Identification of a Homozygous Variant in the CYP21A2 Gene by Next-Generation Sequencing Analysis of Circulating Cell-Free Fetal DNA.

BACKGROUND/

OBJECTIVES

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by mutations in the CYP21A2 gene associated with 21-hydroxylase deficiency and increased levels of adrenal androgens. Affected females are at risk of ambiguous genitalia, while affected males show sexual precocity. Here, we present a case of a newborn female patient, characterized by ambiguous genitalia and previously identified as low risk for common aneuploidies by non-invasive prenatal testing (NIPT).

METHODS

We performed a NIPT, which showed a 46, XX genotype, confirmed by karyotype on the newborn's DNA extracted lymphocytes. For clinical suspicion of CAH, we performed reverse dot blot and Multiple Ligation-dependent Probe Amplification (MLPA) of the CYP21A2 gene on the patients and her parents' DNA. Then, we performed on mother's plasma NGS analysis with an in-house developed panel of genes for monogenic diseases, including the CYP21A2 gene.

RESULTS

Reverse dot blot and MLPA detected the presence of the c.290-13A/C>G (I2 splice) mutation in heterozygosity in the parents and in homozygosity in the child, respectively. NGS detected the c.290-13A/C>G (I2splice) mutation in cell-free fetal DNA (cfDNA) in mother's plasma with a variant allele frequency (VAF) of 67% with a fetal fraction (FF) of 5%. This latter suggests the presence of the variant both in the mother and in newborn cfDNA.

CONCLUSIONS

The study reinforces the hypothesis that cfDNA can be used to identify point mutations, small insertions and/or deletions for the diagnosis of monogenic diseases, reducing the number of invasive tests and the risk of early miscarriages. Early detection of mutations in genes causing sexual development disorders could make it possible to start therapy in the womb.