New insights into X-linked adrenal hypoplasia congenita from a novel splice-site variant of NR0B1 and adrenal CT images

Abstract

Background: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder, often manifesting as primary adrenal insufficiency (PAI) and hypogonadotropic hypogonadism (HH), and caused by variants of NR0B1, most of which are frame-shifting variants, and few splice-site variants.

Methods and results: Here, a novel splice-site variant of NR0B1 (NM_000475.4), c.1169-2A>T (patient 1), and a stop-loss variant of NR0B1 c.1411T>C (patient 2) are described in this study. We perform minigene assays for the splice-site variant (c.1169-2A>T) and determine that the variant causes exon 2 skipping. Moreover, the defect of NR0B1 protein may bring about the severe phenotype of the patient. Through 8 years of follow-up, we compare the CT images from 8 years ago with the latest image, and observe the CT image change of adrenal in patient 2 (from the increased thickness of adrenal to adrenal atrophy).

Conclusion: X-linked adrenal hypoplasia congenita is produced by variants of NR0B1. We report a case that presents a novel splice-site variant, which has been verified that it could lead to the exon 2 skipping in the RNA splicing progress. Moreover, we report the adrenal CT image change of patient 2, which has never been referred to before, and expand the spectrum of X-linked AHC characteristics.

Keywords: CT image change of adrenal; NR0B1 (nuclear receptor subfamily 0 group B member 1); X-linked AHC; infertility issue; minigene; splice-site variant.

FIGURE 1

CT images the X‐linked adrenal hypoplasia congenita patients. Patient 1: adrenal atrophy (a). CT images Patient 2: adrenal with increased thickness (At the age of 17) (b) Patient 2: adrenal atrophy (At the age of 25) (c). The position of adrenal was labeled by the red arrow.

FIGURE 2

Sanger analysis of the X‐linked adrenal hypoplasia congenita patients. The reference sequence of NR0B1 was derived from GenBank and the version number was NM_000475.4. Patient 1: Discovery of NR0B1 gene c.1169‐2A>T variant, which was not detected in the sample of his mother. The red box represented the mutant base, and the green box represented the normal base (a). Patient 2: Discovery of NR0B1 gene c.1411T>C, which was not detected in the sample of his mother. The red box represented the mutant base, and the green box represented normal base (b).

FIGURE 3

Analysis of pcMINI‐C vector assay for the NR0B1 variant (c.1169‐2A>T): Sequencing diagram of minigene construction, “wt” at the top and “mut” at the bottom, the red box revealed the difference. THE “wt” was for wild type. The “mut” was for mutant (a). Agarose gel electrophoresis diagram of RT‐PCR transcription analysis, the results of RT‐PCR showed that the “wild type” was a single band of the expected size (773 bp) both in HeLa and 293T cells and was named the band “a.” The “mutant” was also a single band and was named the band “b.” THE “wt” was for wild type. The “mut” was for mutant (b). Diagram of spliced bands corresponding to sequencing results: the sequencing results showed that the wild‐type band “a” was a regular splicing band with the splicing mode of Exon A (192 bp)‐Exon 2 (402 bp), and the mutant band “b” was an abnormal splicing band with Exon 2 skipping and the splicing mode of Exon A (192 bp)‐vector (c and d). “T7” was for the promoter. Red * indicated variant location.

FIGURE 4

Analysis of pcDNA3.1 vector assay for the NR0B1 variant (c.1169‐2A>T): Sequencing diagram of minigene construction with “wt” at the top and “mut” at the bottom, the red box revealed the difference. THE “wt” was for wild type. The “mut” was for mutant (a). Agarose gel electrophoresis plot of RT‐PCR transcription analysis: the RT‐PCR results showed that the wild type was a single band in HeLa and 293T cells, which was consistent with the expected size (969 bp) and named the band “a.” The mutant was also a single band and named the band “b.” THE “wt” was for wild type. The “mut” was for mutant (b); The sequencing results showed that the wild‐type band “a” was a regular splicing band with the splicing mode of Exon 1 (382 bp)‐Exon 2 (402 bp) and the mutant band “b” was an abnormal spliced band with Exon 2 skipping and the splicing mode of Exon 1 (382 bp)‐vector (c and d). “T7” was for the promoter. Red * indicates variant location.