Bleeding in the heritable connective tissue disorders: Mechanisms, diagnosis and treatment

Blood Reviews 23 (2009) 191–197

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Blood Reviews journal homepage: www.elsevier.com/locate/blre

REVIEW

Bleeding in the heritable connective tissue disorders: Mechanisms, diagnosis and treatment Fransiska Malfait, Anne De Paepe * Centre for Medical Genetics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium

a r t i c l e

i n f o

Keywords: Heritable disorders of connective tissue Ehlers–Danlos syndrome Easy bruising Bleeding tendency Diagnosis Management

s u m m a r y Easy bruising and bleeding are prominent features of some heritable disorders of connective tissue (HDCT), resulting from fragility of capillaries and the perivascular connective tissue rather than clotting or platelet dysfunction. The bleeding tendency is most prominent in the Ehlers–Danlos syndrome (EDS), a heterogeneous group of HDCT sharing clinical manifestations of fragility in skin, ligaments, blood vessels and internal organs. Most EDS-subtypes are caused by mutations in genes encoding fibrillar collagens type I, III or V, or genes encoding enzymes involved in the posttranslational modification of collagens. In the vascular subtype of EDS, caused by defects in type III collagen, fragility of vessel walls can lead to life-threatening bleeding and premature death. Bleeding tendency is also a common feature in other EDS-subtypes, leading to bruising either spontaneously or after minimal trauma. This paper reviews the clinical aspects of bleeding and bruising in different HDCT and covers diagnostic and therapeutic aspects relevant to bleeding in these disorders. Ó 2009 Elsevier Ltd. All rights reserved.

Mechanisms underlying bleeding in the HDCT Components of the extracellular matrix Collagens are the most abundant proteins in the body. They are trimeric proteins that are characterized by the presence of triplehelical domains. To date, 43 collagen genes have been described, the products of which combine to form at least 28 different collagen molecules. The fibril-forming or fibrillar collagens represent the most widespread and abundant class of collagens. They include the collagen types I, II, III, V and XI. They are observed in tissues as long, highly ordered fibrils with a characteristic banding pattern. Type I collagen is the major collagen type in the body and has a widespread tissue distribution. It is a heterotrimer of two a1-chains and one a2-chain, encoded by the COL1A1 (chromosome 17) and the COL1A2 gene (chromosome 7), respectively. Type II and type XI collagen are predominantly found in cartilage. Type III collagen is a homotrimer consisting of three identical a1-chains, encoded by the COL3A1 gene on chromosome 2. It is an essential component of many connective tissues and is found in stretchable tissues such as the blood vessel walls, the gastro-intestinal tractus, the uterus and the skin. Type V collagen is co-expressed with type I collagen in many connective tissues and plays an important role in the fibrillogenesis of this collagen type.1

* Corresponding author. E-mail address: [email protected] (A. De Paepe). 0268-960X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.blre.2009.06.001

Each fibrillar collagen has a central uninterrupted triple-helical domain with short non-helical domains at the carboxy- and amino-terminal end. The presence of glycine, the smallest aminoacid, in every third position of each chain is a prerequisite for the formation of a stable collagen helix. The biosynthesis of fibrillar collagens in the fibroblast is a complex process and starts with the synthesis of soluble precursor molecules, procollagens. These contain globular amino- and carboxy-terminal propeptide extensions, called the N- and the C-propeptide. Intracellular association of three pro-a-chains occurs through interaction and disulphide bonding at the C-propeptide. In this way, correct alignment of the growing polypeptide chain is obtained as required for formation and propagation of the triple-helix from the C- to the N-terminal end of the molecule. During helix propagation, the pro-a-chains undergo extensive enzymatic modifications (i.e. hydroxylation of prolyl and lysyl residues), which cease when the helix is formed. Mature triple-helical procollagen molecules are secreted into the extracellular environment where they are converted to collagen by enzymatic removal of the N- and the C-propeptides. Individual collagen molecules spontaneously assemble in a non-enzymatic process to form fibrils and fibres, which are stabilized by covalent cross-linking. Besides collagen, connective tissues also contain elastic fibers, proteoglycans, and a wide variety of glycoproteins. Elastic fibers are composed of two distinguishable components: an amorphous component, ‘‘elastin”, which contributes to the elasticity of the fiber, and a surrounding sheet of microfibrils. An example of a microfibrillar protein is fibrillin, which is the protein mutated in

