Search

Ehlers-Danlos syndromes (EDS) are a group of inherited connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and variable tissue fragility. However, there are limited published data on the dental manifestations of EDS. This review systematically assessed the spectrum of published dental anomalies in various types of EDS. Twenty-four individual case reports/series and 3 longer case-control studies, reporting on a total of 84 individuals with a clinical diagnosis of EDS, were included in the data analysis. The main dental features listed in classical EDS were pulp calcification and localized root hypoplasia. Common dental abnormalities observed in vascular EDS were pulp shape modifications (52.2%), exceeding root length (34.8%), and molar root fusion (47.8%). Dentinogenesis imperfecta is a consistent finding in osteogenesis imperfecta/EDS overlap syndrome. Data on dental manifestations in other types of EDS are both rare and generally inconclusive.

Ehlers–Danlos Syndrome (EDS) is a group of connective tissue disorders which are both heterogeneous and heritable. The condition is a result of collagen defects, which include genetic variations and collagen protein processing. Collagen is present in fibrous tissues, such as skin, tendons and ligaments, and is also found in cartilage, blood vessels, the cornea, bones and the gut, highlighting its importance and explaining the varied pathophysiology of collagen conditions. These disorders were classified into six major types based upon the genetic and diagnostic variability and pathophysiology which includes skin hyperextensibility, joint hypermobility and tissue friability, but this classification has recently been amended. Determining the type of EDS an individual suffers from is a multifaceted process which relies on clinical examination as well as multiple laboratory techniques. These include: collagen biochemistry; genetic sequencing of the collagen genes, light microscopy, electron microscopy and urinary cross-link analysis. Diagnosis is important to both identify EDS types, allowing management of the condition, and also to aid psychologically where sufferers can appear fit and healthy but in fact be suffering pain and remain undiagnosed. This chapter aims to give an overview of connective tissue disorders, specifically EDS, and to describe and discuss diagnosing EDS in an NHS diagnostic unit. Giving detail on the techniques involved and the interpretation of results obtained.

Ehlers-Danlos syndrome (EDS) encompasses a genetically and clinically heterogeneous group of connective tissue disorders, characterized by joint hypermobility, skin hyperextensibility and tissue fragility. It is a rare condition, and inheritance is either autosomal dominant or recessive. Previously grouped into 11 different subtypes, with increasing knowledge of the underlying molecular defects, it was reclassified in 1997 into 6 major groups, with type VIII excluded from this classification. Type VIII EDS is a very rare subtype, characterized by severe, early-onset periodontitis, skin fragility and abnormal scarring. Voice abnormalities have occasionally been described in other forms of the condition, and may be due to defects in the collagen of the vocal ligament. We report two cases of patients with EDS type VIII and hoarseness.

We report the case of a 59-year-old woman who presented with a persistent papular and nodular cutaneous eruption and new-onset asthma, with normal renal function but persistent haematuria and proteinuria. Investigations revealed eosinophilia, both antineutrophil cytoplasmic antibodies and antiglomerular basement membrane antibodies on serological testing (double-positive vasculitis), and a focal necrotizing glomerulonephritis on renal biopsy. Histological examination of a skin biopsy showed a dense neutrophilic infiltrate with focal fibrinoid necrosis and few eosinophils. The clinical and pathological features suggested a double-positive vasculitis/Churg-Strauss overlap syndrome presenting with a predominantly neutrophilic dermatosis. Specific cutaneous features in patients with double-positive vasculitis have not been documented previously. The patient has responded extremely well to immunosuppressive treatment and her disease is currently in remission.

Sixty-four patients with pseudoxanthoma elasticum (PXE) were investigated in a nationwide study within South Africa and Zimbabwe. Thirty-nine individuals formed a distinct clinical subgroup. These persons were found exclusively among people of Afrikaner descent, whose origins are mainly derived from Dutch and French-Huguenot stock. This disorder was inherited as an autosomal recessive trait and presented mild to moderate cutaneous and cardiovascular manifestations. However, after the third decade of life severe visual impairment developed and culminated in blindness in 8 people by the age of 50. The cause of the visual defect was progressive extension of angioid streaks into the macula with neovascularization and haemorrhage. Laser therapy may have prevented further bleeding in 4 instances. The severity of ocular involvement contrasted with the mildness of the skin changes, and in this respect the condition seems to differ from previously delineated autosomal recessive forms of PXE.

