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Original Article

The Spectrum of CYP21A2 Gene Mutations from 16 Families of Congenital Adrenal Hyperplasia: Genotype-Phenotype Correlation

Sridhar, Subbiah,; Govindhan, Ramajayam1; Soundian, Balasankar2; Poomarimuthu, Maheshkumar1; Nallan, Karuppasamy3; kumar, Santhanakrishnan Ramesh4; Eagappan, Subbiah5; Natarajan, Vasanthiy; Jayaraman, Sangumani6

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Indian Journal of Endocrinology and Metabolism: Nov–Dec 2021 - Volume 25 - Issue 6 - p 532-537
doi: 10.4103/ijem.ijem_442_21
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Congenital adrenal hyperplasia (CAH), OMIM No. 201910 is a group of autosomal recessive disorders, characterized by cortisol deficiency and androgen excess, mainly due to deficiency of steroidogenic enzyme 21-hydroxylase.[1] The worldwide incidence of classic CAH in most studies ranges from 1:14,000 to 1:18,000 live births, whereas nonclassic CAH is more frequent and occurs in one in 200 Caucasian populations.[2]

Mutations in 21-hydroxylase gene (CYP21A2) account for over 95% of cases of CAH.[3] Depending on the extent of enzyme impairment, there are two recognized clinical forms of CAH, classic, and nonclassic. Classic CAH may be subclassified as salt-wasting (SW) and simple virilizing (SV). Females with classic CAH are born with ambiguous genitalia, whereas nonclassic presents with hyperandrogenism at the 2nd decade of life.[1]

CYP21A2 gene is located in chromosome 6p21·3 and consists of 10 exons. The active gene CYP21A2 and its pseudogene CYP21AP present 98% sequence identity.[4] The spectrum of mutations in CYP21A2 gene in 21-hydroxylase deficiency patients had been established from previously published studies around the world. Despite more than 300 CYP21A2 mutations being known to occur in CAH, 50% of classic CAH alleles are due to deletions, conversions, point, and splicing mutations that ablate enzyme activity.[5] There are only six studies that had reported the spectrum and frequency of the CYP21A2 gene mutations in the Indian population.[67891011] Except for the recently published one,[11] there is a lack of data on the clinical profile and spectrum of mutations in the south Indian CAH population. In addition, there was a wide spectrum of clinical presentation among different ethnicities and a lack of phenotype-genotype correlation. Therefore, family studies may be important to assess the parental genotype and transmission of mutation to the offspring. Henceforth, the present study was designed to study the clinical profile of classic CAH and to identify the spectrum of common mutation in CAH patients and their family members.


The study was performed at pediatric endocrine unit of 3,000-bedded tertiary level care hospital, funded by the Department of Health Research/Indian Council of Medical Research through the Multidisciplinary Research Unit (MRU), Madurai Medical College, Madurai. Written informed consent from parents and assent from children (as appropriate) was obtained. The study protocol was approved by the Institute Ethical Committee and registered in the Indian Clinical Trial Registry (CTRI/2020/05/025128).


Clinical diagnosis of CAH was suspected in female newborns, presented with ambiguous or atypical genitalia, clitoromegaly, and hyperpigmentation with or without salt crises and premature pubarche and clitoromegaly in childhood period. Sixteen CAH females, and their parents, along with their available siblings, a total of 58 subjects were included in the study over a period of 3 years (2019–2021). Clinical diagnosis of CAH was made based on the physical examination and hormonal profile. Detailed family history was collected and pedigree chart was made.


Cortisol, total testosterone, Luteinizing Hormone (LH), and Follicle Stimulating Hormone (FSH), were estimated using electrochemiluminescence immunoassay (ECLIA) using kits (Roche Cobas e411, Germany). 17-hydroxyprogesterone (17-[OH] P) was measured in serum by radioimmunoassay (RIA) (Diagnostic system laboratories, Inc., Webster, USA). Karyotype was carried out on peripheral blood, by using standard G-banded metaphase techniques from 72 h culture.

Molecular analysis of CYP21A2 gene

DNA was isolated from anticoagulated blood samples using QiagenDNeasy kit as per the manufacturer's instructions. The presence of six most common gene mutations like Intron 2 (c.293-13A/C>G) (NM_000500.9), c.844G>T (p.Val282Leu) (NM_000500.9), c.1019G>A (p.Arg340His) (NM_000127.2), c.92C>T (p.Pro31Leu) (NM_000500.9), c.955C>T (p.Gln319*) (NM_005650.3), and c.518T>A (p.Ile173Asn) (NM_000500.9)*in CYP21A2 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using specific primers [Table 1]. The specific enzymes used for mutational analysis and the fragment size before and after digestions were mentioned in Table 2. At least one symptomatic classic CAH affected individual was included in every family. The parents and available siblings were genotyped and the pattern of segregation of mutation was analyzed.

