First intragenic inversion of CYP11B1 gene causing 11β-hydroxylase deficiency: a molecular diagnosis easily...

11β-hydroxylase deficiency (11βOHD) is the second most common cause (5%) of congenital adrenal hyperplasia (CAH). The CYP11B1 gene shares 95% of genomic sequence homology with CYP11B2, and therefore Sanger sequencing remains the gold standard. We present a case of 11βOHD due to an intragenic inversion in CYP11B1 that was missed by both the Sanger sequencing and massive parallel sequencing (MPS) methods. The child was born with virilised genitalia at Prader stage 4 and the biological findings showed a hydromineral retention pattern and a pathognomonic increase in steroid precursors suggestive of 11βOHD. Standard trio analysis revealed only one heterozygous pathogenic variation inherited from the father. The study using MPS showed similar outcomes. Careful observation of the alignment BAM files revealed breaks in sequencing depth, incomplete alignments and systematic paradoxical read-pairs orientation. A specifically designed amplification and Sanger protocol confirmed the novel NM_000497.4(CYP11B1):c.[892_1121+7 inv;1121+8_1121+9del]; p.(Glu298HisfsTer113) variant at heterozygous state in the proband and his mother, fulfilling the diagnosis. The present case reports the first short intragenic inversion in CAH and illustrates the pitfalls that must always be kept in mind when using sequencing methods. When the phenotype is unequivocal, a thorough investigation of the locus should be carried out with cross-use of different techniques.