Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency through molecular genetic analysis of the <i>CYP21A2</i> gene

Ji-Hee Yoon; Soojin Hwang; Ja Hye Kim; Gu-Hwan Kim; Han-Wook Yoo; Jin-Ho Choi

doi:10.6065/apem.2346014.007

Ann Pediatr Endocrinol Metab > Volume 29(1); 2024 > Article

Yoon, Hwang, Kim, Kim, Yoo, and Choi: Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency through molecular genetic analysis of the CYP21A2 gene

Original Article

Annals of Pediatric Endocrinology & Metabolism 2024;29(1): 54-59.

Published online: February 29, 2024

DOI: https://doi.org/10.6065/apem.2346014.007

Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency through molecular genetic analysis of the CYP21A2 gene

Ji-Hee Yoon¹

, Soojin Hwang¹

, Ja Hye Kim¹

, Gu-Hwan Kim²

, Han-Wook Yoo³

, Jin-Ho Choi¹

¹Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

²Medical Genetics Center, Asan Medical Center, Seoul, Korea

³Department of Pediatrics, CHA Bundang Medical Center, CHA University, Seongnam, Korea

Address for correspondence: Jin-Ho Choi Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-Gil, Songpa-Gu, Seoul 05505, Korea Email: jhc@amc.seoul.kr

Received January 11, 2023 Revised February 26, 2023 Accepted March 8, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

Deficiency of 21-hydroxylase (21-OHD) is an autosomal recessively inherited disorder that is characterized by adrenal insufficiency and androgen excess. This study was performed to investigate the clinical utility of prenatal diagnosis of 21-OHD using molecular genetic testing in families at risk.

Methods

This study included 27 pregnant women who had previously borne a child with 21-OHD. Fetal tissues were obtained using chorionic villus sampling (CVS) or amniocentesis. After the genomic DNA was isolated, Sanger sequencing of CYP21A2 and multiplex ligation-dependent probe amplification were performed. The clinical and endocrinological findings were reviewed retrospectively.

Results

A total of 39 prenatal genetic tests was performed on 27 pregnant women and their fetal tissues. The mean gestational age at the time of testing was 11.7 weeks for CVS and 17.5 weeks for amniocentesis. Eleven fetuses (28.2%) were diagnosed with 21-OHD. Among them, 10 fetuses (90.9%) harbored the same mutation as siblings who were previously diagnosed with 21-OHD. Among these, 4 fetuses (3 males and 1 female) identified as affected were born alive. All 4 patients have been treated with hydrocortisone, 9α-fludrocortisone, and sodium chloride since a mean of 3.7 days of life. The male patients did not show hyponatremia and dehydration, although they harbored pathogenic variants associated with the salt-wasting type of 21-OHD.

Conclusions

This study demonstrated the diagnostic efficacy and clinical consequences of diagnosis by prenatal genetic testing in families at risk for 21-OHD. All patients identified as affected were treated with hydrocortisone and 9α-fludrocortisone early after birth, which can prevent a life-threatening adrenal crisis.

Keywords: 21-hydroxylase deficiency, Amniocentesis, Chorionic villus sampling, CYP21A2, Prenatal diagnosis

Highlights

· This study demonstrated the clinical outcomes of fetal diagnosis of 21-hydroxylase deficiency (21-OHD) by prenatal genetic testing in families at risk.

· Prenatal genetic diagnosis of 21-OHD can be helpful for early diagnosis and prevention of the life-threatening adrenal crisis in patients at risk.

Introduction

The 21-hydroxylase deficiency (21-OHD) is the most frequent form of congenital adrenal hyperplasia (CAH), which is characterized by adrenal insufficiency and androgen excess [1]. There are 3 types of 21-OHD depending on the residual enzymatic activity: salt-wasting, simple virilizing, and nonclassical [2]. In the salt-wasting type, complete and nearcomplete deficiency of 21-hydroxylase impairs the production of glucocorticoids and mineralocorticoids, resulting in hyponatremia, hyperkalemia, dehydration, and overproduction of adrenal androgens. The virilizing type is characterized by accelerated growth and advanced bone age in the absence of mineralocorticoid deficiency. The nonclassical type is the mildest form of 21-OHD and only causes androgen excess [3].

