Neonatal Screening for Congenital Adrenal Hyperplasia in Indian Newborns with Reflex Genetic Analysis of 21-Hydroxylase Deficiency

Abstract

Congenital adrenal hyperplasia (CAH), screened for in neonates, is the second most common endocrinopathy after congenital hypothyroidism.Newborn screening for CAH due to CYP21A2 deficiency is performed by immunologic assay for 17-hydroxyprogesterone (17-OHP). The second-tier test for confirmation of diagnosis is carried out on recall venous blood sample from screen positives measuring 17-OHP, or other metabolites of steroid metabolism by liquid chromatography–tandem mass spectroscopy. However, as steroid metabolism is dynamic, it can affect these parameters even in the recall sample of a stressed neonate. Moreover, there is some time delay in recalling the neonate for repeat testing. Reflex genetic analysis of blood spot from the initial Guthrie cards of screen positive neonates, if used for confirmatory testing, can avoid this time delay as well as the effect of stress on steroid metabolism. In this study, we used Sanger sequencing and MLPA in a reflex manner for molecular genetic analysis to confirm CYP21A2-mediated CAH. Out of 220,000 newborns screened, 97 were positive on the initial biochemical screen, of which 54 were confirmed true positives with genetic reflex testing, giving incidence of CAH as 1:4074. Point mutations were more common than deletions, indicating that Sanger sequencing should be used ahead of MLPA for molecular diagnosis in India. Amongst the variants detected, the most common was I2G-Splice variant (44.5%), followed by c.955C>T (p.Gln319Ter) (21.2%); Del 8 bp and c.-113G>A were detected with frequencies of 20.3% and 20%, respectively. In conclusion, reflex genetic testing is an effective strategy for identifying true positives in CAH screening in neonates. This will obviate need for recall samples and also aid effective counselling and timely prenatal diagnosis in the future. In Indian newborns, as point mutations are more common than large deletions, Sanger sequencing should be the initial method of choice for genotyping, ahead of MLPA.

Current Status of Newborn Bloodspot Screening Worldwide 2024: A Comprehensive Review of Recent Activities (2020–2023)

“…Reviewing reports from 2020 until now, 2 multi-condition studies have been reported: a 3-year study of CH, CAH, G6PDD, GAL, and PKU in Bengaluru, South India [ 517 ]; and a Canadian collaboration study of CH, CAH, G6PDD, GAL, PKU and BIO in Udupi district of South India [ 518 ]. Reports continue regarding NBS for endocrinopathies: CH screening, prevalence, and etiology in Indian preterm babies [ 519 ]; a report of confirmatory testing from the original DBS to speed diagnosis of CAH (assuming that the DBS and patient match) [ 520 ]; a review of the challenges and opportunities resulting from NBS for CAH [ 521 ]; a case study of a newborn with unnecessary devastating consequences from CAH resulting from no NBS [ 522 ]; a standard operating protocol for routine NBS for CAH in Indian settings [ 523 ]; and a discussion of genetic confirmatory testing for CAH from the original DBS as a means of speeding the diagnostic process [ 520 ].…”

Congenital Adrenal Hyperplasia – A Comprehensive Review of Genetic Studies on 21-Hydroxylase Deficiency from India

“…The risk can be much higher due to the high rates of consanguinity in the country. [ 55 ] Thus, genetic diagnosis in affected probands and carrier testing in their partners before planning pregnancy will be extremely beneficial. Continuous follow-up of both the partners being carriers or CAH-affected individuals marrying a carrier will benefit in better management of CAH-associated pregnancies and drastically reduce the incidence of CAH in India.…”

“…To overcome the challenges with 17-OHP testing by LC-MS/MS, genetic testing for the most common mutations (screening for hotspot mutations) can be considered in those with elevated 17-OHP on first-line NBS with a turnaround time of 5–7 days. [ 55 ] This can be a suitable alternative for the second-tier LC-MS for accurate and cost-effective diagnosis of 21-OHD.…”