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Next-generation sequencing and a novel COL3A1 mutation associated with vascular Ehlers–Danlos syndrome with severe intestinal involvement: a case report
© The Author(s). 2016
Received: 15 March 2016
Accepted: 3 October 2016
Published: 31 October 2016
The vascular type of Ehlers–Danlos syndrome is an autosomal dominant connective tissue disorder caused by a mutation in the COL3A1 gene encoding pro-alpha1 chain of type III collagen. The vascular type of Ehlers–Danlos syndrome causes severe fragility of connective tissues with arterial and intestinal ruptures and complications in surgical and radiological treatments.
We present a case of a 38-year-old Italian woman who was diagnosed as having the vascular type of Ehlers–Danlos syndrome. Genetic testing, conducted by Target Enrichment approach (Agilent Technologies), identified a new mutation c.1493G>A, p.G498D in exon 21 of COL3A1 gene (heterozygous state). This mutation disrupts the normal glycine-X-Y repetitions of type III procollagen by converting glycine to aspartic acid.
We report a new genetic mutation associated with the vascular type of Ehlers–Danlos syndrome. We also describe clinical and genetic findings that are important to understand the genotype/phenotype correlation in patients with the vascular type of Ehlers–Danlos syndrome.
Ehlers–Danlos syndrome (EDS) is a group of heterogeneous connective tissue disorders. It is characterized by abnormalities of the molecules that configure the extracellular matrix, such as collagen or its modifying enzymes . The clinical characteristics of EDS are: hyperextensibility of the skin, hypermobility of the large joints, and easy bruising. On the basis of clinical experience and genetic findings, EDS is divided in six subtypes, (Villefranche classification 1997): (1) classic, which is the most frequent form; (2) hypermobility; (3) kyphoscoliosis; (4) arthrochalasia; (5) dermatosparaxis; and (6) vascular, which is the most dramatic form . Genetic and clinical findings of EDS subtypes point out the genetic heterogeneity of EDS syndrome. Therefore, diagnosing the correct EDS type has important implications for genetic counseling and management. Any subtype of EDS is supported by specific biochemical and molecular investigations .
Vascular type of EDS (vEDS), also known as EDS type IV (NIM#130050), is a rare inherited autosomal dominant disorder with an estimated prevalence of 1 in 150,000. There are four main characteristics of vascular EDS: (1) rupture of blood vessels or internal organs such as the uterus and intestines, (2) an unusual facial appearance, (3) easy bruising, and (4) translucent skin with visible veins . An important clinical event in vEDS is that the systemic arteries, which are rich in type III collagen, may undergo dissection, aneurysm, or rupture. These dramatic events may also occur spontaneously .
In this case report we describe a novel COL3A1 point mutation (c.1493G>A, p.G498D) in a 38-year-old Italian woman. The diagnosis of vEDS was achieved at the Regional Center of Ehlers-Danlos syndrome (Ospedale Maggiore Policlinico, Milan, Italy); in addition to typical clinical characteristics of vEDS she referred an intestinal ischemic perforation of the sigmoid-rectum. The novel COL3A1 mutation c.1493G>A, p.G498D that we describe here for the first time, is associated with profound change in the protein structure, causing vEDS.
A 38-year-old Italian woman presented to our Regional Center of Ehlers-Danlos Syndrome, Ospedale Maggiore Policlinico, Milan, Italy, with a previous diagnosis of vEDS. She referred an intestinal ischemic perforation of the sigmoid-rectum with stercoral peritonitis treated with resection of her sigmoid-rectum and creation of a stoma. She did not have familial history of vEDS. When she was 26-years old and had not yet had a clinical diagnosis of EDS she underwent an orthopedic intervention for alignment of the internal arch plus tarsal sinus arthrodesis of both feet.
List of genes included in the HaloPlex Target Enrichment panel (next-generation gene panel)
EDS type VIIC
EDS progeroid type 1
EDS muscolocontractural type 1
EDS type IV
EDS classic type
EDS classic type
EDS type VI
Lysyl hydroxylase 3 deficiency
EDS due to tenascin X deficiency
Finally, the concentration of each library was measured by TapeStation software and were diluted at 4nmol with TE. The final pool was obtained by mixing different genomic libraries and the ideal final concentration was 8 pM (ideal concentration for cluster density approximately 900/1000 K/mm2). Captured genomic libraries were sequenced to generate 2×150 bp paired end reads using the Illumina MiSeq.