Dean JCS, Gray ES, Stewart KN, Brown T, Lloyd DJ, Smith NC, Pope FM. Restrictive dermopathy: a disorder of skin differentiation with abnormal integrin expression. Clin Genet 1993: 44: 287–291. © Munksgaard, 1993 Clinical features and histological findings in two sibs who died from restrictive dermopathy in the neonatal period are described. Fibroblasts cultured from a skin biopsy from the second sib and fibroblasts from normal neonatal skin were studied using monoclonal antibodies to visualise integrin subunits by immunocytochemistry. Restrictive dermopathy fibroblasts displayed increased expression of the alpha-1 and alpha-2 subunits of integrin, those responsible for collagen binding. The increase was not matrix dependent. Integrins may play an important role in tissue differentiation, and our findings support the hypothesis that restrictive dermopathy is a disorder of skin differentiation.

A large family with dominantly inherited rhegmatogenous retinal detachment, premature arthropathy, and development of phalangeal epiphyseal dysplasia, resulting in brachydactyly was linked to COL2A1, the gene encoding proalpha1(II) collagen. Mutational analysis of the gene by exon sequencing identified a novel mutation in the C-propeptide region of the molecule. The glycine to aspartic acid change occurred in a region that is highly conserved in all fibrillar collagen molecules. The resulting phenotype does not fit easily into pre-existing subgroups of the type II collagenopathies, which includes spondyloepiphyseal dysplasia, and the Kniest, Strudwick, and Stickler dysplasias.

We report elastosis perforans serpiginosa (EPS) arising in three patients with Ehlers-Danlos syndrome, osteogenesis imperfecta and Down's syndrome. These cases illustrate some of the rare but well-recognized disease associations with EPS. The other causes of EPS are reviewed.

Editor—Type I collagen is the major structural protein of skin, bone, tendon, ligaments, and cornea. It is a heterotrimer of two α1(I) chains and one α2(I) chain. Mutations in one of its two structural genes ( COL1A1 , COL1A2 ) underlie the inherited disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome types VIIA and B (EDS VIIA, EDS VIIB).1 Mutations which inhibit the processing of the protein precursor, procollagen, cause the ligamentous laxity and extreme joint hypermobility of EDS VII. Structural abnormalities and/or reduced production of type I collagen lead to the increased bone fragility, thin skin, blue sclerae, dentinogenesis imperfecta, and presenile deafness of osteogenesis imperfecta. The OI phenotype can vary from perinatally lethal to mild disease depending upon the nature of the mutation. Among the mild group are patients who are heterozygous for a non-functional COL1A1 allele.2-5 Homozygosity for a non-functional COL1A1 allele appears to be incompatible with life.6 Several years ago we described a male infant who was totally deficient in α2(I)chains owing to homozygosity for a non-functional COL1A2 allele.7-9 He had the severe, progressively deforming type of osteogenesis imperfecta (OI type III). His heterozygous third cousin parents, however, showed no overt clinical symptoms but were considered to be prematurely osteoporotic following x ray examination. Subsequently, two further patients lacking proα2(I) chains from type I collagen were reported.10 11 However, their clinical phenotypes lacked any of the skeletal abnormalities of osteogenesis imperfecta but included generalised joint hypermobility, skin hyperextensibility, and scarring typical of the Ehlers-Danlos syndrome. We have now identified another patient homozygous for a mutation yielding non-functional COL1A2 alleles whose clinical phenotype showed generalised joint hypermobility and foot deformities reminiscent of Ehlers-Danlos syndrome associated with pale blue sclerae and a mild increase in bone fragility characteristic of osteogenesis imperfecta. The proband is …

