Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2024 Sep 23;30(6):1366–1372. doi: 10.1111/hae.15099

Low vitamin C status and hypermobility‐related disorders in patients with bleeding disorder of unknown cause

Eva Leinøe 1,2,✉, Halla Fridriksdottir 1, Andreas Ørslev Rasmussen 2, Eva Funding 1,3, Anne Louise Tølbøll Sørensen 1, Peter Kampmann 1, Jens Lykkesfeldt 4, Maria Rossing 2,3
PMCID: PMC11659505  PMID: 39311717

Abstract

Introduction

Bleeding disorder of unknown cause (BDUC) is a challenging diagnosis that predominantly affects women. Previous investigations into connective tissue disorders (CTD) and vitamin C have not been conducted.

Aim

To examine the association between hypermobility‐related disorders, vitamin C status and BDUC.

Methods

Patients were selected following laboratory and genetic screening that yielded negative results for known hemostasis disorders. Sixty patients with BDUC and an ISTH BAT score ≥ 10 underwent clinically examination for skin hyperextensibility and for hypermobility assessed by Beighton score. Vitamin C was analyzed by high‐performance liquid chromatography. Genetic screening for causal variants in 42 CTD genes was performed.

Results

The majority of patients were female (56/60). Median ISTH BAT score was 13 (range 10–23). Beighton score was positive in 29/60 patients compared to 1/20 healthy controls (HC) (p < .001). Hyperextensive skin was observed in (18/60) patients, and none (0/20) of the HC (p = .0041). Ten patients met the clinical diagnostic criteria for hypermobile Ehlers–Danlos syndrome (hEDS), and one patient was diagnosed with Noonan syndrome. Genetic screening excluded various subtypes of EDS with known genetic backgrounds. Average vitamin C level was adequate, but lower than in HC (55.9 vs. 70.4 μmol/L; p = .001). Suboptimal, or low vitamin C were identified in 19/60 compared to 1/20 HC (p = .018).

Conclusion

Our study demonstrates that BDUC is frequently associated with hypermobility disorders and low vitamin C status. Our results could pave the way for a randomized study of vitamin C supplementation in patients with BDUC.

Keywords: ascorbic acid, bleeding, connective tissue, whole genome sequencing, women

1. INTRODUCTION

Treatment for X‐linked haemophilia has seen revolutionary advancements, resulting in novel, though expensive, therapeutics. 1 , 2 In contrast, progress in understanding the molecular background and management of autosomal inherited bleeding disorders, which primarily affect women, has significantly lagged. 3 Bleeding disorder of unknown cause (BDUC) is a challenging diagnosis of exclusion, with most patients being women and facing longer diagnostic delays. 4 Recent guidelines from the SSC of the ISTH recommend ruling out coagulation factor deficiencies, platelet dysfunction, medication interfering with platelet function and coagulation, medical conditions associated with increased bleeding, and conducting a clinical work‐up for syndromes and collagen disorders before diagnosing BDUC. 5 When known bleeding disorders are excluded, patients are often not formally registered at a haemophilia treatment centre or routinely offered follow‐up care. 6 Consequently, patients with BDUC have limited access to haemostatic treatment and prophylaxis during surgery and childbirth and suffer from severe menstrual bleeding and a reduced quality of life. Additionally, easy bruising and haematomas, common in these patients, can mistakenly lead to concerns about domestic violence and non‐accidental trauma in paediatric practice. This situation highlights a clear gender gap and underscores the unmet need for improved diagnostics and treatments for patients with BDUC.

Ehlers–Danlos syndrome (EDS) is a group of connective tissue disorders (CTD) associated with collagen and extracellular matrix defects, consisting of 13 subtypes. Hypermobile Ehlers–Danlos syndrome (hEDS) is the most frequent subtype, representing 80%−90% of cases. 7 Similar to BDUC, hEDS is underdiagnosed and primarily affects women, with clinical hallmarks including generalized joint hypermobility, skin hyperextensibility, and soft tissue fragility. 8 Heavy menstrual bleeding affects up to 75% of women with EDS, 9 and the bleeding phenotype typically indicates a defect in primary haemostasis. 10 The cause of bleeding in EDS is due to a fragile vascular wall from abnormal collagen and defective vessel support from the underlying connective tissue, 11 impairing interactions between collagen, platelets, and coagulation factors. Wound healing is also affected. Despite extensive efforts, the genetic background of hEDS, with an estimated prevalence of at least 1:5000, 12 , 13 remains unknown, although pedigree analyses suggest an autosomal dominant inheritance. Notably, a heterozygous missense variant in KLK15 was recently reported to be causative of hEDS in two families as well as prolonged bleeding time in mice. 14 Presently, hEDS is diagnosed clinically based on major and minor criteria outlined in the 2017 International Classification of the Ehlers–Danlos Syndromes. 15 To our knowledge, comprehensive genetic screening for inherited CTD in patients with BDUC has not been published. The prevalence of hEDS in patients examined for bleeding tendency is unknown. Based on previous studies demonstrating high frequencies of bleeding in hEDS, 9 , 16 we suspected an overlap between BDUC and hEDS.

