Clinical presentation of the pedigree
The index case was a 22-year-old female who had a diagnosis of hypertension at the age of 13 (Fig. 1A). There was no obvious masculinization, and her parents stated that there were no obvious abnormalities in vulva development at birth. The patient had undergone spontaneous menarche at the age of 12 and had sparse menstrual flow. On physical examination, the patient’s height was 160 cm (paternal height is 165 cm and maternal height is 160 cm), her body weight was 75 kg, and her body mass index (BMI) was 29.3 kg/m2. She had Tanner stage 3 breast development, with Tanner pubic hair stage 4. Adrenal computed tomography scanning revealed a single mass measuring 3.0 cm×2.4 cm in the left adrenal gland (Fig. 1B). The proband's younger brother is now 14 years old and was diagnosed with hypertension at the age of 10. He also had a history of hypokalemia. His height was 163 cm, his body weight was 73 kg, and his body mass index (BMI) was 27.5 kg/m2. Longitudinal growth seemed to stall at the age of 13. Transthoracic echocardiography showed interventricular septum thickening (13 mm).
Laboratory findings are summarized in Table 1. Overall, the serum levels of ACTH, progesterone, testosterone, were markedly elevated in both siblings. Decreased cortisol and potassium were also noted. Meanwhile, 17-hydroxyprogesterone (697 ng/dl, normal range < 233 ng/dl) and androstenedione (811 ng/dl, normal range 31 - 65 ng/dl) content analyzed by mass spectrometry were increased in the younger brother. Based on the clinical information, a diagnosis of 11β-hydroxylase deficiency was suggested.
CYP11B1 mutation analysis
Using long-range PCR based NGS, we identified a heterozygous missense mutation (NM_000497.4: c.281C>T, p.Pro94Leu) in exon 8 of the CYP11B1 gene in the patients and their father (Fig. 2A-D). The mutation was not present in 1000 Genomes, ESP6500, ExAC, and GnomAD. This variant has been previously reported in medical literature and was classified as likely pathogenic, according to the ACMG guidelines (PS3_Supporting, PM2_Supporting, PM3_Strong, PM5_Supporting). However, the nature of 11β-hydroxylase deficiency is an autosomal recessive inheritance, so we considered that there should be another pathogenic variant in CYP11B1 gene. The chimeric CYP11B2/CYP11B1 gene has been reported to be a less common cause of 11β-OHD. However, no chimeric CYP11B2/CYP11B1 gene was detected via long-range PCR assay (Fig. 3A-B).
Another possible reason is that the missing variant is difficult to identify by long-range PCR-based SRS, such as structural variants. To rule out this possibility, T-LRS was used to identify another causative variation in the patients. In total, 307.80 Mb raw data from LRS was generated for analysis. The mean sequencing depth for the targeted regions was 400.36. The average length of the reads was 3329.66 bp. One SV on chromosome 8 in CYP11B1 gene was identified. This SV was a deletion-insertion mutation and located on chromosome 8 (chr8:143957269-143957579) with the insertion of ‘ACAG’. This mutation covered part sections of intron 5 and exon 6 of CYP11B1 gene (Fig. 4A). According to ACMG guidelines, this mutation has been classified as pathogenic (PVS1+PM2_Supporting+PM3_trans). This variant was in trans with CYP11B1: c.281C>T and has not been described before.
To verify this mutation in CYP11B1 gene, long-range PCR and Sanger sequencing were carried out to confirm the deletion-insertion variation in the family members (Fig. 4B-4E, supplemental Fig. 1). Results showed that compared to the father, the mother and the siblings carried this heterozygous SV.
Follow-up after treatment
The patients were started on dexamethasone supplementation therapy at 0.375 mg once daily to replace the deficient cortisol and reduce excess androgens and mineralocorticoid precursors. To control blood pressure, irbesartan 150mg and amlodipine 5mg were prescribed once daily. Following a 6-month treatment period, the blood pressure and biochemical markers of both patients had nearly reverted to normal levels. The proband resumed spontaneous menstrual bleeding as well.