Non-classical 11β-hydroxylase deficiency caused by compound heterozygous mutations: A case study and literature review

Abstract

Background:
11β-hydroxylase deficiency (11OHD) is extremely rare, and reports of non-classical 11OHD are even rarer. Non-classical 11OHD usually presents as premature adrenarche, hyperandrogenism, menstrual disorders, and hypertension. Because the symptoms of non-classical 11OHD are mild, delayed diagnosis or misdiagnosis as polycystic ovary syndrome or primary hypertension is common.

Case presentation:
This paper introduces a case of a young female patient presenting hypertension and menstrual disorders. Laboratory examination revealed increased androgen levels, mild adrenal hyperplasia, mild left ventricular hypertrophy, and mild sclerosis of the lower limb arteries. 11OHD was confirmed by genetic testing, and the patient was found to carry compound heterozygous mutations in CYP11B1 (c.583 T > C and c.1358G > A). The mutation Y195H is located in exon 3 and has not been reported previously. In silico studies indicated that this mutation may cause reduced enzymatic activity. After treatment with hydrocortisone and spironolactone, blood pressure was brought under good control, and menstruation returned to normal. We also conducted a retrospective review of previously reported cases in the literature (over 170 cases since 1991).

Conclusions:
Early diagnosis of non-classical 11OHD is difficult because its symptoms are mild. The possibility of this disease should be considered in patients with early-onset hypertension, menstrual disorders, and hyperandrogenism to provide early treatment and prevent organ damage due to hypertension and hyperandrogenism. CYP11B1 mutations are known to be race-specific and are concentrated in exons 3 and 8, of which mutations in the former are mostly associated with non-classical 11OHD, whereas mutations in the latter are mostly found in classical 11OHD, characterized by severe loss of enzymatic activity.

Full text

C A S E R E P O R T Open Access
Non-classical 11β-hydroxylase deficiency
caused by compound heterozygous
mutations: a case study and literature
review
Dongdong Wang, Jiahui Wang, Tong Tong and Qing Yang
*
Abstract
Background: 11β-hydroxylase deficiency (11OHD) is extremely rare, and reports of non-classical 11OHD are even
rarer. Non-classical 11OHD usually presents as premature adrenarche, hyperandrogenism, menstrual disorders, and
hypertension. Because the symptoms of non-classical 11OHD are mild, delayed diagnosis or misdiagnosis as polycystic
ovary syndrome or primary hypertension is common.
Case presentation: This paper introduces a case of a young female patient presenting hypertension and menstrual
disorders. Laboratory examination revealed increased androgen levels, mild adrenal hyperplasia, mild left ventricular
hypertrophy, and mild sclerosis of the lower limb arteries. 11OHD was confirmed by genetic testing, and the patient
was found to carry compound heterozygous mutations in CYP11B1 (c.583 T > C and c.1358G > A). The mutation Y195H
is located in exon 3 and has not been reported previously. In silico studies indicated that this mutation may cause
reduced enzymatic activity. After treatment with hydrocortisone and spironolactone, blood pressure was brought
under good control, and menstruation returned to normal. We also conducted a retrospective review of previously
reported cases in the literature (over 170 cases since 1991).
Conclusions: Early diagnosis of non-classical 11OHD is difficult because its symptoms are mild. The possibility of this
disease should be considered in patients with early-onset hypertension, menstrual disorders, and hyperandrogenism to
provide early treatment and prevent organ damage due to hypertension and hyperandrogenism. CYP11B1 mutations
are known to be race-specific and are concentrated in exons 3 and 8, of which mutations in the former are mostly
associated with non-classical 11OHD, whereas mutations in the latter are mostly found in classical 11OHD,
characterized by severe loss of enzymatic activity.
Keywords: 11β-hydroxylase deficiency, Genetic testing, Hypertension, Protein function prediction
Background
Congenital adrenal hyperplasia (CAH) is a common genetic
endocrine metabolic disorder, of which 21-hydroxylase
deficiency (21OHD) is the most common type, accounting
for 90–99% of all CAH cases. The second most common
type of CAH is 11β-hydroxylase deficiency (11OHD), which
accounts for 0.2–8% of cases [1]. Steroid 11b-hydroxylase
defects lead to reduced conversion of 11-deoxycortisol
(S) and 11-deoxycorticosterone (DOC) to cortisol and
corticosterone, thereby leading to accumulation of the
two steroid precursors mentioned above. In addition,
an increase in metabolic products towards sex steroids
corresponds to the typical clinical presentation including
low renin hypertension, hypokalemia, hyperandrogenemia,
and genital ambiguity in affected females. Current reports
in the literature relevant to 11βOHD mostly involve
classical 11OHD, and there are relatively few reports on
non-classical 11OHD because of its mild symptoms.