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patients with Marfan syndrome. Elastin and other structural proteins are woven onto the microfibrillar array to provide the basic meshwork for the connective tissue matrix. Abnormalities of elastin have been associated with other connective tissue disorders, such as cutis laxa. Proteoglycans are core proteins that are bound to glycosaminoglycans. Essentially, proteoglycans are the glue of the connective tissue protein that seal and cement the underlying connective tissue matrix. Macromolecular proteins include the glycoproteins of the basement membrane (type IV collagen, laminin, nidogen) and of the extracellular matrix (fibronectin, tenascin, fibulins). Haemostasis and bleeding in heritable disorders of connective tissue Haemostasis is performed by the concerted interaction of three components: the vessel wall, platelet function, and the clotting cascade. When the integrity of the vascular system is affected, platelets become exposed to and interact with perivascular components of the extracellular matrix, among which type I and type III collagen are of principal importance. Platelet interaction with collagen is a complex phenomenon. The platelets adhere to subendothelial collagens exposed at sites of injury via several specific platelet membrane glycoproteins and non-integrin receptors. Initial entrapment of the platelets on subendothelial collagens requires the plasma protein von Willebrandt factor (vWF) which directly binds to collagen and facilitates transient tethering and rolling of the platelets. Subsequently, collagens bind to platelet receptors, principally integrin a2b1 and GPVI, stimulating platelet signaling, shape change and spreading and the secretion of multiple haemostatic factors.2 The skin and subcutaneous tissues are normally natural barriers against external trauma to the blood vessel. In the HDCT, increased fragility of the connective tissues in skin, subcutaneous tissues and blood vessel walls perturbs the normal anatomic or physiologic barriers against bleeding, resulting in a higher propensity for bruising and bleeding (Table 1). In EDS for example, the bleeding diathesis is explained by an abnormal capillary structure with deficiency of normal perivascular collagen, resulting in poor support of cutaneous blood vessels which rupture when subjected to shearing forces. Different forms of EDS have been associated with more or less well-characterized platelet or coagulation dysfunctions, including platelet aggregation dysfunction and prolonged bleeding time,3–9 platelet delta-storage pool disease,10 deficiency of factor VIII,11,12 factor IX,13 factor XI,5 factor XII14 and factor XIII5 and platelet sensitivity to aspirin.15 Most of these observations however are sporadic and likely to be chance associations, where the platelet or coagulation dysfunctions may have added to the bleeding tendency of an underlying EDS. Table 1 HDCT associated with bruising and bleeding. Skin Ehlers–Danlos syndrome Subcutaneous tissue Ehlers–Danlos syndrome Osteogenesis Imperfecta Marfan syndrome Vascular Ehlers–Danlos syndrome Osteogenesis Imperfecta Marfan syndrome Loeys–Dietz-syndrome Platelet dysfunction / Coagulation protein disorder Pseudoxanthoma elasticum-like (factors II, VII, IX and X)

A recently identified condition, coined as ‘pseudoxanthoma elasticum-like disorder’, is associated with a deficiency of the vitamin-K dependent clotting factors (II, VII, IX and X), due to mutations in the GGCX-gene, involved in the c-carboxylation of clotting factors and matrix gla-proteins.

