ENDOCRINE RESEARCH

Vol. 30, No. 4, pp. 957–964, 2004

A Male Twin Infant with Skull Deformity and ElevatedNeonatal 17–Hydroxyprogesterone: A Prismatic

Case of P450 Oxidoreductase Deficiency

Stefan A. Wudy,1 Michaela F. Hartmann,1 Nicole Draper,2

Paul M. Stewart,2 and Wiebke Arlt2,*

1Steroid Research Unit, Center of Child and Adolescent Medicine,

Justus Liebig University, Giessen, Germany2Division of Medical Sciences, Institute of Biomedical Research, Endocrinology and

Metabolism, The Medical School, University of Birmingham, Birmingham, UK

ABSTRACT

We report on a male twin infant who presented with brachy-turri-cephaly, frontal

bossing, large anterior fontanelle, low set and malformed ears, and mild

arachnodactyly. He had normal male genitalia. There was no evidence for maternal

virilization during pregnancy. The pattern of malformations resembled Antley–

Bixler–Syndrome (ABS). However, sequencing analysis of the fibroblast growth

factor receptor 2 gene (FGFR2) did not reveal mutations. The boy’s twin sister did not

show any somatic or endocrine abnormalities. In the boy, neonatal screening for

congenital adrenal hyperplasia was positive with moderately elevated 17–hydroxy-

progesterone. Sequence analysis of his CYP21 gene did not reveal any mutations. The

short synacthen test revealed an exaggerated 17–hydroxyprogesterone and a blunted

cortisol response. Urinary steroid profiling by gas chromatography-mass spectrometry

(GC-MS) revealed a unique steroid metabolome suggestive of impaired activity of

both 17–hydroxylase and 21–hydroxylase. Clinical and metabolic findings therefore

*Correspondence: Dr. Wiebke Arlt, Division of Medical Sciences, Institute of Biomedical

Research, Endocrinology and Metabolism, Rm 233, The Medical School, Univesity of

Birmingham, Birmingham, B15 2TT, United Kingdom.

957

DOI: 10.1081/ERC-200044174 0743-5800 (Print); 1532-4206 (Online)

Copyright D 2004 by Marcel Dekker, Inc. www.dekker.com

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were compatible with the recently described variant of congenital adrenal hyperplasia,

P450 oxidoreductase deficiency (ORD). Subsequently, sequencing analysis of CPR,

the gene encoding P450 oxidoreductase (OR), revealed a homozygous mutation in the

patient, resulting in an amino acid exchange in position 284 of the OR protein (A284P).

Both the female twin sister and the parents were heterozygous for the A284P mutation.

P450 oxidoreductase deficiency represents a novel autosomal recessively inherited

form of congenital adrenal hyperplasia. Its characteristic steroid metabolome can

readily be detected by GC-MS analysis of spot urine. Clinical features may include an

ABS phenotype, ambiguous genitalia (virilization in girls, feminization in boys), and

glucocorticoid deficiency. If required, hydrocortisone replacement should be provided.

Key Words: P450 oxidoreductase; P450c17; P450c21; Antley –Bixler; P450

oxidoreductase deficiency; Glucocorticoid deficiency.

INTRODUCTION

Herein we describe a male twin infant, who—unlike his twin sister—postnatally

exhibited a skull deformity and had elevated 17–hydroxyprogesterone in the neonatal

screening test. An association between these two findings was felt to be unlikely.

However, it was not possible to delineate a known variant of congenital adrenal

hyperplasia by hormone analysis. Furthermore, his skull deformity could not be classified

to the most common skull deformity syndromes such as Apert’s syndrome, Crouzon’s

syndrome or Pfeiffer’s syndrome (1). Finally, it was current progress in metabolic and

genetic steroid research that allowed establishing the definitive diagnosis.

CASE REPORT

The boy was born as first twin to nonconsanguinous caucasian parents after 34 weeks

of gestation at a county hospital. The mother, a 34 year old healthy 1st gravida/1st para,

underwent Cesarian section because of HELLP syndrome. The course of the twin

pregnancy had been otherwise uneventful and the mother had not received any drug

treatment during pregnancy. The boy’s birth weight was 2405 g (90th percentile), length

was 48 cm (2 cm > 90th percentile) and head circumference 33 cm (1 cm > 90th

percentile). He was noted to have frontal bossing, a large anterior fontanelle, low set and

malformed ears, and mild arachnodactyly. The external genitalia had normal male

appearance with bilaterally descended testes. There were no skin abnormalities, in

particular no evidence of hyperpigmentation. The boy’s healthy and apparently unaffected

twin sister had a birth weight of 2140 g (50th–90th percentile), her length was 44 cm

(50th percentile) and her head circumference was 31 cm (50th–90th percentile).

