A TNXB splice donor site variant as a cause of hypermobility type Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia

Abstract

Background: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disease of steroidogenesis that affects 1 in 15,000. Approximately, 10% of the CAH population also suffer from CAH-X, a connective tissue dysplasia consistent with hypermobility type Ehlers-Danlos syndrome (EDS). Most patients with CAH-X carry a contiguous gene deletion involving CYP21A2 encoding 21-hydroxylase and TNXB encoding tenascin-X (TNX), but some are of unknown etiology.

Methods: We conducted clinical evaluation and medical history review of EDS-related manifestations in subjects from two unrelated CAH families who carry a heterozygous TNXB c.12463+2T>C variant that alters the splice donor site of intron 42. A next generation sequencing (NGS) based EDS panel composed of 45 genes was performed for index patients from each family. TNX expression in patient skin biopsy tissues and dermal fibroblasts was assessed by qRT-PCR and Sanger sequencing.

Results: All three evaluated CAH patients carrying the TNXB splice site variant had moderate EDS manifestations. An NGS panel excluded involvement of other known EDS-related variants. RNA assay on skin biopsies and dermal fibroblasts did not detect splicing errors in TNX mRNA; however, the removal of intron 42 was less efficient in the allele harboring the splice site variant as evidenced by the existence of a premature TNX RNA form, leading to an allele specific decrease in TNX mRNA.

Conclusions: Carrying a TNXB c.12463+2T>C variant at the intron 42 splice donor site causes an allele specific decrease in TNX expression, which can be associated with moderate EDS in CAH patients.

Keywords: TNXB; CAH; EDS; Ehlers Danlos syndrome; congenital adrenal hyperplasia.

FIGURE 1

CAH‐X genetic pedigree and phenotype. (a) TNXB variants present in two unrelated families with CAH. Phenotypes of CAH and hypermobile EDS (hEDS) are presented in the pedigree. Plus (+) sign indicates present, negative (−) sign indicates absence, and n.a. indicates not available. Black arrows indicate subjects with phenotypic CAH‐X that are positive in both CAH and hypermobile EDS. (b) Examples of clinical EDS findings included thin skin and wide scars in subject A‐II‐2 and joint hypermobility in subject B‐II‐1

FIGURE 2

Effects of TNXB c.12463+2T>C (IVS42+2T>C) variant on tenascin‐X (TNX) expression in skin tissue. (a) Haplotypes of TNXB exons 35–44 locus of two carrier families are shown including the c.12463+2T>C variant (green oval) and other single nucleotide polymorphisms (SNPs; pink ovals). (b) Sanger chromatograms showing a decrease in TNX mRNA abundance from the allele of the splice site variant. RNA heterozygosity was confirmed in all subjects and red arrows mark the reduced chromatogram signals of the SNPs specific to the TNXB splice site variant alleles. (c) Sanger chromatograms of TNX mRNA exons 42–43 junction without a splicing error in all subjects. d RT‐PCR of TNX mRNA exons 35–44, clean single banded PCR products free of satellite bands suggested absence of alternative splicing or premature RNA. Subjects A‐II‐1 and B‐I‐1 are index for carrier and non‐carrier of TNXB c.12463+2T>C, respectively, and a commercial sourced adult skin total RNA sample is shown as a control. Skin RNA samples from all patients were extracted within 2 hours of skin biopsy

FIGURE 3

Effects of TNXB c.12463+2T>C (IVS42+2T>C) variant on tenascin‐X (TNX) expression in dermal fibroblasts. (a) RT‐PCR of TNX exons 42–44. Subjects A‐II‐1 and B‐II‐1 were selected as index for their respective families, Subject B‐I‐1 and an unrelated CAH patient were used as controls. A premature TNX RNA with moderate density (marked with red star) was found in the fibroblasts carrying the TNXB splice site variant but was absent from the control group; Sanger sequencing revealed that it failed to remove intron 42. A faint trace satellite band, revealed as nascent TNX RNA by Sanger (marked with red asterisk), was found in subject A‐II‐1. Both nascent and premature TNX forms were exclusively transcribed from the allele hosting the TNXB c.12463+2T>C variant (marked with red arrow), as revealed by Sanger. The primer recognition sites were marked with black arrows. RNA schemes are illustrated on the right. (b) A moderate decrease in TNX mRNA abundance commonly observed in dermal fibroblasts derived from TNXB c.12463+2T>C carriers. TNX mRNA abundance normalized to GAPDH are shown in three genotype categories: CAH‐X CH‐1 of TNX haploinsufficiency (green), CAH patient/carrier with TNXB c.12463+2T>C variant (red), CAH patients and carriers without TNXB defects (black). Normalization to other references, including β‐actin, α‐actin and α‐tubulin, obtained similar results (data not shown). Notably subject B‐II‐1 was 3 years old at the time of biopsy and there was no available age‐matched control