To understand the generated FASTQ files (a file that stores a biological sequence and its quality score), a workflow was set up by Galaxy software (https://usegalaxy.org/) . FASTQ files were evaluated by quality and they were trimmed, aligned, and mapped to the human reference genome (GRCh38/hg38) using Burrows-Wheeler Aligner [6, 7]. Alignments including sorting, merging, indexing, and generating alignments in a per-position format were manipulated by SAM tools [8, 9] to generate a BAM (binary format for storing sequence) file. Local realignment, base quality recalibration, and variant calling were performed using Genome Analysis Toolkit (GATK) version 2.0 (Broad Institute, Cambridge, MA, USA)  applying these quality parameters: coverage>20, base quality score>30, and mapping quality>20. Variants that did not pass these quality values were removed.
Finally, the Variant Calling Files (VCF) were filtered through different parameters such as genotype quality filters, gene feature filters (missense, splicing, frame shift), and functional impact filters using KGGSeq software . The variants found with NGS target enrichment method were confirmed by Sanger method using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Life Technologies) followed by capillary electrophoresis on an Applied Biosystems 3130xl (Applied Biosystems). Electropherograms were analyzed by Sequencher software v5.1.
To investigate the pathogenetic properties of a missense variant, different types of software were used: SIFT, PROVEAN, PolyPhen-2, Mutation Taster, Grantham distance, and Align GVGD; these types of software are based on sequence homology, the physiochemical similarity between the alternate amino acids, effect of an amino acid substitution on the structure and function of a protein, and conservation level of the amino acid residue among species [12–15].
Resequencing by target enrichment approach (HaloPlex technique) conducted using our custom panel (Table 1) showed the presence of an undescribed mutation in exon 21 of COL3A1 gene at position 1493 leading to a nucleotide replacement (c.1493G>A, p.G498D) (www.le.ac.uk/ge/collagen/). It was confirmed by Sanger method on a second DNA extraction. Moreover, a complementary DNA (cDNA) analysis extracted from fibroblasts culture was not feasible due to the extreme fragility of the clinical state of our patient. 343 Gly-X-Y repetitions (X and Y, any other amino acids)
In the current case report we described a case of a 38-year-old Italian woman with typical symptoms of vEDS. She referred an ischemic perforation of her sigmoid-rectum with stercoral peritonitis treated with resection of sigmoid-rectum and creation of a stoma. The stoma was not closed to avoid surgical complications. Her intestinal problems worsened her clinical state. A physical examination revealed that her skin was slightly smooth and translucent with visible veins. Genetic analysis did not confirm any familial history.
Using a target NGS approach (HaloPlex protocol), a pathogenetic mutation c.1493G>A, p.G498D was discovered in the COL3A1 gene (www.le.ac.uk/ge/collagen/). Up to now, more than 200 mutations in the COL3A1 gene have been identified , all of which lead to the synthesis of an abnormal type III procollagen protein (Fig. 1). Approximately, two thirds of the mutations are single nucleotide substitutions that result in change for Gly residues in the triple helical domain of the proα1(III) chain. Most of the rest are splice site mutations that result in exon skipping, although some have more complex outcomes and a smaller number are larger genomic deletions . There are no correlations between the nature or location of the mutation and the type or frequency of major complications .
The mutation c.1493G>A was detected in heterozygosity in the COL3A1 gene, which resulted in a substitution of a Gly, a neutral amino acid, to aspartic acid, a polar amino acid. The meta-analysis, conducted by PolyPhen-2, SIFT, PROVEAN, and Align GVGD [12–15] showed that this point mutation was pathogenetic and was the cause of vEDS phenotype. The Gly substitution negatively influences the normal assembly of COL3A1 collagen fibers. In fact, the collagen characteristic is represented by the 343 repetitions of Gly-X-Y (X and Y, any other amino acids) . The Gly amino acid is very important because it is the smallest amino acid and determines the link between the three peptide chains.
