Supplementary Material for: Next-Generation Sequencing Identifies Different Genetic Defects in 2 Patients with Primary Adrenal Insufficiency and Gonadotropin-Independent Precocious Puberty

<b><i>Background:</i></b> The development of gonadotropin-independent (peripheral) precocious puberty in male children with primary adrenal insufficiency (PAI) is consistent with a defect in the genes encoding for the enzymes involved in steroid hormone biosynthesis. <b><i>Methods:</i></b> Two young boys presented with peripheral precocious puberty followed by PAI. In both patients, the analysis of <i>CYP21A2</i> gene encoding 21-hydroxylase was normal. As a second step, a targeted next-generation sequencing (NGS) was performed in both patients using a customized panel of congenital endocrine disor ders. <b><i>Results:</i></b> Case 1 had a new homozygous variant in the <i>CYP11B1</i> gene (c.1121+5G&gt;A). Mutations of this gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency, an essential enzyme in the cortisol biosynthesis pathway. Case 2 showed a new hemizygous mutation in the <i>NR0B1</i> gene (c.1091T&gt;G), which encodes for DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita [AHC] and critical region on the X chromosome gene 1). <i>NR0B1</i> mutations cause X-linked AHC<i></i> and hypogonadotropic hypogonadism. Pathogenicity prediction software defined both mutations as probably damaging. <b><i>Conclusions:</i></b> Peripheral precocious puberty was the atypical presentation of 2 rare genetic diseases. The use of NGS made the characterization of these 2 cases with similar clinical phenotypes caused by 2 different genetic defects possible.