The 21-OHD is caused by mutations in the CYP21A2 gene, which is located on the short arm of chromosome 6p213 [4]. Most mutations (~95%) arise from 2 types of intergenic recombination between CYP21A2 and CYP21A1P, microconversions and large gene rearrangements that are generated by unequal meiotic crossing-over [5,6]. CYP21A1P, the pseudogene of CYP21A2, shares a high degree of homology with CYP21A2, which leads to gene conversions and deletions due to homologous recombination [4]. In intergenic recombinations, 70%–75% of mutations are derived from CYP21A1P by microconversion. The remaining 20%–25% of mutations are caused by large deletions or duplications of the CYP21A2 or CYP21A1P/CYP21A2 chimeric genes [7-9]. The remaining 1%–2% of variants are de novo mutations [10]. Therefore, molecular analysis of CYP21A2 must include an assessment of copy number variations and Sanger sequencing [7,8]. More than 200 pathogenic or likely pathogenic variants in CYP21A2 have been identified (https://www.ncbi.nlm.nih.gov/clinvar/).

Newborn screening for 21-OHD aims to identify newborns affected with 21-OHD by measuring the 17α-hydroxyprogesterone (17-OHP) level using an immunoassay to prevent a life-threatening salt-wasting crisis and long-term morbidity [11,12]. However, the serum 17-OHP level can be elevated in healthy neonates during the first 1 to 2 days of life, as well as in low-birth-weight infants, sick or stressed infants, and premature neonates; therefore, false positive results are not uncommon [13,14]. The first prenatal diagnosis of 21-OHD was based on measurement of 17-OHP level using amniotic fluid obtained through amniocentesis [15]. Although measurement of the 17-OHP level in amniotic fluid is a reliable method for prenatal diagnosis of the salt-wasting type of 21-OHD, it can be normal in patients with simple virilizing or nonclassical type of 21-OHD [16-18]. In contrast, prenatal genetic testing provides more accurate diagnosis and genetic counseling for pregnant women with a family history of 21-OHD [19].

A number of studies has reported the usefulness of prenatal genetic diagnosis using molecular analysis of CYP21A2 [20-23]. In these studies, prenatal genetic diagnosis of 21-OHD was performed to prevent virilization of affected female fetuses through prenatal treatment with dexamethasone [22,23], although this is not recommended by current clinical practice guidelines [14]. If prenatal genetic testing identifies a fetus with the genotype of the salt-wasting type of 21-OHD, a life-threatening adrenal crisis can be prevented in the affected newborn with early treatment. Therefore, this study was performed to investigate the usefulness of the prenatal diagnosis of 21-OHD using molecular genetic testing in families at risk.

Materials and methods

1. Participants

This study included 27 pregnant women who had a family history of 21-OHD and who requested to undergo prenatal genetic testing of CYP21A2 between January 2006 and December 2022 at Asan Medical Center, Seoul, Republic of Korea. The exclusion criteria were as follows: (1) prenatal genetic testing was performed because of abnormal prenatal ultrasound findings, such as ambiguous genitalia, but without a family history of 21-OHD and (2) lack of clinical information about the fetus and the family members.

2. Methods

The medical records were reviewed retrospectively to collect clinical data including family history, molecular analysis results in families with 21-OHD, the type of 21-OHD, and endocrine findings of serum levels of sodium, potassium, 17-OHP, adrenocorticotropic hormone (ACTH), cortisol, plasma renin activity, aldosterone, testosterone, and androstenedione.

