Home | About us | Editorial board | Search | Ahead of print | Current issue | Archives | Submit article | Instructions | Subscribe | Contacts | Advertise | Login 
Search Article 
Advanced search 
  Users Online: 35 Home Print this page Email this page Small font sizeDefault font sizeIncrease font size  

Table of Contents
Year : 2015  |  Volume : 19  |  Issue : 5  |  Page : 644-648

Clinical profile and inheritance pattern of CYP21A2 gene mutations in patients with classical congenital adrenal hyperplasia from 10 families

1 Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India
2 Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, India

Date of Web Publication19-Aug-2015

Correspondence Address:
Arundhati Sharma
Department of Anatomy, All India Institute of Medical Sciences, New Delhi - 110 029
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2230-8210.163191

Rights and Permissions

Context: Congenital adrenal hyperplasia (CAH) is an autosomal recessive metabolic disorder caused by mutations in the CYP21A2 gene. Genetic diagnosis of 21-OH deficiency causing CAH is more complicated than any other monogenic disorder due to high variability of the locus. The disease has a wide spectrum of clinical variants making it difficult to establish a genotype-phenotype correlation. Therefore, family studies are necessary to ascertain parental genotype and segregation of the mutant allele among the offspring. Aim: The present study aimed to identify CYP21A2 gene mutations and analyze the segregation pattern in CAH trios (patients and their parents). Materials and Methods: A total of ten families having at least one CAH child were recruited. Results: Out of 31 children from ten families, 15 were affected with CAH and 13 of /them (12 females and 1 male) were available for genetic testing. One family had all the children affected with CAH. Compound heterozygous mutations were identified in seven patients (53.8%) whereas p.P30L, In2 and δ8 bp mutations were present in homozygous state in three (23.1%), two (15.3 %) and one (7.6%) patient respectively. Conclusions: In majority of the families, mutant alleles observed in the patients were inherited from the parents whereas three families showed sporadic mutations without any paternal or maternal origin. This indicated their novel occurrence due to misalignment of the parental genes and /or large deletion of the gene. Female preponderance was noted in the CAH families and also among the patients raising the possibility of survival advantage among females.

Keywords: Adrenal crisis, ambiguous genitalia, CYP21A2 gene mutations, female preponderance, precocious puberty

How to cite this article:
Yadav S, Birla S, Marumudi E, Sharma A, Khadgawat R, Khurana M L, Ammini A C. Clinical profile and inheritance pattern of CYP21A2 gene mutations in patients with classical congenital adrenal hyperplasia from 10 families. Indian J Endocr Metab 2015;19:644-8

How to cite this URL:
Yadav S, Birla S, Marumudi E, Sharma A, Khadgawat R, Khurana M L, Ammini A C. Clinical profile and inheritance pattern of CYP21A2 gene mutations in patients with classical congenital adrenal hyperplasia from 10 families. Indian J Endocr Metab [serial online] 2015 [cited 2021 Sep 28];19:644-8. Available from: https://www.ijem.in/text.asp?2015/19/5/644/163191

Sarita Yadav∗, Shweta Birla∗, Eunice Marumudi∗
∗These authors contributed equally to this work

   Introduction Top

Congenital adrenal hyperplasia (CAH) is one of the hereditary diseases with autosomal recessive inheritance resulting from a deficiency of steroid 21-hydroxylase (21-OH). It is characterized by cortisol deficiency, with or without aldosterone deficiency, and androgen excess. [1] The incidence varies according to ethnicity and geographical area. [2],[3],[4],[5],[6],[7],[8],[9],[10]

The 21-OH (CYP21A2/CYP21B) active gene, (OMIM #201910), is located on chromosome 6p21·3 within the histocompatibility complex and shares >95% homology with an inactive pseudogene (CYP21A1P/CYP21A) which is a result of an ancestral gene duplication. Genetic diagnosis of 21-OH deficiency is more complicated than any other monogenic disorder due to the high variability of the locus. In most of the CAH cases, inactivating CYP21A2 mutations are generated by unequal crossing-over or gene conversion events. The disease has a wide spectrum of clinical variants making it difficult to establish genotype-phenotype correlation. Therefore, family studies are necessary to ascertain parental genotype and transmission of mutant alleles to the offspring. The present study was planned to identify CYP21A2 gene mutations and to identify the inheritance pattern from parents to patients.