* Correspondence: yangq@sj-hospital.org
Obstetrics and Gynecology Department of Shengjing hospital, China Medical
University, Shenyang 110001, People’s Republic of China
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wang et al. Journal of Ovarian Research (2018) 11:82
https://doi.org/10.1186/s13048-018-0450-8
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Case presentation
The patient was a 23-year-old female (46, XY karyotype)
diagnosed with hypertension (180/120 mmHg) since age
14 and a BMI of 20.8 kg/m
2
. There was no obvious
masculinization, and her parents stated that there were no
obvious abnormalities in vulva development at birth. Anti-
hypertensive drug therapy (nifedipine sustained-release tab-
lets) had been taken continuously, and blood pressure was
controlled to 130–140/80–90 mmHg. The patient sought
treatment at our hospital due to menstrual disorders. The
patient is the only child of non-consanguineous healthy
parents from Northeast China. The study was approved
by the ethics committees of China Medical University,
and informed consent was obtained from the patient and
her parents.
Clinical examination and testing
Imaging examinations included an ultrasonic cardio-
gram, a colour Doppler ultrasound of the carotid artery
and lower limb arteries, a pelvic colour Doppler ultra-
sound (SSA660A, Toshiba), and a contrast-enhanced
adrenal computer tomography scan (16-slice computer
tomography machine, GE Lightspeed). Laboratory tests
included measurements of serum potassium, natrium,
testosterone, free testosterone, androstenedione, dehydro-
epiandrosterone sulphate, adrenocorticotropic hormone,
cortisol, 17-hydroxyprogesterone, renin, and aldosterone
using chemiluminescence immunoassays and biochemical
assays.
Genetic analysis
Peripheral blood samples from the patient and her par-
ents were collected for gene analysis. Direct sequencing
was performed on all the exons and the exon–intron
boundaries of CYP21A2 (NM_000500) and CYP11B1
(NM_000497.3).
In silico analysis
PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2,Protein
ID for CYP11B1 is NP_000488.3 or P15538) and SIFT/
Provean (http://sift.jcvi.org/) were used to predict whether
an amino acid substitution affects protein function.
The alignment in CYP11 families was performed using
CYP11B1 sequences from different species and other
human steroidogenic P450 cytochromes. PolyPhen-2
and DNAMAN software was used for multiple amino
acid sequence alignment. CYP11B2 (PDB entry: 4DVQ.A),
which shares 93.6% sequence identity with CYP11B1,
was selected as the template for model building of
CYP11B1. The structural representations were generated
using PyMOL 2.0.6.
Results
Clinical characteristics and serum hormone levels
The patient’s blood pressure at admission was 140/
100 mmHg. The laboratory examination results are shown
in Table 1. Blood potassium was normal, and androgen
levels were increased. Adrenal CT indicated mild hyper-
plasia of both adrenal glands. Echocardiography indicated
mild hypertrophy and slight enlargement of the left
ventricle. Vascular ultrasound indicated mild sclerosis
of the arteries in the lower limbs. Ultrasound of the
uterine adnexa did not reveal any abnormality.
Genetic analysis
No mutations in CYP21A2 were found in the patient.
Exons 3 and 8 of CYP11B1 harboured a compound hetero-
zygous mutation (c.583 T > C and c.1358G > A) leading to
the conversion of tyrosine at amino acid position 195 to
histidine (Y195H) and arginine at amino acid position 453
to glutamine (R453Q) (Fig. 1a). Each parent of the patient
carried one of these heterozygous mutations.
Bioinformatics and in silico analysis of Y195H
Homology alignments indicate that the Tyr195 residue
in CYP11B1 is highly conserved among different species,
but compared to other human CYP family members, it is
only the same in CYP11B2 (Table 2). In silico analysis by
both PolyPhen-2 and SIFT/Provean predicted a pathogenic
effect of the novel mutation Y195H. The amino acid
residue Y195 is localized in the E helix (Fig. 1b).
Table 1 Summary of laboratory data for the affected subject
with steroid 11β-hydroxylase deficiency
Parameter Result Reference
range
Basal
level
3 months after treatment
with hydrocortisone
ACTH (pg/ml) 48.9 16.59 7.2–63.3
Cortisol (nomol/L) 322 391 171–536
Aldosterone (ng/ml) 0.13 0.12 0.07–0.30
Renin (ng/ml) 0.08 0.44 0.93–6.56
Serum K
+
(mmol/L) 3.64 4.13 3.50–5.30
Serum Na
+
(mmol/L) 141.2 139.2 137.0–147.0
Testosterone (nmol/L) 6.07 0.76 0.69–2.77
Androstendione (nmol/L) > 35 6.8 2.09–10.82
DHEA-S (umol/L) 5.02 3.31 0.95–11.67
Free Testosterone (pmol/L) 42.27 13.24 0.77–33.03
Estradiol (pmol/L) 202 –73.4–587
Progestogen (nmol/L) 4.67 –0.64–3.6
17OHP (nmol/L) 11.6 –0–30
ACTH Adrenocorticotropic Hormone, K+ Potassium, DHEA-S
Dehydroepiandrosterone sulfate, 17OHP 17-hydroxyprogesterone
Wang et al. Journal of Ovarian Research (2018) 11:82 Page 2 of 6
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Intervention and outcome
After diagnosis of non-classical 11OHD was confirmed, the
patient was given hydrocortisone twice a day (hydrocorti-
sone 10 mg in the morning and 5 mg in the afternoon) and
spironolactone (40 mg each day). Her blood pressure
was brought under good control gradually (120/70 mmHg),
and menstruation returned to normal with decreased
androgen levels (Table 1).