Empirical clinical practice suggests that bruising associated with EDS may improve with high‐dose oral vitamin C supplementation, 17 an essential co‐factor for synthesizing procollagen chains that form triple‐helical collagen molecules. 18 Severe long‐term vitamin C deficiency can result in scurvy, characterized by symptoms such as skin fragility, petechiae, and spontaneous bruising. 19 Previously, we found decreased vitamin C levels in a cohort of patients with bleeding tendencies and germline variants of unknown significance in collagen‐related genes. 20

We hypothesized that BDUC is associated with poor vitamin C status. The primary aim of our study was to examine vitamin C status in patients with BDUC. The secondary aim was to evaluate the outcome of clinical evaluation for hEDS and genetic screening for CTD.

2. MATERIALS AND METHODS

2.1. Patients

From 2015 to 2023, 297 patients were referred to our haemophilia treatment centre due to suspected inherited bleeding disorders. These patients were evaluated using the International Society of Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH‐BAT), with significant bleeding defined as an ISTH‐BAT score ≥ 5 for women and ≥ 3 for men. The ISTH BAT score was determined at the initial time of diagnosis of BDUC for all patients. Initial laboratory screening included standard coagulation tests (platelet count, APTT, INR, fibrinogen, coagulation factors VIII, IX, XI, XIII, von Willebrand factor antigen, VWF ristocetin activity) and thromboelastographic screening for hyperfibrinolysis. For evaluation of platelet function defects, an accredited flow cytometry analysis was performed. Aggregation (PAC‐1), degranulation (CD62P, CD63), and phosphatidylserine expression (lactadherin) were analysed following stimulation with ADP, collagen, and TRAP. Light transmission aggregometry analysis was not available. From 2015 to 2019, germline DNA was screened using whole exome sequencing (WES), and from 2019 to 2023, using whole genome sequencing (WGS) and a continuously updated in‐house bleeding gene panel (121 genes, Table S1). No laboratory findings explained the individual bleeding phenotypes in 230/297 patients (77%). For the present study, we invited patients with BDUC, and strong bleeding phenotypes defined as an ISTH‐BAT score ≥ 10 to participate (n = 102). Following informed consent to further examinations including vitamin C status, Beighton score, skin hyperextensibility, and genetic screening for CTD, sixty patients were included in the study. Forty‐two patients were non‐available or did not consent to further examinations. Twenty healthy, age‐ and gender‐matched healthcare workers from the hospital served as HC.

2.2. Clinical evaluation

Patients and HC were examined for generalized hypermobility using the Beighton score, which measures joint hypermobility on a 9‐point scale. 21 A Beighton score of a least five points was considered positive. A skin hyperextensibility test was conducted by pulling the skin on the forearm and the lateral side of the neck until resistance was met. The test was considered positive (abnormal) if the skin extended more than 2 cm in both sites. To prevent interpersonal variability, the same medical student performed the Beighton score and skin extensibility tests for all patients and HC. Medical history, including family disposition, was obtained by the treating physician. Patients were also assessed by a trained physician for the presence of a clinical phenotype consistent with hEDS.

2.3. Vitamin C analysis

Participants received oral and written guidelines regarding vitamin C intake before their blood sample collection. These guidelines instructed participants to avoid vitamin C supplements for 3 days prior to sampling and to refrain from consuming vitamin C‐rich foods on the day of their appointment. Plasma samples were immediately acidified with 10% meta‐phosphoric acid to ensure the stability of ascorbic acid and were subsequently analysed using high‐performance liquid chromatography, as previously described. 22

Severe vitamin C deficiency was defined as <11 μM, deficiency as <23 μM, suboptimal status as 23–49 μM and adequate status as >49 μM. 23 Both the European Food Safety Association (EFSA) and the Nordic Nutrition Recommendations (NNR) have concluded that a target concentration of 50 μM would be considered adequate for vitamin C. 24 Vitamin C levels are affected by a number of factors of which gender, diet and smoking are the most important. 25 Consequently, patient's smoking status was also recorded.

