hydroxylysine and Ehlers-Danlos-Syndrome

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.

Topics: Animals; Arthrogryposis; Connective Tissue Diseases; Ehlers-Danlos Syndrome; Fibrillar Collagens; Glycosylation; Humans; Hydroxylation; Hydroxylysine; Hydroxyproline; Lysine; Osteogenesis Imperfecta; Proline; Protein Folding

Topics: C-Peptide; Chemical Phenomena; Chemistry; Collagen; Collagen Diseases; Copper; Ehlers-Danlos Syndrome; Genetic Variation; Humans; Hydroxylysine; Marfan Syndrome; Menkes Kinky Hair Syndrome; Mutation; Osteogenesis Imperfecta; Phenotype; Procollagen; Protein Biosynthesis; Protein-Lysine 6-Oxidase; Transcription, Genetic

Topics: Adolescent; Adult; Child; Collagen; Ehlers-Danlos Syndrome; Female; Fibronectins; Humans; Hydroxylation; Hydroxylysine; Male; Middle Aged; Pepsin A; Periodontal Diseases; Procollagen; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Protein-Lysine 6-Oxidase; Proteoglycans; Skin; X Chromosome

Topics: Collagen; Ehlers-Danlos Syndrome; Epidermolysis Bullosa; Glucocorticoids; Humans; Hydroxylysine; Hydroxyproline; Microbial Collagenase; Scleroderma, Systemic; Skin; Skin Diseases; Skin Neoplasms

The collagens are the major structural glycoproteins of connective tissues. A unique primary structure and a multiplicity of post-translational modification reactions are required for normal fibrillogenesis. The post-translational modifications include hydroxylation of prolyl and lysyl residues, glycosylation, folding of the molecule into triple-helical conformation, proteolytic conversion of precursor procollagen to collagen, and oxidative deamination of certain lysyl and hydroxylysyl residues. Any defect in the normal mechanisms responsible for the synthesis and secretion of collagen molecules or the deposition of these molecules into extracellular fibers could result in abnormal fibrillogenesis; such defects could result in a connective tissue disease. Recently, defects in the regulation of the types of collagen synthesized and in the enzymes involved in the post-translational modifications have been found in heritable diseases of connective tissue. Thus far, the primary heritable disorders of collagen metabolism in man include lysyl hydroxylase deficiency in Ehlers-Danlos syndrome type VI, p-collagen peptidase deficency in Ehlers-Danlos syndrome type VII, decreased synthesis of type III collagen in Ehlers-Danlos syndrome type IV, lysyl oxidase deficency in S-linked cutis laxa and Ehlers-Danlos syndrome type V, and decreased synthesis of type I collagen in osteogenesis imperfecta.

Topics: Aortic Diseases; Bone and Bones; Brain Diseases; Collagen; Connective Tissue; Cutis Laxa; Ehlers-Danlos Syndrome; Fascia; Genetic Linkage; Homocystinuria; Humans; Hydroxylysine; Hydroxyproline; Joints; Marfan Syndrome; Microbial Collagenase; Osteogenesis Imperfecta; Protein Biosynthesis; Protein Conformation; Protein-Lysine 6-Oxidase; Sex Chromosomes; Skin Abnormalities

Topics: Animals; Collagen; Ehlers-Danlos Syndrome; Humans; Hydroxylysine; Microscopy, Electron, Scanning; Protein Biosynthesis; Protein Conformation; Protein Precursors

Topics: Adolescent; Chemical Phenomena; Chemistry; Collagen; Collagen Diseases; Complement C1; Ehlers-Danlos Syndrome; Female; Humans; Hydroxylysine; Infant; Ketoglutaric Acids; Lysine; Mixed Function Oxygenases; Phenotype; Solubility

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.

Topics: Animals; Collagen; Disease Models, Animal; Ehlers-Danlos Syndrome; Gait; Hydroxylysine; Mice; Mice, Inbred Strains; Mice, Knockout; Muscle Hypotonia; Phenotype; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Skin Diseases; Tissue Distribution