Bleeding and bruising in the HDCT: clinical overview The Ehlers–Danlos syndrome The Ehlers–Danlos syndrome (EDS) comprises a clinically and genetically heterogeneous group of connective tissue diseases of which the principle clinical features are skin hyperextensibility, delayed wound healing with atrophic scarring, joint hypermobility, bleeding tendency and generalized connective tissue fragility.16 These clinical manifestations are present, to varying degrees, in each subtype of the condition. In EDS, the bleeding tendency manifests itself mainly as easy bruisability, bleeding of gums, prolonged bleeding after dental or surgical procedures and prolonged menstrual episodes. In children with EDS, excessive bruising is often the presenting complaint to the paediatrician. If pronounced, it can cause suspicion of a haematological disorder, a malignancy, or child abuse. Careful evaluation of the medical and family history, and rigorous clinical examination with special attention to skin features that are characteristic for EDS, are mandatory to distinguish between a HDCT and other causes of bruising. Epistaxis, petecchiae, hematuria, hemophtysis and hemarthrosis are usually not observed, except when rupture of a specific blood vessel is associated. Arterial rupture leading to life-threatening complications is a hallmark of the vascular subtype of EDS, which is discussed below. Skin hyperextensibility means that the skin extends easily and snaps back after release, and should be differentiated from cutis laxa, were skin is redundant, hangs in loose folds and only slowly returns to its former position. Widened atrophic scarring occurs mainly over knees, elbows, shins, forehead and the chin. It is characterized by splitting of the skin following relatively mild trauma, and formation of wide and thin ‘‘cigarette-paper-like” scars. In areas of repetitive trauma hemosiderin deposition may lead to dark and unaesthetic discoloration of the skin. Joint hypermobility is often general, affecting both large and small joints and usually comes to attention when a child starts to walk. It frequently leads to joint subluxation or dislocation and chronic musculoskeletal pain. The latest classification of EDS, the Villefranche Nosology, recognizes six genetic subtypes, which differ in clinical symptoms, inheritance pattern and the nature of the underlying biochemical and molecular defect(s).17 Several EDS subtypes are caused by mutations in the genes for collagen type I (arthrochalasis type), type III (vascular type) or type V (classic type) or in the genes involved in the processing of type I collagen (kyphoscoliosis and dermatosparaxis type) (Table 2). The classic, hypermobile and vascular type of EDS are the most common, while the kyphoscoliosis, arthrochalasis and dermatosparaxis type represent very rare conditions. The Villefranche classification is important to help the clinician in establishing the accurate subtype of EDS, which is very important in terms of management and counselling to the patient and his/her family. Besides the recognized subtypes however, there are many unclassified EDS variants, in which the underlying molecular defect is usually not known. Vascular Ehlers–Danlos syndrome The vascular type of EDS, previously called EDS type IV or the arterial-ecchymotic type of Sack–Barabas,18,19 is an autosomal

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F. Malfait, A. De Paepe / Blood Reviews 23 (2009) 191–197 Table 2 The different types of EDS according to the Villefranche Nosology and their underlying defect. Type

Old nomenclature

Inheritance pattern

Protein

Gene

Classic Hypermobility

Type I/II Type III

AD AD

Vascular Kyphoscoliosis Arthrochalasia Dermatosparaxis

Type Type Type Type

AD AR AD AR

Type V collagen (50%) Tenascin-X (5%) Unknown Type III collagen Lysyl hydroxylase Type I collagen Procollagen N-proteinase

COL5A1, COL5A2 (50%) TNX-B (5%) Unknown COL3A1 PLOD1 COL1A1, COL1A2 ADAMST2

IV VI VII A/B VIIC

AD: autosomal dominant. AR: autosomal recessive.

