A total of 72 subjects (49 paediatric and 23 adults) were included in the study, of whom 66 subjects were clinically suspected of having 21-OH deficiency while six were suspected for 11β -OH deficiency. Sixty-seven were from the southern part of the country, and five were from northern India, with 32 males and 40 females.
Among the subjects with 21-OH deficiency, 60.6% (n=40) were of Salt-Wasting (SW) phenotype, 31.8% (n=21) with Simple Virilizing (SV) phenotype and 7.6% (n=5) with Non-Classical (NC) CAH. The age of diagnosis varied from 1 to 95 days in SW type, two weeks to 8 years in SV type, and 3.5 to 26 years in NC CAH. The consanguinity proportions were 42.5%(SW), 30%(SV), and 50%(NC). The mean basal 17-OHP values were 127.9, 36.1, and 21 ng/ml in SW, SV, and NC phenotype respectively. Subjects with SW phenotype presented with mean sodium of 126.4 meq/l and mean potassium of 7.1 meq/l. A short synacthen test confirmed poor cortisol response among 17 patients with SW phenotype. In four males and two female subjects suspected of 11β -OH deficiency (5 Paediatric and 1 adult), the age of diagnosis varied from 2.5 to 14 years with a mean basal 17-OHP of 10.7 ng/ml, and 40% of the patients were born of consanguineous marriages.
Long range PCR and MLPA
The long-range PCR yielded specific amplification of the functional and pseudogene in 62/66 samples with appropriate restriction digestion patterns. Subjects C15, C62, and C71 had only the functional gene amplified with a restriction digestion pattern of the pseudogene. On the other hand, subject C29 with only the pseudogene amplification gave a restriction digestion pattern of the functional gene. Based on these results, MLPA was carried out, which showed a homozygous large deletion involving 5’ of CYP21A1P and 3’ of CYP21A2 in subjects C15, C62, and C71. These three subjects were homozygous positive for all eight hotspot mutations in ASPCR. MLPA results also confirmed a large gene conversion involving 5’ of CYP21A2 and 3’ of CYP21A1P in subject C29.
Allele Specific PCR for hotspots screening
Utilizing the in-house designed ASPCR approach, CYP21A2 hotspot mutations were identified in 55/62 subjects - 33 SW, 19 SV, and 3 NC CAH. Out of 33 subjects with SW phenotype, 25 (75.8%) had biallelic mutations, seven (21.2%) had multiple heterozygous mutations, while one subject (C3) was positive only for a heterozygous Q318X mutation. Among the 19 subjects with SV phenotype, 10 (52.6%) had biallelic mutations, eight (42.1%) had multiple heterozygous mutations, and one subject (C33) was heterozygous for the 8BP deletion in exon 3. Out of five non-classical subjects, two were positive for biallelic mutations (40%) and one subject (C65) had a single heterozygous V281L hotspot mutation. The remaining seven subjects were negative for ASPCR.
Next Generation Sequencing for a targeted panel of 5 genes in CAH
The NGS assay for a single gene CYP21A2 was carried out for all the samples with 21-OH deficiency - both positive and negative for ASPCR. Five gene NGS panel was utilized for those subjects negative for ASPCR and those with suspected 11β -OH deficiency. The Multiplex PCR - NGS assay covered the complete coding and splice site regions of five genes included in the panel. The mean base coverage for five genes was 700X and >99 % of the target had a minimum coverage of 20X. Further, with NGS, no additional samples were positive for the eight hotspot mutations corroborating the sensitivity and specificity of the ASPCR assay.
Additional homozygous CYP21A2 variants were identified in five out of seven subjects who were negative for ASPCR. These variants include c.1451G>C(p.Arg484Pro) in three subjects, c.143A>G(p.Tyr48Cys) in one subject and a novel c.1274G>T(p.Gly425Val) variant in one subject
Interestingly, subjects C31 and C32 with SW phenotype and subject C47 with SV phenotype were positive for homozygous CYP21A2:c.1451G>C variant. The younger sibling of subject C31 with a similar clinical presentation of SW phenotype was also homozygous positive for this recurrent variant. This variant has been previously reported in patients with both SW and SV phenotype [14, 15]. Subject C3, one year old female with SW phenotype, was heterozygous positive for Q318X mutation identified through ASPCR and heterozygous positive for CYP21A2:c.1042G>A(p.Ala348Thr) variant identified through NGS. This subject was compound heterozygous for the above variants and inherited the Q318X mutation from the mother and A348T variant from the father. In subjects C33 and C65, who only had single heterozygous hotspot mutation identified in ASPCR, no additional CYP21A2 variants were identified through NGS. Subject C63 with a non-classical phenotype was negative for variants in all the five genes screened.