Type VI Ehlers-Danlos syndrome is a disease characterized by disturbed lysine hydroxylation of collagen. The disease is caused by mutations in lysyl hydroxylase 1 gene and it affects several organs including the cardiovascular system, the joint and musculoskeletal system, and the skin. The skin of type VI Ehlers-Danlos syndrome patients is hyperelastic, scars easily, and heals slowly and poorly. We hypothesized that providing functional lysyl hydroxylase 1 gene to the fibroblasts in and around wounds in these patients would improve healing. In this study we tested the feasibility of transfer of the lysyl hydroxylase 1 gene into fibroblasts derived from rats and a type VI Ehlers-Danlos syndrome patient (in vitro) and into rat skin (in vivo). We first cloned human lysyl hydroxylase 1 cDNA into a recombinant adenoviral vector (Ad5RSV-LH). Transfection of human type VI Ehlers-Danlos syndrome fibroblasts (about 20% of normal lysyl hydroxylase 1 activity) with the vector increased lysyl hydroxylase 1 activity in these cells to near or greater levels than that of wild type, unaffected fibroblasts. The adenoviral vector successfully transfected rat fibroblasts producing both beta-galactosidase and lysyl hydroxylase 1 gene activity. We next expanded our studies to a rodent model. Intradermal injections of the vector to the abdominal skin of rats produced lysyl hydroxylase 1 mRNA and elevated lysyl hydroxylase 1 activity, in vivo. These data suggest the feasibility of gene replacement therapy to modify skin wound healing in type VI Ehlers-Danlos syndrome patients.

Topics: Adenoviridae; Ehlers-Danlos Syndrome; Fibroblasts; Galactosidases; Gene Transfer Techniques; Humans; Hydroxylysine; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; RNA, Messenger; Skin

We report on the unprecedented combination of two recessively inherited disorders, the kyphoscoliosis type of Ehlers-Danlos syndrome (EDS type VI) and cystic fibrosis (CF), in two sibs born to consanguineous Turkish parents. Because of failure to thrive and bronchitis CF was diagnosed in the index patient early whereas EDS VI was recognized only very late. Both patients had marked muscular hypotonia at birth, delayed gross motor development, progressive kyphoscoliosis, joint dislocations, Marfanoid habitus, hypertrophic and atrophic scars, and osteopenia. EDS VI was proven by collagen studies and the pathognomonic pattern of urinary pyridinolines. Because the genes coding for the two disorders are located on different chromosomes and a chromosomal rearrangement was excluded, we conclude that their combination is a chance association. The cardiopulmonary impairment common to both diseases makes the prognosis dismal.

Topics: Amino Acids; Collagen; Consanguinity; Cystic Fibrosis; Ehlers-Danlos Syndrome; Female; Genes, Recessive; Hand Deformities, Congenital; Humans; Hydroxylysine; Infant, Newborn; Nuclear Family; Pedigree; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Prognosis; Radiography; Scoliosis; Turkey

Ehlers-Danlos syndrome type VI (EDS VI) is an autosomal recessive disorder of connective tissue characterized by hyperextensible, friable skin and joint hypermobility. Severe scoliosis and ocular fragility are present in some patients. This disease is caused by defective collagen lsyl hydroxylase, a vitamin C-dependent enzyme that converts lysyl residues to hydroxylysine on procollagen peptides. Hydroxylysine is essential for the formation of the covalent pyridinium cross-links pyridinoline (Pyr) and deoxypyridinoline (Dpyr), among mature collagen molecules. Pyr derives from three hydroxylysyl residues, whereas Dpyr derives from one lysyl and two hydroxylysyl residues. Patients with EDS VI have high urinary excretion of Dpyr, resulting in a high ratio of Dpyr-Pyr. In this study, we evaluate content and production of pyridinium cross-links in the skin and cultured fibroblasts from patients with EDS VI. The skin of normal controls contained both Pyr and Dpyr, with a marked predominance of Pyr as observed in normal urine. The skin of patients with EDS VI had reduced total content of pyridinium cross-links, with the presence of Dpyr but not Pyr. Long-term cultures of control fibroblasts produced both Pyr and Dpyr, with a pattern resembling that of normal skin. By contrast, cross-links were not detected in dermal fibroblasts cultured from patients with EDS VI. Vitamin C, which improves the clinical manifestations of some patients with EDS VI, decreased Dpyr accumulation though only minimally affecting Pyr content in control cells. By contrast, addition of vitamin C to fibroblasts from patients with EDS VI stimulated the formation of Dpyr more than that of Pyr and greatly increased total pyridinium cross-link formation. These results indicate that qualitative and quantitative alterations of pyridinium cross-links occur in skin and in cultured dermal fibroblasts of patients with EDS VI and may be responsible for their abnormal skin findings. The vitamin C-stimulated production of Dpyr and Pyr in fibroblasts from patients with EDS VI may explain at least in part the therapeutic effects of this vitamin in EDS VI.