dominant disorder that is caused by defects in the proa1(III) chain of type III collagen, encoded by the COL3A1 gene. Of all EDS subtypes, it has the worst prognosis because of the risk of potentially fatal vascular and intestinal complications. Unlike other types of EDS, the skin is not hyperextensible, but rather thin and translucent, showing a visible venous pattern over the chest, abdomen and extremities.16,20 Excessive bruising is the most common sign and is often the presenting complaint. Bleeding from gums following teeth brushing, or profuse bleeding after tooth extraction, are also frequent signs. The bleeding tendency may instigate extensive haematological evaluation, with the usual result that no abnormality is identified. The Rumpel–Leede (or Hess) test may be positive, indicating capillary fragility.5,20 Other early manifestations include premature rupture of the membranes, congenital club foot or congenital hip dislocation, inguinal hernia, pneumothorax and recurrent joint dislocation or subluxation.21 Patients with vascular EDS often display a characteristic facial appearance, with prominent eyes (due to lack of subcutaneous adipose tissue around the eyes), a thin, pinched nose and small lips, hollow cheeks and lobeless ears. Hypermobility is usually limited to the small joints of the hands. Excessive wrinkling and thinness of the skin over hands and feet may produce an old-looking appearance, referred to as ‘‘acrogeria”. The generalized vascular fragility largely dominates the clinical picture. Apart from excessive bruising and bleeding, it may cause precocious and severe varicosities and arterial rupture, potentially resulting in sudden death, usually in the third or the fourth decade of life. The vascular fragility affects large as well as small blood vessels, and bleeding may occur at every possible site in the body. The most common location of arterial bleeding is the abdominal cavity due to rupture of medium-sized arteries, such as the renal or splenic arteries, rather than of the aorta itself. Acute myocardial infarction due to coronary dissection or rupture is a rare complication. Some affected individuals may harbour predisposing lesions such as aneurysms or arterio–venous fistulae, but in other patients ruptures occur at locations that appear completely normal by angiography.16 Besides the vascular ruptures, dangerous internal complications such as spontaneous rupture of the bowel (usually the colon, sometimes the intestine), the gravid uterus, and hemorrhagic pneumothorax may occur.22–26 Although uncommon, EDS type IV is a cause of stroke in young adults. The mean age of intracranial aneurismal rupture, spontaneous carotid-cavernous sinus fistula and cervical artery aneurysm is 28 years.27 Obstetrical complications are frequent and include vascular, intestinal or uterine rupture, vaginal lacerations, prolapse of uterus and bladder, and premature delivery because of cervical insufficiency or fragility of the membranes. Patients with vascular EDS who are pregnant should be enrolled in a high-risk obstetrical program. The clinical appearance of patients with vascular EDS may deviate from the typical picture, and especially the facial and cutaneous features, such as the acrogeria, may be very subtle or even absent. In the absence of a positive family history or a major vascular or

intestinal complication, clinical diagnosis is difficult, especially in children. The diagnosis of vascular EDS is based on clinical findings. Confirmation of a suspected diagnosis is possible by biochemical demonstration of a type III collagen deficiency which is present in virtually all patients with vascular EDS. Biochemical testing involves SDS–polyacrylamide gel electrophoresis (SDS–PAGE) of radioactively labelled collagens extracted from skin fibroblast cultures. This analysis probably identifies more than 95% of individuals harbouring a defect of type III collagen. It allows detecting quantitative (reduced amounts of collagen type III) or qualitative (structurally abnormal type III collagen with altered electrophoretic mobility) defects of type III collagen. Molecular genetic testing to identify mutations in the COL3A1 gene is available to patients with a biochemically confirmed diagnosis of vascular EDS. To date more than 250 COL3A1 mutations have been identified,28 most of which are point mutations leading to substitutions for glycine in the triple-helical region of the collagen molecule. Other types of mutations include splice site mutations, partial gene deletions, and, rarely, mutations resulting in COL3A1 haplo-insufficiency.16,29 Parental mosaicism for COL3A1 mutations has been documented in vascular EDS30–33 and may explain unexpected recurrences in families where a ‘new’ dominant mutation was identified. Prenatal and pre-implantation diagnosis based on direct demonstration of the mutation in embryonic tissues can be offered in at-risk pregnancies. Genotype-phenotype correlations have been investigated extensively in vascular EDS. Missense mutations located at the extreme carboxyl-terminal end of the molecule usually cause the so-called ‘‘acrogeric” form of EDS, associated with severe vascular problems and early death. This relationship is however not absolute and severe clinical phenotypes have been reported with more amino-terminal-located mutations as well. Bleeding and bruising in other EDS-subtypes Easy bruising is, to a variable degree, a common complaint in all subtypes of EDS. Apart from the vascular subtype, it is most prominent in the dermatosparaxis subtype, an autosomal recessive condition, characterized by extreme fragility and laxity of the skin, premature rupture of the membranes, large fontanels, umbilical hernia, short stature and characteristic dysmorphic facies, and an increased risk for bladder rupture. No severe vascular complications have been reported yet.34–36 The condition is caused by a deficient activity of procollagen-N-proteinase due to mutations in the gene encoding ADAMTS-2.37,38 This enzyme is mainly responsible for cleavage of the N-terminal propeptide of procollagen type I,39 but has also been shown to process procollagens type II, III40 and V.41 Easy bruising is also a prominent feature in an autosomal recessive HDCT caused by mutations in the gene encoding the non-collagenous protein tenascin X.42 Tenascin-X is a large extracellular matrix glycoprotein which plays a role in the maturation and/or

