Keywords 21-hydroxylase deficiency - hyponatremia and hyperkalemia - hyperpigmentation - molecular
genetic diagnosis - infant - timely therapeutic intervention - follow-up
Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders,
with approximately 90 to 95%[1 ] of cases attributable to 21-hydroxylase deficiency (21-OHD) caused by CYP21A2 mutations. 21-OHD is characterized by cortisol deficiency with or without aldosterone
deficiency and androgen excess. Based on the severity of 21-OHD, CAH is classified
into classic and non-classic forms, with a strong emphasis on genotype–phenotype correlations.[2 ] The classic form is further subdivided into the salt-wasting (SW) and simple virilizing
(SV) forms. In the SV form, the primary clinical manifestations include virilization
of the external genitalia in female newborns, as exemplified by a reported case of
a 17-year-old girl presenting with virilized external genitalia and primary amenorrhea,[3 ] and precocious puberty in boys attributable to androgen overproduction. By contrast,
untreated SW caused by aldosterone deficiency can lead to life-threatening complications
such as hypovolemia, shock, hyponatremia, hyperkalemia, dehydration, and adrenal crisis.[1 ]
[4 ]
21-OHD is a genetic disorder characterized by the impaired biosynthesis of cortisol
and aldosterone, accompanied by the accumulation of 17-hydroxyprogesterone (17-OHP)
and adrenal androgens.[5 ] Most classic CAH cases are identified during infancy through the detection of 17-OHP
levels in newborn screening programs. In China, the implementation of nationwide newborn
screening for CAH commenced in the 1990s, and universal coverage has been achieved
across almost all counties. A comprehensive meta-analysis of newborn CAH screening
data in China revealed an overall incidence rate of 0.43 per 10,000 live births (95%
confidence interval = 0.39–0.48).[6 ] Notably, significant regional variations in disease prevalence were observed, with
Zhejiang, Guangdong, Hubei, and Shaanxi provinces demonstrating lower incidence rates
than the national average, whereas other regions exhibited higher prevalence rates
than the national estimate.[6 ]
Meanwhile, 21-hydroxylase is encoded by CYP21A2 , located on the short arm of chromosome 6. CYP21A1P , a homologous pseudogene, is situated approximately 30 kb upstream of the functional
gene, and it shares >95% sequence homology with CYP21A2 while maintaining 65 nucleotide differences.[7 ] The close proximity and high sequence similarity between CYP21A2 and CYP21A1P make these genes prone to frequent recombination events, resulting in the transfer
of pseudogene-derived mutations to the functional gene. These recurrent mutations
include large deletions/gene conversions, as well as specific point mutations, such
as p. Pro31Leu, c.293-13A/C>G, p. Gly111ValfsTer21, p. Ile173Asn, E6 cluster mutations,
p. Val282Leu, p. Leu308PhefsTer6, p. Gln319Ter, and p. Arg357Trp.[2 ]
[8 ] More than 200 pathogenic or likely pathogenic variants in CYP21A2 have been identified.[9 ] In this study, we presented a case with longitudinal follow-up from birth to 12
months of age in which comprehensive genetic analysis through whole-exome sequencing
(WES), Sanger sequencing, and multiplex ligation-dependent probe amplification (MLPA)
revealed compound heterozygosity in CYP21A2 . The patient inherited a large deletion spanning exons 1–7 from the maternal allele
and compound heterozygous mutations from the paternal allele. Although the patient
was diagnosed with 21-OHD, his phenotypes were controlled effectively. In this report,
we described the genetic profile associated with the clinical manifestations of hyperpigmentation
and elevated adrenocorticotropic hormone (ACTH) levels and 17-OHP concentrations,
as well as the long-term clinical outcomes.
Case Presentation
The index case was a male infant, the first child of non-consanguineous parents, delivered
via cesarean section at 393/7 weeks of gestation with a birth weight of 3,700 g. The pregnancy was uneventful.
