Bixler syndrome (42, 43). Despite a concerted effort to provide families with an etiological diagnosis for PAI using targeted molecular analysis, we were unable to arrive at a precise diagnosis in six patients. These patients are candidates for study of other loci linked to ACTH resistance, such as the 8q region and a novel gene on chromosome 21, coding for the melanocortin 2 receptor accessory protein, or MRAP (44). In conclusion, as our understanding of adrenal failure improves, molecular diagnoses are leading to changes in clinical practice. Confirmation of the precise etiology of primary adrenal failure has the potential to markedly improve our genetic counseling of patients and their families. Future studies will uncover additional molecular etiologies of PAI and should include great attention to genotype-phenotype correlations in an effort to better predict the natural history of this potentially fatal endocrinopathy.
Acknowledgments
We extend our gratitude to our collaborators: Dr. Adrian Clark (London, UK) for ACTHR sequencing; Dr. Angela Huebner (Dresden, Germany) for AAAS sequencing; Dr. Olle Ka mpe (Uppsala, Sweden) for antibody panels; Dr. Guy Lepage (Montreal, Canada) for very long chain fatty acids; Dr. Edward McCabe (Los Angeles, CA) for NROB1 sequencing; and Dr. Jacques Simard (Quebec City, Canada) for HSD3B2 sequencing. Received January 6, 2004. Accepted March 3, 2005. Address all correspondence and requests for reprints to: Cheri Deal, Ph.D., M.D., Endocrinology Service, Sainte-Justine Hospital, 3175 SainteCatherine Road, Montreal, Quebec, Canada H3T 1C5. E-mail: cheri.l.deal@ umontreal.ca. This work was supported by the Research Center of the Hospital Sainte-Justine. R.P. was supported by a fellowship from the Department of Pediatrics of the University of Montreal. O.K. was supported by the Vaugrenier Foundation (Geneva, Switzerland) and the Leon Fredericq Foundation (Liege, Belgium) during her time in the laboratory of C.D. C.D. is a Chercheur-boursier from the Fonds de Recherche en Sante du Que bec.

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