AIMS—To compare the clinical and molecular genetic features of two phenotypically distinct subgroups of families with type 1 Stickler syndrome. BACKGROUND—Stickler syndrome (hereditary arthro-ophthalmopathy, McKusick Nos 108300 and 184840) is a dominantly inherited disorder of collagen connective tissue, resulting in an abnormal vitreous, myopia, and a variable degree of orofacial abnormality, deafness, and arthropathy. Stickler syndrome is the commonest inherited cause of rhegmatogenous retinal detachment in childhood with a risk of giant retinal tear (GRT) which is commonly bilateral and a frequent cause of blindness. METHOD—Pedigrees were identified from the vitreoretinal service database and subclassified according to vitreoretinal phenotype. Ophthalmic, skeletal, auditory, and orofacial features were assessed. Linkage analysis was carried out with markers for the candidate genes COL2A1, COL11A1, and COL11A2. The COL2A1 gene was amplified as five overlapping PCR products. Direct sequencing of individual exons identified mutations. RESULTS—Eight families exhibiting the type 1 vitreous phenotype were studied. Seven were consistent for linkage to COL2A1, with lod scores ranging from 2.1 to 0.3. In most instances linkage to COL11A1 and COL11A2 could be excluded. One family was analysed without prior linkage analysis. Three of the families exhibited a predominantly ocular phenotype with minimal or absent systemic involvement and were found to have mutations in exon 2 of COL2A1. Five other pedigrees with an identical ocular phenotype plus orofacial, auditory, and articular involvement had mutations in others regions of the COL2A1 gene. None of the pedigrees exhibited the characteristic lenticular, retinal pigment epithelial, or choroidal changes seen in Wagner syndrome. CONCLUSIONS—These data confirm that type 1 Stickler syndrome is caused by mutations in the gene encoding type II collagen (COL2A1). In addition, data are submitted showing that mutations involving exon 2 of COL2A1 are characterised by a predominantly ocular variant of this disorder, consistent with the major form of type II procollagen in non-ocular tissues having exon 2 spliced out. Such patients are all at high risk of retinal detachment. This has important implications for counselling patients with regard to the development of systemic complications. It also emphasises the importance and reliability of the ophthalmic examination in the differential diagnosis of this predominantly ocular form of Stickler syndrome from Wagner's vitreoretinopathy.

A detailed morphological study was carried out using light and electron microscopy on 36 bone specimens from patients suffering from osteogenesis imperfecta (OI) and 20 age- and site-matched control bone specimens. The findings were grouped into the clinical types of OI according to the Sillence classification. The morphological and ultrastructural alterations observed in OI bone correlate well with clinical severity. Thus, OI type I, the mildest type, showed the least abnormalities in bone ultrastructure. OI type IV closely resembled type I, with only minor abnormalities in the bone cells and osteoid. OI type III showed abnormalities in the structure and distribution of osteoid collagen fibrils, whilst OI type II, the lethal form, revealed many varied abnormalities such as thin cortical bone, sparse trabecular bone, increased numbers of osteoclasts and osteocytes, thin osteoid with thin collagen fibrils, and patchy mineralization.

We describe a 16-year-old patient with Ehlers-Danlos syndrome (EDS) type I and recurrent urinary retention caused by giant bladder diverticulum and review the literature on this association.

We have recently analysed by histological, protein and molecular DNA techniques 23 mutations of the collagen III gene (COL3A1), most of which cause premature arterial fragility, thin skin and variants of vascular Ehlers-Danlos syndrome. There were 14 glycine substitutions between residues 637 and 1021, eight exon skips between exons 7 and 45 and one small inframe deletion. The glycine substitutions produce a gradient of increasingly abnormal clinical phenotypes from exons 36 to 49 while the clinical severity of exon skips is much more variable. Each mutation is private for the affected family or individual concerned having the potential for early prenatal diagnosis and prevention.

Osteogenesis imperfecta (OI) is a rare, heterogeneous, inherited connective tissue disorder frequently caused by abnormalities of type I collagen. It is characterized by bone fragility, osteopenia, and progressive skeletal deformities. Electron microscopy of three OI type II fetal bone samples revealed numerous large osteocyte lacunae. In addition, there was a perilacunar osteoid-like band of collagen surrounding the osteocytes, which was unmineralized and morphologically unusual. Furthermore, large osteocyte lacunae contained fine particles and filamentous material similar to the expected ultrastructural appearance of proteoglycans. More detailed examination was carried out using histochemical and immunogold localization of proteoglycans at light and ultrastructural levels. These tests and the use of electron probe X-ray microanalysis confirmed that the material in the osteocyte lacunae was proteoglycan. In contrast, in the age- and site-matched normal fetal bone, all the osteocyte lacunae appeared negative for proteoglycan. Proteoglycans are regarded as inhibitors of calcification. Our observation of substantial amounts of proteoglycan in abnormally enlarged osteocytic lacunae of some OI fetal bone suggests association with the abnormal bone of this particular subtype of OI type II.