Topics: Adolescent; Amino Acids; Ascorbic Acid; Cells, Cultured; Child; Collagen; Cross-Linking Reagents; Ehlers-Danlos Syndrome; Female; Fibroblasts; Humans; Hydroxylysine; Lysine; Pyridinium Compounds; Skin

Two Turkish sibs with clinical features of Ehlers-Danlos syndrome type VI-B are presented. The hydroxylysine contents of dermis and gel electrophoresis of type I and type III collagen produced by fibroblasts were normal. Ultrastructural studies of skin collagen and elastic fibers showed discrete abnormalities. Other syndromes with similar clinical, biochemical and ultrastructural features are discussed.

Topics: Child, Preschool; Collagen; Diagnosis, Differential; Ehlers-Danlos Syndrome; Elastin; Eye Abnormalities; Female; Fibroblasts; Humans; Hydroxylysine; Infant; Joint Instability; Male; Pedigree; Sclera; Scoliosis; Skin

Three variants of the Ehlers-Danlos syndrome type VI are described: a severe form with skeletal, dermal and ocular manifestations associated with a lack of hydroxylysine in skin and little lysyl hydroxylase activity in cultured fibroblasts; a similarly affected form with a nearly normal hydroxylsine content in skin, but with only little enzyme activity in cultured fibroblasts; and a predominantly ocular form with no biochemical abnormality in skin or cultured skin fibroblasts. The activities of prolyl 4-hydroxylase and the two hydroxylysyl glycosyltransferases were normal in all cases, and the failure to find lysyl hydroxylase activity was not due to altered solubility characteristics of the enzyme or to the presence of an enzyme inhibitor. The collagen produced in cell culture, however, was hydroxylated to a markedly higher extent than that found in skin. In both the mutant and control cells hydroxylation of lysyl residues was less sensitive to ascorbate deficiency than that of prolyl residues.

Topics: Adolescent; Adult; Cells, Cultured; Child; Child, Preschool; Collagen; Ehlers-Danlos Syndrome; Female; Fibroblasts; Humans; Hydroxylation; Hydroxylysine; Male; Mixed Function Oxygenases; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Skin

Topics: Collagen; Ehlers-Danlos Syndrome; Humans; Hydroxylysine

Several abnormalities in collagen biosynthesis have been described in patients with Ehlers-Danlos syndrome. Examples of collagen structural mutations as well as post-translational enzymatic defects have been detected. Patients with hydroxylysine-deficient collagen disease (Ehlers-Danlos type VI) have diminished lysyl hydroxylase activity. One mutant enzyme has been characterized which is thermally labile and had an altered affinity for ascorbate. Another mutant enzyme had a normal requirement for cofactors but activity was diminished. Type VII Ehlers-Danlos syndrome is associated with altered processing of procollagen to collagen. Most often the disorder is associated with deficient procollagen aminoprotease activity. One patient appears to represent a structural mutation of pro alpha 2 (I) resulting in incomplete cleavage of the amino terminal propeptide. One family with x-linked Ehlers-Danlos syndrome (type V) has been described with altered lysyl oxidase activity. Other patients with this disorder have had normal lysyl oxidase activity. The ecchymotic form of Ehlers-Danlos syndrome (type IV) has defective type III collagen-synthesis. Patients have been described with absent synthesis, diminished synthesis and diminished secretion.

Topics: Collagen; Ehlers-Danlos Syndrome; Female; Humans; Hydroxylysine; Male

Lysyl hydroxylase was isolated as an essentially homogeneous protein from human fetal tissues and as a homogeneous protein from placental tissue by a procedure involving ammonium sulfate fractionation, affinity chromatography on concanavalin A-agarose, affinity chromatography on collagen linked to agarose and gel filtration. The specific activity of the best enzyme preparations from human fetal tissues was about 80,000 times, and from human placenta about 63,000 times that in the 15,000 X g supernatant of the corresponding tissue homogenate. The molecular weight of lysyl hydroxylase from both sources was about 190,000 by gel filtration, and that of the enzyme subunit about 85,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and molecular properties reported are very similar to those of pure chick-embryo lysyl hydroxylase, and the Km values for type I protocollagen substrate, and synthetic peptide substrate and all the co-substrates of the human placenta enzyme are likewise very similar to those of the chick-embryo enzyme. No difference in the Km values for type I protocollagen or any of the co-substrates was found between the human placenta enzyme and a crude lysyl hydroxylase from the skin fibroblasts of a patient with the type VI variant of the Ehlers-Danlos syndrome.

