Comprehensive Overview of Congenital Adrenal Hyperplasia and its Genetic Diagnosis Among Children and Adolescents

CASE SCENARIOS

Case 2

A 27-year old primigravida who was diagnosed with CAH at 2.5 years of age presented for consultation after confirming her pregnancy at 6 weeks gestation. She was born to second-degree consanguineous parents. As a toddler, she was evaluated for failure to thrive with recurrent vomiting when she was noted to have clitoromegaly with normal blood pressure, and biochemical tests showed elevated 17OHP of 630 ng/mL (ref 0–1.1). Electrolytes were normal (sodium 135 mmol/L, and potassium 5.5 mmol/L) with normal plasma renin activity (PRA) 9.9 µIU/mL/h (ref 4.4–46.1). Serum dehydroepiandrosterone-sulfate (DHEAS) 21 µg/dL (ref 6–79) was also in the normal range. She was diagnosed with CAH at 2.5 years of age and initiated treatment on oral hydrocortisone and low dose fludrocortisone. Her weight centile improved dramatically on treatment, and she underwent a clitoral reduction surgery at 7 years of age. She had regular follow-up, attained menarche at 11.5 years of age and was otherwise well, attaining a final adult height of 154 cm. Her care was transitioned from pediatric to adult endocrinology services and she had good control throughout.

At this visit after confirming her pregnancy, a detailed discussion was held with the family and genetic testing was undertaken and her treatment was changed to dexamethasone. Her mother was homozygous positive for CYP21A2 c.518T>A (p.I172N) missense change which is classified as group B of CAH mutations with 2% enzyme activity. Her husband was a heterozygous carrier for 30 kb deletion carrying multiple pseudogene-derived mutations on one allele. The chorion villus sample (CVS) on the fetus done at 12 weeks of gestation confirmed the heterozygous carrier state of CYP21A2: c.518T>A (p.I172N) on one allele. The karyotype was 46,XX on the fetus [Figure 1]. After the availability of these reports, dexamethasone was changed back to hydrocortisone. The baby was well after birth and further genetic testing on the newborn sample confirmed the carrier status of the baby.

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Figure 1:

Sanger chromatogram showing CYP21A2: c.518T>A (p.I172N) in homozygous state in the mother (proband) and heterozygous state in the fetus. CVS: Chorion villus sample.

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INTRODUCTION

CAH comprises an extensive group of autosomal recessive disorders that result from a defect in the adrenal steroidogenesis pathway.^[1] 21-Hydroxylase deficiency (21OHD) is the most common form of CAH and one of the most common inborn errors of metabolism. It is estimated that 90–99% of CAH results from this single enzyme deficiency which causes cortisol and aldosterone insufficiencies.^[2,3] In this article 21OHD form of CAH alone is being discussed and hence used interchangeably with CAH. The clinical result of this deficiency is life-threatening salt wasting which needs to be identified early and managed effectively for a good outcome. The deficiency occurs from mutations in the CYP21A2 gene, the majority of which occur due to the unequal crossing over and gene conversion events between the functional CYP21A2 gene and non-functional CYP21A1P pseudogene. There are more than 230 pathogenic variants known to cause the biochemical deficiency posing a unique challenge in its genetic diagnosis.^[4]

In this article, we will try and answer some of the common questions regarding CAH and aspects of its genetic diagnosis.

WHAT ARE THE COMMON FORMS OF 21OHD PRESENTING DURING CHILDHOOD AND ADOLESCENCE?

CAH from 21OHD is classified into three groups—classic salt wasting (SW-CAH), classic simple virilizing (SV-CAH), and non-classic (NC-CAH).^[5] The combined estimated incidence of classic 21OHD is 1 in 14,000–18,000 live births, with approximately 75% classified as SW-CAH and 25% classified as SV-CAH. NC-CAH is more common than the classic forms, with an estimated incidence of 1 in 200.^[6]

In SW-CAH, there is little or no residual enzymatic activity, resulting in extreme cortisol and aldosterone deficiencies. In addition, there is excess adrenal androgen synthesis as increased precursor steroids are diverted to the unaffected androgen pathways. If not recognized or treated, these infants present with “salt wasting crises” characterized by hyponatremia, hyperkalemia, acidosis, and shock.