Chorionic villus sampling (CVS) or amniocentesis was used to obtain fetal tissues for prenatal genetic testing at 10–12 weeks or 15–18 weeks of gestation, respectively [24]. Genomic DNA was isolated from chorionic villi samples or amniocytes using the QuickGene DNA blood kit (Fujifilm, Tokyo, Japan). Contamination of the maternal cell was ruled out by comparing the short tandem repeat markers (AmpFLSTR, Identifiler PCR amplification kit, Applied Biosystems, Foster, CA, USA) of the fetuses and mothers. Allele-specific polymerase chain reaction and Sanger sequencing were performed using 2 pairs of primers specific for CYP21A2 and CYP21A1P, as previously described [25]. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect deletions or duplications of CYP21A2 using the SALSA MLPA kit (P050B CAH; MRC Holland, Amsterdam, The Netherlands) according to the manufacturer's protocols.

3. Ethical statement

This study was approved by the Institutional Review Board (IRB) of Asan Medical Center (IRB No. 2022-0499). The need to obtain informed consent was waived by the IRB, as the clinical data were obtained retrospectively without molecular analysis.

Results

1. Prenatal genetic diagnosis

A total of 39 prenatal genetic tests was performed on 27 pregnant women. Nine pregnant women were tested more than twice. The mean age of the pregnant mothers at the time of testing was 32.3±2.6 years (range, 28–39 years). Twentysix probands (96.3%) were identified as the salt-wasting type, while the remaining one child was identified to have the simple virilizing type of 21-OHD. CVS was performed in 32 cases (82.1%) from 23 women at 11.7 weeks of gestational age (range, 11–12.7 weeks), while amniocentesis was performed in 7 cases (17.9%) from 7 women at 17.5 weeks of gestation (range, 11.3– 23.4 weeks).

Among the 39 prenatal genetic tests, 11 fetuses (28.2%) from 11 families were diagnosed with 21-OHD (Table 1). Fifteen fetuses (38.5%) were heterozygous carriers of mutations in CYP21A2. In contrast, mutations in CYP21A2 were not identified in the remaining 13 fetuses (33.3%). Among the 11 fetuses who were diagnosed with 21-OHD, 4 were born alive at a gestational age of 38.1–39.6 weeks, while 7 fetuses were lost to follow-up. Four fetuses (3 males and 1 female) from 4 families were confirmed to have 21-OHD postnatally via endocrine tests and Sanger sequencing of CYP21A2 using genomic DNA extracted from peripheral blood leukocytes.

Among the 11 fetuses diagnosed with 21-OHD, 7 (63.6%) harbored homozygous CYP21A2 mutations, and 4 (36.4%) were compound heterozygotes. The c.293-13A>G mutation was the most frequent (7 of 22, 31.8%), followed by p.R357W (6 of 22, 27.3%) and large deletions (2 of 22, 9.1%). The genotype was identical to that of the proband in 10 fetuses (90.9%). One fetus (9.1%), who was homozygous for p.Q139*, harbored different mutations from those of a sibling who was compound heterozygous for p.P30L and p.Q319*.

2. Clinical outcomes of 4 fetuses with 21-OHD who were diagnosed by prenatal genetic testing

The 4 fetuses who were identified as 21-OHD patients by prenatal genetic tests were born at term. Their probands were referred to Asan Medical Center because of high 17-OHP level in newborn screening. These babies were ultimately diagnosed with a salt-wasting type of 21-OHD through endocrine and genetic testing. The mean age at diagnosis was 21.3±7.8 days (range, 15–30 days). One of the probands developed adrenal crisis with severe hyponatremia (111 mmol/L), hyperkalemia (7.3 mmol/L), and hypotension before the start of treatment.