At 7 hours after birth, the newborn was admitted to another hospital presenting with
“shortness of breath for 18 minutes.” Physical examination revealed no significant
abnormalities excluding generalized hyperpigmentation, notably in the scrotum and
penis. The infant exhibited normal male external genitalia, and he did not experience
any episodes of hypoglycemia during the neonatal period. Initial blood electrolyte
analysis indicated elevated serum potassium and reduced sodium levels, but no specific
values were documented. His serum cortisol level was 17.33 μg/dL. Based on these clinical
findings, a provisional diagnosis of CAH was made. However, the family history was
negative for CAH. During hospitalization, the infant developed abdominal distension,
which was managed with a 2-day regimen of fasting, omeprazole for acid suppression,
enema, and intravenous nutrition. Following this treatment, normal feeding was successfully
resumed. Abdominal ultrasonography revealed distension, with evidence of multiple
and slightly dilated intestinal loops, and a left colonic diameter of approximately
2.1 cm.
At 11 days old, he was admitted to our hospital for CAH. At the first evaluation in
our hospital, the infant exhibited normotensive anterior fontanel and hyperpigmentation
of the skin, specifically the armpit, perineum, and scrotum, with normal testis feel
in the scrotum. The laboratory examination results are presented in [Table 1 ]. The patient's serum electrolytes results included a high potassium level (7.06 mmol/L
[normal range, 4.2–5.9 mmol/L]), low sodium (125.10 mmol/L [135–150 mmol/L]), low
calcium (1.09 mmol/L [2.1–2.8 mmol/L]) and chlorine levels (96.60 mmol/L [100–116 mmol/L]).
The patient also exhibited extremely high level of ACTH (611.00 pg/mL [0–46 pg/mL]),
testosterone (TSTO; 713.37 ng/dL [0–30 ng/dL]), androstenedione (AND; >10.00 ng/mL
[0.6–3.1 ng/mL]), and 17-OHP (319.91 nmol/L [0–30 nmol/L]), whereas his dehydroepiandrosterone
(DHEAS) level was normal (291.00 μg/dL [80–560 μg/dL]). Notably, the patient's serum
17-OHP level was consistent with the newborn screening value (312.13 nmol/L), further
supporting the diagnosis of CAH.
Table 1
Laboratory examination results of the case by follow-up for 12 months
Age
Cut-off value
At admission
At discharge
Follow-up
11 days
23 days
1 month
2 months
4 months
6 months
9 months
12 months
AND (ng/mL)
0.6–3.1
>10.00
>10.00
1.90
<0.30
<0.30
<0.30
<0.30
<0.30
ACTH (pg/mL)
0–46
611.00
121.00
175.00
–
57.60
9.58
376.00
–
TSTO (ng/dL)
<7.00
713.37
94.24
–
–
–
–
<7.00
15.63
17-OHP (nmol/L)
0–30
319.91
158.51
8.30
5.48
–
7.10
133.71
–
DHEAS (μg/dL)
80–560
291.00
60.90
19.00
<15.00
<15.00
<15.00
<15.00
<15.00
Potassium (mmol/L)
4.2–5.9
7.06
4.73
5.47
4.66
3.40
3.48
5.47
4.84
Sodium (mmol/L)
135–150
125.10
136.40
138.20
139.60
140.70
140.00 (134–143)
133.80 (134–143)
134.40 (134–143)
Chlorine (mmol/L)
100–116
96.60
104.20
101.20
105.10
100.80
103.90 (98–110)
98.70 (98–110)
99.50 (98–110)
Calcium (mmol/L)
2.1–2.8
1.09
2.63
2.59
2.58
2.73
2.54
2.75
2.47
Cortisol (8 a.m.) (μg/dL)
5.27–22.45
–
–
–
–
–
–
1.59
2.82
Abbreviations: 17-OHP, 17-hydroxyprogesterone; ACTH, adrenocorticotropic hormone;
AND, androstenedione; DHEAS, dehydroepiandrosterone sulfate; TSTO, testosterone.
“–” means no data available. The reference range was changed for sodium (134–143 mmol/L)
and chlorine (98–110 mmol/L) at the ages of 6, 9, and 12 months.
Note: Bold numerical values indicate values outside the reference range.
