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Challenges of Molecular Analysis of Congenital Adrenal Hyperplasia Caused Due to Steroid 21 Hydroxylase Deficiency

Sudhisha Dubey1, Renu Saxena1, Vinu Narayan2, Ratna Dua Puri1, Ishwar C Verma1
1Institute of Medical Genetics and Genomics, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi, India
2Rainbow Children’s Hospital, Marathahalli, Bengaluru, Karnataka, India
Correspondence to: Dr Sudhisha Dubey      Email: sudhishadubey@gmail.com

1 Abstract

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disorder which results from inherited defects in the steroid 21-hydroxylase enzyme encoded by the CYP21A2 gene. Molecular analysis of CYP21A2 is important for confirming the diagnosis, carrier screening, providing accurate genetic counseling, and calculating risk of recurrence in each pregnancy. An interesting feature of the CYP21A2 gene is its location in the variable genomic regions called RCCX and presence of its highly homologous CYP21A1P pseudogene that makes molecular analysis quite challenging as compared to other monogenic disorders. Here we discuss the complexity of the CYP21A2 gene and the importance of comprehensive molecular analysis of CYP21A2 for accurate interpretation of the results citing molecular analysis of two interesting CAH cases.

Keywords: Congenital adrenal hyperplasia, CYP21A2 gene, CYP21A1P, pseudogene, variants, MLPA, deletion, duplication

2 Introduction

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (OMIM# 201910), is an autosomal recessive disorder caused by inherited deficiency of steroid 21-hydroxylase (21OH) enzyme in the steroid biosynthesis pathway in the adrenal cortex. 21OH enzyme acts on progesterone and 17-hydroxyprogesterone (17OHP) and converts these to deoxycortisosterone and 11-beta-hydroxylase respectively, which are further converted into aldosterone and cortisol by other enzymes in the steroidogenic pathway. Deficiency of 21OH enzyme results in shunting of 17OHP and progesterone into the adrenal pathway resulting in excessive production of androgens and deficiency of aldosterone and cortisol (Figure 1). Excessive androgens lead to prenatal virilization in females and rapid somatic growth in both sexes (White & Speiser, 2000). Deficient cortisol level disrupts the negative feedback to the anterior pituitary that results in constant secretion of adrenocorticotropic hormone (ACTH) that overstimulates the adrenal cortex to secret more of cortisol. Due to 21OH deficiency in the adrenal pathway, the cortisol is not secreted and adrenals become hyperplastic due to overstimulation of ACTH in fetal life. That is how this condition obtained its name as “congenital adrenal hyperplasia”.


Figure 1: Steroid pathways for biosynthesis of progesterone, aldosterone, cortisol, androgens (testosterone and dihydrotestosterone), and estrogens (estradiol) are arranged from left to right. The enzymatic activities catalyzing each bioconversion are written in boxes. For those activities mediated by specific cytochromes P450, the systematic name of the enzyme (“CYP” followed by a number) is listed in parentheses. CYP11B2 and CYP17 have multiple activities. The planar structures of cholesterol, aldosterone, cortisol, dihydrotestosterone, and estradiol are placed near the corresponding labels (adapted from White & Speiser, 2000).

CAH is divided into classic and non-classic (NC) CAH. Classic CAH is again divided into salt-wasting (SW) and simple virilizing (SV) forms. SW-CAH is a severe form characterised by deficiency of both cortisol and aldosterone and found in about 75% of patients. Aldosterone deficiency predisposes SW-CAH patients to develop hyponatremic dehydration which is fatal if not treated with glucocortcoids in time. SV-CAH is a milder form found in about 25% of CAH patients. Aldosterone levels are adequate to maintain sodium balance in the SV form and hence there is normally no salt wasting. The NC form is asymptomatic at birth and presents with various degrees of late-onset hyperandrogenism (White & Speiser, 2000). Prenatal virilisation may or may not be present in the mild NC form but is always present in the SW or SV classic forms.

The overall incidence of CAH in the general population worldwide is between 1 in 10,000 to 1 in 20,000 live births for the classic form of CAH (Therrell et al., 2001). However, the prevalence of classic CAH in India is 1 in 5762 according to a recent survey (ICMR task force, 2018). Non-classic CAH is one of the most common autosomal recessive disorders in humans and affects approximately 1 in 1,000 individuals (Speiser et al., 1985).