The postnatal clinical features and molecular analysis of the 4 patients are summarized in Table 2. All patients were confirmed by Sanger sequencing of CYP21A2 postnatally. Two males were homozygous for c.293-13A>G, while 1 male was homozygous for p.R357W. The 3 male patients had high 17-OHP and ACTH levels. Two of them manifested hyperpigmentation. However, none of the male patients developed the salt-wasting 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). The female patient who was homozygous for p.R357W had ambiguous genitalia with Prader stage III. The female patient, who was born at an outside hospital, developed hyponatremia (132 mmol/L) and hyperkalemia (8.1 mmol/L) on the seventh day of life; she was later referred to Asan Medical Center.

Discussion

In this study, prenatal genetic testing identified 28.2% of fetuses with 21-OHD among 39 fetuses from 27 pregnant women who had previously bore a child with 21-OHD. Among them, 10 of 11 fetuses diagnosed with 21-OHD (90.9%) harbored the same mutation as the probands with 21-OHD. In the present study, 1 fetus (1 of 11, 9.1%) harbored a different mutation from its sibling. Due to the intergenic recombination caused by the highly homologous pseudogene CYP21A1P, the fetus may have different genetic variants from those of siblings [26]. Therefore, prenatal genetic testing requires a comprehensive evaluation of CYP21A2, including Sanger sequencing and MLPA analysis [26]. Four of the 11 fetuses who were diagnosed with 21-OHD were born alive. None of the prenatally diagnosed patients developed severe hyponatremia or dehydration because treatment was initiated at an early age. Therefore, a prenatal genetic diagnosis can be helpful for early diagnosis and treatment of neonates with 21-OHD to prevent a life-threatening adrenal crisis.

Newborn screening programs that measure the 17-OHP level via dried blood spots are usually performed between 2 and 4 days after birth [27]. However, the 17-OHP level is normally high after birth and decreases rapidly during the first few postnatal days in a healthy newborn; therefore, this measurement is not reliable within the first 2 days of life in patients with 21-OHD [14]. In addition, as the 17-OHP level increases over time in infants with classical 21-OHD, newborn screening tests for 21-OHD performed within 72 hours of life miss nearly 30% of cases of 21-OHD [14,28]. Therefore, diagnosis of 21-OHD might be delayed until hyponatremia, hyperkalemia, and severe dehydration occur [29]. Thus, molecular genetic analysis is useful for accurate diagnosis of families at risk, enabling early treatment in affected newborns.

A number of studies has evaluated prenatal genetic diagnosis of 21-OHD using molecular analysis of CYP21A2. One previous study showed that 116 of 532 fetuses were diagnosed with 21-OHD using amniocentesis or CVS by molecular analysis of CYP21A2. Among them, 105 fetuses were affected with classical 21-OHD and 11 with nonclassical type [20]. In India, 6 of 15 fetuses with affected siblings were diagnosed with 21-OHD by Sanger sequencing and MLPA analysis using CVS or amniocentesis. Among them, only one affected male child was born alive [21]. Noninvasive prenatal genetic testing using maternal plasma DNA identified 7 patients (3 males and 4 females) with 21-OHD among 14 fetuses from 12 families at risk [22]. However, there was no information regarding the postnatal clinical outcomes of the fetuses diagnosed with 21-OHD by noninvasive prenatal genetic testing.

Female fetuses with 21-OHD are virilized in utero because of increased exposure to adrenal androgens. Prenatal treatment using dexamethasone can reduce androgen excess to block virilization of the external genitalia of female fetuses [30]. However, prenatal treatment with dexamethasone cannot reduce the risk of life-threatening salt-wasting crises. In addition, there are maternal and fetal safety concerns regarding the use of dexamethasone during pregnancy, such as edema and striae in mothers [31], and the risk of fetal orofacial cleft [32], compromised fetal brain growth, poor cognitive performance [33,34], and insulin resistance in neonates [35]. Therefore, prenatal therapy with dexamethasone is considered experimental [14].

Although prenatal genetic testing for 21-OHD was intended to provide proper genetic counseling for families at risk and early management for affected newborns before life-threatening events occur, there are ethical issues surrounding artificial abortion. Previously, 5 families with 6 affected fetuses decided to terminate the pregnancies [21]. Thus, the choice to pursue prenatal genetic testing should be made carefully, and those families who do undergo testing need comprehensive information on the outcomes of 21-OHD and psychological support.