Neonatal screening for congenital adrenal hyperplasia (21–hydroxylase deficiency)

on the 4th day of life showed a moderately elevated 17–hydroxyprogesterone of

34 ng/ml (cut off 20 ng/ml). This was confirmed by a recall sample obtained on the 9th

day of life (30 ng/ml). A urinary sample was sent for GC-MS analysis and showed

normal excretion of glucocorticoid metabolites and mildly elevated markers of 21–

hydroxylase deficiency (17–hydroxypregnanolone, 15b–OH–pregnanolone, pregnane-

triol, and pregnanetriolone) suggesting mild enzyme deficiency. Serum analysis by

958 Wudy et al.

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radioimmunoassay was further suggestive of 21–hydroxylase deficiency with elevated

levels of 17–hydroxyprogesterone (11.75 ng/ml; normal range 0.20–3.00 ng/ml) and

21–deoxycortisol (2.65 ng/ml; normal range 0.02–0.15 ng/ml). Serum sodium was

normal on several occasions. However, serum potassium was noted to be within the

upper normal range or even slightly elevated (up to 5.4 mmol/L).

At the age of 2 months, the boy was referred to a pediatric endocrinology unit for

further evaluation. Endocrine work up revealed lower but still elevated basal values of

17–hydroxyprogesterone (6.16 ng/ml) and 21–deoxycortisol (0.63 ng/ml). 11–Deoxy-

cortisol was normal (0.12 ng/ml). The elevated serum ACTH (259 pg/ml) was inter-

preted to be caused by stress. Serum sodium was normal, serum potassium was again

slightly elevated (5.52 mmol/L). However, complete sequencing of the CYP21 gene did

not reveal any mutations and thus failed to confirm underlying 21–hydroxylase

deficiency. A chromosomal analysis showed a normal male karyotype (46,XY).

Thereafter, at the age of 5 months, the boy (Fig. 1) was referred to our pediatric

endocrinology unit. A short synacthen test revealed a blunted cortisol response (0 min

6 mg/dL, 60 min 9 mg/dL) while 17–hydroxyprogesterone pathologically increased after

ACTH stimulation (0 min 6.60 ng/ml; 60 min 47.68 ng/ml). Plasma renin and serum

aldosterone were normal. Abdominal ultrasound revealed adrenals of normal size.

Furthermore, ultrasound of brain and heart were normal, too. Further skeletal ab-

normalities could not be detected neither clinically nor radiologically. Bone age was

concordant with chronological age. The parents were advised to give hydrocortisone

replacement in cases of fever and stress and, in the meanwhile, the parents have

reported that while receiving hydrocortisone during febrile illness the boy copes much

better, appearing less weak than he used to do under these conditions.

Figure 1. Left panel, index patient (left) and his healthy twin sister at the age of 11 months. Right

panel, face of index patient. In the patient the facial disproportion with a relatively large

neurocranium, midfacial hypoplasia, epicanthal folds, low set and malformed ears, as well as a

umbilical hernia are notable. Mobility of joints and appearance of external genitalia was normal,

testes were descended.

Prismatic Case of P450 Oxidoreductase Deficiency 959

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At the age of 7 months the boy was presented to a neurosurgeon as the parents

reported a progressive flattening of the occiput since the 2nd month. Examination

revealed brachy-turri-cephaly with a plagiocephalic component and mild exorbitism (no

ocular complications). A CT scan ruled out synostosis of the sutures, no brain mal-

formations were noted. The boy received helmet therapy with a plastic helmet with

foam lining and since then the deformity has improved considerably. Regular fundo-

scopic evaluations never showed signs of papilledema. Head circumference had always

followed the 75th percentile. To exclude Crouzon’s syndrome sequencing analysis of

the fibroblast growth factor receptor 2 gene FGFR2 was performed but did not reveal

mutations. Concerning the patient’s further developmental milestones, a slight re-

tardation has been noted. He started walking freely at the age of 15 months, he can

speak a few words since the age of 18 months.

Urinary Steroid Analysis

Urinary steroids were profiled using GC-MS and selected ion monitoring (SIM)

analysis as previously described (2). In brief, steroids were enzymatically hydrolysed,

extracted by solid phase extraction, and derivatized prior to GC-MS analysis.

The initial urinary steroid profile (Fig. 2A) was obtained at the age of 2 weeks.