Steroid 21OH enzyme, is encoded by the CYP21A2 gene located on chromosome 6 (6p21.3) in the HLA class III of the major histocompatibility (MHC) region (Yang et al., 1999). About 30 kb upstream a non-functional pseudogene CYP21A1P is located that shares about 98% sequence homology to CYP21A2. About 95% of the pathogenic variants are pseudogene derived and are transferred from CYP21A1P to CYP21A2 by gene conversion events (Higashi at al., 1986). The remaining 5% are new/rare and unique for single families or considered as population specific (White & Speiser, 2000; Stikkelbroeck et al., 2003). A compilation of 233 pathogenic variants and their clinical classification have been done recently (Concolino & Costella, 2018).

CYP21A2 gene is a part of the genetic unit comprising of RP2-C4B-CYP21A2-TNXB genes known as the RCCX module. Each chromosome bears two RCCX modules; one with the functional CYP21A2 gene and other with the non-functional CYP21A1P as shown in Figure 2. Majority of the individuals have a bimodular haplotype i.e., two modules present on each chromosome. However, three modules have also been reported to be present on one chromosome which is known as the trimodular haplotype. In the trimodular haplotype either two CYP21A1P and one CYP21A2 or one CYP21A1P and two CYP21A2 are present on one chromosome (Figure 2). The later has two copies of functional gene on a chromosome resulting in duplication of the CYP21A2 gene that complicates the molecular analysis of the CYP21A2 gene.


Figure 2: Schematic diagram of the organization of the RCCX modules. The most common is the bimodular haplotype with two RCCX modules, one with pseudogene CYP21A1P and other functional CYP21A2 gene. Trimodular haplotype with three RCCX modules can result in duplication of the CYP21A2 gene. C4 (C4A and C4B) gene encodes the fourth component of the serum complement. RP2, a truncated copy of RP1, encodes the threonine kinase enzyme and TNXB encodes tenascin-X an extracellular matrix protein. TNXA is a non-functional homologue of the TNXB gene (adapted from Sweeten et al., 2008).

In about 20-30% of cases, the large 30kb deletion extends from somewhere between exon 3 of CYP21A1P through C4B to the corresponding point in CYP21A2 yielding a single copy with 5 end of CYP21A1P and 3 end of CYP21A2, also known as the chimeric gene. Nine different chimeras have been reported depending on the extent of deletion involved (Chen et al., 2012). Extent of the deletion also helps in determining the genotype -phenotype correlation (Narasimhan et al., 2019).

3 Materials and methods

Written informed consent was obtained from the parents of both patients. About 100 ng of each genomic DNA was subjected to selective amplification of CYP21A2 into two large fragments with two sets of primers highly specific to the active i.e., CYP21A2 gene (Figure 3A). Absence of bands indicate the deletion of 8 bp of exon 3 or whole of the active gene which is confirmed by MLPA. These fragments were purified using the Qiagen kit (QIAamp PCR Clean-up, Qiagen GmbH, Hilden, Germany) and quantified with MassRuler (Fermentas Life Sciences, Thermo Fisher Scientific, Waltham MA, USA) (Figure 3B) (Dubey et al., 2017). Purified products were subjected to direct sequencing using ABI 3500 Genetic Analyser (PE Applied Biosystems, Thermo Fisher Scientific, Waltham MA, USA). Pathogenic variants were screened using Chromas v2.4 and SeqScape v2.1.1 (Applied Biosystems) against the NCBI reference sequence NM_000500 and transcript ID ENST00000418967. Multiple ligation dependent probe amplification (MLPA) was done using Salsa MLPA Kit P050-C1 (MRC-Holland, Amsterdam, The Netherlands) to detect deletions and duplications.


Figure 3: A. PCR amplification of the CYP21A2 gene into two fragments; fragment A (1130bp) and fragment B (2127bp). M- DNA Ladder; Lanes 2 & 6-Fragment A; Lanes 3 & 7 – Fragment B; Lane 4-5, 8-9 – Absence of bands or amplification indicating gene deletion. B. Purified PCR products of fragment A and B with MassRuler (MR). (Dubey et al., 2017)

4 Patient description and results

Patient 1: A five-years-old female child presented with ambiguous genitalia at birth. She had complete labial fusion and clitoral hypertrophy. Her karyotype was normal female (46, XX) and ultrasound-abdomen revealed bilateral ovaries. She had elevated levels of 17 OHP (greater than 37 ng/mL), renin (greater than 500 ng/mL/hour), potassium (7.8 mEq/L) and low level of sodium (116 mEq/L). She was reported to have seven mutations i.e., I2g (c.293-13A/C>G) (intron2), c.332_339delGAGACTAC (exon 3), c.515T>A (exon 4), c.710T>A (exon 6), c.713T>A (exon 6), c.719T>A (exon 6), and c.923_924insT (exon 7) by NGS. All mutations were in heterozygous form except splice site mutation I2g (c.293-13A/C>G) in intron 2 of the CYP21A2 gene. Snapshots of Integrative genome viewer (IGV) software and MLPA ratio chart were also provided that clearly illustrated presence of these mutations.