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the maintenance of the dermal collagen and elastin network. Its complete absence results in a classic EDS-like phenotype with extensive bruising, skin- and joint hypermobility, and occasionally increased laxity of the genito-urinary tract causing uterine and vaginal prolapse, and increased risk for post-partum haemorrhage.43 EDS type VIII or periodontic type of EDS is a distinct entity that is characterized by severe early-onset periodontal disease in conjunction with the features of EDS, such as increased tendency to bruising on mild trauma, skin hyperextensibility and fragility and mild to moderate joint hypermobility, especially of the digits. Discrete, chronically inflamed pretibial plaques, reminiscent of necrobiosis lipoidica, are often present.44–46 The existence of EDS type VIII as a distinct entity was uncertain, until a genome-wide linkage analysis established linkage of EDS type VIII to a region on chromosome 12q13 in a large Swedish family.47 The existence of EDS type X as a distinct entity is questionable.17 It was first described by Arneson et al. in 1980 in a family with a recessively inherited variant of EDS (joint hypermobility, thin skin with atrophic scarring, easy bruising, petechiae) and a platelet aggregation defect. This defect can be corrected in vitro by the addition of normal human fibronectin. On the other hand, the patient’s plasma failed to support the aggregation of gelfiltered platelets from controls in response to collagen. Since the measured levels of immunoreactive fibronectin were normal and immunohistochemical studies showed fibronectin to be present in the platelet a-granules, the authors assumed a minor structural abnormality of the fibronectin molecule to be present. Of note, altered production, assembly and distribution of fibronectin have been observed in cultured fibroblasts from patients with several types of EDS.48–50 On the other hand, decreased levels of fibronectin with a normal electrophoretic mobility in eight members of a three-generation family, did not result in abnormalities of platelet aggregation or clinical signs of EDS.51 Although arterial dissection or rupture is mainly restricted to the vascular subtype of EDS, it can rarely occur is some other EDS-forms. Spontaneous rupture of large arteries, along with intracranial aneurysms and arteriovenous fistulae, may occur in the rare individuals with a severe form of classic EDS. Mutations in the genes encoding type V collagen (COL5A1 and COL5A2) are responsible for approximately 50% of patients with classic EDS. Rupture of middle-sized arteries have also been observed in a series of adults with a classic/vascular EDS-overlap syndrome, caused by specific arginine-to-cysteine mutations in COL1A1, the gene encoding the proa1(I) chain of type I collagen.52 Both aortic dilatation and dissection, and rupture of mediumsized arteries have been observed in patients with the rare autosomal recessive kyphoscoliotic type of EDS. This condition is caused by a deficient activity of procollagen-lysine-2-oxoglutarate 5 dioxygenase-1 (PLOD-1 or lyslyhydroxylase-1), a collagen-modifying enzyme. Other features of this condition include severe progressive kyphoscoliosis, joint hypermobility and ocular fragility with retinal detachment and bleeding and rupture of the ocular globe. Other HDCT Easy bruising can occasionally be seen in other heritable collagen disorders, such as Osteogenesis Imperfecta. This condition is characterized by a variable degree of bone fragility, with multiple fractures and sometimes bone deformities, short stature, blue sclerae and hearing impairment. It is caused by mutations in the COL1A1 and COL1A2 gene, encoding type I collagen. Rarely, individuals with OI may suffer from vascular dissection.53 In patients presenting with aortic dilatation or dissection, Marfan syndrome (MFS) should be considered. Usually MFS can easily be distinguished from vascular EDS by clinical evaluation. The

