ESPE Abstracts

Background: Steroid 11-β hydroxylase deficiency is the second most frequent cause of adrenal hyperplasia (CAH) with autosomal recessive inheritance. Girls have importantly virilized external genitalia at birth, and boys display precocious pseudopuberty. Unlike others enzymatic deficiency involved in CAH, there is no salt wasting during infancy but patients develop hypertension. Because of more than 90% of homologous sequence between CYP11B1 gene (11-β hydroxylase) and CYP11B2 gene (aldosterone synthase), Sanger sequencing remains the gold-standard method. Here, we report a patient with an 11-β hydroxylase deficiency in whom classical Sanger failed to identify a structural molecular variant, revealed by additional investigation using massively parallel sequencing (MPS) and an adapted Sanger technique.

Case: The patient is the second child of non-consanguineous parents, born with atypical external genitalia without palpable gonads (Prader IV), and karyotype 46,XX. The high level of 11-deoxycortisol (206.5 nmol/L) was pathognomonic of the 11-β hydroxylase deficiency (> 40 nmol/L). Surgery of genitals was undertaken, as well as a treatment by hydrocortisone and nicardipin.

Molecular Characterization: Sanger sequencing revealed only one heterozygous short indel (NM_000497.4:c.331_332delinsC ; p.(Met111Argfs*22)), inherited from the father. In the absence of identification of a second mutation, CYP11B1 was sequenced by targeted DNA sequencing (custom design SeqCap EZ (Roche NimbleGen®) on a Nextseq 500 (Illumina®)). The in-house bioinformatic pipeline also failed in the identification of a second variation. Alignments files visualization by IGV® revealed incompletely aligned and abnormally distant read pairs. By looking at the non-aligned base pairs, we identified a short intragenic inversion of 598bp, involving exons 5 and 6 (NM_000497.4:c.892_1121+7inv;1121+8_1121+9del). We designed a specific Sanger sequencing method flanking the breakpoint regions in the inverted allele to confirm the variant.

Discussion: We here report the first short intragenic inversion in CYP11B1. It may conduce to an aberrant truncated protein or absence of protein. Classical pre-sanger molecular amplification dropped out the inversion-carrying allele as did the bioinformatic pipeline used for MPS. In the face of evidence of a defect in CYP11B1 (based on clinical and hormonal data), the specific visualization of the reads obtained by MPS and imperfectly aligned on CYP11B1 was necessary to reveal the molecular mechanism missed by classical technologies. A specific strategy by Sanger sequencing allowed its characterization. Our analysis thus allow personalized genetic counseling.