Topics: Animals; Chick Embryo; Chromatography, Affinity; Ehlers-Danlos Syndrome; Electrophoresis, Polyacrylamide Gel; Female; Fetus; Fibroblasts; Humans; Hydroxylysine; Kinetics; Mixed Function Oxygenases; Molecular Weight; Placenta; Pregnancy; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase

Topics: Collagen; Collagen Diseases; Ehlers-Danlos Syndrome; Extracellular Space; Genes; Humans; Hydroxylysine; Hydroxyproline; Osteogenesis Imperfecta; Procollagen; Protein Biosynthesis; RNA, Messenger

Topics: Aging; Animals; Colchicine; Collagen; Collagen Diseases; Ehlers-Danlos Syndrome; Glucosyltransferases; Growth; Homocystinuria; Humans; Hydroxylysine; Hydroxyproline; Marfan Syndrome; Menkes Kinky Hair Syndrome; Penicillamine; Procollagen; Procollagen-Proline Dioxygenase; Protein Biosynthesis; Protein-Lysine 6-Oxidase; Scurvy; Skin; Transcription, Genetic; Vascular Diseases

It has been previously shown that dermis from subjects with hydroxylysine-deficient collagen contains approximately 5% of normal levels of hydroxylysine and sonicates of skin fibroblasts contain less than 15% of normal levels of collagen lysyl hydroxylase activity. However, cultures of dermal fibroblasts from two siblings with hydroxylysine-deficient collagen (Ehlers-Danlos Syndrome Type VI) compared to fibroblasts from normal subjects synthesize collagen containing approximately 50% of normal amounts of hydroxylysine. The lysyl hydroxylase deficient cultures synthesize both Type I and Type III collagen in the same proportion as control cultures. Both alpha 1(I) and alpha 2 chains are similarly reduced in hydroxylysine content. Collagen prolyl hydroxylation by normal collagen lysyl hydroxylation is the same with or without ascorbate supplementation. In mutant cells the rate of prolyl hydroxylation measured after release of inhibition by alpha, alpha'-dipyridyl is the same as in control cells. The rate of lysyl hydroxylation is reduced in mutant cells but only to approximately 50% of normal.

Topics: Ascorbic Acid; Collagen; Ehlers-Danlos Syndrome; Fibroblasts; Humans; Hydroxylation; Hydroxylysine; Hydroxyproline; Lysine; Proline; Skin

A patient is described with congenital hypotonia, lax joints, friable skin, hemorrhagic scars, high-arched palate, and borderline microcornea. Acid hydrolyzed whole skin collagen had a reduced hydroxylysine content of 0.5 residues per 1,000 as compared to 5.1 +/- 0.7 in control skin. Collagen lysyl hydroxylase in dialyzed subcellular fractions of cultured skin fibroblasts required L-ascorbate as a principal cofactor. Activity of this enzyme in cultured skin fibroblasts derived from this patient, his father, and mother were 17%, 66%, and 39% of control values, respectively. Collagen prolyl hydroxylase activity was normal. Pharmacologic amounts of oral vitamin C (4 gm/day) produced an increase and withdrawal resulted in abrupt diminution of urinary excretion of hydroxylysine. Over a two-year period the patient's wound healing and muscle strength improved and corneal diameter increased. Hydroxylysine content of the skin did not increase.

Topics: Ascorbic Acid; Child; Clinical Enzyme Tests; Ehlers-Danlos Syndrome; Female; Fibroblasts; Humans; Hydroxylysine; Male; Mixed Function Oxygenases; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Skin

Topics: Child; Collagen; Ehlers-Danlos Syndrome; Heterozygote; Humans; Hydroxylysine

Skin fibroblasts from two siblings with hydroxylysine-deficient collagen collagen (Ehlers-Danlos syndrome, type VI) contained normal levels of collagen prolyl hydroxylase activity but were markedly deficient in collagen lysyl hydroxylase activity. The deficiency was evident in all fractions of cell lysates, in low and high ionic strength buffers, and in detergent. Assays of mixtures of wild-type and mutant cell lysates indicated no activation of mutant enzyme by factors in wild-type cells or inhibition of normal enzyme by material in mutant cells. Wild type or mutant cells cultured with ascorbic acid (50 mug/ml of culture medium, added daily) contained approximately the same level of lysyl hydroxylase activity as cells cultured without ascorbate, but prolyl hydroxylase activity without ascorbate was depressed in both an average of 41%. The mutant lysyl hydroxylase was less stable at 37 degrees C than the wild type and did not form high molecular weight aggregates in low ionic strength buffers, as did the control enzyme. The activity of the mutant enzyme was maximally stimulated after dialysis against buffer solutions containing 10 mM dithiothreitol. When assayed in 100 muM dithiothreitol, the mutant enzyme exhibited a higher apparent Km for ascorbate (20 muM) than the wild type (4 muM). In 1.0 mM dithiothreitol the mutant enzyme's apparent Km for ascorbate was reduced to 5 muM. Wild type and mutant enzymes had the same apparent Km for alpha-keto-glutarate (20 muM). The properties of prolyl hydroxylase in wild type and mutant cells were identical: apparent Km's for ascorbate and alpha-ketoglutarate were 100 muM and 20 muM, respectively. If mutant enzyme protein with altered kinetic properties is the only enzyme functioning to hydroxylate lysyl residues in collagen, the variations in hydroxylysine content observed in collagen from different tissues in the subjects reported here could be in part due to differences in cofactor concentrations and in rate and sequence of events in collagen synthesis in different tissues.