Table 1:
Primer Sequence used CYP21A2 gene mutation analysis
Table 2:
Characteristics of mutation analysis in the CYP21 gene


The age at presentation varied from birth to 4 years. All CAH patients were female and confirmed by blood karyotype (46 XX). Consanguinity history was present in 13 families. Based on the clinical manifestations and hormonal evaluation, all the classic CAH patients were classified into SW form (n = 12, 75%) and SV form (n = 4, 25%). Among the varied clinical manifestations, the most common were clitoromegaly (100%), ambiguous genitalia (75%), hyperpigmentation (75%), and premature pubarche with heterosexual precocious pseudo puberty (25%) [Figure 1a-c]. We found elevated level of 17(OH) P in the range of >10,000 ng/dL (>250 nmol/L) in all classic SW CAH, whereas in simple virilizing CAH 17(OH) P level was between 1,500 and 9,400 ng/dL, (37.5–272.5 nmol/L). There was a good correlation between the 17(OH) P levels and the type of classic CAH presentation, SW, and SV form. In addition to hyperpigmentation, most SW CAH patients also had hyponatremia, hyperkalemia, and elevated total testosterone with low cortisol.

Figure 1:
(a) Ambiguous genitalia with clitoromegaly and hyperpigmentation in classic SW CAH (b and c) Severe form of clitoromegaly in SV CAH

In genetic analysis of 58 individuals including 16 symptomatic CAH patients, the most common mutation observed in CAH patient was Intron 2 (c.293-13A/C>G) (100%) followed by p.Pro31Leu (98%), p.Gln319* (93%), p.Val282Leu (91.4%), and p.Ile173Asn (19%) [Figure 2] while p.Arg340His mutation was not observed in this study. Moreover, Intron 2 (c. 293-13A/C>G) homozygous was observed in 31.3% cases, p.Ile173Asn homozygous and p.Pro31Leu homozygous were observed in 18.8% and 12.5% cases, respectively. Interestingly, p.Ile173Asn homozygous was found to be associated with SW form and completely absent in SV form.

Figure 2:
Frequency distribution of CYP21 gene mutations in CAH patients

Table 3 and Figure 3 highlight the frequency and type of mutations reported in the present study among patients and their families. Family 9 and 11 probands, had simultaneous presence of two homozygous mutations of p.Ile173Asn and Intron 2 (c.293-13A/C>G), with clinical presentation of severe SW CAH phenotype and biochemical 17(OH) P levels of more than 100,000 ng/dL. First sibling of family 11, male child, died 1 week after birth, probable of SW CAH. Mother of family 11 proband, had homozygous mutation p.Ile173Asn, had regular cycles, no ambiguity, and normal fertility. Family 13 proband also had two homozygous mutations of p.Pro31Leu, p.Ile173Asn, and Intron 2 (c.293-13A/C>G) with SW CAH and 17(OH) P levels of 60,200 ng/dL with good phenogenotype correlation.

Table 3:
Segregation of CYP21 gene mutation among CAH families
Figure 3:
Frequency distribution of CYP21 gene mutations in CAH families


To our knowledge, the present cohort study is the largest single-center experience of CAH patients due to 21-hydroxylase deficiency and their family members from southern India. In the present study, all the affected newborn and children were female. It is because all female classic CAH patients are symptomatic at birth with either ambiguous genitalia or clitoromegaly that makes early recognition. In India, absence of neonatal screening, CAH diagnosis is missed in male gender and recognized only in the later part of life when signs of adrenal insufficiency like hyperpigmentation, hypotension, or precocious pseudo puberty appear.[1213]

Serum cortisol and 17(OH) P are the most important biochemical investigations in all suspected cases of CAH due to 21-hydroxylase deficiency. Rarely 17(OH) P may be elevated in CAH due to 11B hydroxylase deficiency, 3B hydroxysteroid dehydrogenase deficiency, and P450 oxidoreductase deficiency.[3] From the Endocrine Society Clinical Practice guidelines, 17(OH) P level of greater than 1,000 ng/dL is more in favor of classic CAH.[1] Similarly, in the present study, 17(OH) P levels of more than 1,000 ng/dL were noted on SW CAH patients and levels were seen in between 1,500 and 9,400 ng/dL in SV CAH. Hence without corticotropin (ACTH) stimulation test, baseline 17(OH) P levels correlated well with severity of CAH and the clinical presentation. The percentage of 21-hydroxylase enzyme activity is correlated with different clinical presentations, complete inactivation is associated with SW CAH, 2% enzyme activity is with SV CAH, and 20%–60% residual enzymatic activity is associated with nonclassic CAH.[14] Other laboratory features more in favor of SW CAH are hyponatremia, hyperkalemia variable levels of elevated total testosterone with or without cortisol deficiency. Severe clitoromegaly, premature pubarche, and heterosexual precious pseudo puberty is the most common clinical presentation of SV CAH in our cohort, due to late presentation and referral.

