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Year : 2022  |  Volume : 2  |  Issue : 1  |  Page : 47-51

Implications of CYP21A2 gene duplications in carrier screening and prenatal diagnosis of congenital adrenal hyperplasia due to 21 Hydroxylase deficiency

Molecular Genetics Laboratory, Institute of Medical Genetics and Genomics, Sir Ganga Ram Hospital, New Delhi, India

Date of Submission06-Jul-2021
Date of Decision16-Dec-2021
Date of Acceptance04-Feb-2022
Date of Web Publication25-Feb-2022

Correspondence Address:
Dr. Sudhisha Dubey
Institute of Medical Genetics and Genomics, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi - 110 060
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ipcares.ipcares_211_21

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Background: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that presents as salt wasting or simple virilization (SV). It is due to biallelic mutations in the CYP21A2 gene that encodes the 21-hydroxylase enzyme. This gene is susceptible to deletions and duplications due to the presence of a homologous pseudogene and its location in the RCCX module. This complicates the interpretation of molecular analysis of the CYP21A2 gene. Clinical Description: During preconception counseling and subsequent workup of a couple, the wife (who had been diagnosed with simple virilizing CAH at the age of 14 years, based on clinical and metabolic profile) was identified with c.373C >T variant on one and a deletion on the other allele of CYP21A2. Her asymptomatic husband harbored a novel c. 939+5G>A variant in intron 7 of CYP21A2. Prenatal diagnosis by Sanger sequencing revealed the presence of both maternal (c.373C>T) and paternal (c. 939+5G>A) variants in the fetus, indicative of SV form. After genetic counseling, the parents decided to continue with the pregnancy. Management and Outcome: A baby boy was born who underwent investigations according to the standard protocol. However, a diagnosis of CAH could not be established conclusively. The molecular diagnosis of both baby and parents was revisited. It was found that the baby harbored a duplication of CYP21A2 (inherited from his father) along with a novel variant. The duplication neutralized the paternal variant, and thus the baby was not affected, but a carrier. Conclusion: Evaluation of duplication in parents is crucial before prenatal testing, as duplications have important bearing on the carrier status.

Keywords: CYP21A1P, multiplex ligation-dependent probe amplification, pseudogene, RCCX, variant

How to cite this article:
Dubey S, Saxena R, Puri RD, Verma IC. Implications of CYP21A2 gene duplications in carrier screening and prenatal diagnosis of congenital adrenal hyperplasia due to 21 Hydroxylase deficiency. Indian Pediatr Case Rep 2022;2:47-51

How to cite this URL:
Dubey S, Saxena R, Puri RD, Verma IC. Implications of CYP21A2 gene duplications in carrier screening and prenatal diagnosis of congenital adrenal hyperplasia due to 21 Hydroxylase deficiency. Indian Pediatr Case Rep [serial online] 2022 [cited 2022 May 27];2:47-51. Available from: http://www.ipcares.org/text.asp?2022/2/1/47/338479

The P450 enzyme 21-hydroxylase, hydroxylates progesterone, and 17-hydroxyprogesterone to yield 11-deoxycortisone (required for the synthesis of aldosterone) and 11-deoxycortisol (required for the synthesis of cortisol), respectively. Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, is a common autosomal recessive disorder, with impaired steroidogenesis resulting in the deficiency of cortisol and aldosterone, and the overproduction of adrenal androgens. The latter occurs because the excess 17-hydroxyprogesterone (which accumulates due to the enzymatic block) is diverted into the pathway for androgen biosynthesis. This results in increased levels of androstenedione, that in turn is converted to testosterone outside the adrenal gland. These derangements start by the 8th–10th week of gestation.

Thus, both classical forms of 21-hydroxylase deficiency (severe salt-wasting [SW] and mild simple virilizing [SV]) manifest as ambiguous genitalia in newborn females, and progressive postnatal virilization in both genders. The 21-hydroxylase enzyme is completely inactivated in the SW form resulting in severe renal salt loss, and a critical presentation that requires emergency management (vomiting, dehydration, weight loss, hypotension, hypoglycemia, hyponatremia, and hyperkalemia). In contrast, since the SV form has normal aldosterone levels and salt loss is absent. The nonclassical form is milder; affected individuals are asymptomatic at birth and present later with various degrees of hyperandrogenism.[1] Worldwide, the overall incidence of CAH ranges from 1 in 10,000 to 1 in 20,000 in the general population.[2] In India, a recent survey reported a prevalence of 1 in 5762.[3]

The CYP21A2 gene is present in the major histocompatibility complex at chromosome locus 6p21.3, along with a highly homologous pseudogene CYP21A1P located 30 kb downstream. About 95% of the pathogenic variants encountered in CAH are present in this pseudogene. Thus, approximately 70% of CYP21A2, disease-causing variants are pseudogene derived, i.e., they are transferred from the pseudogene (CYP21P) to the functional gene (CYP21A2) by nonreciprocal transfer of deleterious mutations, a phenomenon referred to as gene conversion.