Subject C46, who was initially suspected of having a 21- OH SV phenotype, was negative for mutations in the CYP21A2 gene but was found to be positive for an aromatase gene CYP19A1:c.1142T>A(p.Asp381Val) variant. Born to second-degree consanguineous parents, this subject had ambiguous genitalia and clitoromegaly at birth with an elevated 17-OHP of 15.4 ng/ml. Deficiency in the aromatase enzyme due to CYP19A1 mutations has been reported to cause ambiguous genitalia in 46XX females . A recent report has also shown that aromatase deficiency can mimic SV CAH . The majority of the in silico tools support a pathogenic prediction, and only two heterozygous alleles have been reported in South Asians so far (MAF - 0.0000653, GnomAD exomes). However, there is a need for further investigations and family screening to confirm its pathogenicity, and based on ACMG 2015 guidelines, this variant has been classified as a Variant of Uncertain Significance (VUS).
Out of six subjects with suspected 11β -OH deficiency, four had homozygous, and two had compound heterozygous variants; four novel and two reported variants were identified in total (CYP11B1:c.1201-1G>A, c.1200+1delG, c.412C>T, c.623G>A, c.1024C>T, and c.1012dupC). Subject C56, an eight year old female born of second degree consanguineous marriage, presenting with hyperpigmentation, clitoromegaly, and hypertension, was homozygous positive for CYP11B1:c.412C>T (p.Arg138Cys). In vitro studies of this variant had demonstrated partially impaired CYP11B1 activity . Subject C53 is a 13-year-old male, presenting with hypertension and hypokalemia from 2.5 years of age and was found to be compound heterozygous for two reported missense variants CYP11B1:c.412C>T (p.Arg138Cys) and CYP11B1:c.623G>A (p.Arg208Gln). Subject C54, a 12-year-old male, with hypertension and hyperpigmentation from five years of age, was compound heterozygous for a novel nonsense mutation CYP11B1:c.1024C>T (p.Gln342Ter) and a novel duplication CYP11B1:c.1012dupC (Gln338ProfsTer16) resulting in a frameshift at codon 338 followed by premature termination at codon 353 instead of 504.
Two unrelated subjects born to second degree consanguineous parents (C51 and C55) were homozygous positive for a novel splice variant CYP11B1:c.1201-1G>A. Subject C51, a 19-year-old male, presented with precocious puberty at two years of age, followed by recurrent hypokalemic paralysis and hypokalemic cardiomyopathy. Subject C55 had enlarged clitoris from birth and hypokalemic paralysis with hypertension at four years of age. Subject C52, presenting with hypertension, true puberty, and aggressive behavior, was positive for a novel homozygous splice variant CYP11B1:c.1200+1delG. The in silico analysis for these splice variants predict aberrant splicing and requires functional assays to confirm its pathogenicity.
No mutations were identified in the CYP17A1 and POR genes. The details of the individual variants identified through NGS assay are mentioned in Table 2a and 2b, and the complete workflow with the results is depicted in Fig 2.
Family screening was carried out using Sanger sequencing and the carrier status was confirmed in 18 out of 21 available paediatric parental samples. De novo homozygous mutations were identified in three probands as their parents were negative for those mutations. Family screening is incomplete in the other study subjects due to the unavailability of one or both parental samples.
Majority of the subjects with SW phenotype in this study had null or group A mutations which are known to result in <1% of the enzymatic activity, and I2G hotspot was the predominant genotype identified. The SV phenotype had most of its genotype falling in group B (1-2 % enzyme activity), with the predominant genotype being I172N. Two out of five NCCAH subjects were positive for I2G mutation in the homozygous state under Group A. All the subjects with null mutations presented with SW phenotype. 9% of the classical subjects were positive for P30L, which usually is predicted to result in NCCAH. I2G, a SW genotype was identified in 28% of the subjects with SV phenotype. I172N, usually reported in SV phenotype was seen in 16 % of the subjects with SW phenotype. Eleven subjects had multiple homozygous and heterozygous mutations, indicating smaller gene conversions involving multiple exons that are frequently observed in 21-OH deficiency . The frequency of the mutated alleles and their associated phenotype is depicted in Fig 3. Table 3 explains the different groups of genotypes identified and their correlation with clinical phenotypes.