   Materials and Methods Top

The study was approved by the Institute ethics committee. A total of 13 patients from ten families clinically diagnosed with classical CAH along with their available family members were recruited from the outpatient Department of Endocrinology Clinic between 2007 and 2012. All patients underwent a physical examination, and the diagnosis was based on the hormonal profile. [2] Detailed family history in the form of pedigree charts was collected, and 5 ml of peripheral blood sample was drawn in ethylenediaminetetraacetic acid for genetic screening from the available family members after taking informed consent.


Hormonal analysis

Levels of cortisol, ACTH, testosterone, and DHEAS were estimated using electrochemiluminescence immunoassay using commercial kits (Roche, Germany). Radioimmunoassay kit based method was used for estimating 17 OHP levels. (Diagnostic Systems Laboratories, Inc., Webster, TX, USA).

Cytogenetic analysis

Conventional cytogenetic analysis was carried out on peripheral blood using standard techniques. [11] Karyotyping was carried out on G-banded metaphases obtained from 72-h cultures.

Molecular analysis

Genomic DNA was extracted from peripheral blood leukocytes using a standard protocol. [12] Polymerase chain reaction (PCR) amplification was done for analyzing eight most common mutations in CYP21A2 gene. The gene was amplified in two segments, S1 and S2 with primers that selectively amplify CYP21A2. S1 and S2 segments were used as templates for nested PCR to amplify the desired region harboring the known mutations as described previously. [2] Amplification was done using 70-100 ng DNA, 1.5 mM MgCl 2 , 0.25 mM of each of the dNTPs, 10 pM of each primer, and 0.5 units of Taq Polymerase (Invitrogen, Carlsbad, CA, USA) in a 25 μl volume mixture using thermocycler (ABI 9700; AB, Foster City, CA).

The PCR products were subjected to digestion using respective restriction enzymes as described previously [2] for rapid genotyping. The digested products were subjected to electrophoresis in 10% polyacrylamide gels, at 100 V for 3 h at room temperature. Gels were stained with ethidium bromide and visualized under UV light. All samples were analyzed to check for the presence of any of the eight mutations commonly present in CYP21A2 gene namely p.P30L, In2, Δ8 bp, p.I172N, p.V281L, p.Q318X, p. R356W, and p.P453S.

   Results Top

Molecular analysis identified mutant alleles in the parents and their transmittance to the offspring [Figure 1] and [Figure 2]. Of 31 children from 10 CAH families, 15 children (12 girls and 3 boys) were diagnosed to have CAH [Table 1]. The age at presentation varied from 0.6 to 21 years. Parental consanguinity was documented in one family. Three families had a history of infant/fetal death. All the children were affected with CAH in one family whereas four families had more than 1 affected child. Two of the female patients were reared as males and five of the 13 (38.5%) patients had a history of hospitalization due to adrenal crisis. Of these 15 CAH children, 13 were available for genetic testing. Normal genotype was observed in two of the parents from different families, rest all the individuals had one or more mutant alleles present either in heterozygous or homozygous state. The most prevalent mutation was p.P30L found in three patients (23.1%), followed by In2 in two patients (15.4%), and Δ8 bp in one (7.6%) patient. Remaining seven patients were compound heterozygous (53.8%) for different mutations. Genetic analysis showed that the patients inherited the parental mutant alleles. However, in 3 families (2, 5, and 6), mutations In2, p.I172N, p.Q318X, and p.R356W were identified in the offspring, which were not present in the parents. This indicated their novel occurrence, which may be due to misalignment of the parental genes or the large deletion of the gene.
Figure 1: Frequency of CYP21A2 mutations in congenital adrenal hyperplasia patients

Click here to view
Figure 2: Pedigree charts of the patients showing the segregation of mutations

Click here to view
Table 1: Clinical presentation and genotype of the congenital adrenal hyperplasia patients and family members

Click here to view

   Discussion Top

In the present study, a segregation analysis of the eight common CYP21A2 mutations in ten CAH families was performed. All the recruited families had at least one CAH affected individual. The parents were genotyped, and the pattern of segregation of mutations was analyzed.

In the absence of CAH neonatal screening, several studies have shown female preponderance as presence of ambiguous genitalia raises clinical suspicion of CAH among girls whereas in boys the diagnosis may be missed till they present with features of adrenal insufficiency leading to preventable deaths among them.