CYP21A2 is a part of the larger genetic unit comprising RP2-C4B-CYP21A2-TNXB genes and known as the RCCX module. Most individuals have bimodular haplotype, i.e., two modules present on each chromosome. However, on occasion monomodular and trimodular chromosomes resulting from the deletion or duplication of RCCX module, respectively, exist. In the trimodular haplotype, either two CYP21A1P and one CYP21A2, or one CYP21A1P and two CYP21A2 genes are present on each chromosome.[4] In the second scenario, the presence of two copies of the functional gene on a chromosome result in duplication of the CYP21A2 gene. In this case report, we discuss the clinical implications of duplication of the functional CYP21A2 gene by describing the clinical outcome of an expectant couple, in which the wife was affected with SV CAH, and her asymptomatic husband was found to harbor a variant of the CYP21A2 gene.

This report highlights a very important message regarding the role of pediatricians in providing continuity of care to children and their families with rare inheritable genetic disorders throughout their life course. The index case had been diagnosed with CAH during adolescence, and her family had been offered genetic counseling. We describe the sequence of events that occurred when the couple was planning to conceive and end with the quandary that emerged when the protocol for postnatal diagnosis of CAH was undertaken.

  Clinical Description Top

A 33-year-old primigravida was referred to us for preconceptional counseling. She had been diagnosed with SV CAH at the age of 14 years when she had sought medical attention for hirsutism and irregular menstrual cycles. Genetic testing had not been included in the work-up. At that time, the family had undergone genetic counseling in which the cause of the disorder, clinical manifestations, course of illness, treatment, complications, etc., had been explained.

Now that she had come for a different purpose, the primary goal of counseling was to calculate the risk of recurrence of disease in the family, which is essential for any autosomal recessive genetic disorder. In this case, the first step would be to evaluate the proband and her spouse for pathogenic variants in the causative gene. The next step would be to investigate the fetal deoxyribonucleic acid (DNA) for any mutations that were found in the parents. A detailed algorithmic approach of genetic testing of an affected individual and prenatal testing in a family at high risk of CAH is depicted in [Figure 1].
Figure 1: Flowchart showing approach to genetic analysis of congenital adrenal hyperplasia. *Heterozygous cases where the second mutation is not found but they have manifestation of disease.[11] **Further evaluation includes i.e., re-phenotyping or alternative diagnosis, 17-OHP levels, possibility of de novo variant

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We proceeded to evaluate both partners for pathogenic variants in the CYP21A2 gene. Multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing of the CYP21A2 gene in the mother revealed c. 373C>T (p.Arg125Cys) variant on one allele [Figure 2]a, and a deletion of exon 1–6 on the other allele. This variant c. 373C>T (dbSNP rs371412889) has been previously reported, is documented to exhibit 16% enzymatic activity, and is associated with the SV clinical phenotype.[5] Her asymptomatic husband was evaluated for carrier status for CAH, and found to harbor a c. 939+5G>A variant in intron 7 of the CYP21A2 gene [Figure 2]b. This variant has not been included in the genomic databases that are available in the public domain, i.e., http://www.cypalleles.ki.se/cyp21.htm, https://www.ncbi.nlm.nih.gov/SNP/; and http://evs.gs.washington.edu/EVS/. It was also absent in GenomeAsia 100K (https://genomeasia100k.org), but present in gnomAD (https://gnomad.broadinstitute.org), with a reported frequency of 0.00003654. As per various in silico tools (Mutation Taster, TRAP, DANN, NetGene2, MaxENTScan, BDGP splice site prediction tool, and Splice Site Score Calculation, dbscSNV Database), the variation c. 939+5G>A found was deemed likely pathogenic, as it alters the donor site and affects the normal splicing of proteins. Thus, in this specific clinical scenario, with the affected mother harboring two variants, and the father identified as a carrier of a variant, the possible risk to the fetus for mild CAH was up to 50% for every pregnancy. This was discussed with the parents, and the option of prenatal testing was explained and offered.
Figure 2: Partial electropherogram showing variants identified in couple and fetus. (a) c.373C>T (p.Arg125Cys) variant in exon 3 of CYP21A2 gene in mother. (b) Father showing heterozygous c. 939+5G>A variant in intron 7 of CYP21A2 gene. (c) Fetal sample showing heterozygous maternal c. 373C>T (p.Arg125Cys) variant. (d) Fetal sample showing heterozygous paternal c. 939 + 5G>A variant. All variants are indicated by red arrows