diagnosis of MFS requires the presence of a combination of clinical manifestations in different organ systems, among which the skeletal system, the eyes and the cardiovascular system. Patients with MFS often present a typical ‘‘marfanoid habitus” with a long and slender build, mild joint hypermobility which can be generalized, scoliosis and/or pectus deformities and arachnodactyly. The most typical ocular manifestation is bilateral lens dislocation. Most importantly however these patients are at-risk for developing dilatation and dissection of the aorta. There is no increased fragility of capillaries and small-to-middle-size arteries and veins. Some patients with MFS however complain of easy bruising. Several causal factors may be at work, e.g. patients with MFS may have a thinner skin, and less subcutaneous fat, and hence less protective cushioning for minor traumata. Also, joint laxity and poor visual acuity may contribute to a predisposition to contusions in some patients. Some patients with MFS present mild hyperextensibility of the skin, reminiscent of EDS. MFS is an autosomal dominant disorder, caused by mutations in fibrillin-1. Fibrillins polymerize extracellularly as parallel bundles and form microfibrils in association with latent transforming growth factor beta binding proteins (LTBPs) among many others. Microfibrils provide structural support and can associate with elastin to form elastic fibers, providing elasticity to the tissues in a time and tissue-dependent manner.54 For a long time, it was believed that the pathophysiology of MFS was entirely based on severely reduced and fragmented elastic fibers in affected tissues. This observation led the hypothesis that structural deficiency of the fibrillin-1 protein was the most important player in the etiology this condition. While this hypothesis offered an explanation for the connective tissue weakness, it did not reconcile the observation of long bone overgrowth, thickening of the cardiac valves, muscle hypoplasia or alveolar septation defects. The creation and analysis of fibrillin-1 mutant mouse lines that faithfully recapitulate the MFS spectrum has recently challenged this ‘mechanistic’ view. The earliest observations in aneurysm formation showed that fibrillins are needed for the maintenance, rather than the assembly of elastic fibers.55 Also, it became increasingly evident that microfibrils have an essential role in the regulation of growth factors that effect tissue development and homeostasis. Mouse models have shown that structural fibrillin-1 deficiency leads to increased activation of the sequestered cytokine, transforming growth factor beta (TGFb).56 Enhanced activation of the TGFb pathway was shown to contribute to the development of emphysema, aortic aneurysms and muscle hypoplasia seen in MFS. In murine models these changes can be effectively blocked by the administration of TGFb antibodies.56–58 Further evidence of perturbed TGFb signaling in aortic aneurysm came from the identification of the Loeys–Dietz syndrome, caused by heterozygous mutations in the TGFBR1 and TGFBR2 genes, encoding the TGFbeta receptors 1 and 2. Loeys–Dietz syndrome (LDS) is a novel autosomal dominant aortic aneurysm syndrome characterized by a triad of hypertelorism, bifid uvula/cleft palate and arterial tortuosity with ascending aortic aneurysm/dissection. The main differences with Marfan syndrome are the absence of long bone overgrowth and lens dislocations, and the presence of multiple other findings, including craniosynostosis, Chiari malformation, club feet, patent ductus arteriosus, and aneurysms and dissections throughout the arterial tree. In contrast to this typical presentation, which was later coined as Loeys–Dietz syndrome type I, some patients show less craniofacial abnormalities but more prominent joint and skin manifestations, reminiscent of vascular EDS. This subset of patients (referred to as Loeys–Dietzsyndrome type 2) is characterized by easy bruising, a velvety translucent skin, widened atrophic scars, uterine rupture, severe peripartal bleeding, and arterial aneurysm/dissection throughout the arterial circulation.