The proband was referred to us for validation and segregation of pathogenic variants in her parents, her paternal aunt and the aunt’s husband, as her aunt was pregnant and the family wanted prenatal diagnosis (PND) to be done. Sequencing of the proband was carried out to validate the seven reported pathogenic variants. However, only I2g pathogenic variant was found in homozygous state and all other mutations were clearly absent (Figure 4). To know whether this mutation was in homozygous or hemizygous form, MLPA was carried out for detection of deletion. Half dosage was seen in the probes covering exon 3,4,6 and 7 indicating heterozygous deletion from exon 3 to 7. I2g (intron 2 splice) mutation was found in homozygous state by the two probes included in the MLPA kit P050-C1 for detection of I2g mutation. (Figure 5).


Figure 4: Partial electropherogram showing homozygous I2g (c.293-13A/C>G) mutation detected in the proband (Patient 1).


Figure 5: MLPA analysis using Coffalyser software showing heterozygous deletion of exon 3- 7 of CYP21A2 gene in Patient 1. Deletions of exons 3 and 7 are marked by red circle. SALSA MLPA kit P050-C1 was used to detect deletion in our patients. Normal alleles A and C at I2g showing zero value (shown by arrows) indicate absence of both A and C alleles and presence of homozygous allele G. Normalized peak height ratio between 0.7 and 1.3 was considered as normal in patient DNA w.r.t. control DNA.

Her parents were analysed for segregation of mutations by Sanger sequencing and MLPA. Mother was found to carry the I2g mutation as expected but father was negative for the same. He was then checked for deletion by MLPA that showed normal dosage for all probes indicating that he was negative for the deletion which was unexpected.

Paternal aunt (sister of proband’s father) was checked for deletion and duplication by MLPA. She was found to harbor a heterozygous duplication shown by 3 copies of CYP21A2 (Figure 6). After analysing results of paternal aunt, MLPA results of the father were reinterpreted and it was inferred that father harboured both a duplication and a deletion together, due to which he was showing normal dosage. And his sister had inherited the duplicated allele but not the deletion, and hence was not a carrier of CAH. Her husband too was checked and he was found to be negative for deletion and duplication.


Figure 6: MLPA analysis using Coffalyser software showing heterozygous duplication indicated by the red circle. All probes fall above the normal ratio (1.5) indicating three copies of CYP21A2 gene in the paternal aunt of patient 1. SALSA MLPA kit P050-C1 was used to detect deletion in our patients. Normalized peak height ratio between 0.7 and 1.3 was considered as normal in patient DNA w.r.t. control DNA.

Hence it was confirmed that the proband was compound heterozygous for whole gene deletion and I2g mutation. The deletion was inherited from the father and the I2g mutation from the mother. Proband’s aunt and uncle were counseled about the insignificant risk of having a child affected with CAH.

Patient 2: A five-years-old female child clinically confirmed to have CAH was referred to our genetic clinic for molecular analysis. Her mother was 18 weeks pregnant and the family wanted PND to be done.

Deletions being more common in the CYP21A2 gene, MLPA was first done that showed half dosage of exon 4,6, and 7 indicating heterozygous deletion from exon 4-7 (Figure 7). To look for second mutation, Sanger sequencing was done and the proband was found to harbour c.515T>A (p.Ile172Asn) in exon 4, E6 cluster [c.710T>A (p.Ile236Asn); c.713T>A (p.Val237Glu); c.719T>A (p.Met239Lys)] in exon 6, c.923_924insT (p.Leu306+T) in exon 7, and c.955C>T (p.Gln319Ter) in exon 8, all in heterozygous form (Figure 8).


Figure 7: MLPA analysis using Coffalyser software showing half ratios of exon 4-7 indicating heterozygous deletion from exon 4-7 of CYP21A2 gene in Patient 2. SALSA MLPA kit P050-C1 was used to detect deletion in our patients. Normalized peak height ratio between 0.7 and 1.3 was considered as normal in patient DNA w.r.t. control DNA.


Figure 8: Partial electropherograms showing mutations detected in Patient 2. A. Mutation c.515T>A in exon 4; B. E6 cluster mutation (c.710T>A, 713T>A, 719T>A) in exon 6; C. c.923_924 insT in exon 7; and D. c.955C>T in exon 8 of CYP21A2 gene. All mutations are shown by arrows.

Her parents were then checked for segregation analysis to confirm whether these mutations were present in cis or trans. Mother was found to have E6 cluster [p.Ile236Asn, p.Val237Glu, p.Met239Lys], p.Leu306+T and p.Glu319Ter mutations, and father was heterozygous for the p.Ile172Asn mutation. Hence it was confirmed that the child was compound heterozygous for the point mutations.