2.4. WES and WGS

WES (N = 16) and WGS (N = 44) was performed using Illumina platform sequencing (TruSeq DNA LT and Illumina DNA PCR‐Free library preparation protocols, respectively). Bioinformatic processing was performed using the newest version of a continuously updated pipeline. Sequencing reads were mapped to the hg38/GRCh38 reference genome using BWA. 26 Single nucleotide variant calling was performed using GATK HaploTypeCaller (following best practices). 27 For samples processed with WGS, structural variant calling was performed using a combined approach of multiple tools: Manta, Delly2, Lumpy 28 and CNVpytor. Annotation and filtering of variants was done using VarSeq 2.5.0 (Golden Helix, Inc., Bozeman, MT, www.goldehelix.com). Variants were filtered using a continually updated in silico gene panel consisting of 121 relevant genes associated with platelet and bleeding disorders 29 and genes known to be associated with classical and vascular EDS (Table S1). For this study we further filtered using an in silico gene panel consisting of 42 genes associated with Mendelian disorders with EDS symptomatology and syndromes with EDS‐like features, including genes associated with Noonan syndrome, osteogenesis imperfecta, Loyes–Dietz syndrome and cutis laxa (Table S2). Variants were interpreted and classified according to ACMG/AMP guidelines 30 and guidelines from the Clinical Genomics Resource Sequence Variant Interpretation Working Group (SVI‐WG) (https://clinicalgenome.org/working‐groups/sequence‐variant‐interpretation/). Variants of uncertain significance were further scrutinized for relevance by considering/applying the following criteria: genotype‐phenotype correlation, previous literature reports, allele frequency in gnomAD v4.0 and in silico score using REVEL. 31 Heterozygous VUS in genes with a known recessive inheritance pattern were included as recent studies have indicated that minor effects on the phenotype may be observed in heterozygotes for several inherited haemostasis disorders. 32

2.5. Statistics

Descriptive statistics in the form of median and ranges described continuous variables. Continuous vitamin C data were normally distributed by Anderson–Darling test and had homogenous variations between groups by F‐test. Patients and HC were compared by unpaired t‐test. Chi‐squared or Fisher's Exact test (if n < 5 in any category) was used to compare categorical variables. Correlation between vitamin C levels and BAT score was tested by Spearman correlation. All p values were two‐sided, and p values < .05 denoted statistical significance. Statistical analyses were performed using GraphPad Prism (version 10.2.1 (394)).

3. RESULTS

3.1. Patient clinical characteristics

Most patients were female (56 out of 60), with a median age of 48 years (range 21−75). The median BAT score was 13 (range 10−23). Forty‐three patients reported a positive family history of bleeding in at least one first‐degree relative. The most common bleeding manifestations were menorrhagia (55 out of 60) and bleeding related to surgery (49 out of 60), followed by cutaneous bleeding (46 out of 60) and epistaxis (41 out of 60) (Figure 1). Due to menorrhagia, 16 out of 56 women had undergone a hysterectomy. Excessive bleeding, characterized by the occurrence of at least one symptom (gastrointestinal, menorrhagia, postpartum or surgery) with BAT score 4, was found in 30 out of 60 patients. Prophylactic haemostatic treatment was not provided during surgery, tooth extraction or childbirth prior to referral. The Beighton score was positive (≥5 points) in 29 out of 60 patients compared to one out of 20 HC (p = .0004). The BAT score was higher in patients with hypermobility (median 14) compared to patients with a negative Beighton score (median 12) (p = .03) (Figure 2). The test for hyperextensive skin was positive in 18 out of 60 patients, but in none of the 20 HC (p = .0041).

FIGURE 1.

FIGURE 1

Open in a new tab

Bleeding symptoms evaluated by ISTH BAT score at the time of referral in sixty patients with bleeding disorder of unknown cause. Menorrhagia was the most frequent symptom found in 55/56 women, followed by bleeding during surgery and cutaneous bleeding.

FIGURE 2.

FIGURE 2

Open in a new tab

ISTH BAT scores in hypermobile versus non‐hypermobile patients. Hypermobile are shown in orange and non‐hypermobile are shown in blue. ISTH BAT scores were significantly higher in patients with hypermobility compared to non‐hypermobile patients (Median 14 vs. 12; p = .03). Eleven patients diagnosed with a heritable connective tissue disorder syndrome (Ten patients with hypermobile Ehlers‐Danlos syndrome and one patient with Noonan syndrome) are highlighted with squares.

