Molecular analysis improves newborn screenings for congenital adrenal hyperplasia

BOSTON — Molecular testing served as a useful tool in identifying false-positive results for congenital adrenal hyperplasia in newborns in Brazil, helping to prevent unnecessary follow-up testing after inconclusive results, according to study findings presented here.

“We found that [the molecular test] was very useful in symptomatic patients who presented with high [17-hydroxyprogesterone] levels,” Mirela Costa de Miranda, MD, a professor at University of Sao Paulo, Brazil, told Endocrine Today. “We could rule out the classical form [of congenital adrenal hyperplasia] and prevent unnecessary treatment that prolongs the follow-up in these patients.”

Mirela Costa de Miranda

Costa de Miranda and colleagues analyzed DNA samples from 86 infants who tested positive for congenital adrenal hyperplasia (CAH) during newborn screenings between 1999 and 2014 in Goias, Brazil. Researchers performed molecular analysis of the DNA samples and measured 17-hydroxyprogesterone (17OHP) levels with immunofluorometric assay, with cutoffs adjusted for birth weight. Confirmatory tests included serum 17OHP, androstenedione, testosterone and cortisol measurements. Researchers used allele-specific polymerase chain reaction and large gene rearrangements by MLPA technique to determine CYP21A2 genotypes; when needed, researchers performed entire gene sequencing.

Researchers found that 40 of the 86 infants who tested positive had classical forms of CAH (21 boys); 33 of the 40 had the more severe salt-wasting CAH. Within the cohort, 22 infants carried the I2 splice as the less severely affected allele; 16 of the 22 presented with slight or moderate hypernatremia in the first days of life; precocious treatment was introduced in the remaining six infants and clinical form distinction was not possible.

All patients carrying the p.I172N mutation, in the less affected allele, had the simple virilizing form of CAH. Genotyping identified mutations in 100% of classical alleles. Among the 30 asymptomatic newborns (15 boys) with persistently increased serum 17OHP levels, genotyping predicted the nonclassical form in 15, and the other 15 with non-affected genotypes (including 6 heterozygotes) were discharged.

The mean 17OHP level in classic CAH patients (confirmed by genotyping) was 327 ng/mL vs. 137 ng/mL in nonclassical newborns. Researchers found that affected newborns exhibited mutations derived from gene conversion events in 89% of the alleles; the most frequent were I2 splice (35%), p.Q318X (23%) and p.R356W (19% of alleles). Mutations not derived from pseudogene were found in 11% of the alleles.

The researchers noted that a good genotype/phenotype correlation was helpful in predicting clinical forms of CAH in asymptomatic newborns.

“We have to implement [newborn screening programs] in Brazil,” Costa de Miranda said. “We have to think about the best way to do this across the whole country without increasing costs. Molecular tests are expensive, but you rule out the false-positive results.” – by Regina Schaffer

Reference :

Costa de Miranda M, et al. PP08-3. Presented at: The Endocrine Society Annual Meeting; April 1-4, 2016; Boston.

Disclosure: Costa de Miranda reports no relevant financial disclosures.