The mutation c.1493G>A, p.G498D was located on the triple helical region and it was theoretically conceivable that this type of mutation may change the characteristic triple helical structure and generate a severer phenotype. In fact, substitution of Gly in this region of the protein may strongly interfere with helix formation, delay the triple helix assembly, and lead to a severe modification. The negative effect was a dramatic decrease in the quantity and quality of COL3A1 protein. We did not have an opportunity to study the effect of this mutation on the three-dimensional protein structure because, in the literature, there is no sequence homology.
We conclude that c.1493G>A, p.G498D identified in COL3A1 gene was the cause of vEDS. The particular position of Gly substitution suggested a correlation between genotype and phenotype. The functional properties of c.1493G>A, p.G498D are still unknown, further studies on in vitro cell culture will be necessary to understand the link between this new variant and vEDS phenotype. The discovery of this new variant in vEDS underlines the importance of genetic tests as NGS in rare genetic diseases.
Thanks to Dr Agostino Seresini who constructed the workflow for NGS data by Galaxy software (https://usegalaxy.org).
Availability of data and materials
There are no additional data available for this study.
Concept and design: FC, BM, and BDG. Manuscript writing: all authors. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Ethics approval and consent to participate
The study was performed in agreement with the principles of the Declaration of Helsinki and the local Ethics Committee was notified in compliance with Italian legislation on retrospective studies. Written informed consent was obtained from all participants.
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- De Paepe A, Malfait F. The Ehlers-Danlos syndrome, a disorder with many faces. Clin Genet. 2012;82(1):1–11.View ArticlePubMedGoogle Scholar
- Beighton P, De Paepe A, Steimann B, Tsipouras P, Wenstrup RJ. Ehlers-Danlos syndromes: Revised Nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos support group (UK). Am J Med Genet. 1998;77:31–7.View ArticlePubMedGoogle Scholar
- Steimann B, Royce PM, Superti-Furga A. The Ehlers-Danlos syndromes. In: Royce PM, Steinmann B, editors. Connective Tissue and its Heritable Disorders: Molecular, Genetic and Medical Apsects. 2nd ed. New York: Wiley-Liss; 2002. p. 431–524.View ArticleGoogle Scholar
- Wenstrup RJ, Hoechstetter LB. Ehlers-Danlos syndromes. In: Cassady SB, Allanson JE, editors. Management of Genetic Syndromes. 2nd ed. Hoboken: Wiley; 2005. p. 211–23.Google Scholar
- Cuccuru G, Orsini M, Pinn A, Sbardellati A, Soranzo N, Travaglione A, et al. Bioinformatics. 2014;30(13):1928–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26(5):589–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011;27(21):2987–93.View ArticlePubMedPubMed CentralGoogle Scholar
- McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297–303.View ArticlePubMedPubMed CentralGoogle Scholar
- Li MX, Gui HS, Kwan JS, Bao SY, Sham PC. A comprehensive framework for prioritizing variants in exome sequencing studies of Mendelian diseases. Nucleic Aicds Res. 2012;40(7):e53.View ArticleGoogle Scholar
- Adzhubei I, Jordan MD, Sunyaev SR. Predicting Functional Effect of Human UNIT 7.20 Missense Mutations Using PolyPhen-2. Protoc HumGenet. 2013;76(7):20.1–7. 20.41.Google Scholar
- Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4(7):1073–81.View ArticlePubMedGoogle Scholar
- Tavtigian SV, Deffenbaugh AM, Yin L, Judkins T, Scholl T, Samollow PB, et al. Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet. 2006;43(4):295–5.View ArticlePubMedGoogle Scholar
- Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One. 2012;7(10):e46688.View ArticlePubMedPubMed CentralGoogle Scholar
- Watanabe A, Takashi S. Vascular type of Ehlers-Danlos syndrome. J Nippon Med Sch. 2008;75(5):254–61.View ArticlePubMedGoogle Scholar
- Pepin M, Schwarze U, Superti-Furga A, Byers PH. Clinical genetics features of Ehlers-Danlos syndrome type IV, the vascular type. N Engl J Med. 2000;342:673–80.View ArticlePubMedGoogle Scholar