Based on the laboratory findings, a comprehensive management plan was implemented
upon admission, including the maintenance of body warmth, continuous ECG monitoring,
meticulous skin and oral care, and appropriate feeding regimens. For hyperkalemia,
intravenous calcium, insulin–glucose solution, and sodium bicarbonate were administered
to reduce serum potassium levels and prevent myocardial toxicity. Hormonal therapy
with intravenous hydrocortisone (oral hydrocortisone after 3 days) and oral fludrocortisone
was initiated 2 days after admission. In addition, hyponatremia was addressed with
oral 10% sodium chloride supplementation (15 mL in milk). Color Doppler echocardiography
revealed a left-to-right shunt caused by a patent foramen ovale with normal left ventricular
function. Cranial ultrasound revealed bilateral subependymal cysts (right: 1.5 cm × 0.5 cm;
left: 0.8 cm × 0.5 cm), which resolved by 4 months of age. Abdominal ultrasound confirmed
bilateral adrenal gland enlargement with morphological abnormalities, supporting the
diagnosis of CAH.
Furthermore, molecular genetic diagnosis was performed using WES of the peripheral
blood leukocytes from this infant. Proband-only WES identified the homozygous typical
CYP21A2 variant c.293-13A/C>G (in intron 2; [Table 2 ]), which was classified as a pathogenic variant using standard PM3_VeryStrong + PS3 + PP4
according to the American College of Medical Genetics and Genomics (ACMG) guidelines[10 ] and confirmed by Sanger sequencing ([Fig. 1A ]). Parental genotyping revealed that the father was a heterozygous carrier of the
c.293-13A/C>G variant, whereas the mother was wild-type ([Supplementary Data ]). Notably, Sanger sequencing of the father revealed double peaks from 51-bp downstream
of the c.293-13A/C>G locus ([Supplementary Data ] [available in the online version only]), suggesting an additional variant. Based
on the polymorphism of CYP21A2 , specific MLPA (P050, MRC-Holland, Amsterdam, the Netherlands) for CYP21A2 was conducted for both the proband and his mother. MLPA identified a large deletion
spanning exons 1–7 of CYP21A2 in the mother, confirming a single-copy loss in this region ([Fig. 1B ]). In addition, the proband exhibited a heterozygous deletion of exons 1–7 in CYP21A2 (pathogenic: PVS1 + PM3_Strong +PM2), consistent with inheritance from the mother
([Fig. 1C ] and [Table 2 ]). Additionally, a second pathogenic variant (PVS1 + PM3_VeryStrong + PM2), namely
c.332_339 deletion (p. Gly111Valfs*21, in exon 3), was identified in the proband,
corresponding to the father's double peaks and indicating a compound heterozygous
state attributable to exons 1–7 deletion ([Table 2 ]). The results of Sanger sequencing for both the proband and parent are presented
in [Supplementary Data ]. The paternal origin of the c.332_339 deletion variant was further validated by
genotyping the paternal grandparents ([Fig. 1D ]). The result illustrated that the c.332_339 deletion was inherited from grandparent
I1, whereas grandparent I2 was wild-type. The family pedigree is summarized in [Fig. 1E ]. Both the c.293-13A/C>G and c.332_339 deletion loci were inherited in cis. The Sanger
sequencing results for the two variants in the grandparents are presented in [Supplementary Data ].
Fig. 1 The genetic analysis of CYP21A2 in the family. (A ) Sanger sequencing validation of the c.293-13A/C>G variant in the proband. (B, C ) MLPA analysis revealed a heterozygous deletion spanning exons 1–7 of CYP21A 2 in both the proband (C ) and the mother (B ). (D ) Sanger sequencing confirmation of the c.332_339 deletion in paternal grandparent
I1. (E ) Pedigree analysis of CYP21A2 mutations in the family. Paternal grandparent I1 carried both c.293-13A/C>G and c.332_339
deletion in cis, while I2 was normal. Individual II2 carried the exons 1–7 deletion,
and II1 was compound heterozygous for c.293-13A/C>G and c.332_339 deletion (in cis).
The proband inherited the exons 1–7 deletion from the mother (II2) and the compound
heterozygous c.293-13A/C>G and c.332_339 deletion from the father (II1). MLPA, multiplex
ligation-dependent probe amplification.