The SV-CAH group with residual enzyme activity of 1–5% has slightly more aldosterone and hence is less likely to suffer acute salt wasting crisis. They present with varying degrees of virilization in girls and hyperandrogenic features in boys due to markedly elevated adrenal androgen production.^[4] They also have cortisol deficiency necessitating treatment.^[2] The genital appearance of affected 46,XX infants can give the appearance of male genitals with penis and scrotum but empty of gonads, an important clinical finding that confirms virilization. Müllerian duct formation is preserved although abnormality in urogenital sinus formation results in varying degrees of conjoined urethra and vagina. Thus, the reproductive potential exists in females despite atypical external genitalia.

In NC-CAH, the residual enzyme activity of 20–50% is noted resulting in late-onset or non-classical presentation. These children have normal basal cortisol and aldosterone production with mildly increased adrenal androgens. But nearly one-third still demonstrate suboptimal cortisol response on ACTH stimulation.^[5] Children in this group present with symptoms from hyperandrogenism such as premature adrenarche, acne, and advanced skeletal maturation but many, especially males, are asymptomatic. Adolescent girls or adult women may present with hirsutism, oligomenorrhea, acne, and subnormal fertility.^[5] The estimated prevalence of this milder type is nearly 1 in 200 in the Caucasian population.^[6]

Table 2 summarizes the age-wise clinical pointers to a presumptive diagnosis of CAH.

WHAT DO WE NEED TO KNOW ABOUT NEWBORN SCREENING (NBS) FOR CAH?

WHAT IS THE DISEASE BURDEN OF CAH AND THE SCOPE OF NBS FOR CAH IN INDIA?

Although the exact disease burden of CAH in India is unclear, a recent study by the Indian Council of Medical Research (ICMR) Taskforce is suggestive of a significant burden in our country. In this large multicentric study, 18 of the 104,066 neonates screened were positive for CAH. There was a striking regional preponderance noted in South India with a 1 in 2036 prevalence as compared to 1 in 9983 in the western part of India.^[19] Similar data have been confirmed by other Indian studies as well.^[20] This could possibly be due to the high rate of consanguinity noted in South Indian population.

In our country, there are nearly 70,000 births every day with an annual birth rate of 17.3/1000 reported in 2021. CAH qualifies for newborn screening in Indian context as it is a prevalent disease with treatment available and timely treatment being able to prevent significant sequelae, fulfilling the Wilson and Jungner criteria. The practical and logistic difficulties with enormous births and significant number having home deliveries remains a huge challenge as highlighted extensively by Dabas et al.^[21]

WHAT ARE THE DIFFERENT TESTS/ TECHNOLOGY AVAILABLE FOR GENETIC DIAGNOSIS OF CAH?

Although baseline and ACTH-stimulated 17OHP measurements are routinely used as the biochemical markers for CAH, these results are often unreliable with false positivity requiring secondary confirmation with LC-MS and so far, cannot specify whether an individual is a homozygote or heterozygote.^[22] Therefore, genetic testing in CAH is highly advantageous to confirm the diagnosis of CAH and provide carrier screening. Genotyping CYP21A2 gene, implicated in 21OHD, the most common form of CAH is known to be extremely challenging due to a highly homologous pseudogene CYP21A1P present in proximity. Most of these mutations are pseudogene-derived due to meiotic crossing-over errors and gene conversion events. To overcome these challenges, several molecular techniques have been adapted in clinical and research setting.