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The natural history of LDS type I and II is far more aggressive than MFS or even vascular EDS, with a mean age of death of 26 years. Aortic dissections occur in young childhood and/or at smaller aortic dimensions, and the incidence of pregnancy-related complications is high.59 While patients with pseudoxanthoma elasticum are usually not subject to a bleeding diathesis, a recently described pseudoxanthoma elasticum-like condition was shown to be associated with a deficiency of the vitamin-K dependent clotting factors.60 This condition can easily be distinguished from EDS upon clinical examination of the skin. Affected patients present however clinical overlap with classic PXE, with manifestations of yellowish papules as observed in classic PXE, however the severity of the skin symptoms, with spreading toward the trunk and limbs and the occurence of thick, leathery skin folds is also reminiscent of autosomal dominant cutis laxa syndrome. Some affected individuals were reported to have a bleeding tendency, including epistaxis, gingival bleeding, vaginal or post-partum hemorrhage and haematemesis. This condition is caused by specific mutations in the GGCX-gene encoding a vitamin-K dependent carboxylase which is essential for c-carboxylation of clotting factors, enabling them to attach to the phospholipid bilayer of membranes as a prerequisite for blood coagulation. Diagnosis The evaluation of the patient presenting bleeding or bruising in whom a HDCT is suspected should start with a detailed clinical and family history coupled with a thorough physical examination. Laboratory investigation of platelet aggregation, clotting factors and bleeding time in patients with EDS or other HDCT is usually normal. In certain EDS subtypes, biochemical and molecular analyses can be very helpful to confirm the diagnosis. To this purpose, a skin biopsy is required to obtain cultured skin fibroblasts. Ultrastructural examination of the skin, performed by electron microscopy, usually reveals abnormalities of collagen fibrillogenesis. These include irregular, disrupted collagen fibrils (‘‘collagen-flowers”) and variability within the diameter of the individual collagen fibrils. However, these abnormalities are found in several EDS variants and are usually insufficient to discriminate between individual EDS subtypes. Only in the dermatosparaxis subtype of EDS, a pathognomonic ultrastructural aspect of the collagen fibril architecture is observed. Collagen fibrils in the dermatosparaxis subtype lose their normal cross-sectional circular aspect and have instead an irregular, branched, ‘‘hieroglyphic” appearance (Pierard, 1976; Malfait, 2004). Biochemical study of the collagen types I, III and V includes SDS–polyacrylamide gel electrophoresis of radio-labelled collagens, extracted from the cultured fibroblasts. In the vascular subtype of EDS, biochemical analysis of type III procollagen identifies more than 95% of all patients. Molecular screening of the COL3A1 gene identifies virtually all mutations. Biochemical analysis can also be helpful in the diagnosis of the arthrochalasis, kyphoscoliosis, dermatosparaxis, and some other rare subtypes of EDS, where distinct abnormal migration patterns of type I (pro)-collagen chains are observed. These findings guide further molecular analysis of the correct genes. For the other HDCT discussed in this paper, sequence analysis of the casual genes is available when the clinical diagnosis is strongly suspected. Management of the bleeding tendency in EDS No causal therapy is available for EDS, however a series of ‘‘preventive” guidelines are applicable to all forms of EDS.

