Clinical and genetic analysis of classical Ehlers-Danlos syndrome patient caused by synonymous mutation in COL5A2

Abstract

Background: Classical Ehlers-Danlos syndrome (cEDS) is a heterogeneous connective tissue disorder that mainly results from the germline mutation of COL5A1 and COL5A2. The majority of the COL5A2 mutations reported to date represent structural mutations, including missense or in-frame exon-skipping splice mutations. The only reported synonymous mutation was expected to affect on splicing of exon 29 by prediction programs which should be further confirmed.

Methods: Whole exome sequencing was performed to identify the genetic variants of a Chinese boy who was characterized by skin hyperextensibility, abnormal scarring, hypermobile joints and scoliosis. Sanger sequencing was used to validate the variants in his parents. Reverse transcription polymerase chain reaction (RT-PCR) was performed to analyze the functional effects of the variant.

Results: A de novo heterozygous synonymous variant (NM_000393.5:c.1977 G>A) of COL5A2 gene was identified in the patient. The results of RT-PCR revealed that the synonymous variant led to skipping of exon 29 in the RNA transcript.

Conclusions: Our study supplies further supporting evidence that the synonymous COL5A2 mutation c.1977 G>A can cause skipping of exon 29 in the RNA transcript, thus resulting in the production of mutant α2(V)-chains and clinical phenotype of cEDS. This result highlights the need to include splicing-altering synonymous mutations into the screening for cEDS.

Keywords: COL5A2; classic Ehlers Danlos syndrome; splicing; synonymous mutation; whole-exome sequencing.

FIGURE 1

Clinical pictures of the patient. (a) X‐ray photographs of the thoracoabdominal of the patient. The patient presented marked scoliosis at age of 28 months. (b) Extreme joint hypermobility: the thumb can be passively moved to touch the ipsilateral forearm. (c) Extreme joint hypermobility: metacarpal‐phalangeal joint of the fifth finger can be hyperextended more than 90° with respect to the dorsum of the hand. (d) Atrophic scars of the knee. (e) Extreme joint hypermobility: elbow extends more than 10°

FIGURE 2

Effects of COL5A2 mutations on splicing. (a) Sanger sequencing of the c.1977 G>A (pointed by red arrows) mutation of COL5A2 in this family. Parents are free of the mutation. (b) RT‐PCR of RNA from the patient. The cDNA was amplified with primers in exons 27 and 33 and the expected 385 bp product is indicated. The smaller band of patient results from skipping of exon 29. There is an additional heteroduplex in the subject sample. (c) The sequence analysis of the 331‐bp PCR product presented in (a). As indicated by the cDNA sequences of exons 28 and 30 of COL5A2, exon 29 is clearly absent from this transcript. C, control individual; P, subject

FIGURE 3

Pathology and Ultrastructural Collagen of patient and normal human. (a) Irregularly organized collagen bundles are intensely stained and have a cloudy appearance. (b) The overall aspect of the connective tissue is loose. (c) Regularly and tightly organized collagen bundles are intensely stained in normal human skin. (d) Cross‐sectioned collagen fibrils of variable diameter and loosely‐packed. (e) Longitudinal section of a collagen bundle with several thickened rope‐like and twisted fibrils (arrows)