Topics: Ascorbic Acid; Chloromercuribenzoates; Collagen; Dithiothreitol; Ehlers-Danlos Syndrome; Fibroblasts; Humans; Hydroxylysine; In Vitro Techniques; Kinetics; Mercaptoethanol; Mixed Function Oxygenases; Mutation; Procollagen-Proline Dioxygenase

Two siblings suffered from Ehlers-Danlos syndrome characterized by skin fragility, joint laxity and dermal hyperelasticity. The association with microcornea and muscle hypotonia allowed the preliminary classification into type VI according to McKusick. Ultrastructure analysis of skin biopsies revealed poor integration of collagen fibrils into fibres; accordingly, the texture of the connective tissue appeared irregular. Lysyl hydroxylase activity of cultured skin fibroblasts was markedly reduced in the cells of the two patients. Preliminary studies revealed intermediate activity in the cells cultured from the skin of the parents. This finding suggested an autosomal recessive mode of inheritance. Unexpectedly and in contrast to the 3 cases reported in the literature, the hydroxylysine deficit in the patients' skin was, for reasons not yet understood, only mild. Therefore, amino acid analysis of skin is not adequate for the diagnosis of lysyl hydroxylase-deficient Ehlers-Danlos syndrome type VI.

Topics: Adolescent; Adult; Cells, Cultured; Collagen; Ehlers-Danlos Syndrome; Female; Fibroblasts; Humans; Hydroxylysine; Ketoglutaric Acids; Lysine; Male; Mixed Function Oxygenases; Pedigree; Skin

Topics: Adult; Collagen; Ehlers-Danlos Syndrome; Female; Genes, Recessive; Humans; Hydroxylysine

Two sibs with the Ehlers-Danlos syndrome, one of whom was shown to have hydroxylysine-deficient collagen, are described. In addition to the usual features of the Ehlers-Danlos syndrome (loose-jointedness and excessively stretchable, fragile, and bruisable skin), these patients had severe scoliosis and fragility of ocular tissues leading to rupture of the globe or retinal detachment. This combination of symptoms was tentatively classified as Ehlers-Danlos syndrome, Type VI. The condition is inherited as an autosomal recessive. The activity of lysyl hydroxylase was present at a reduced level in fibroblasts cultured from the patient's skin.

Topics: Chromosome Aberrations; Chromosome Disorders; Collagen Diseases; Ehlers-Danlos Syndrome; Female; Fibroblasts; Genes, Recessive; Humans; Hydroxylysine; Ketoglutaric Acids; Lysine; Male; Middle Aged; Mixed Function Oxygenases; Pedigree; Procollagen-Proline Dioxygenase

Cell culture studies were performed on members of a family in which two sisters, ages 9 and 12, have a similar disorder characterized clinically by severe scoliosis, joint laxity and recurrent dislocations, hyperextensible skin, and thin scars. The skin collagen from the sisters was markedly deficient in hydroxylysine, but other amino acids were present in normal amounts. Hydroxylysine in collagen from fascia and bone was reduced to a lesser extent. Since the most likely explanation for the hydroxylysine deficiency was a reduction in enzymatic hydroxylation of lysine residues in protocollagen, we measured the activity of lysyl-protocollagen hydroxylase in crude lysates of cultured skin fibroblasts. Enzyme activities in the two affected children were 14 and 10% of controls, whereas the activity was about 60% of normal in the mother, a pattern most consistent with autosomal recessive inheritance. The mutant enzyme demonstrated the same cofactor requirements as that from normal cells. Deficiency of lysyl-protocollagen hydroxylase is the first inborn error of human collagen metabolism to be defined at the biochemical level.

Topics: Adolescent; Adult; Amino Acids; Cells, Cultured; Child; Collagen; Consanguinity; Ehlers-Danlos Syndrome; Female; Fibroblasts; Humans; Hydrogen-Ion Concentration; Hydroxylysine; L-Lactate Dehydrogenase; Lysine; Male; Metabolism, Inborn Errors; Methyltransferases; Mixed Function Oxygenases; Procollagen-Proline Dioxygenase; Proline; Protein Precursors; Serine; Skin; Time Factors