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General measures In EDS it remains important to control the risk of vascular damage by avoidance of well known risk factors of atherosclerotic cardiovascular disease such as smoking, hypertension, dyslipidemia, obesity and diabetes mellitus. A healthy life style applies to all affected individuals and this includes a balanced diet with fruit and vegetables, a reduced intake of saturated fats, daily physical exercise, and avoidance of overweight and smoking, as well as moderation of alcohol consumption. Patients, especially children, with pronounced skin fragility should wear protective pads or bandages over the forehead, knees and shins, in order to avoid skin lacerations. Dermal wounds should be closed without tension, preferably in two layers. Deep stitches should be applied generously. Cutaneous stitches should be left in place twice as long as usual, and additional fixation of adjacent skin with adhesive tape can help prevent stretching of the scar. Patients with pronounced bruising are advised to avoid contact sports and heavy exercise (Pepin and Byers, 2002). Protective pads and bandages can be useful also in the prevention of bruises and haematomas. Supplementation of ascorbic acid, a cofactor for cross-linking of collagen fibrils, can ameliorate the tendency towards bruising in some patients.16 Potential hazards from drugs or clinical procedures in vascular EDS For the vascular type of EDS, some prophylactic measures are of special importance. Drugs that interfere with the haemostatic process should especially be avoided by patients with this subtype. Drugs that interfere with platelet function include aspirin (acetylsalicylic acid, ASA), dipyridamole, clopidogrel, and non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and diclofenac. These drugs affect platelet function, thereby prolonging the bleeding time. Aspirin, clopidogrel and dipyridamole are frequently prescribed as therapeutic agents in patients with coronary artery disease, cerebrovascular disease, and peripheral arterial disease. Ibuprofen and diclofenac are popular as pain killers and anti-inflammatory agents. Paracetamol (acetaminophen) and COX-1 sparing NSAIDS, such as celecoxib, do not influence haemostasis and can be considered safe. By definition, anticoagulant drugs will increase the bleeding tendency. These include the traditional oral vitamin-K antagonists (coumarins) such as acenocoumarol, fenprocoumon and warfarin; heparin and low molecular weight heparins (LMWH), pentasaccharides, and the newer oral thrombin inhibitors and pentasaccharides. Invasive vascular procedures such as arteriography and catheterization should be avoided because of the risk of vascular ruptures which cause significant morbidity and may have fatal outcome.61,62 They should rather be replaced by ultrasonography and/or subtraction angiography. Surgical interventions are generally discouraged because of increased vascular fragility and conservative therapy is recommended. If surgery is unavoidable, thorough investigation of platelet function and clotting is appropriate, as affected persons are already subject to bleeding from ruptured vessels or organs and an intrinsic clotting defect may complicate clinical outcome. Manipulation of vascular and other tissues should be done with extreme care. Although no effective preventive treatment yet exists for vascular EDS, the use of b-adrenergic blockade is now under study, in analogy with the prophylactic efficacy of b-blockers in slowing the rate of aortic dilation and reducing the development of aortic complications in some patients with Marfan syndrome.63

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Vasopressin analogue DDAVP and recombinant factor VIIa DDAVP may help to reduce a bleeding tendency temporarily in subjects undergoing a dental or surgical procedure. The vasopressin analogue DDAVP (desmopressin acetate, 1-Desamino-8-D-Arginine Vasopressin) increases the concentrations of Factor VIII-vWf temporarily, whether administered intravenously, subcutaneously, or by nasal spray. This effect is ascribed to induced release of these factors from storage sites in vascular endothelial cells. Administration of vasopressin has been applied successfully in subjects with mild hemophilia, von Willebrand disease and in other patients with a variety of qualitative platelet abnormalities such as uremia or liver cirrhosis. It is important to test the effect of vasopressin before a procedure is carried out. Vasopressin can be of use for patients with EDS who do not have a haemostatic defect but who have a prolonged bleeding time due to an abnormality in the vessel wall. Two children, one with the periodontitis type EDS and one with the kyphoscoliosis type EDS, both with a prolonged bleeding time, had a normalized bleeding time after administration of vasopressin. Both children successfully underwent surgical procedures without bleeding complications. One patient continued using intranasal vasopressin to avoid nasal and gum bleedings.64 Perioperative use of recombinant factor VIIa may be useful in management of continued bleeding after surgical repair of ruptured medium-sized vessels in vascular type EDS. A case report describes the successful use of recombinant factor VIIa in a patient with vascular type EDS, in whom continued bleeding was successfully stopped after intravenously administered recombinant factor VIIa. Of note, the platelet count, prothrombin time and activated partial thromboplastin time were all normal.65 Practice points  Easy bruising is a common finding in patients with EDS, and may be the presenting symptom in children  EDS is extremely heterogeneous, both at the clinical and molecular level  Genetic counselling is an essential part of EDS management  Patients with vascular EDS require a special approach, with avoidance of surgery and invasive vascular procedures Research agenda  Study of the efficacy of b-adrenergic blockade as a preventive treatment for vascular EDS  Many EDS-variants cannot be classified into the current classification system. Elucidation of their natural history, and/or their underlying biochemical and molecular defect will improve early diagnosis, management and genetic counselling, and will allow for the development of more effective therapies Conflict of interest statement No conflict of interest. Acknowledgments This study was supported by the Fund for Scientific Research, Flanders (Belgium) (Grant No. G.0171.05) and Ghent University (Grants No. 12051203 and 01M01108). References 1. Birk DE. Type V collagen: heterotypic type I/V collagen interactions in the regulation of fibril assembly. Micron 2001;32:223–37.

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