Two closely spaced missense COL3A1 variants in cis cause vascular Ehlers‐Danlos syndrome in one large Chinese family

Abstract Vascular Ehlers‐Danlos syndrome (vEDS) is a rare and severe hereditary connective tissue disease arising from a mutation in the type III collagen alpha I chain (COL3A1) gene, with a poor prognosis due to exceptional vascular ruptures and premature death. Herein, starting from a 36‐year‐old Chinese male patient with a complaint of upper abdominal pain, we collected clinical data of and performed a genetic analysis of a total of 20 family members. We identified two closely spaced COL3A1 missense variants in cis, p.Leu734Phe (c.2199_2200TC>AT) and p.Gly741Ser (c.2221G>A), as the cause of vEDS in this family. p.Gly741Ser, a glycine substitution mutation, has been previously reported, whereas p.Leu734Phe, a non‐glycine substitution mutation, is novel. We analysed their independent and combined effects on the COL3A1 level in transfected skin fibroblast cells by means of Western blotting. We found that both variants independently led to a reduced COL3A1 level and, when combined, led to an even more reduced COL3A1 level compared to the wild type. Thus, each missense variant can be independently classified as a pathogenic variant, albeit with a synergetic effect when occurring together. Moreover, our genetic findings provide an explanation for four previous sudden deaths and identified two high‐risk carriers in the family.


Abstract
Vascular Ehlers-Danlos syndrome (vEDS) is a rare and severe hereditary connective tissue disease arising from a mutation in the type III collagen alpha I chain (COL3A1) gene, with a poor prognosis due to exceptional vascular ruptures and premature death.
Herein, starting from a 36-year-old Chinese male patient with a complaint of upper abdominal pain, we collected clinical data of and performed a genetic analysis of a total of 20 family members. We identified two closely spaced COL3A1 missense variants in cis, p.Leu734Phe (c.2199_2200TC>AT) and p.Gly741Ser (c.2221G>A), as the cause of vEDS in this family. p.Gly741Ser, a glycine substitution mutation, has been previously reported, whereas p.Leu734Phe, a non-glycine substitution mutation, is novel. We analysed their independent and combined effects on the COL3A1 level in transfected skin fibroblast cells by means of Western blotting. We found that both variants independently led to a reduced COL3A1 level and, when combined, led to an even more reduced COL3A1 level compared to the wild type. Thus, each missense variant can be independently classified as a pathogenic variant, albeit with a synergetic effect when occurring together. Moreover, our genetic findings provide an explanation for four previous sudden deaths and identified two high-risk carriers in the family.

K E Y W O R D S
aneurysm, COL3A1 gene, Ehlers-Danlos syndrome vascular type, missense variant

| INTRODUC TI ON
Vascular Ehlers-Danlos syndrome (vEDS, also known as Ehlers-Danlos type IV; OMIM# 130050) is a rare inherited autosomal dominant disorder (prevalence, 1/50,000-200,000) that is mainly characterized by fatal arterial and gastrointestinal complications. 1-4 vEDS is usually caused by heterozygous mutations in the COL3A1 gene that codes for the pro-alpha 1 chain of type III procollagen. [5][6][7][8][9] The clinical diagnosis of vEDS is typically based on the standards laid down by an expert group in 1997. 2 Thus, ecchymosis, thin skin with an evident venous pattern and typical facial characteristics lead to the diagnosis. Nonetheless, in most cases, the diagnosis is not suspected until the time an aneurysm and/or grooves in the arteries, crevices in the bowel or rupture of organs occurs. Significant phenotypic heterogeneity is also exhibited within family members in terms of disease onset and severity and affected organs. Additionally, the clinical features of vEDS may overlap with Loeys-Dietz syndrome and Marfan syndrome, further complicating the clinical diagnosis. 3,4 Therefore, genetic tests are often required to establish a formal diagnosis.
Type III collagen is a critical structural protein in the walls of the vascular system and in the walls of hollow organs. Structural defects or lower levels of type III procollagen resulting from COL3A1 mutations thus underlie the escalated ecchymosis, bowel and arterial frailty, and vaginal, uterine and cervical frailty in pregnancy as well as in delivery in vEDS patients. To date, a diverse range of loss-of-function variants in the COL3A1 gene have been reported in the literature, with most of them being heterozygous glycine substitutions that occurred within the [Gly-X-Y] 343 repeat of the type III procollagen. 6 In this regard, Bowen and colleagues recently created two mouse models of vEDS, each carrying a heterozygous glycine substitution mutation (i.e., p. Gly209Ser or p. Gly938Asp) in Col3a1. They showed that Col3a1 structural deficiencies conferred by the glycine substitution mutations led to signalling abnormalities in the PLC/IP3/PKC/ERK (phospholipase C/inositol 1,4,5-triphosphate/protein kinase C/extracellular signalregulated kinase) pathway that in turn mediated the risk of vascular rupture. 10 Apart from these typical glycine substitution missense mutations, atypical non-glycine substitution missense mutations (e.g., glutamic acid to lysine (Glu>Lys) substitutions) in the COL3A1 gene have also been increasingly recognized to cause the disease. 6,11 In the present study, we describe the identification and functional analysis of two closely spaced COL3A1 missense variants in cis, one being a typical glycine substitution missense mutation and the other being an atypical non-glycine substitution missense mutation, as the cause of vEDS in a large Chinese family that affected more than 10 members across four generations. We show that the two missense variants had a synergetic effect on reducing the level of COL3A1 in transfected cells, providing novel insights into the pathogenesis of vEDS.