In the present study also, female prevalence was observed among the number of children born in these ten families (10 males: 21 females) and the number of children with CAH (1 male: 12 females). The findings highlight two important observations; first, lack of neonatal screening in our set up, which could enable earlier diagnosis and reduce the number of deaths. This speculation is further strengthened by the observation of a report from Sweden [13] where neonatal screening for CAH is mandatory, the male: female ratio among CAH patients is nearly 1. Second, female prevalence in the number of children born raises the question of advantage for girls even prenatally.

Different mutations confer different effect on the activity of the enzyme, and thus severity of the disease is directly proportional to the degree of enzyme activity compromised by the mutation. [14] In2 mutation is known to affect 98-99% of the activity whereas p.I172N mutation leads to the residual enzyme activity <10%. The other three mild point mutations (p.V281L, p.P453S, and p.P30L) cause 7-75% of residual enzyme activity. Thus, the presence of homozygous and/or compound heterozygous changes with other mutations governs the resulting phenotype.

Clinical expression of the disease is determined by the presence of any one of the mild mutations on one allele despite the presence of a severe mutation in the other allele leading to varied phenotype among the affected. Mutation p.P30L has a special status in the group of mild mutations as its in vitro expression is high, but it does not translate in corresponding in vivo activity. [8] It was evident by studies that the mild p.P30L mutation along with an additional severe mutation or a gene conversion could explain the salt wasting or simple virilizing (SV) phenotype in patients. [15] The finding is replicated in the present report where p.P30L, considered to be commonly found in NC CAH was identified in classic CAH patients.

The high frequency of p.P30L mutations in this study is similar to previous reports on a larger sample size. [16] This mutation was identified in combination with severe mutations in five patients. Similar pattern was reported in another study on SV CAH patients from Brazil. [17]

On the contrary, several studies found p.P30L mutation to be less prevalent in their patient populations [18],[19],[20],[21],[22],[23],[24],[25],[26] and also suggested a correlation between genotype and phenotype. [8],[20],[27],[28]

In contrast, the present study identified unusual presentation in some of the classic CAH families, both in terms of the mutation spectrum and clinical features indicating genotype/phenotype discrepancy.

An interesting pattern of disease expression was observed on genetic and pedigree analysis. Analysis of eight common CYP21A2 mutations in the CAH families confirmed transmission from parents. Four probands exhibited additional mutations not seen in the parents, which is in accordance with literature as about 1-2% of affected alleles are known to be spontaneous mutations not carried by either parent. Mothers (2.1, 2.5, 2.6) with heterozygous p.P30L and In2 mutations were not affected, but their offspring with the same genotype expressed the CAH phenotype. In families (2.4, 2.6), the fathers with homozygous p.P30L and In2 mutations, respectively, were expected to show a severe phenotype, but they presented as normal individuals. Molecular analysis of these families confirmed the variability of presentation in carriers of different mutations thereby posing difficulties in decision making regarding therapy and genetic counseling.

   Conclusions Top

India has a diverse population with people belonging to different ethnic groups. Novel mutations in the offspring not identified in the parents indicated large deletions, and/or misalignment of parental genes. The present study also showed female preponderance in terms of number of children born and the number of children diagnosed with CAH raising the possibility of survival advantage for girls, even prenatally. There is a need for more genetic studies to substantiate these observations and delineate the pathophysiology of the genotype and the resulting phenotype.