Table 2
Genetic analysis results of CYP21A2 variants identified by whole-exome sequencing and multiplex ligation-dependent probe
amplification of the proband
Gene symbol
Location (GRCH38)
Mutation
gnomAD MAF
ACMG variant grade
Disease
Inheritance pattern
Zygote type
CYP21A2
Chr6: 32039081
NM_000500.9: exon3: c.293-13A/C>G
0.00226067
Pathogenic: PM3_VeryStrong +PS3 + PP4
Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency/hyperandrogenism,
non-classic type, due to 21-hydroxylase deficiency
[MIM: 201910]
AR
Homozygous (parental)
Chr6: 32039133-32039140
NM_000500.9: exon3: c.332_339 deletion (p. Gly111Valfs*21)
–
Pathogenic: PVS1 + PM3_VeryStrong + PM2
Homozygous (parental)
Chr6
NM_000500.9: exon (1–7) deletion
–
Pathogenic: PVS1 + PM3_Strong + PP4 + PM2
Heterozygous (maternal)
Abbreviations: ACMG, American College of Medical Genetics and Genomics; LOF, loss-of-function.
PM3_VeryStrong: For recessive disorders, detected in trans with a pathogenic variant,
and has been reported in the literature that this variant was detected in several
patients with congenital adrenal hyperplasia (CAH) along with other variants, and
the homozygous variant has been detected in multiple patients with CAH. PS3: Well-established
in vitro or in vivo functional studies supportive of a damaging effect on the gene
or gene product. PP4: Patient's phenotype or family history is highly specific for
a disease with a single genetic etiology. PM2: 2 Absent from controls (or at extremely
low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or
Exome Aggregation Consortium. PVS1: Null variant (nonsense, frameshift, canonical
±1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where
LOF is a known mechanism of disease.
At discharge, the infant's serum electrolyte levels (potassium, sodium, calcium, and
chloride) had normalized, and his serum ACTH, TSTO, and 17-OHP levels were significantly
lower than those at admission. He was discharged on a regimen of oral hydrocortisone
(1 mg three times daily), fludrocortisone (0.05 mg twice daily), and 0.9% sodium chloride
(15 mL three times daily), with adjustments based on the results of follow-up evaluations.
In addition, monthly follow-up visits for the first 3 months and quarterly thereafter.
At 3 months of age, the patient's hyperpigmentation was limited to the scrotal region
only. His laboratory findings during the 12-month follow-up are summarized in [Table 1 ]. The patient's serum electrolytes remained stable from 1 to 12 months of age. Although
his potassium level slightly fluctuated at 4 and 6 months of age, it remained within
the normal range at the next two assessments (at 9 and 12 months old). The indicators
of AND and DHEAS levels were consistently below the lower cutoff from 2 to 12 months
of age. The patient's ACTH levels remained elevated except at 6 months, whereas his
17-OHP levels were within the normal range at all time points, excluding at 9 months.
TSTO and morning cortisol levels (8 a.m.) were measured only at 9 and 12 months. His
TSTO level was within normal limits at 9 months, whereas his cortisol remained below
the lower detection limit. Then, he continued to follow-up at our Pediatric Health
Care Center, demonstrating normal physical development, including head circumference,
height, and weight. Ongoing monitoring of his physical and laboratory parameters has
been recommended to optimize the management of CAH.
Discussion
Our case illustrates the classic presentation of CAH, which was characterized by hyponatremia,
hyperkalemia, elevated 17-OHP and ACTH levels, and hyperpigmentation, as well as a
risk of adrenal crisis. Molecular genetic diagnoses confirmed compound heterozygous
mutations in CYP21A2 , comprising a c.293-13A/C>G variant and a c.332_339 deletion (p. Gly111Valfs*21)
variant inherited in cis from the father, along with a large deletion spanning exons
1–7 inherited from the mother. Both parents were heterozygous carriers, eventually
resulting in loss of 21-hydroxylase and CAH development in the proband. Molecular
genetic techniques, including WES, MLAP, and Sanger sequencing, were employed to diagnose
CAH in this male infant with normal male external genitalia, consistent with the genetic
findings. According to the genotype − phenotype correlations, all three variants were
classified as pathogenic and associated with the severe SW form.[11 ]
[12 ] However, the patient did not develop severe hyponatremia or dehydration because
treatments were initiated at an early age. Over the 1-year follow-up period, the patient
received daily treatment with hydrocortisone and fludrocortisone, which reduced high
androgen levels and replaced adrenal functions. Through this early intervention and
treatment, normal plasma volume and physiological balance were maintained to prevent
adrenal crisis attributable to adrenal insufficiency. In addition, he underwent continuous
monitoring through regular physical examinations, endocrine function assessments,
and blood electrolyte measurements, which ensured effective disease management. The
long-term dosage of hydrocortisone and fludrocortisone can also be adjusted according
to the results of regular check-ups. Therefore, a newborn screening program can detect
17-OHP levels within 72 hours after birth in newborns, which can improve the diagnosis
rate and permit immediate treatment for classic CAH.