Discussion and conclusions
11βOHD is an autosomal recessive genetic disease. Over
100 mutations of CYP11B1 have been reported in the
literature to date. There are more homozygous than
compound heterozygous mutations (69.1% vs. 29.8%),
and there are few single heterozygous mutations (1.1%),
possibly because mutations in other exons have not yet
been discovered [2,3]. In the past, it was believed that
mutations that cause 11βOHD are mostly concentrated
in exons 2, 6, 7, and 8 [2,4,5]. However, our statistics
revealed that most causative mutations are located in
exons 3 and 8 (40%), and that the distribution in the
other exons is actually more average. Patients carrying
mutations in exon 8 account for the highest proportion
among all patients, and patients carrying the R448H mu-
tation are the most numerous (Fig. 2a)[5–12]. Because
the highly conserved amino acid sequence near C450 is
located in exon 8, the normal structure of this region is
essential for maintaining normal enzymatic activity [2].
Thus, most point mutations in exon 8 result in severe
reduction of enzymatic activity, thereby resulting in classical
11βOHD. Although there are many mutations in exon 3,
most cause a partial reduction in enzymatic activity and
thus result in non-classical 11βOHD (Fig. 2c)[5,7,13–23].
Thus, the probability of a mutation appearing in each exon
is similar, but because the mutations reduce enzymatic
activity to different degrees, different severities of clinical
presentation are observed, and there is a high probability of
misidentifying the disease for mutations in certain exons.
The R448H mutation and 8 other mutations are the most
frequently reported and account for approximately 40% of
all cases (Fig. 2b). CYP11B1 mutation shows significant race
specificity. For example, the R448H mutation mentioned
above is common among Moroccan Jews [6]. Tunisians
often carry the two mutations, G379 V and Q356X [24], of
which Q356X is also common among sub-Saharan Africans
and African-Americans [2,3,24–26]. The T318 M muta-
tion is most common among Yemenis [2,27,28], and some
Fig. 1 CYP11B1 sequencing results and 3D molecular schematic representation of the mutation site. aCompound heterozygous mutation
(c.583 T > C and c.1358G > A) that leads to the conversion of tyrosine at amino acid position 195 to histidine (Y195H) and arginine at amino acid
position 453 to glutamine (R453Q). bThree-dimensional model structure of CYP11B1. Green, E helix; red, L helix. The side chains of amino acid
residues Y195 (on the E helix) and R453 (on the L helix) are depicted
Table 2 Homology alignments between CYP family members
Protein ID (UniProtKB/Swiss-Prot) Sequence framing Tyr195
P15538 (HUMAN CYP11B1) TLDVQPSIFH YTIEASNLAL
F7GMV0 (Macaca mulatta CYP11B1) TLDVQPSIFH YTIEASNLAL
F6XJ24 (Equus caballus (Horse) CYP11B1) TLDARPSIFH YTIEASNLAL
P51663 (Ovis aries (Sheep) CYP11B1) TLDIAPSVFR YTIEASTLVL
Q29552 (Sus scrofa (Pig) CYP11B1) TLDIKPSIFR YTIEASNLVL
P15150 (Bos taurus (Bovine) CYP11B1) TLDIAPSVFR YTIEASNLVL
Q3TG86 (Mus musculus (Mouse) CYP11B1) SMDFQSSVFN YTIEASHFVL
P15393 (Rattus norvegicus (Rat) CYP11B1) SINIQSNMFN YTMEASHFVI
P19099 (HUMAN CYP11B2) TLDVQPSIFH YTIEASNLAL
P05108 (HUMAN CYP11A1) SGDISDDLFR FAFESITNVI
P05093 (HUMAN CYP17A1) IDNLSKDSLV DLVPWLKIFP
P08686 (HUMAN CYP21A2) SLLTCSIICY LTFGDKIKDD
P11511 (HUMAN CYP19A1) AESLKTHLDR LEEVTNESGY
Wang et al. Journal of Ovarian Research (2018) 11:82 Page 3 of 6
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new mutations such as c.53_54insT, G206 V, W260X,
R448P, and H465L are often found in Saudi Arabs [29–31].