Regarding markers of 21–hydroxylase deficiency, 17–hydroxypregnanolone, pregna-

netriol and 15b–OH–pregnanolone were slightly elevated. The metabolite of serum

21–deoxycortisol, 11–ketopregnanetriol, however showed a much more marked

increase. The unusually high excretion of 5–pregnene metabolites, particularly 16–

OH–pregnenolone was noted, but was attributed to prematurity. Excretion of cor-

ticosterone metabolites was not conspicuous. As neonatal cortisol metabolites were

normal, a mild form of 21–hydroxylase deficiency was thought to be still possible and

analysis of a control sample was recommended. Further urinary samples were obtained

in close intervals. Interestingly, they showed up to the age of 5 months (Fig. 2B) a

changing pattern of steroid excretion with rising concentrations of metabolites of

pregnenolone (particularly pregnenediol) and corticosterone (particularly aTHB),

indicative of impaired 17–hydroxylase activity. Markers of 21–hydroxylase deficiency

were slightly increasing, too. Cortisol metabolites remained normal, though in the lower

normal range.

Table 1 summarizes the results of typical urinary steroid metabolite product/

precursor ratios as suggested for the diagnosis of the steroid metabolome characteristic

for P450 oxidoreductase deficiency (3). High values were obtained when the urinary

metabolites of the important adrenal precursor steroids pregnenolone or progesterone

were related to cortisol metabolites. Pregnadienol, which is considered a key analyte in

the diagnosis of this entity, was present in excessive amounts in all urinary specimens.

Abnormally high ratios of pregnanetriol to cortisol metabolites were indicative for 21–

hydroxylase deficiency. Furthermore, the ratios of corticosterone metabolites to cor-

tisol metabolites were consistently elevated, thus revealing impaired 17–hydroxylation.

The ratios for C19–steroids (androsterone + etiocholanolone) to pregnanetriol were

normal and did not reflect impaired 17,20–lyase activity. Regarding our neonatal

specimen, we can confirm an excessively increased ratio between the typical neonatal

steroids 16a–OH–pregnenolone to 16a–OH–DHEA, a ratio indicating attenuated 17–

hydroxylation/17,20–lyase activity. As shown in Table 1, heterozygous individuals

960 Wudy et al.

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only showed slight differences to normals and thus may not be readily identified by

urinary steroid analysis.

Sequencing Analysis of the Coding Region of P450 Oxidoreductase

Specific primer pairs were used to amplify for all 15 exons and adjacent exon/

intron junctions of CPR, the gene encoding P450 oxidoreductase, with subsequent

direct sequencing analysis, as described previously (4). Sequences were compared with

the genomic sequence of human P450 oxidoreductase as reported in the Genbank entry

for human chromosome 7 (accession number NT 007933); mutation numbering refers

to the amino acid position in the protein (GenBank accession number P16435). Genetic

Figure 2. GC-MS urinary steroid profiles (scan runs) of our patient with P450 oxidoreductase

deficiency. 5 ml of spot urine were extracted, a tenth of the extract derivatized and a 1/250 aliqot

of the derivative was subjected to GC-MS analysis, respectively. Internal standards are indicated

by arabical numbers (1: 5a–Androstane–3a,17a–diol; 2: Stigmasterol; 3: 5–Cholestene–3b–ol

N–butyrate). The neonatal urinary steroid profile (Panel A)—obtained at the age of 2 weeks—is

dominated by the excessive excretion of 16a–hydroxypregnenolone (16OH–P5o; retention time

(RT) 28.35 min. The ratio of this metabolite to 16a–hydroxydehydroepiandrosterone (16OH–

DHA; RT 23.49) is disproportionately high. The steroid profile obtained at the age of 6 months

(Panel B) shows elevated metabolites of pregnenolone (P5D, pregnenediol; RT 27.68 min), 17–

hydroxyprogesterone (PT, pregnanetriol, RT 26.99 min; 17–HP, 17–hydroxypregnanolone, RT

24.69 min), and of corticosterone (aTHB, allo– tetrahydrocorticosterone, 32.12 min; THA,

tetrahydro–11–dehydrocorticosterone, 31.29 min). Furthermore, pregnadienol (A), the artifact of

pregnenediol disulfate, was present in all specimens.

Prismatic Case of P450 Oxidoreductase Deficiency 961

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Ta

ble

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(OR

D):

dia

gn

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top

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rm

etab

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Ind

exp

atie

nt

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Ref

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(n=

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ge

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s0

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12

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50

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–0

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OH

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OH

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Pre

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Th

era

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met

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are

dia

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–d

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HA

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dro

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trah

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(a–

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met

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s(F

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17

–h

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sea

ctiv

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hig

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neo

nat

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ids

16a

–h

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rox

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ind

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of

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Pre

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+tr

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ts.