Although newborn screening for 17-OHP is an effective strategy for the early detection
of classic CAH, its utility is limited by high rates of false positives and negatives.
These inaccuracies arise from various influencing factors, including gender, birth
weight, postnatal age, and methodological differences.[13 ] In China, 17-OHP detection primarily relies on enzyme-linked immunosorbent assay
or dissociation-enhanced lanthanide fluorescence immunoassay, both of which are associated
with high false-positive rates and low positive predictive values.[6 ] By contrast, secondary screening using liquid chromatography-tandem mass spectrometry
(LC-MS/MS) has demonstrated significantly improved sensitivity and specificity for
CAH diagnosis. In prior research, LC-MS/MS in the secondary screening of raw dried
blood spots significantly increased the positive predictive value of CAH screening
to 25 − 70%,[14 ]
[15 ] indicating the potential of LC-MS/MS to enhance the accuracy and reliability of
CAH screening programs.
As previously described, the high recombination rate (gene conversion) between CYP21A2 and its pseudogene (CYP21A1P ) contributes to the high frequency of mutations in CYP21A2 . Variants of c.293-13A/C>G (intron 2: g.655C/A>G) and c.332_339 deletion (exon 3:
g.707_714, p.Gly111Valfs*21) are derived from CYP21A2 and its pseudogene. The c.293-13A/C>G variant, associated with abnormal splicing
attributable to the activation of an upstream cryptic splice acceptor site, results
in fewer than 5% of residual 21-hydroxylase enzyme activity.[8 ] Meanwhile, this variant is the most common mutation in China,[16 ]
[17 ]
[18 ] as well as in populations from South America, North America, and Europe.[16 ] In addition, the c.293-13A/C>G variant is the most frequent mutation in cohorts
in southern China, specifically compared with different regions in China, including
Southeast Asia.[18 ]
[19 ]
[20 ] Conversely, the c.332_339 deletion, an 8-bp frameshift deletion in exon 3, leads
to a premature stop codon and complete loss of 21-hydroxylase activity,[5 ] but it occurs less frequently than c.293-13A/C>G.[18 ] Both variants exhibited strong genotype–phenotype correlations, and they were associated
with classic CAH and female genital ambiguity.[2 ]
[8 ]
[16 ]
[21 ] Additionally, large deletions spanning exons 1–7 of CYP21A2 have been reported as high frequency mutation in Chinese populations, including a
small cohort of 37 patients with CAH[17 ] and a larger cohort of 166 patients with classical 21-OHD from southern China.[18 ] These findings underscore the genetic heterogeneity and regional variability of
CYP21A2 mutations in classical CAH.
In this case, we initially utilized WES for genetic diagnosis, identifying a homozygous
CYP21A2 variant (c.293-13A/C>G) in the proband. However, the scope of WES in detecting CYP21A2 polymorphisms is limited. First, WES only detects the exonic region and ±20-bp intronic
region, whereas the variants in introns and large deletions across exons (such as
exon 1–7 deletion in our case) cannot be effectively detected. Meanwhile, it is difficult
to identify the real mutations existing in CYP21A2 or CYP21A1P , highlighting the risk of missing detections. Considering these limitations, we further
employed MLPA and Sanger sequencing to confirm the diagnosis. Through this approach,
two additional deletions (c.332_339 deletion and exon 1–7 deletion) were identified
in the proband. Sanger sequencing results are accurate, reliable, and highly sensitive.