Mutations in 21-hydroxylase gene (CYP21A2) account for over 95% of cases of CAH.[5] Due to the high variability of active gene CYP21A2 and its pseudogene CYP21AP, genetic diagnosis of 21-hydroxylase deficiency is more complicated than any other monogenic disorder.[45] Among the six analyzed mutations, the most common homozygous was Intron 2G (31.3%) followed by p.Ile173Asn (18.7%), among CAH patients in the present cohort. According to the reported literature worldwide, p.Ile173Asn as well as Intron 2 (c.293-13A/C>G) mutations lead to residual enzyme activity less than 5% and all are expressed in homozygous state, leading to SW severe CAH phenotype. The presence of homozygous p.Ile173Asn and Intron 2 (c.293-13A/C>G) mutations has a good phenotype-genotype correlation in the present familial cohort study. Moreover, the prevalence of the above mutations is similar to the previously published studies from India.[891011]

All the analyzed CAH patients, families, siblings had some form of compound heterozygous mutations in the present study. The most common heterozygous mutation noted in the present study was p.Pro31Leu found in 98% of study cohorts. Unexpectedly in two of the families, both parents had homozygous. Ile173Asn mutations were expected to show a severe phenotype, but they were asymptomatic with normal fertility. In all four SV CAH patients of present cohort, no homozygous mutation was detected. The compound heterozygous mutations of p.Val282Leu and p.Gln319* were detected in all SV CAH. From the reported literature from other series, p.Val282Leu was the most common mutation in SV CAH and nonclassic CAH.[15] In contrast, in a few of the series SV form of CAH is most often caused by the p.Ile173Asn followed by p.Val282Leu. Although comparing phenogenotype among CAH, SW form has good correlation than SV CAH. None of the 58 individuals showed p.Arg340His mutation. Significantly 17(OH) P levels correlated with classic CAH. All homozygous mutations are associated with severe CAH phenotype and very high 17(OH) P levels. Previous history of early neonatal death was noticed in one of the homozygous mutation-positive families.

The large majority of mutations detected in the present study were found in compound heterozygous, whereas homozygous mutations were observed in 22.4% (13 out of 58 analyzed, including patients and parents) of the entire study cohort. The importance of genetic analysis in CAH is different mutations confer different effects on 21-hydroxylase enzyme activity. For a better understanding of pathophysiology, to predict the severity, and its implications in diagnosis, premarital and genetic counseling, implementation of National Preventive Health Program, mutation analysis will be helpful. Among the analyzed mutations, the point mutation like p.Val282Leu, p.Pro31Leu, are associated with >50% of residual enzyme activity and mild disease.

Compared with previous northern Indian published studies, none of our present cohorts had p.Arg340His mutation.[910] The high frequency of p.Pro31Leu mutations in this study is similar to the other published literature from India. The most common mutations identified in the other published Indian studies were Intron 2 (c.293-13A/C>G) mutation, p.Pro31Leu, and p.Ile173Asn.[891011] More than 90% of mutant alleles carry one or more of a discrete number of mutations, if no mutations are detected, we may assume that the individual is unaffected. The reasons for difference in mutation prevalence among different studies were due to the degree of consanguinity, ethnicity, availability of newborn screening, varied time of presentation as SW/SV, and nonclassic nature of disease.[161718] The segregation analysis of present cohort confirmed the variability of presentation in carriers of different mutations, and genetic analysis alone is insufficient to initiate treatment in CAH when there are no clinical manifestations. The strength of the present study is that patients and family members were analyzed from a single center in south India.

Study limitations

Due to the absence of newborn screening in India, in the present cohort, males are unreported and untreated. This is a preliminary study aimed at targeted gene sequencing by PCR-RFLP rather than extensive screening of Exons of CYP21A2 gene by next-generation sequencing (NGS) or multiplex ligation-dependent probe amplification (MLPA), hence novel mutations were not reported.


We analyzed the spectrum of CYP21A2 gene mutations of 16 patients and their 42 related family members of SW and SV CAH with varied clinical manifestations. Our results suggested a high prevalence CYP21A2 gene mutations in CAH patients and a heterogeneous spectrum of mutations among their family members. The outcomes afford valuable evidence for premarital screening as well as planning suitable programs to prevent the development of CAH in the south Indian population. There is a need for more genetic analysis in India, due to the complicated nature of CYP21A2 gene and pseudo gene, diverse population, and different ethnicity.

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

This work was supported by the Department of Health Research, Ministry of Health and Family Welfare, Government of India [No. V25011/464/2015/HR].

Conflicts of interest

There are no conflicts of interest.


For the support of this study, we acknowledge Multidisciplinary Research Unit, Madurai Medical College, Madurai, Tamil Nadu, India funded by the Department of Health Research, Government of India. We are also thankful to all the patients and their parents included in this study.


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21-hydroxylase deficiency; ambiguous genitalia; clitoromegaly; congenital adrenal hyperplasia; CYP21A2 gene mutations; India

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