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The parents consented to chorionic villus sampling that was performed at 11 weeks of gestation. The extracted fetal DNA was subjected to Sanger sequencing, which detected the presence of both the paternal c.939+5G>A and maternal c. 373C>T variants [Figure 2]c and [Figure 2]d. On the basis of this, the fetus was predicted to have a SV phenotype like the mother, since this particular combination correlates with the milder variant of classical CAH.[4] The couple was counseled and explained about the expected phenotype spectrum in affected males and affected females. The couple opted to continue with the pregnancy. A baby boy was born at 38 weeks of gestation through a cesarean section, the indication being a primigravida with breech presentation.

  Management and Outcome Top

The neonate had an Apgar score of 8/10 and 9/10 at 1 min and 5 min, respectively. The vital parameters were normal and oxygen saturation (SpO2) was 98% on room air. Anthropometry performed after stabilization was normal. Hyperpigmentation was not seen. The external genitalia appeared to be that of a boy, without evidence of any ambiguity. The remaining general physical and systemic examinations were normal. We proceeded to investigate the baby as per the protocol for determining the diagnosis of CAH.

Serum 17-hydroxylaseprogesterone on day 1 of life was 9.05 ng/mL (0.49–4.10 ng/ml). However, this can be elevated in a small proportion of unaffected newborns who are preterm or sick. Although our patient was neither, we repeated the test after one week, the morning level of which was normal, 2 ng/mL. Electrolytes remained in the normal range; sodium (139 mEq/L) and potassium (5.7 mEq/L). Cortisol was low (8.55 ug/dL, normal 10–20 ug/dL), and testosterone was normal 115.3 ng/dL (75–400 ng/dL), as expected in a male baby with SV CAH. The adrenocorticotropic hormone (ACTH) test that detects levels of 17-hydroxyprogesterone before and after stimulation with ACTH was normal. The neonate remained hemodynamically stable without any of the aforementioned symptoms or metabolic derangements. At discharge, the parents were counseled regarding the clinical red flags including vomiting and lethargy and instructed to report immediately if they occurred.

The baby was kept under close follow-up. Karyotype confirmed the physical male phenotype of a boy with a 46, XY genotype. The variants that had been identified in the prenatal sample were rechecked in the postnatal sample, and the biallelic variants were confirmed. As the investigative workup was not consistent with CAH, we decided to revisit the molecular analysis. MLPA identified duplication of CYP21A2 in the neonate [Figure 3], as well as in the father. Thus, the final diagnosis was that the infant was a carrier, like his father, and not affected with CAH like his mother.
Figure 3: MLPA ratio chart showing three copies of exons 3-7 or duplication of CYP21A2 gene in fetal DNA. Three copies of exons 3-7 of CYP21A2 are encircled by a red circle, indicating duplication of the entire gene. Normalized peak height ratio between 0.7 and 1.3 was considered as normal in the proband's DNA with respect to control DNA. The X-axis labels denote the length in the base pair of each probe generated during multiplex ligation-dependent probe amplification polymerase chain reaction

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  Discussion Top

The CYP21A2 gene is susceptible to deletions and duplications resulting from misalignment during meiosis. This is due to its location in the highly variable RCCX region that contains pseudogenes and tandem repeat sequences. Duplications of CYP21A2 were previously considered rare,[6] until they were reported in 1.6% of the Swedish population that equals the carrier frequency of CAH.[7] Duplications were also reported in 14% of normal Caucasian subjects, wherein 12.3% of subjects carried duplication of CYP21A1P and 1.7% carried duplication of CYP21A2.[8] Usually, only one of the duplicated CYP21A2 gene carries the pathogenic variant, but a few cases in which both copies of CYP21A2 harboring the pathogenic variant have been reported.[6],[9]

Although there are no functional studies available on duplicated alleles, duplications are usually considered to exhibit normal function as they are observed to be more prevalent in the healthy general population than in the CAH patients.[10] They are only considered pathogenic if both the duplicated genes have variants. Therefore, it is important to check whether the pathogenic variant is located on the allele with one CYP21A2, or on the allele with duplicated CYP21A2, to correctly determine the carrier status of an individual. That is why family segregation studies are very important, as they determine whether the occurrence of a given trait shared by members of a family, cannot be readily accounted for by chance.