5 Discussion

Molecular genetic diagnosis of CAH is more complicated than for many other monogenic disorders due to the location of the CYP21A2 gene in the highly variable genomic region with more than one RCCX repeat unit on the same chromosome. Presence of a non-functional pseudogene further complicates the amplification of the functional gene. The 11 most common mutations known to cause CAH are present in the pseudogene too. Due to this reason, it is extremely important that the functional gene should only be amplified in the background of pseudogene. It is quite difficult as there is not much difference in the sequence between the two genes. The most significant difference is the 8 base pairs GAGACTAC present in exon 3 of CYP21A2 and these 8 base pairs are deleted in exon 3 of CYP21A1P. This ‘8bp site’ has been exploited extensively to design primers for selective amplification of the active gene. To be twice as sure, two primers - forward as well as reverse, were designed at the wild type sequence of the “8bp site” to amplify the CYP21A2 gene into two large fragments. This ensures that amplification occurs only when both primers bind on the wild type sequence at the ‘8bp site’. Absence of amplification indicates the absence of the active gene or presence of the homozygous 8 bp deletion or presence of only the pseudogene (Figure 3A). The extent of deletion can then be analysed by MLPA.

It is important to know that due to presence of the pseudogene, the capture-based NGS approach is not considered appropriate as it may interfere with the analysis and give erroneous results. Recently, a customized work flow involving selective amplification of CYP21A2 followed by NGS has been used to correctly detect variants in CAH patients (Gangodkar et al., 2020).

In Patient 1, all pathogenic variants except I2g were reported in heterozygous form by NGS. These pathogenic variants appeared in IGV as heterozygous state as half reads were generated from the active gene and half reads from the pseudogene that harboured the corresponding mutant allele. I2g variant was seen in homozygous form as there was no wild type allele present in the proband. MLPA Kit P050-C1 probes are complimentary to the sequences encompassing the pathogenic variants present in different exons, thus their ratios indicate deletions as well as zygosity of the variants present in the sample. In this patient, half ratios of the probes were wrongly interpreted as heterozygous variants. Since MLPA results were concordant with NGS results, all variants were reported without validating by Sanger sequencing. However, these ratios were actually indicating deletions in exons 1-7. Sanger validation in this patient could have avoided the erroneous interpretation.

The scenario for Patient 2 was completely opposite to that of Patient 1. In Patient 2, MLPA was first performed and heterozygous deletion of exon 3 to 7 was detected. Only after performing Sanger sequencing, the proband was found to harbour 4 pathogenic variants, [p.Ile172Asn, E6 cluster, p.Leu306+T, p.Glu319Ter], all in heterozygous state. Since there is no probe available for exon 8 in the MLPA Kit P050-C1 used, p.Glu319Ter a common pathogenic variant present in exon 8 was not picked up by MLPA. Therefore, one should keep in mind while analysing the MLPA results that half ratio (0.5) or zero ratio observed in any exon indicates heterozygous or homozygous deletion of the corresponding exon respectively. However, these ratios could also indicate the presence of heterozygous/ homozygous variant in that exon as seen in Patient 2. Thus, MLPA results should always be complemented with Sanger sequencing. On the contrary, whenever homozygous variants are detected by Sanger sequencing, MLPA should be done to verify whether the pathogenic variant is homozygous or hemizygous.

Therefore, for molecular analysis of the CYP21A2 gene, more than one method should be used for comprehensive analysis. For example, while performing PND for the I2g variant, microsatellite linkage analysis should also be performed in addition to direct DNA sequencing and MLPA, as this variant is known to have a high rate of allele drop out (Tsai & Lee, 2012)

Secondly, mutant alleles must be segregated in the parents to verify their presence on different alleles for correct interpretation of the molecular genetics results, as observed in Patient 2. In Patient 1, we could have missed carrier status of the father if we had not analysed the proband’s aunt. Duplications and deletions of the CYP21A2 gene are now being detected relatively frequently due to the use of MLPA, a valid alternative to Southern blotting. However, the interpretation of MLPA results requires extensive knowledge of CYP21A2 gene rearrangements (Concolino et al., 2009). Duplications have been reported to be quite frequent in Caucasians (Parajes et al., 2008), however, no data is available from Indian subjects. Duplications have great impact on the carrier status of an individual therefore they represent a significant pitfall in the molecular diagnosis of steroid 21-hydroxylase deficiency (Koppens et al., 2002). Hence, it is imperative to screen duplications in all couples referred for preconceptional carrier screening.


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