This study has several limitations. As a retrospective study with a small number of patients, there were missing data on the clinical course of some fetuses. In addition, parental DNA samples were not available for segregation analysis in most probands. However, the current study is the first to investigate the clinical utility of prenatal diagnosis of 21-OHD using molecular genetic testing in Republic of Korea. Early diagnosis by prenatal genetic testing enables prevention of the saltwasting phenomenon in neonates with 21-OHD.

In conclusion, this study demonstrated the clinical consequences of fetuses who were diagnosed with 21-OHD by prenatal genetic testing in families at risk. All patients identified through the prenatal genetic tests were treated with hydrocortisone and 9α-fludrocortisone early after birth; thus, life-threatening salt-wasting crises were avoided. Therefore, prenatal genetic diagnosis of 21-OHD can facilitate early diagnosis and prevention of a life-threatening salt-wasting crisis in affected patients.

Notes

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Funding

This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT) (No. NRF2021R1F1A104593011).

Data availability

The data that support the findings of this study can be provided by the corresponding author upon reasonable request.

Author contribution

Conceptualization: JHY, JHC; Data curation: JHY, J Choi, SH, JHK, GHK, HWY; Formal analysis: JHY, J Choi, SH, JHK, GHK, HWY; Funding acquisition: JHC; Methodology: JHY, JHC; Project administration: JHY, JHC; Visualization: JHY, JHC; Writing - original draft: JHY; Writing - review & editing: JHY, J Choi, SH, JHK, GHK, HWY

Table 1.

Genotype of fetuses who were diagnosed with 21-hydroxylase deficiency by prenatal genetic tests

No.	Family No.	Specimen	Gestational age (wk)	Allele 1	Allele 2
1	1	CVS	11	c.[515T>A(;)955C>T(;)1069C>T]	(p.[I172N(;)Q319*(;)R357W])
2	2	Amniocentesis	23+3	c.1069C>T (p.R357W)	c.1069C>T (p.R357W)
3	3	CVS	11+3	c.293-13A>G	Large deletion
4	6	CVS	11+5	c.293-13A>G	c.293-13A>G
5	11	CVS	11+1	c.293-13A>G	c.293-13A>G
6	16	CVS	11+6	c.1069C>T (p.R357W)	c.1069C>T (p.R357W)
7	18	CVS	12+3	c.955C>T (p.Q319*)	c.955C>T (p.Q319*)
8	22	CVS	12+1	c.492del (p.E164fs)	c.1118G>A (p.S373N)
9	24	Amniocentesis	22+5	c.293-13A>G	c.293-13A>G
10	25	CVS	11+2	c.1069C>T (p.R357W)	c.1069C>T (p.R357W)
11	26	CVS	12	c.1064G>C (p.R355H)	Large deletion

CVS, chorionic villus sampling.

Table 2.

Initial biochemical and hormonal profiles of 4 patients who were diagnosed with 21-hydroxylase deficiency by prenatal genetic tests

Family No.	Sex	Allele 1	Allele 2	Na (mmol/L)	K (mmol/L)	17-OHP (ng/mL)	ACTH (pg/mL)	Plasma renin activity (ng/mL/hr)	Aldosterone (ng/dL)	Testosterone (ng/mL)	Androstenedione (ng/mL)
2	F	c.1069C>T (p.R357W)	c.1069C>T (p.R357W)	132	8.1	333	396	37	81.2	6.9	>11
6	M	c.293-13A>G	c.293-13A>G	135	6.2	51.2	255	25.1	ND	5.7	ND
16	M	c.1069C>T (p.R357W)	c.1069C>T (p.R357W)	139	5.0	192	787	20	1050	57	ND
24	M	c.293-13A>G	c.293-13A>G	143	4.1	135	391	11.1	57.2	24	>10.8

17-OHP, 17α-hydroxyprogesterone; ACTH, adrenocorticotropic hormone; ND, not done.