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analysis of the patient’s genomic DNA revealed a homozygous mutation with a single

base pair change (GCT > CCT) in exon 8 of CPR, thereby confirming the diagnosis of

P450 oxidoreductase deficiency (ORD). The clinically unaffected twin sister and both

parents were heterozygous for the same mutation. The mutation can be predicted to

result in a single amino acid exchange (Ala > Pro) in position 284 of the P450

oxidoreductase protein. Previous functional analysis of this mutations employing

bacterial expression and cytochrome c reduction assays had already established the

inactivating nature of this mutation (4).

DISCUSSION

Apparent combined P450c17 and P450c21 deficiency is a fascinating variant of

congenital adrenal hyperplasia first reported in 1985 (5) but only recently its underlying

molecular pathogenesis has been revealed (4,6). This variant of CAH is caused by

inactivating in P450 oxidoreductase, that provides electrons and thus facilitates

enzymatic activity of both P450c17 and P450c21. P450 oxidoreductase also serves as

electron donor for 14a–lanosterol demethylase and squalene epoxidase, two enzymes

involved in sterol biosynthesis, and it has been suggested that impairment of their

activities may be responsible for the bone malformation syndrome characteristically

observed in some but not all patients with P450 oxidoreductase deficiency, also

typically seen in our patient. This malformation pattern resembles a milder variant of

Antley–Bixler syndrome (MIM 207410), a congenital malformation syndrome first

described in 1975 (7) and typically associated with mutations in the FGFR2 gene,

which had not been found in our patient.

Our case highlights that P450 oxidoreductase deficiency needs to be included into

the differential diagnosis of congenital adrenal hyperplasia, in particular in patients

with elevated serum 17–hydroxyprogesterone levels in the neonatal screening but no

evidence of CYP21 mutations. The diagnosis is readily established with GC-MS,

characteristically providing evidence of both impaired 17–hydroxylase and 21–hy-

droxylase activity. The concurrent presence of malformations resembling the Antley–

Bixler phenotype is most suggestive of underlying P450 oxidoreductase deficiency,

however, bone malformations may be completely absent. Similarly, most affected

patients are characterized by ambiguous genitalia (feminization in boys and notably

virilization in girls) but this may not be present as our case illustrates. In most cases

with ambiguous genitalia circulating androgens and urinary androgen metabolites are

characteristically low. Of note, glucocorticoid deficiency may be present and patients

need to be carefully screened and should receive glucocorticoid replacement at least for

periods of increased stress and febrile illness, which may have life-saving

consequences. Unrecognized glucocorticoid deficiency may have contributed to the

previously reported high mortality rate in patients with Antley–Bixler syndrome (8).

ACKNOWLEDGMENTS

The authors are indebted to Dr. Egbert Schulze (University of Heidelberg,

Germany) for performing sequence analysis of the gene for 21–hydroxylase. Steroid

Prismatic Case of P450 Oxidoreductase Deficiency 963

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radioimmunoassays were carried out at the Steroid Laboratory of the Institute of

Pharmacology of the University of Heidelberg, Germany. Genetic analysis of the

FGFR–2 gene was performed at the institute of human genetics (Goethe University,

Frankfurt). WA is an MRC Senior Clinical Fellow.

REFERENCES

1. Ridgway EB, Weiner HL. Pediatr Clin N Am 2004; 51:359–387.2. Wudy SA, Homoki J. Diagnostics of Endocrine Function in Children and

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3. Shackleton C, Marcos J, Malunowicz EM, Szarras-Czapnik M, Jira P, Taylor NF,

Murphy N, Crushell E, Gottschalk M, Hauffa B, Cragun DL, Hopkin RJ, Adachi M,

Arlt W. Am J Med Genet 2004; 128A:223–231.4. Arlt W, Walker EA, Draper N, Ivison HE, Ride JP, Hammer F, Chalder SM,

Borucka-Mankiewicz M, Hauffa BP, Malunowicz EM, Stewart PM, Shackleton

CHL. Lancet 2004; 363:2128–2135.5. Peterson RE, Imperato-McGinley J, Gautier T, Shackleton C. N Engl J Med 1985;

313:1182–1191.6. Fluck SE, Tajima T, Pandey AV, Arlt W, Okuhara K, Verge CF, Jabs EJ, Mendonca

BB, Fujieda K, Miller WL. Nat Genet 2004; 36:228–230.7. Antley R, Bixler D. Birth Defects 1975; 11:397–401.8. Lee H-J, Cho D-Y, Tsai F-J, Shen W-C. Pediatr Neurosurg 2001; 34:33–39.

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