In the second kindred, a 6-year-old child (patient 4) with female phenotype presented for incidentally diagnosed increased blood pressure during a school health check. Her weight and height were 49 kg (3 SDs above the mean) and 136 cm (2 SDs above the mean), respectively. The bone age of patient 4 was approximately 6 years. In kindred 3 (patient 5), the parents noted that the child had with a vulva, but no vagina. However, testicular masses were palpable in the respective inguinal regions. The child underwent surgical exploration, which revealed that a vagina was absent and right and left masses were confirmed to be testes.
Blood tests were performed according to standard methods. Blood hormone, Adrenocorticotropic hormone (ACTH), plasma renin activity (PRA), aldosterone (ALD), 17alpha-hydroxyprogesterone (17OHP) AMH (anti-Mullerian hormone), and inhibin B (INB) were measured by chemiluminescence; cortisol (F) was measured by electrochemiluminescence; dihydrotestosterone (DHT), and androstenedione (AD) were measured by liquid chromatography-mass spectrometry; dehydroepiandrosterone (DHEA), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), testosterone (T), progesterone (P), and prolactin (PRL) levels were measured by enzyme-linked immunosorbent assay (Table 1).
With the consent of the patient's parents, we collected the peripheral blood of the patients and their parents in three families. In addition, in family 1, peripheral blood of the proband's sister was collected. Later, DNA was extracted, next-generation sequencing (NGS) sequencing was performed for the probands. The observed variants in the probands were confirmed and the family members were screened for the respective variants by Sanger's method. A minimum of 3 mug DNA was used to prepare indexed Illumina libraries (MyGenostics, Beijing, China), according to the manufacturer's protocol. The exons of genes associated with congenital adrenal hyperplasia (all the genes including ABCD1 AIRE ARMC5 CDKN1C CYP11A1 CYP11B1 CYP11B2 CYP17A1 CYP21A2 DHCR7 GK GK2 GLCCI1 GNAS H6PD HESX1 HSD11B1 HSD3B2 LHX4 MC2R MCM4 MEN1 MKS1 MRAP NNT NR0B1 NR3C1 NR5A1 PCSK1 PDE11A PDE8B POMC POR PRKACA PRKAR1A PROP1 REN RXRA RXRB SOX3 STAR TBX19 TP53 TXNRD2) were targeted via a gene capture strategy, using the GenCap custom enrichment kit (MyGenostics), according to the manufacturer's protocol. The enriched libraries were sequenced on a NextSeq 500 sequencer (Illumina, San Diego, CA, USA) to generate paired-end reads of 150 bp. Single-nucleotide polymorphisms, insertions, and deletions were identified using Genome Analysis Toolkit software.