The R454C mutation has only been reported among the
Chinese [8,10,32].
The case reported in this paper is a patient carrying
the compound heterozygous mutations Y195H and
R453Q located in exons 3 and 8, respectively, which are
mutation hotspots. Of these mutations, R453Q has been
reported in one individual of European descent and 3
Chinese individuals [8,23,33], and is likely to be more
common among the Chinese [33]. The residue R453 is
located in the L-helix and is adjacent to the Cys-pocket
motif. This domain is highly conserved in the P450 family
of enzymes, and causes 11-hydroxylase activity to decrease
to approximately 1% of the wild-type activity [23]. The
Y195H mutation has not been reported previously. Y195
is located in the E helix. Homology alignments indicate
that this amino acid sequence is relatively conserved, and
predictions using PolyPhen-2 and SIFT/Provean indicate
that this mutation may impair protein function. A mutation
in the neighbouring amino acid T196 has been reported
previously (T196A). This mutation can lead to a 30–50%
loss in enzymatic activity and can result in non-classical
11OHD [15]. Three-dimensional structural models show
that Y195 and T196 are located in the middle segment of
the E helix and are close to L463-L464 in the L helix, which
is involved in heme binding [34]. This suggests that Y195H
and T196A are similar and may affect enzymatic activity by
indirectly affecting the structure of the L helix, but to a
small degree; it is thus a mutation that causes non-classical
11OHD.
Because the clinical presentation of non-classical
11OHD is atypical and highly variable, its prevalence
may be underestimated and it may be misdiagnosed.
Some patients may be misdiagnosed as having polycystic
ovary syndrome because of the mildly elevated androgen
levels [13,16], and there are patients that only present
with hypertension with mineralocorticoid features [15].
After the ACTH stimulation test, the degree of increase
in 17-hydroxyprogesterone can be used to differentiate
Fig. 2 Characteristics of previously reported CYP11B1 mutations in the literature. aDistribution of CYP11B1 mutations. bThe 8 most common
types of mutations. cEffect of known mutation sites on 11OH enzymatic activity
Wang et al. Journal of Ovarian Research (2018) 11:82 Page 4 of 6
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from non-classical 21OHD, but there is still no consensus
on the diagnostic standards for non-classical 11OHD
[4,17,21]. Baseline and stimulated 11-deoxycortisol
measurements, 11β-hydroxylase activity assays, and urinary
steroid profiling using LC-MS/MS are recommended to
avoid missing the diagnosis of non-classical 11OHD
[16,35]. However, 11-deoxycortisol measurement and
ACTH medications are currently unavailable in most
Chinese hospitals, which hinders the diagnosis of 11OHD,
especially non-classical 11OHD. As the cost of genetic diag-
nosis decreases, genetic testing of CYP11B1 in suspected
patients may make the diagnosis of non-classical 11OHD
convenient and accurate.
Non-classical 11OHD patients may develop hypertension
[14,16], but this is highly variable, and its incidence and
extent are still not clear [35]. The patient in our case devel-
oped severe hypertension at an early age, but because her
diagnosis was never confirmed, antihypertensive therapy
was irregular and she developed left ventricular myocardial
hypertrophy and arteriosclerosis-like changes in the lower
limbs, indicating the importance of early diagnosis and
antihypertensive therapy. The use of mineralocorticoid
receptor antagonists such as spironolactone or eplerenone
for antihypertensive therapy is recommended in such
patients [14]. The patient in this case was given spirono-
lactone therapy, which controlled the blood pressure to
normal.
In summary, the present study reports the case of a
Chinese patient with non-classical 11OHD and presenting
with early-onset hypertension, and carrying compound
heterozygous mutations in CYP11B1, of which one muta-
tion is reported for the first time. We also analysed and
summarised over 170 cases that were previously reported
in the literature and found that exons 3 and 8 were
mutation hotspots. Mutations in exon 3 often result in
non-classical 11OHD, whereas mutations in exon 8 more
often result in complete loss of enzymatic activity and
classical 11OHD. The possibility of non-classical 11OHD
should be considered in hypertensive patients with hyper-
androgenism or elevated mineralocorticoids. Such patients
should be carefully identified and given an early diagnosis
and treatment to avoid the adverse outcomes caused by
hyperandrogenism or long-term hypertension.
Availability of data and materials
The datasets obtained and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Authors’contributions
DDW and JHW performed the molecular genetic studies; DDW and TT
participated in the sequence alignment and drafted the manuscript. DDW
participated in the sequence alignment. QY participated in the design of the
study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The Hospital Ethics Committee of the Shengjing hospital of China Medical
University approved the study.
Consent for publication
The patient and her family provided written informed consent for
publication of their data.
Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Received: 12 May 2018 Accepted: 26 August 2018
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