In this case, the infant had no clinical, metabolic, or biochemical parameters suggestive of CAH. This can be explained by the fact that he was carrying the c.373C>T variant on the chromosome with the single CYP21A2, and the c.939+5G>A variant on other chromosome with the duplicated CYP21A2 gene. That is why the pathological effect of the c.939+5G>A variant was getting masked by the second normal copy of CYP21A2, and normal enzyme was being produced. Hence, the infant was a carrier and not affected with CAH. This reiterates the fact that it is very important to investigate whether the pathogenic variant is present on the allele with the single CYP21A2 gene, or the allele with the duplicated CYP21A2 gene to avoid misdiagnosis. Since the CYP21A2 locus is variable and complex, there is an increased likelihood of error. The recent guidelines of CYP21A2 genotyping have extensively addressed the issues relating to the complexities of the CYP21A2 locus. It is recommended to use multiple and appropriate methodologies for accurate and comprehensive analysis of the CYP21A2 gene,[11] as was done in this case.

To conclude, the presence of the closely placed highly homologous pseudogene, and two copies of the CYP21A2 gene on one chromosome complicates the molecular analysis and the interpretations of results. As duplications are largely restricted to c.955C>T (p.Gln319Ter) pathogenic variant, it is of paramount importance to look for duplications whenever this variant is encountered. However, this case highlights the fact that duplications should also be suspected with variants other than c.955C>T (p.Gln319Ter). Notably, it is imperative to rule out duplications in preconception work up in couples at risk of CAH, as their identification has important implications on confirmation of carrier or affected status, prenatal diagnosis, and genetic counseling.

Pediatricians play various important roles in context to CAH; ensuring that babies undergo newborn screening in which the early diagnosis of CAH can lead to timely initiation of specific treatment, keeping a high index of suspicion for features of SW in newborns, and of late-onset virilization in older children, diagnosing CAH as per the recommended protocols, offering or referring families at risk for genetic counseling, and ensuring liaison between multiple professionals so that there is continuity of care of the affected individuals and families, throughout the life course.


We are thankful to Dr Deepti Gupta, Scientist at Institute of Medical Genetics and Genomics for her valuable support in helping with the in-silico prediction tools.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245-91.  Back to cited text no. 1
Therrell BL. Newborn screening for congenital adrenal hyperplasia. Endocrinol Metab Clin North Am 2001;30:15-30.  Back to cited text no. 2
ICMR Task Force on Inherited Metabolic Disorders. Newborn screening for congenital hypothyroidism and congenital adrenal hyperplasia. Indian J Pediatr 2018;85:935-40.  Back to cited text no. 3
Hannah-Shmouni F, Chen W, Merke DP. Genetics of congenital adrenal hyperplasia. Endocrinol Metab Clin North Am 2017;46:435-58.  Back to cited text no. 4
Krone N, Rose IT, Willis DS, et al. Genotype-phenotype correlation in 153 adult patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Analysis of the United Kingdom Congenital adrenal Hyperplasia Adult Study Executive (CaHASE) cohort. J Clin Endocrinol Metab 2013;98:E346-54.  Back to cited text no. 5
Wedell A, Stengler B, Luthman H. Characterization of mutations on the rare duplicated C4/CYP21 haplotype in steroid 21-hydroxylase deficiency. Hum Genet 1994;94:50-4.  Back to cited text no. 6
Koppens PF, Hoogenboezem T, Degenhart HJ. Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: Characteristics of three unusual haplotypes. Hum Genet 2002;111:405-10.  Back to cited text no. 7
Blanchong CA, Zhou B, Rupert KL, et al. Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in caucasians. The load of RCCX genetic diversity on major histocompatibility complex-associated disease. J Exp Med 2000;191:2183-96.  Back to cited text no. 8
Dubey S, Tardy V, Chowdhury MR, 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;145:194-202.  Back to cited text no. 9
[PUBMED]  [Full text]  
Parajes S, Quinteiro C, Domínguez F, et al. High frequency of copy number variations and sequence variants at CYP21A2 locus: Implication for the genetic diagnosis of 21-hydroxylase deficiency. PLoS One 2008;3:e2138.  Back to cited text no. 10
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.  Back to cited text no. 11


  [Figure 1], [Figure 2], [Figure 3]


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