3.2. Patients meeting hypermobile Ehlers Danlos clinical criteria

Following clinical examinations, medical history assessments, and gathering information on family disposition, nine BDUC patients with a Beighton score ≥ 5 fulfilled the 2017 International Classification Criteria for hEDS. Additionally, a 54‐year‐old female with a Beighton score of 4, which is significant for individuals over 50 years old, also met the criteria for hEDS. 15

3.3. Vitamin C status

Vitamin C levels ranged from 7.8 to 88.0 μmol/L in patients and from 48.7 to 96.5 μmol/L in HC. Although patients' average vitamin C level was adequate, it was significantly lower than that of HC (55.9 vs. 70.4 μmol/L; p = .001). The mean vitamin C level in patients with a positive Beighton score was 55.5 μmol/L similar to the mean level in all patients (Figure 3). A suboptimal level or severe deficiency of vitamin C was found in 19 out of 60 patients (18 with suboptimal levels and one with severe deficiency) compared to one out of 20 HC (p = .018; Figure 4). Among patients with a positive Beighton score, 11 out of 29 had suboptimal vitamin C levels. In this group, the frequency of low vitamin C was higher compared to HC but not significantly different from patients without hypermobility (p = .016 and p = .41, respectively; Figure 4). In patients fulfilling the 2017 diagnostic criteria for hEDS, two out of 10 had suboptimal vitamin C levels. A positive smoking status that could negatively affect vitamin C levels was equally represented in four out of 60 patients and one out of 20 HC (p > .5). No associations were identified between low vitamin C levels, a positive Beighton score (p = .71) or a BAT score above the median (p = .46).

FIGURE 3.

FIGURE 3

Open in a new tab

Vitamin C levels in patients and healthy controls. Severe vitamin C deficiency is defined as < 11 μM, deficiency as < 23 μM, suboptimal status as 23–49 μM and adequate status as > 49 μM. The average vitamin C level in patients with bleeding disorder of unknown cause (BDUC) was in the normal range (55.9 μmol/L) but significantly lower compared to healthy controls (HC) (70.4 μmol/L) (p = .001). The average vitamin C level in BDUC patients with hypermobility was not different from BDUC patients (55,52 μmol/L).

FIGURE 4.

FIGURE 4

Open in a new tab

Proportion of patients and healthy controls with adequate Vitamin C level. Vitamin C status is shown for healthy controls (HC), patients with bleeding disorder of unknown cause (BDUC) and patients with BDUC and hypermobility (HM) (Beighton score > 5). The proportion of HC and patients with adequate vitamin C is shown in blue and suboptimal or severe deficiency in orange. Suboptimal or severe deficiency of vitamin C were found in 1/20 HC and 19/60 patients with BDUC (p < .01). Suboptimal Vitamin C levels were found in 11/29 BDUC patients with HM compared to 1/20 HC (p < .005).

3.4. Outcome of genetic screening for hereditary CTD

Filtered WES and WGS data were reviewed for genes underlying Mendelian disorders with EDS symptomatology and syndromes with EDS‐like features. The genetic screening resulted in a diagnosis of Noonan syndrome associated with a pathogenic variant in PTPN11 c.922A > G, (p.Asn308Asp) in a 67‐year old woman, whose syndrome had remarkably remained undetected until her seventh decade of life. Diagnoses of vascular, classical, and classical‐like EDS were excluded in all patients since no significant variants were identified in the relevant genes (COL3A1, COL1A1, COL5A1, COL5A2, AEBP1, TNXB). However, in a patient, fulfilling the 2017 hEDS criteria, a variant of unknown significance was identified in COL1A1 (c.Gly697Thr), which may be associated with osteogenesis imperfecta/EDS overlap disorder 33 or arthrochalasia EDS. Among the eleven patients diagnosed with a hypermobility‐associated disorder (10 with hEDS and one with Noonan syndrome), the median BAT score was 14 (range 10–21), which was higher compared to the 49 patients without a hypermobility‐associated diagnosis (median 12, range 10−23) (p = .03). For all patients, the screening of 42 CTD‐related genes identified found variants of interest that warrants further studies.

