ObjectiveThe study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a singleCYP21A2gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functionalCYP21A2gene context (trimodular RCCX haplotype).Methods38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay.ResultsBoth MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a singleCYP21A2in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicatedCYP21A2with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3’-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH).ConclusionThe employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a singleCYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.