References

1. Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med 2003;349:776-88.

2. New MI, White PC. Genetic disorders of steroid hormone synthesis and metabolism. Baillieres Clin Endocrinol Metab 1995;9:525-54.

3. New MI, Wilson RC. Steroid disorders in children: congenital adrenal hyperplasia and apparent mineralocorticoid excess. Proc Natl Acad Sci U S A 1999;96:12790-7.

4. White PC, Werkmeister J, New MI, Dupont B. Steroid 21-hydroxylase deficiency and the major histocompatibility complex. Hum Immunol 1986;15:404-15.

5. Higashi Y, Tanae A, Inoue H, Fujiikuriyama Y. Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene - implications for steroid 21-hydroxylase deficiency. Am J Hum Genet 1988;42:17-25.

6. White PC, Tusie-Luna MT, New MI, Speiser PW. Mutations in steroid 21-hydroxylase (CYP21). Hum Mutat 1994;3:373-8.

7. Concolino P, Mello E, Toscano V, Ameglio F, Zuppi C, Capoluongo E. Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of CYP21A2 gene deletions/duplications in congenital adrenal hyperplasia: first technical report. Clin Chim Acta 2009;402:164-70.

8. Tolba A, Mandour I, Musa N, Elmougy F, Hafez M, Abdelatty S, et al. Copy number variations in genetic diagnosis of congenital adrenal hyperplasia children. Front Genet 2022;13:785570.

9. Tusie-Luna MT, White PC. Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms. Proc Natl Acad Sci U S A 1995;92:10796-800.

10. Speiser PW, Dupont J, Zhu D, Serrat J, Buegeleisen M, Tusie-Luna MT, et al. Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 1992;90:584-95.

11. Joint LWPES/ESPE CAH Working Group. Consensus statement on 21-hydroxylase deficiency from the Lawson Wilkins Pediatric Endocrine Society and the European Society for Paediatric Endocrinology. J Clin Endocrinol Metab 2002;87:4048-53.

12. Honour JW, Torresani T. Evaluation of neonatal screening for congenital adrenal hyperplasia. Hormone Res 2001;55:206-11.

13. Merke DP, Auchus RJ. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. N Engl J Med 2020;383:1248-61.

14. Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merke DP, et al. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an endocrine society clinical practice guideline. J Clin Endocr Metab 2018;103:4043-88.

15. Jeffcoate TN, Fliegner JR, Russell SH, Davis JC, Wade AP. Diagnosis of the adrenogenital syndrome before birth. Lancet 1965;2:553-5.

16. Pang S, Pollack MS, Loo M, Green O, Nussbaum R, Clayton G, et al. Pitfalls of prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia. J Clin Endocrinol Metab 1985;61:89-97.

17. Gueux B, Fiet J, Couillin P, Rauxdemay MC, Mornet E, Galons H, et al. Prenatal-diagnosis of 21-hydroxylase deficiency congenital adrenal-hyperplasia by simultaneous radioimmunoassay of 21-deoxycortisol and 17-hydroxyprogesterone in amniotic-fluid. J Clin Endocr Metab 1988;66:534-7.

18. Forest MG. Pitfalls in prenatal-diagnosis of 21-hydroxylase deficiency by amniotic-fluid steroid analysis - a 6 years experience in 102 pregnancies at risk. Ann Ny Acad Sci 1985;458:130-47.

19. Prado MJ, de Castro SM, Kopacek C, de Mello MP, Rispoli T, Grandi T, et al. Development of CYP21A2 genotyping assay for the diagnosis of congenital adrenal hyperplasia. Mol Diagn Ther 2017;21:663-75.