4. DISCUSSION

The aims of the study were to examine if low vitamin C status is more prevalent among patients with BDUC, and to investigate the association between hypermobility disorders and BDUC, both of which are more prevalent among women. Clinical, biochemical and genomic examinations substantiated this association. We identified a significant decrease in vitamin C levels among patients with BDUC compared to matched controls. Our data indicate that Vitamin C levels are lower in BDUC patients compared to age ‐and gender matched controls, and a large proportion of BDUC patients have inadequate vitamin C levels. We found a high frequency of hypermobility, with almost half of the patients having a positive Beighton score. Following clinical examinations and family history assessments, we determined that 10 patients met the 2017 clinical diagnostic criteria for hEDS. 15 Additionally, one patient was diagnosed with Noonan syndrome. In line with our findings, Jackson et al reported a high prevalence of symptomatic joint hypermobility in bleeding disorder patients; 24% versus 2% in controls. 34 We observed higher ISTH BAT scores in patients with hypermobility compared to those with a negative Beighton score. Considering that up to 20% of the background population can be hypermobile depending on age, ethnicity, and gender, 35 asymptomatic hypermobility may not necessarily be associated with bleeding symptoms but could indicate a predisposition for bleeding. Our results suggest that hEDS is one of many underlying causes of BDUC. Further supporting our findings, a recent retrospective study of 2149 patients clinically diagnosed with hEDS showed that 22% self‐reported bleeding and clotting problems. 36 To further assess the prevalence of significant bleeding in patients with hEDS, a prospective study implementing the ISTH BAT score is warranted. Eighteen patients with hypermobility did not fulfil the hEDS clinical criteria, A hypermobility spectrum disorder could perhaps contribute to their bleeding phenotype. Since BDUC is a diagnosis by exclusion wherein the only positive criterion is that the patient must have a significant bleeding history, we focused our study on patients with ISTH BAT score ≥ 10. Therefore, the median ISTH BAT score of 13 was higher than previously published ISTH BAT scores in BDUC cohorts. 37

In patients with hEDS, impaired collagen homeostasis may increase the consumption of vitamin C. Pre‐procollagen is formed in fibroblasts and processed in the endoplasmic reticulum through the hydroxylation of its proline and lysine residues, which allows for proper protein folding. This process requires vitamin C as an essential cofactor for lysyl oxidase. 38 Moreover, hEDS is associated with joint ‐and muscle pain as well as a chronic inflammatory state 39 which could lead to increased turnover of vitamin C beyond the body`s recycling capacity. 40 Additionally, a severe bleeding tendency could theoretically reduce quality of life, potentially resulting in a less healthy lifestyle and consequently lower vitamin C levels. Collectively, it seems plausible that poor vitamin C status is linked to bleeding and hypermobility disorders. Further studies are required to elucidate the exact mechanisms and causality of this association. Due to the lack of prospective trials, there are no established treatment recommendations for bleeding in patients with BDUC or hEDS. Haemostatic treatment options are limited to tranexamic acid, hormones, topical haemostatics, desmopressin, and platelet transfusions. Following a diagnosis of BDUC, patients were treated with desmopressin and tranexamic acid before major procedures and tranexamic acid before minor procedures, unless specific contraindications were present. Based on the present results, it would be relevant to investigate whether patients with BDUC and hEDS could benefit from oral vitamin C supplementation to reduce bleeding, as vitamin C is a non‐toxic compound without known side effects in the physiologically relevant dose range.

Our in‐house comprehensive WGS gene panel for inherited bleeding disorders includes genes associated with classical and vascular EDS. The addition of a gene panel covering various hypermobility‐related CTD did not improve WGS diagnostic outcomes, except for one patient diagnosed with Noonan syndrome. Systematic investigations of this category of genes have not previously been performed in patients with BDUC. Our results support ISTH SCC recommendations for clinical examination for hypermobility‐related syndromes in the work up of BDUC. They also suggest that future identification of causal hEDS genes could improve diagnostic yields of WGS in BDUC.

A limitation of our study is that the healthy age‐ and gender‐matched control group consisted of healthcare workers, who may have been biased towards a healthier lifestyle, including higher intake of fruits and vitamin C supplements. We attempted to mitigate this bias by providing study participants with guidelines to avoid vitamin C supplements for 3 days before blood sampling and to refrain from consuming vitamin C‐rich foods on the day of the appointment. Another limitation is that we matched 60 patients with only 20 HC. A strength of the study is the comprehensive genetic screening which allowed us to examine for inherited bleeding ‐and CTD with known genetic causes.