The initial process is to amplify the functional gene without co-amplification of the pseudogene. This is usually achieved with long range PCRs for locus specific amplification of CYP21A2 gene that has consistently proven to be specific across several studies.^[23,24] Following this, large 30 kb deletion and rearrangements can either be picked up with specifically designed long range PCRs or multiplex ligation-dependent probe amplification (MLPA). If these large deletions and rearrangements are ruled out, pseudogene-derived common mutations such as P30L, I2G, 8BPDEL, I172N, E6 CLUS, V281L, Q318X, and R356W can be identified [Figure 2] with genotyping techniques like ASPCR, that utilizes primer sets with 3’ terminal mismatches for wild type and mutant allele, restriction digestion with restriction enzyme specific for these mutations, real-time PCR based assays, and sequencing of the hotspot mutation regions. If negative for these hotspot mutations, sequencing the entire gene with Sanger or next generation sequencing (NGS) is carried out.

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Figure 2:

CYP21A2 gene with the most common pseudogene-derived point mutations. The exons are shown in dark grey boxes numbered from 1 to 10. The protein change of these mutations is marked on the exonic regions in the different zones of phenotypic severity that corresponds to the retained enzymatic activity depicted on the Y axis. The single nucleotide polymorphism database ID of these mutations is mentioned below the horizontal line. SW: Salt wasting, SV: Simple virilizing, NC: Non-classic.

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The molecular complexity of CYP21A2 gene analysis is that one single technique cannot narrow down to the actual extent of mutation in 21OHD.^[25,26] While long range PCRs are efficient in amplifying the functional gene and to rule out large rearrangements, genotyping techniques are limited to the hotspot mutations. MLPA used for gene deletions and duplications cannot provide the actual extent of chimeras that arise due to rearrangements between CYP21A2 and CYP21A1P gene.^[27]

Several studies in India have been published regarding the molecular techniques used in CAH diagnosis as highlighted in Table 3.^[28-33] At Christian Medical College and Hospital, Vellore, we used comprehensive approach ASPCR followed by NGS in those affordable or negative on the ASPCR.^[28] This method helped diminish costs tremendously such that we are currently offering the ASPCR for Rs. 2500 (INR) only.

On identification, understanding the extent and severity of these mutations is required to correlate clinically and optimize therapeutic measures. Based on this experience and published data with excellent diagnostic yield, we would recommend a stepwise comprehensive approach with multiple techniques to delineate the underlying genetic cause and enable effective family screening [Figure 3]. A combination of long-range PCR with one of the genotyping assays can identify large rearrangements and point mutations along with specific chimeras. On the other hand, a primary requisite for NGS analysis of CYP21A2 gene is the development of computational pipelines with stringent filters that will restrict alignment of the reads to the reference region of CYP21A2 gene and not CYP21A1P gene.

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Figure 3:

Algorithmic representation of genetic tests that can be applied in a stepwise manner for comprehensive and cost-effective genetic diagnosis of congenital adrenal hyperplasia.

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HOW DO WE CHOOSE A SPECIFIC GENETIC TEST/TECHNIQUE FOR A GIVEN CHILD WITH SUSPECTED GENETIC FORM OF CAH?

Based on the clinical suspicion and biochemical findings, if the subject presents with a classical salt wasting or simple virilizing form, the first step is to identify pseudogene-derived long rearrangements and point mutations. 85–95% of the subjects can be genetically confirmed of 21OHD at this point. This is also cost effective and less time consuming in a developing country like India where many can still not afford single or multigene sequencing for CAH.

If negative, proceed with sequencing the complete CYP21A2 gene. For subjects with non-classic forms of CAH, or subjects with other forms of CAH, followed by the first step, a targeted NGS panel of the most implicated genes— CYP11A1, CYP11B1, CYP17A1, HSD3B2, POR and StAR can be utilized. If found negative for these genes, a clinical exome or a whole exome sequencing can be considered. The flow chart explains the proposed detailed algorithm of CAH genetic testing that has been in use in our clinical setting that has proven to be comprehensive and cost effective for Indian scenario [Figure 3].

IS THERE A SPECIFIC GENOTYPE-PHENOTYPE CORRELATION WITH VARIOUS GENETIC FORMS KNOWN SO FAR?