20. New MI, Carlson A, Obeid J, Marshall I, Cabrera MS, Goseco A, et al. Prenatal diagnosis for congenital adrenal hyperplasia in 532 pregnancies. J Clin Endocrinol Metab 2001;86:5651-7.

21. Dubey S, Tardy V, Chowdhury MR, Gupta N, Jain V, Deka D, et al. Prenatal diagnosis of steroid 21-hydroxylase-deficient congenital adrenal hyperplasia: experience from a tertiary care centre in India. Indian J Med Res 2017;145:193-201.

22. Ma DY, Yuan Y, Luo CY, Wang YS, Jiang T, Guo FY, et al. Noninvasive prenatal diagnosis of 21-hydroxylase deficiency using target capture sequencing of maternal plasma DNA. Sci Rep 2017;7:7427.

23. New MI, Tong YK, Yuen T, Jiang PY, Pina C, Chan KCA, et al. Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-free fetal DNA in maternal plasma. J Clin Endocr Metab 2014;99:E1022-30.

24. Chorionic villus sampling and amniocentesis: recommendations for prenatal counseling. centers for disease control and prevention. MMWR Recomm Rep 1995;44:1-12.

25. Choi JH, Jin HY, Lee BH, Ko JM, Lee JJ, Kim GH, et al. Clinical phenotype and mutation spectrum of the CYP21A2 gene in patients with steroid 21-hydroxylase deficiency. Exp Clin Endocrinol Diabetes 2012;120:23-7.

26. Baumgartner-Parzer S, Witsch-Baumgartner M, Hoeppner W. EMQN best practice guidelines for molecular genetic testing and reporting of 21-hydroxylase deficiency. Eur J Hum Genet 2020;28:1341-67.

27. White PC. Optimizing newborn screening for congenital adrenal hyperplasia. J Pediatr 2013;163:10-2.

28. Chan CL, McFann K, Taylor L, Wright D, Zeitler PS, Barker JM. Congenital adrenal hyperplasia and the second newborn screen. J Pediatr 2013;163:109. -13. e1.

29. Swerdlow AJ, Higgins CD, Brook CGD, Dunger DB, Hindmarsh PC, Price DA, et al. Mortality in patients with congenital adrenal hyperplasia: a cohort study. J Pediatr 1998;133:516-20.

30. Bachelot A, Grouthier V, Courtillot C, Dulon J, Touraine P. Management of endocrine disease: congenital adrenal hyperplasia due to 21-hydroxylase deficiency: update on the management of adult patients and prenatal treatment. Eur J Endocrinol 2017;176:R167-81.

31. Fernandez-Balsells MM, Muthusamy K, Smushkin G, Lampropulos JF, Elamin MB, Abu Elnour NO, et al. Prenatal dexamethasone use for the prevention of virilization in pregnancies at risk for classical congenital adrenal hyperplasia because of 21-hydroxylase (CYP21A2) deficiency: a systematic review and meta-analyses. Clin Endocrinol 2010;73:436-44.

32. Carmichael SL, Shaw GM, Ma C, Werler MM, Rasmussen SA, Lammer EJ. Maternal corticosteroid use and orofacial clefts. Am J Obstet Gynecol 2007;197:585.e1. -7. discussion 683-4, e1-7.

33. Damsted SK, Born AP, Paulson OB, Uldall P. Exogenous glucocorticoids and adverse cerebral effects in children. Eur J Paediatr Neurol 2011;15:465-77.

34. Hirvikoski T, Nordenstrom A, Lindholm T, Lindblad F, Ritzen EM, Wedell A, et al. Cognitive functions in children at risk for congenital adrenal hyperplasia treated prenatally with dexamethasone. J Clin Endocr Metab 2007;92:542-8.

35. Dalziel SR, Walker NK, Parag V, Mantell C, Rea HH, Rodgers A, et al. Cardiovascular risk factors after antenatal exposure to betamethasone: 30-year follow-up of a randomised controlled trial. Lancet 2005;365:1856-62.