In conclusion, this study demonstrates that BDUC is frequently associated with low vitamin C, hypermobility, and hEDS. Based on the presented data, patients with BDUC have lower vitamin C status compared to HC. The potential causality of this association should be investigated in future controlled intervention studies with Vitamin C supplementation.

AUTHOR CONTRIBUTIONS

Eva Leinøe designed the study. Eva Leinøe, Halla Fridriksdottir, Anne Louise Tølbøll Sørensen, Eva Funding, and Peter Kampmann enrolled and treated patients, and collected data. Eva Leinøe, Andreas Ørslev Rasmussen, Maria Rossing, and Jens Lykkesfeldt analysed the data. All authors participated in interpreting the data, writing the manuscript, and approving the final submitted version.

CONFLICT OF INTEREST STATEMENT

Eva Leinøe: Received research grants from Novo Nordisk and CLS Behring. Served as consultant for Novo Nordisk. Maria Rossing: Received personal fees from AstraZeneca and MSD and serving on the advisory board of MSD outside the submitted work. Eva Funding: None ALS: Speaker for BMS and Advisory Board for Bayer. HF: None Jens Lykkesfeldt: Is director of the LifePharm Centre that is partly funded by Novo Nordisk A/S. Peter Kampmann: AbbVie: Pod cast interview; expert panel attendance. BioMarin: Congress fee; speakers bureau; advisory board. CSL Behring: Advisory board, congress fee, travel expenses, educational session on sponsored symposium. Novo Nordisk: Congress fee, travel expenses, advisory board, educational session for company employees. Roche: Educational session for company employees. Takeda: Educational session for company employees, expert panel attendance. Bayer: Educational session on sponsored symposium. SOBI: Congress fee, travel expenses, educational session on sponsored symposium.

ETHICS STATEMENT

Patients received oral and written information about WES/WGS as well as prior information regarding the risk of incidental findings. All patients and healthy controls signed informed consent form to publication of their data in concordance with the Helsinki Declaration. The study was approved by the Regional Ethics Committee, Copenhagen, Denmark (H‐15011677) and the Danish Data Registry (30‐1470).

Supporting information

Supporting Information

Additional supporting information can be found online in the Supporting Information section at the end of this article

HAE-30-1366-s001.pdf (380.8KB, pdf)

ACKNOWLEDGEMENTS

This study was supported by grants from Novo Nordisk and CLS Behring. We thank the Danish National Genome Center for providing raw data for the WGS analyses.

Leinøe E, Fridriksdottir H, Rasmussen AØ, et al. Low vitamin C status and hypermobility‐related disorders in patients with bleeding disorder of unknown cause. Haemophilia. 2024;30:1366–1372. 10.1111/hae.15099

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available upon reasonable request from the corresponding author. Regarding WGS data, all raw data files are stored at the High Performance Computer of the Danish National Genome Center. Raw data files used for the analysis in the published article are not publicly available due to institutional restrictions. A subset of the processed files can be made available to qualified researchers through application to the Danish National Genome Center. Please contact: kontakt@ngc.dk or corresponding author.

