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 DubeyEmail: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.
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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