This study is being supported by Department of Biotechnology (Ref.No.BT/PR7548/Med/12/593/2013), Government of India.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Merke DP, Bornstein SR. Congenital adrenal hyperplasia. Lancet 2005;365:2125-36.  Back to cited text no. 1
Oriola J, Plensa I, Machuca I, Pavía C, Rivera-Fillat F. Rapid screening method for detecting mutations in the 21-hydroxylase gene. Clin Chem 1997;43:557-61.  Back to cited text no. 2
Loidi L, Quinteiro C, Parajes S, Barreiro J, Lestón DG, Cabezas-Agrícola JM, et al. High variability in CYP21A2 mutated alleles in Spanish 21-hydroxylase deficiency patients, six novel mutations and a founder effect. Clin Endocrinol (Oxf) 2006;64:330-6.  Back to cited text no. 3
Rabbani B, Mahdieh N, Ashtiani MT, Larijani B, Akbari MT, New M, et al. Mutation analysis of the CYP21A2 gene in the Iranian population. Genet Test Mol Biomarkers 2012;16:82-90.  Back to cited text no. 4
Skordis N, Kyriakou A, Tardy V, Ioannou YS, Varvaresou A, Dracopoulou-Vabouli M, et al. Molecular defects of the CYP21A2 gene in Greek-Cypriot patients with congenital adrenal hyperplasia. Horm Res Paediatr 2011;75:180-6.  Back to cited text no. 5
Tardy V, Menassa R, Sulmont V, Lienhardt-Roussie A, Lecointre C, Brauner R, et al. Phenotype-genotype correlations of 13 rare CYP21A2 mutations detected in 46 patients affected with 21-hydroxylase deficiency and in one carrier. J Clin Endocrinol Metab 2010;95:1288-300.  Back to cited text no. 6
Soardi FC, Barbaro M, Lau IF, Lemos-Marini SH, Baptista MT, Guerra-Junior G, et al. Inhibition of CYP21A2 enzyme activity caused by novel missense mutations identified in Brazilian and Scandinavian patients. J Clin Endocrinol Metab 2008;93:2416-20.  Back to cited text no. 7
Krone N, Braun A, Roscher AA, Knorr D, Schwarz HP. Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab 2000;85:1059-65.  Back to cited text no. 8
Chan AO, But WM, Ng KL, Wong LM, Lam YY, Tiu SC, et al. Molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Hong Kong Chinese patients. Steroids 2011;76:1057-62.  Back to cited text no. 9
Loke KY, Lee YS, Lee WW, Poh LK. Molecular analysis of CYP-21 mutations for congenital adrenal hyperplasia in Singapore. Horm Res 2001;55:179-84.  Back to cited text no. 10
Barch MJ, Knutsen T, Spurbeck JL. The AGT Cytogenetics Laboratory Manual. 3 rd ed. Philadelphia: Lippincott-Raven Publishers; 1997.  Back to cited text no. 11
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.  Back to cited text no. 12
Gidlöf S, Falhammar H, Thilén A, von Döbeln U, Ritzén M, Wedell A, et al. One hundred years of congenital adrenal hyperplasia in Sweden: A retrospective, population-based cohort study. Lancet Diabetes Endocrinol 2013;1:35-42.  Back to cited text no. 13
New MI, Abraham M, Gonzalez B, Dumic M, Razzaghy-Azar M, Chitayat D, et al. Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 2013;110:2611-6.  Back to cited text no. 14
Dolzan V, Stopar-Obreza M, Zerjav-Tansek M, Breskvar K, Krzisnik C, Battelino T. Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: A novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease. Eur J Endocrinol 2003;149:137-44.  Back to cited text no. 15
Marumudi E, Sharma A, Kulshreshtha B, Khadgawat R, Khurana ML, Ammini AC. Molecular genetic analysis of CYP21A2 gene in patients with congenital adrenal hyperplasia. Indian J Endocrinol Metab 2012;16:384-8.  Back to cited text no. 16
Araujo RS, Billerbeck AE, Madureira G, Mendonca BB, Bachega TA. Substitutions in the CYP21A2 promoter explain the simple-virilizing form of 21-hydroxylase deficiency in patients harbouring a P30L mutation. Clin Endocrinol (Oxf) 2005;62:132-6.  Back to cited text no. 17
Wedell A, Thilén A, Ritzén EM, Stengler B, Luthman H. Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: Implications for genetic diagnosis and association with disease manifestation. J Clin Endocrinol Metab 1994;78:1145-52.  Back to cited text no. 18
Ezquieta B, Oliver A, Gracia R, Gancedo PG. Analysis of steroid 21-hydroxylase gene mutations in the Spanish population. Hum Genet 1995;96:198-204.  Back to cited text no. 19
Jääskeläinen J, Levo A, Voutilainen R, Partanen J. Population-wide evaluation of disease manifestation in relation to molecular genotype in steroid 21-hydroxylase (CYP21) deficiency: Good correlation in a well defined population. J Clin Endocrinol Metab 1997;82:3293-7.  Back to cited text no. 20
Bachega TA, Billerbeck AE, Madureira G, Marcondes JA, Longui CA, Leite MV, et al. Molecular genotyping in Brazilian patients with the classical and nonclassical forms of 21-hydroxylase deficiency. J Clin Endocrinol Metab 1998;83:4416-9.  Back to cited text no. 21
Ferenczi A, Garami M, Kiss E, Pék M, Sasvári-Székely M, Barta C, et al. Screening for mutations of 21-hydroxylase gene in Hungarian patients with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1999;84:2369-72.  Back to cited text no. 22
Tajima T, Fujieda K, Nakayama K, Fujii-Kuriyama Y. Molecular analysis of patient and carrier genes with congenital steroid 21-hydroxylase deficiency by using polymerase chain reaction and single strand conformation polymorphism. J Clin Invest 1993;92:2182-90.  Back to cited text no. 23
Wilson RC, Mercado AB, Cheng KC, New MI. Steroid 21-hydroxylase deficiency: Genotype may not predict phenotype. J Clin Endocrinol Metab 1995;80:2322-9.  Back to cited text no. 24
Koyama S, Toyoura T, Saisho S, Shimozawa K, Yata J. Genetic analysis of Japanese patients with 21-hydroxylase deficiency: Identification of a patient with a new mutation of a homozygous deletion of adenine at codon 246 and patients without demonstrable mutations within the structural gene for CYP21. J Clin Endocrinol Metab 2002;87:2668-73.  Back to cited text no. 25
Koppens PF, Hoogenboezem T, Degenhart HJ. CYP21 and CYP21P variability in steroid 21-hydroxylase deficiency patients and in the general population in the Netherlands. Eur J Hum Genet 2000;8:827-36.  Back to cited text no. 26
Stikkelbroeck NM, Hoefsloot LH, de Wijs IJ, Otten BJ, Hermus AR, Sistermans EA. CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: Six novel mutations and a specific cluster of four mutations. J Clin Endocrinol Metab 2003;88:3852-9.  Back to cited text no. 27
Speiser PW, New MI, Tannin GM, Pickering D, Yang SY, White PC. Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Hum Genet 1992;88:647-8.  Back to cited text no. 28