REFERENCES

  • 1. Abdelgawad HAH, Foster R, Otto M. Nothing short of a revolution: novel extended half‐life factor VIII replacement products and non‐replacement agents reshape the treatment landscape in hemophilia A. Blood Rev. 2023;64:101164. [DOI] [PubMed] [Google Scholar]
  • 2. Hermans C, Krumb E, Rotellini D, Pierce GF. The underevaluated impacts of the therapeutic revolution of hemophilia on women and girls. J Thromb Haemost. 2023;22(4):915‐918. [DOI] [PubMed] [Google Scholar]
  • 3. Mezzano D, Quiroga T. Diagnostic challenges of inherited mild bleeding disorders: a bait for poorly explored clinical and basic research. J Thromb Haemost. 2019;17(2):257‐270. [DOI] [PubMed] [Google Scholar]
  • 4. Atiq F, Saes JL, Punt MC, et al. Major differences in clinical presentation, diagnosis and management of men and women with autosomal inherited bleeding disorders. eClinicalMedicine. 2021;32:100726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Baker RI, Choi P, Curry N, et al. Standardization of definition and management for bleeding disorder of unknown cause: communication from the SSC of the ISTH. J Thromb Haemost. 2024;22:2059‐2070. [DOI] [PubMed] [Google Scholar]
  • 6. Berkowitz C, Kirkland K, Ma A, Key NS. Bleeding disorder of unknown cause & unclassified bleeding disorders at US hemophilia treatment centers. Res Pract Thromb Haemost. 2024;8(1):102296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. What are the Ehlers‐Danlos syndromes? The Ehlers Danlos Society (2022, January 26) Retrieved February 22 2024, from https://www.ehlers‐danlos.com/what‐is‐eds/ [Google Scholar]
  • 8. Malfait F, Francomano C, Byers P, et al. The 2017 international classification of the Ehlers–Danlos syndromes. Am J Med Genet C Semin Med Genet. 2017;175(0):8‐26. [DOI] [PubMed] [Google Scholar]
  • 9. Kumskova M, Flora GD, Staber J, Lentz SR, Chauhan AK. Characterization of bleeding symptoms in Ehlers‐Danlos syndrome. J Thromb Haemost. 2023;21(7):1824‐1830. [DOI] [PubMed] [Google Scholar]
  • 10. Jesudas R, Chaudhury A, Laukaitis CM. An update on the new classification of Ehlers‐Danlos syndrome and review of the causes of bleeding in this population. Haemophilia. 2019;25(4):558‐566. [DOI] [PubMed] [Google Scholar]
  • 11. Key NS, DE Paepe A, Malfait F, Shovlin CL. Vascular haemostasis. Haemophilia. 2010;16(5):146‐151. [DOI] [PubMed] [Google Scholar]
  • 12. Colombi M, Dordoni C, Chiarelli N, Ritelli M. Differential diagnosis and diagnostic flow chart of joint hypermobility syndrome/ehlers‐danlos syndrome hypermobility type compared to other heritable connective tissue disorders. Am J Med Genet C Semin Med Genet. 2015;169C(1):6‐22. [DOI] [PubMed] [Google Scholar]
  • 13. Demmler JC, Atkinson MD, Reinhold EJ, Choy E, Lyons RA, Brophy ST. Diagnosed prevalence of Ehlers‐Danlos syndrome and hypermobility spectrum disorder in Wales, UK: a national electronic cohort study and case‐control comparison. BMJ Open. 2019;9(11):e031365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Gensemer C, Beck T, Guo L, et al. Variants in the Kallikrein gene family and hypermobile Ehlers‐Danlos syndrome. Res Sq. 2024. doi: 10.21203/rs.3.rs-4547888/v1 [DOI] [Google Scholar]
  • 15. Malfait F, Francomano C, Byers P, et al. The 2017 international classification of the Ehlers‐Danlos syndromes. Am J Med Genet C Semin Med Genet. 2017;175(1):8‐26. [DOI] [PubMed] [Google Scholar]
  • 16. Artoni A, Bassotti A, Abbattista M, et al. Hemostatic abnormalities in patients with Ehlers‐Danlos syndrome. J Thromb Haemost. 2018;16(12):2425‐2431. [DOI] [PubMed] [Google Scholar]
  • 17. Bowen JM, Sobey GJ, Burrows NP, et al. Ehlers–Danlos syndrome, classical type. Am J Med Genet C Semin Med Genet. 2017;175(1):27‐39. [DOI] [PubMed] [Google Scholar]
  • 18. Libby P, Aikawa M. Vitamin C, collagen, and cracks in the plaque. Circulation. 2002;105(12):1396‐1398. [DOI] [PubMed] [Google Scholar]
  • 19. Pope M, Elder J. Scurvy: an elusive diagnosis. Clin Case Rep. 2023;11(6):e7418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Fager Ferrari M, Zetterberg E, Rossing M, et al. Collagen remodelling and plasma ascorbic acid levels in patients suspected of inherited bleeding disorders harbouring germline variants in collagen‐related genes. Haemophilia. 2021;27(1):e69‐77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Juul‐Kristensen B, Schmedling K, Rombaut L, Lund H, Engelbert RHH. Measurement properties of clinical assessment methods for classifying generalized joint hypermobility‐a systematic review. Am J Med Genet C Semin Med Genet. 2017;175(1):116‐147. [DOI] [PubMed] [Google Scholar]
  • 22. Lykkesfeldt J. Determination of ascorbic acid and dehydroascorbic acid in biological samples by high‐performance liquid chromatography using subtraction methods: reliable reduction with tris[2‐carboxyethyl]phosphine hydrochloride. Anal Biochem. 2000;282(1):89‐93. [DOI] [PubMed] [Google Scholar]