| Chip capture high-throughput sequencing
Chip capture high-throughput sequencing for an eight-gene panel (i.e., ADAMTS2, B4GALT7, COL5A1, COL5A2, PLOD1, COL3A1, SLC39A13 and COL1A1) was performed on the proband. In brief, genomic DNA was isolated, and the library was prepared; target gene-coding In the year when the proband was hospitalized.

| Genotyping of the two COL3A1 missense variants
The two COL3A1 missense variants were analysed in all blood relatives of the proband by means of Sanger sequencing. The forward and reverse primers used for PCR amplification were 5′-GAACGTGGACCTCCTGGAT-3′ and 5′-TGAAAATCAGCCAAGAAGAGG-3′ respectively.

| Semiquantification of blot findings and statistical analysis
All computations were performed using the Image Processing and Analysis program in Java (ImageJ

| Genetic findings
Genetic exploration performed on the proband (III:9; Figure 1) using next-generation sequencing identified two missense variants in the Protein function prediction was performed using SIFT and PolyPhen software as previously described. 15 Both variants were predicted to be harmful.

| Functional characterization of the two missense variants in transfected skin fibroblasts
We employed a Western blot assay to study the expression of the wild-type and variant COL3A1 proteins in transfected skin fibroblasts. Each variant in isolation led to a significantly reduced level of COL3A1, and the two variants in combination led to a more profound reduction in the level of COL3A1 in the transfected cells compared to the wild type ( Figure 5).

| DISCUSS ION
Ehlers-Danlos syndromes constitute a pathologically and genetically heterogeneous class of inheritable connective tissue defects, mani- p. Gly741Ser, a typical glycine substitution missense mutation, has previously been reported in vEDS patients. 6 In contrast, p.
Leu734Phe, a non-glycine substitution missense mutation, is novel. Since the two variants are located on the same chromosome, we analysed their independent and combined effects on the COL3A1 level in transfected cells by means of Western blotting. We found that both variants independently led to a reduced COL3A1 level and, when combined, led to an even more reduced The two closely spaced COL3A1 missense variants in cis were found not only in all clinically affected members but also in two healthy family members, IV8 and IV12 (Figure 1). It should, however, be pointed out that while IV8 was less than 1 year old, IV12 was 16 years old. In this regard, it is worth mentioning that in a European cohort study, only 17% of vEDS patients had experienced an initial complication by the age of 20. 6 Thus, genetic counselling should be given to IV8 and IV12 about their risk of developing vEDS manifestations.
In summary, we have for the first time reported a combination of a typical glycine substitution missense mutation and an atypical nonglycine substitution missense mutation as the cause of vEDS in an unusually large Chinese family. In fact, this is the first report of such a combination ever described to cause vEDS in the literature. More importantly, we demonstrated that both variants are independently functional and, when combined, confer a more severe effect on the structure/function of COL3A1.

ACK N OWLED G EM ENT
We thank the family members for participating in this study.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.