CAH being an autosomal recessive disorder, the phenotype severity is dictated by the allele carrying the least severe mutation. Typically, we classify CAH to be classic (either SWCAH or SV-CAH) and NC-CAH. We know that complete inactivation results in SW-CAH phenotype, enzyme activity of around 2% causes SV-CAH phenotype and activity between 10% and 75% results in NC-CAH phenotype.^[34]

Excellent work by New et al. on a heterogenous population brought in a lot of light to the genotype-phenotype correlations in CAH.^[35] By computational modeling humanizing the crystal structure of bovine CYP21A2 was established and it was noted that mutations affecting critical enzyme functions like membrane anchoring, heme binding, substrate binding, or that alter enzyme stability caused a complete non-functionality of the enzyme resulting in SW-CAH. On the other hand, mutations that affected the transmembrane region or conserved hydrophobic patches resulted in about a 98% reduction in enzyme activity and caused SV-CAH. Alteration in oxidoreductase interactions, salt-bridge and hydrogen bonding networks, and nonconserved hydrophobic clusters resulted in the milder NCCAH. This data established genotype-phenotype correlation comprehensively and has been highlighted in the text box below [Box 1].

Further, well established in vivo studies have described the effect of these pseudogene-derived hotspot mutations.^[36] Based on the extent of loss of the enzyme activity, the mutations have been classified into four groups.^[37]

• The Null group involving large deletions, gene conversion, point mutations such as E6 cluster in exon 6 (I235N, V236E, and M238K), 8 bp deletion in exon 3, p.Gln319Ter (Q318X) and p.Arg357Trp (R356W) in exon 8 result in zero enzyme activity leading to SWCAH phenotype

• Group A consists of the intron 2 splice mutation (I2G) that results in 0–1% of the enzyme activity also expected to result in SW-CAH phenotype

• Group B with p.Ile173Asn (I172N) in exon 4 results in 1–2% of the enzyme activity usually result in SV-CAH phenotype

• Group C consists of p.Pro31Leu (P30L) in exon 1, p.Val282Leu (V281L) in exon 8 and p.Pro454Ser (P453S) in exon 10 with 20–60% enzyme activity and lead to NCCAH phenotype.

Around 90% of the cases show good genotype-phenotype correlation in 21OHD.

Although the nature of these mutations has been well described, there exists a genotype-phenotype discordance in some of the cases in particular to I2G and I172N mutations that alternate phenotypes between SW-CAH and SV-CAH.^[26] P30L, on the other hand, has been reported to result in SV-CAH forms in few cases. For the other non-pseudogene-derived mutations (>230 in HGMD database) establishing clear genotype-phenotype correlations is an enormous task often requiring molecular modeling functional investigations.

WHICH ARE THE STANDARD LABORATORIES OFFERING GENETIC DIAGNOSIS OF GENETIC FORMS OF CAH IN INDIA? WHAT IS THE APPROXIMATE COST?

Several common private labs offer genetic testing of CAH, the names have been kept anonymous for the purpose of this article. The approximate cost of the genetic tests for the diagnosis of CAH is given in [Table 4].

Institute of Genomics and Integrative Biology, New Delhi, Centre for Human Genetics, Bangalore, Institute of Medical Genetics at Sir Gangaram Hospital, New Delhi and Christian Medical College, Vellore, are few of the other leading organizations offering genetic testing for congenital adrenal hyperplasia. In CMC Vellore, ASPCR for the common 8 mutations is offered at Rs. 2500 (INR).

HOW DO WE COUNSEL THE FAMILY WHEN A CHILD IS DIAGNOSED WITH A GENETIC FORM OF CAH?