  • 23. Lykkesfeldt J, Poulsen HE. Is vitamin C supplementation beneficial? Lessons learned from randomised controlled trials. Br J Nutr. 2010;103(9):1251‐1259. [DOI] [PubMed] [Google Scholar]
  • 24. European Food Safety Authority Panel on Dietetic Products Nutrition and Allergies . Scientific opinion on dietary reference values for vitamin C. EFSA J Eur Food Saf Authority 2013;11(11):3418 (68 pp). doi: 10.2903/j.efsa.2013.3418 [DOI] [Google Scholar]
  • 25. Carr AC, Lykkesfeldt J. Factors affecting the vitamin C dose‐concentration relationship: implications for global vitamin C dietary recommendations. Nutrients. 2023;15(7):1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Li H, Durbin R. Fast and accurate short read alignment with Burrows‐Wheeler transform. Bioinformatics. 2009;25(14):1754‐1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next‐generation DNA sequencing data. Genome Res. 2010;20(9):1297‐1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Layer RM, Chiang C, Quinlan AR, Hall IM. LUMPY: a probabilistic framework for structural variant discovery. Genome Biol. 2014;15(6):R84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Megy K, Downes K, Simeoni I, et al. Curated disease‐causing genes for bleeding, thrombotic, and platelet disorders: communication from the SSC of the ISTH. J Thromb Haemost. 2019;17(8):1253‐1260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405‐423. http://www.nature.com/doifinder/10.1038/gim.2015.30 Available from [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Ioannidis NM, Rothstein JH, Pejaver V, et al. REVEL: an ensemble method for predicting the pathogenicity of rare missense variants. Am J Hum Genet. 2016;99(4):877‐885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Stefanucci L, Collins J, Sims MC, et al. The effects of pathogenic and likely pathogenic variants for inherited hemostasis disorders in 140 214 UK Biobank participants. Blood. 2023;142(24):2055‐2068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Morlino S, Micale L, Ritelli M, et al. COL1‐related overlap disorder: a novel connective tissue disorder incorporating the osteogenesis imperfecta/Ehlers‐Danlos syndrome overlap. Clin Genet. 2020;97(3):396‐406. [DOI] [PubMed] [Google Scholar]
  • 34. Jackson SC, Odiaman L, Card RT, Van der Bom JG, Poon MC. Suspected collagen disorders in the bleeding disorder clinic: a case‐control study. Haemophilia. 2013;19(2):246‐250. [DOI] [PubMed] [Google Scholar]
  • 35. Nicholson LL, Simmonds J, Pacey V, et al. International perspectives on joint hypermobility: a synthesis of current science to guide clinical and research directions. J Clin Rheumatol. 2022;28(6):314‐320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Petrucci T, Barclay SJ, Gensemer C, et al. Phenotypic clusters and multimorbidity in hypermobile Ehlers‐Danlos syndrome. Mayo Clin Proc Innov Qual Outcomes. 2024;8(3):253‐262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Mehic D, Schwarz S, Shulym I, Ay C, Pabinger I, Gebhart J. Health‐related quality of life is impaired in bleeding disorders of unknown cause: results from the Vienna Bleeding Biobank. Res Pract Thromb Haemost. 2023;7(6):102176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Manon‐Jensen T, Kjeld NG, Karsdal MA. Collagen‐mediated hemostasis. J Thromb Haemost. 2016;14(3):438‐448. [DOI] [PubMed] [Google Scholar]
  • 39. Scicluna K, Formosa MM, Farrugia R, Borg I. Hypermobile Ehlers‐Danlos syndrome: a review and a critical appraisal of published genetic research to date. Clin Genet. 2022;101(1):20‐31. [DOI] [PubMed] [Google Scholar]
  • 40. Mikkelsen SU, Gillberg L, Lykkesfeldt J, Grønbæk K. The role of vitamin C in epigenetic cancer therapy. Free Radic Biol Med. 2021;170:179‐193. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

Additional supporting information can be found online in the Supporting Information section at the end of this article

HAE-30-1366-s001.pdf (380.8KB, pdf)

Data Availability Statement

The data that support the findings of this study are available upon reasonable request from the corresponding author. Regarding WGS data, all raw data files are stored at the High Performance Computer of the Danish National Genome Center. Raw data files used for the analysis in the published article are not publicly available due to institutional restrictions. A subset of the processed files can be made available to qualified researchers through application to the Danish National Genome Center. Please contact: kontakt@ngc.dk or corresponding author.

Articles from Haemophilia are provided here courtesy of Wiley

RESOURCES