However, it is difficult to detect gene copy number variation, short read length,
and small throughput using this technique, and it is not suitable for screening structural
variations, such as deletions or duplications.[22 ] However, long-read sequencing (LRS, also known as single-molecule real-time sequencing)
has emerged as a powerful tool for the genetic diagnosis of rare diseases, including
CAH and spinal muscular atrophy. LRS offers the unique advantage of generating reads
exceeding 200 kb in length without the need for PCR amplification, thereby reducing
the rate of mismatches during amplification.[23 ]
[24 ] A distinctive feature of LRS is its capability to determine the allelic configuration
(in trans or cis) without a parental comparison, thereby enhancing the diagnostic
precision and efficiency. CYP21A2 comprises 10 exons. However, MLPA is limited to probes targeting exons 1–7, and interference
by the pseudogene can affect the accuracy of the probes, which makes it difficult
to find mutations across exons 8–10 and decreases the reliability of the results.
Moreover, MLPA cannot detect copy number neutral inversions or translocations. By
contrast, LRS offers significant advantages in detecting mutant events within exons
8–10, and greater utility in detecting large segmental variations such as structural
deletions, duplications, inversions and translocations, and microtransformations.
Although LRS has incomparable advantages in detecting genetic structural variations,
its high economic cost limits its wide application in clinical practices.
Prenatal diagnosis plays a pivotal role in the early identification and management
of CAH, particularly in families with a known history of the disorder. In this case,
the identification of compound heterozygous mutations in CYP21A2 in the proband highlighted the potential utility of prenatal genetic testing in at-risk
families, although the parent denied a family history of CAH. Prenatal diagnosis of
CAH can be achieved through chorionic villus sampling or amniocentesis, followed by
molecular genetic analysis of CYP21A2 .[25 ]
[26 ] This approach permits the early detection of affected fetuses, thereby enabling
timely counseling and intervention. For families with a history of CAH, prenatal diagnosis
provides critical information for reproductive decision-making and pregnancy management.
In cases where an affected fetus is identified, prenatal treatment with dexamethasone
can be considered to mitigate the virilization of external genitalia in female fetuses,
a hallmark of classical CAH.[27 ] Furthermore, prenatal diagnosis facilitates the preparation of health care teams
for the immediate postnatal management of affected newborns, including the prompt
initiation of glucocorticoid and mineralocorticoid replacement therapy to prevent
life-threatening adrenal crises.[9 ]
Early initiation of treatment, including glucocorticoid and mineralocorticoid replacement
therapy, prevented the development of severe hyponatremia, dehydration, and adrenal
crisis in the patient. Prior research identified four patients as 21-OHD via prenatal
genetic diagnosis in four patients born at term. Three patients were boys, none of
whom developed SW phenomenon or dehydration because they were treated with hydrocortisone,
9α-fludrocortisone, and sodium chloride starting at a mean of 3.7 days after birth
(range, 2–7 days).[9 ] Meanwhile, the female infant patient exhibited ambiguous genitalia because of increased
exposure to adrenal androgens in utero. In our case, the patient was diagnosed with
21-OHD within 11 days, and subsequent treatment effectively prevented the development
of severe phenotypes. Hormone replacement therapy is the primary treatment for CAH.
However, as this is a long-term therapy, patients require regular monitoring of electrolyte
levels, hormone levels, and growth/development parameters to prevent serious adverse
effects.[28 ] Our case underscored the importance of timely diagnosis and intervention to rescue
phenotypes and improve clinical outcomes. The goal of treatment is to correct adrenal
insufficiency crisis and inhibit androgen synthesis, ensuring that growing individuals
have as normal linear growth and puberty as possible. For patients who have developed,
normal reproductive function needs to be maintained to the greatest extent possible.[29 ]
Conclusion
In conclusion, we presented a case with detailed clinical manifestations, follow-up
through 12 months, and genetic diagnosis of classic CAH (SW form) attributable to
21-OHD and revealed the importance of early genetic diagnosis combined with timely
therapeutic intervention and long-term monitoring. Through these approaches, the patient's
phenotypes were effectively controlled and rescued. We further emphasized the incorporation
of comprehensive genetic diagnostics (including WES, MLPA, Sanger sequencing, and
LRS) in prenatal or postnatal diagnosis and provided classic educational value for
CAH in infancy care.