Genetic counseling should be provided to parents at the time of birth of a child with CAH as well as adolescents at the time of transition to adult care, according to the Endocrine Society Clinical Practice Guidelines.^[38]

Heterozygotes cannot be distinguished from the general population using biochemical assays. Thus, genotyping is helpful for genetic counseling and is a useful tool for determining the carrier status in an individual.^[35]

Several crucial aspects should be considered when providing genetic counseling:

1. Genetic testing should be performed in a reputable lab and reports analyzed by a qualified professional

2. Pre-conception period is the optimal time for determination of the genetic risk, carrier status, or treatment

3. Testing of reproductive partners of probands for carrier status is recommended when one or both disease-causing mutations have been identified in the proband

4. Before testing, caregivers should be fully informed about the risks and benefits. They should be aware of the prognosis, morbidity, mortality, and potential effects of a positive genetic screen on the patient or their family.^[39]

WHAT IS THE ROLE OF GENETIC DIAGNOSIS IN PREMARITAL, PRE-PREGNANCY, ANTENATAL, AND PERINATAL GENETIC COUNSELING?

TREATMENT OF GENETIC FORMS OF CAH – DOES IT MAKE A DIFFERENCE?

Specific genotypes have been proven to be associated with SW, SV, or NC forms of CAH, with an excellent genotypephenotype correlation. With less severe disease, the genotype-phenotype link becomes less significant.^[45]

Gene deletions and large conversions—8 bp deletion, E6 cluster, p.Gln319Ter (Q318X) and p.Arg357Trp (R356W) are commonly associated with classic SW-CAH, the most severe form of the disease, warranting treatment with glucocorticoids and fludrocortisone. Missense pathogenic variant I172N (p.[Ile173Asn]) confers around 1–2% 21-hydroxylase activity. This produces nearly normal aldosterone production, thereby leading to the SV-CAH necessitating only glucocorticoid supplementation. p.Pro31Leu (P30L), p.Val282Leu (V281L) and p.Pro454Ser (P453S) are pathogenic variants commonly associated with NC-CAH.^[46]

Therefore, CYP21A2 genotyping of the fetus is predictive of his/her clinical outcome. As a result, many institutions provide CYP21A2 genotyping in conjunction with prenatal diagnosis and dexamethasone therapy.^[45] Dexamethasone is given to pregnant woman beginning in the 6^th–7^th week of gestation as part of the prenatal therapy of this illness to ameliorate the symptoms of virilization in affected female fetuses.^[47] However, it is crucial to note that prenatal therapy is still viewed as experimental because of conflicting information, notably concerning the treatment’s outcome and the safety of both mothers and their unborn children.^[45] Since the discovery of cell-free fetal DNA in the mother’s bloodstream, it is now possible to identify the gender of the fetus by the seventh week of pregnancy. Thus, adopting this novel technique could potentially prevent treating male babies and healthy female fetuses while partially addressing some ethical issues with prenatal therapy.^[39] Preimplantation genetic testing in conjunction with in vitro fertilization techniques have been attempted in at-risk pregnancies. Only genetically transferable embryos — those free of the disease — are transferred to the woman’s uterus after molecular genotyping and haplotyping procedures. This limits the spread of a disease within a family.^[44]

Many of these have internal ethical concerns and the scope of this paper does not allow to address that. The authors have highlighted the possibilities contextually and do not confirm to the use of genetically transferrable embryos. Antenatal gender identity again will have legal bearing based on the country of residence and hence the experimental dexamethasone therapy remains as a decision made on a case-to-case basis by an experienced endocrinologist after detailed discussion with family and considering the local practices and rules.

CONCLUSION

1. There is significant burden of CAH in India as highlighted by the recent newborn screening data published by the ICMR Taskforce

2. Genetic testing in 21OHD is challenging and complex due to a highly homologous pseudogene CYP21A1P present near the to the CYP21A2 gene

3. An algorithmic approach to genetic testing utilizing multiple molecular techniques as highlighted in Figure 2 will help in comprehensive and cost-effective analysis, a felt need in our country

4. The common genetic mutations can be classified as Null and A-D based on functional enzyme studies. This awareness will help clinicians tailor therapy accordingly

5. Majority of the mutations in the severe group SW-CAH show excellent genotype-phenotype correlation

6. Genetics do not depose biochemical testing, rather add an additional benefit of phenotypic prediction, and assist with genetic counseling.