  [Figure 1], [Figure 2]

  [Table 1]

This article has been cited by
1 First insights into the genetics of 21-hydroxylase deficiency in the Roma population
Mirjana Kocova,Violeta Anastasovska,Aleksandar Petlichkovski,Henrik Falhammar
Clinical Endocrinology. 2021;
[Pubmed] | [DOI]
2 Assessment of the most common CYP21A2 point mutations in a cohort of congenital adrenal hyperplasia patients from Egypt
Mona Essawi,Inas Mazen,Lubna Fawaz,Heba Hassan,Nagham ElBagoury,Michael Peter,Khadiga Gaafar,Mahmoud Amer,Wajeet Nabil,Gisela Hohmann,Hala Soliman,Wolfgang Sippell
Journal of Pediatric Endocrinology and Metabolism. 2020; 0(0)
[Pubmed] | [DOI]
3 Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India
Priyanka Gangodkar,Vaman Khadilkar,P. Raghupathy,Rakesh Kumar,Archana Arya Dayal,Devi Dayal,Ahila Ayyavoo,Tushar Godbole,Rahul Jahagirdar,Kavitha Bhat,Neerja Gupta,Sadishkumar Kamalanathan,Sujatha Jagadeesh,Shatakshi Ranade,Nikhil Lohiya,Rashmi Lote Oke,Karthik Ganesan,Kavita Khatod,Meenal Agarwal,Nikhil Phadke,Anuradha Khadilkar
Endocrine. 2020;
[Pubmed] | [DOI]
4 The distribution of intrafamilial CYP21A2 mutant alleles and investigation of clinical features in Turkish children and their siblings in Southeastern Anatolia
Murat Karaoglan
Journal of Pediatric Endocrinology and Metabolism. 2019; 0(0)
[Pubmed] | [DOI]
5 Common CYP21A2 Gene Mutations in South Indian Congenital Adrenal Hyperplasia Patients
R. Nageshwari,M. Dhivakar,K. Balakrishnan,Sivan Arul Selvan,V. Kumaravel
International Journal of Human Genetics. 2017; 17(3): 103
[Pubmed] | [DOI]
6 Precocious puberty
Taymour Mostafa
Human Andrology. 2016; 6(2): 31
[Pubmed] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

   Abstract Introduction Materials and Me... Results Discussion Conclusions Article Figures Article Tables
  In this article

 Article Access Statistics
    PDF Downloaded343    
    Comments [Add]    
    Cited by others 6    

Recommend this journal