The study group included 100 hEDS patients of Polish origin, women (84) and men (16), aged 17–63 years (median: 31 years). Patients were enrolled in the study by experienced clinical geneticists, according to the 2017 International Classification of the Ehlers-Danlos syndrome diagnostic criteria [1 (link)]. Joint hypermobility was evaluated on the Beighton scale. The control group consisted of 100 volunteers from the general Polish population matched by age and sex with the investigated group, who were healthy (including lack of EDS) at the time of the investigation and without a history of EDS in the family.
All hEDS patients or their parents provided informed consent to participate in the study. Consent to publish clinical/genetic data was also obtained from the patients or their parents.
The study was approved by the Ethics Committee of Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland (KB485/2013).
The analysis was performed on genomic DNA (gDNA), which was extracted from leukocytes (fibroblasts were not available) by QIAamp DNA Mini Kit (Qiagen, Germany) using standard procedures. In all patients, other types of EDS or other connective tissue disorders were excluded by testing them with NGS technology (Illumina NextSeq 550). The connective tissue disorder customer panel included COL5A1, COL5A2, COL3A1, COL1A1, COL1A2, TNXB, ADAMTS2, PLOD1, FKBP14, ZNF469, PRDM5, B4GALT7, B3GALT6, SLC39A13, CHST14, DSE, COL12A1, C1R, C1S, SEC23A, SEC24D, COL6A1, COL6A2, COL6A3, COL9A1, COL9A2, FBN1, FBN2, FLNA, and FLNB. Copy number variant (CNV) analysis was also performed. Sequencing data were aligned to the hg19 human reference genome. Based on the guidelines of the American College of Medical Genetics and Genomics, a minimum depth coverage of 30X was considered suitable for analysis. All patients negative for the NGS test were analysed for alterations in the SERPINH1 gene. Molecular analysis of the SERPINH1 gene was performed by Sanger sequencing (ABI3130XL) according to standard procedure (primer sequences available upon request). The pathogenicity of detected variants was assessed according to the ACMG guideline by Varsome [19 (link), 20 (link)].
All hEDS patients or their parents provided informed consent to participate in the study. Consent to publish clinical/genetic data was also obtained from the patients or their parents.
The study was approved by the Ethics Committee of Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland (KB485/2013).
The analysis was performed on genomic DNA (gDNA), which was extracted from leukocytes (fibroblasts were not available) by QIAamp DNA Mini Kit (Qiagen, Germany) using standard procedures. In all patients, other types of EDS or other connective tissue disorders were excluded by testing them with NGS technology (Illumina NextSeq 550). The connective tissue disorder customer panel included COL5A1, COL5A2, COL3A1, COL1A1, COL1A2, TNXB, ADAMTS2, PLOD1, FKBP14, ZNF469, PRDM5, B4GALT7, B3GALT6, SLC39A13, CHST14, DSE, COL12A1, C1R, C1S, SEC23A, SEC24D, COL6A1, COL6A2, COL6A3, COL9A1, COL9A2, FBN1, FBN2, FLNA, and FLNB. Copy number variant (CNV) analysis was also performed. Sequencing data were aligned to the hg19 human reference genome. Based on the guidelines of the American College of Medical Genetics and Genomics, a minimum depth coverage of 30X was considered suitable for analysis. All patients negative for the NGS test were analysed for alterations in the SERPINH1 gene. Molecular analysis of the SERPINH1 gene was performed by Sanger sequencing (ABI3130XL) according to standard procedure (primer sequences available upon request). The pathogenicity of detected variants was assessed according to the ACMG guideline by Varsome [19 (link), 20 (link)].
