Novel TNXB Variants in Two Italian Patients with Classical-Like Ehlers-Danlos Syndrome

Abstract

TNXB-related classical-like Ehlers-Danlos syndrome (TNXB-clEDS) is an ultrarare type of Ehlers-Danlos syndrome due to biallelic null variants in TNXB, encoding tenascin-X. Less than 30 individuals have been reported to date, mostly of Dutch origin and showing a phenotype resembling classical Ehlers-Danlos syndrome without atrophic scarring. TNXB-clEDS is likely underdiagnosed due to the complex structure of the TNXB locus, a fact that complicates diagnostic molecular testing. Here, we report two unrelated Italian women with TNXB-clEDS due to compound heterozygosity for null alleles in TNXB. Both presented soft and hyperextensible skin, generalized joint hypermobility and related musculoskeletal complications, and chronic constipation. In addition, individual 1 showed progressive finger contractures and shortened metatarsals, while individual 2 manifested recurrent subconjunctival hemorrhages and an event of spontaneous rupture of the brachial vein. Molecular testing found the two previously unreported c.8278C > T p.(Gln2760*) and the c.(2358 + 1_2359 - 1)_(2779 + 1_2780 - 1)del variants in Individual 1, and the novel c.1150dupG p.(Glu384Glyfs*57) and the recurrent c.11435_11524+30del variants in Individual 2. mRNA analysis confirmed that the c.(2358 + 1_2359 - 1)_(2779 + 1_2780 - 1)del variant causes a frameshift leading to a predicted truncated protein [p.(Thr787Glyfs*40)]. This study refines the phenotype recently delineated in association with biallelic null alleles in TNXB, and adds three novel variants to its mutational repertoire. Unusual digital anomalies seem confirmed as possibly peculiar of TNXB-clEDS, while vascular fragility could be more than a chance association also in this Ehlers-Danlos syndrome type.

Keywords: Classical-like; Ehlers-Danlos syndrome; TNXB; haploinsufficiency; tenascin-X.

Figure 1

Selected clinical features of individual 2, and schematic diagram showing the genomic structure of TNXB and the secondary structure of tenascin-X. (a) Markedly hyperextensible skin of the dorsum of the hands. (b) Residual scar from recurrent molluscoid pseudotumors of the elbow. (c) Piezogenic papules of the heel. (d) Coding regions are highlighted with white boxes and introns with black horizontal lines. Tenascin-X is characterized structurally from N-terminus to C-terminus by: (i) an N-terminus with a series of repeats that resemble epidermal growth factor (EGFL repeats); (ii) a stretch of fibronectin type III repeats (FBIII1-31); and (iii) a large C-terminal domain structurally related to fibrinogen (Fibrinogen C). Previously identified variants associated with TNXB-clEDS are represented in black. Variants identified in the individuals 1 and 2 are represented in blue and red, respectively.

Figure 2

Molecular findings of individuals 1 and 2. (a) Electropherograms showing DNA sequencing analysis of PCR product amplified with primers targeting exon 8 of TNXB. Nucleotide sequences are provided. The position of the identified variant is indicated with an asterisk. (b) Protein sequence alignment of TNXB generated by Clustal Omega showed that the affected Gln 2760 residue of tenascin-X is evolutionary conserved. (c) Bar chart generated by Coffalyser.net- MLPA analysis software. MLPA was performed on DNA from the individual 1, her unaffected parents and controls (data not showed). A probe ratio of 1 indicates a normal DNA copy number; a probe ratio of 0.5 indicates a heterozygous deletion. MLPA analysis reveals a deletion of exon 5 of TNXB in individual 1. (d) Profiles of qPCR assay performed to map the deletion breakpoints within the region encompassing the exons 3 to 8 of TNXB. Relative DNA quantity of each exon was determined for the patient (red), her asymptomatic mother and father, (green and purple, respectively), and a pool of DNA controls (CNTs, orange). (e) Results of chromosomal microarray analysis in the Individual 1. Intensity data (Summarized log 2 ratio value) of each probe is drawn along chromosome 6 from 32,000 to 32,080 kb (USCS Genome Browser build February 2009, hg19). The red box indicates the interstitial microdeletion (62 probes with decreased signal) identified, encompassing the exons 5 and 6 of the TNXB gene (lower panel). (f) Electropherograms showing cDNA Sanger sequencing of a transcript region of TNXB amplified with primers targeting exon 3 to 8 of Individual 1 and her mother. (g) Sanger sequence of a PCR product amplified with primers targeting exon 3 of TNXB of individual 2 and her unaffected parents. The position of the identified variant is indicated with an asterisk. (h) Bar chart generated by Coffalyser.ne-MLPA analysis performed on DNA from the individual 2, her unaffected father and controls (data not showed). MLPA analysis reveals the common partial deletion of exon 35 of TNXB in individual 2.