Summary Although catastrophic vascular complications in vascular Ehlers–Danlos Syndrome (EDS) are well recognized, other complications such as flexion contractures and tendon nodules are rarely reported and poorly characterized. We report a young man with vascular EDS, who developed flexion contractures and tendon nodules, causing considerable disability. Limited management strategies are available for these complications, which have continued to prove a challenge to management.

To strengthen the evidence for genetic linkage to COL7A1, we have studied 26 generalised recessive dystrophic epidermolysis bullosa (EB) families of British, Italian, Irish, and South African origin. We chose two linkage markers, a COL7A1 PvuII intragenic polymorphism and a highly informative anonymous microsatellite marker, D3S1100, which maps close to the COL7A1 locus at 3p21.1-3. Diagnosis was established by family history, clinical examination, immunofluorescence, and ultrastructural studies. The PvuII marker was informative in 16 families with a maximum lod score (Zmax) of 3.51 at recombination fraction (theta) = 0. The D3S1100 microsatellite was informative in 24 out of 25 families with Zmax = 6.8 at theta = 0.05 (Z = 4.94 at theta = 0) and no obligatory recombination events. These data strongly suggest that COL7A1 mutations cause EB in these families and, combined with previous studies, indicate locus homogeneity. The importance of anchoring fibrils for dermal-epidermal adhesion is further underlined. D3S1100 may later prove useful in prenatal diagnosis of this disease, if used in combination with other markers.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable disorders of connective tissue. The type III variety is characterized by joint hypermobility and minor hyperextensibility and softness of the skin. While collagen fibril structure has been shown to be abnormal in such patients, the underlying molecular defect(s) has not been determined. Here we characterize the first mutation found in a family with EDS III. Analysis of cultured fibroblasts from the affected family revealed intracellular retention of type III collagen. This is usually a biochemical characteristic of EDS IV, caused by mutations of COL3A1. Analysis of the cDNA sequence in this EDS III family revealed a glycine to serine mutation at amino acid residue 637 of the type III collagen molecule. This was confirmed by allele-specific oligonucleotide hybridization against amplified genomic DNA. Thus mutations of type III collagen can cause either EDS IV or EDS III. Two affected family members had virtually normal skin and so more closely resembled the phenotype of articular hypermobility syndrome.

Ehlers-Danlos syndrome type IV is usually caused by mutations in COL3A1, the gene coding for type III collagen. In a woman with a milder form of this disease, analysis of type III collagen synthesised by her cultured skin fibroblasts showed an apparently shorter form of the protein. Amplification of overlapping cDNAs, encoding the triple helical region of the molecule, showed a deletion near the 5' end of the gene. Sequencing showed that exon 7 was missing from the cDNA sequence. Analysis of genomic DNA showed that this was the result of a T+6 to C+6 mutation in the donor splice site of intron 7. The proband's parents and 35 normal controls were homozygous for T+6 at this position, indicating that the C+6 mutation was causative.

The clinical and biochemical observations in a patient with a mild form of Ehlers-Danlos syndrome (EDS) type IV are described. The patient's skin fibroblasts produced markedly diminished amounts of type III collagen. SDS-polyacrylamide gel electrophoresis of collagens produced by cells obtained from other, non-cutaneous tissues showed two forms of collagen alpha 1(III) chains, a normal and a slow migrating, mutant form. Further analysis confirmed that the type III collagen molecules containing mutant alpha chains which were overmodified had a lower thermal stability and were poorly secreted into the extracellular medium. The protein defect was mapped by in situ cyanogen bromide digestion and was located in alpha 1(III) CB9, the C-terminal peptide of the collagen triple helix. This study shows that non-cutaneous connective tissues can be a useful source for the study of type III collagen defects in patients with EDS type IV.

We have investigated a family with an autosomal dominantly inherited connective-tissue defect causing extreme joint hypermobility, premature osteoporosis and late-onset fractures. Analysis of collagenous proteins from affected individuals showed a deletion in some alpha 2(I) chains. Peptide mapping localized this to the CB peptide alpha 2CB4, which covers the N-terminal one-third of the protein chain. Polymerase chain reaction amplification and sequencing of cDNA derived from this region of the mRNA identified a heterozygous deletion of the 54 bp comprising exon 9. Similar analysis of the genomic DNA revealed an 11-bp deletion from bp3 to bp13 of IVS-9. This disrupts the consensus 5' splice signal (GTAAGT) and leads to exon skipping. In a family study of 13 affected and unaffected family members using both heteroduplex formation and direct analysis for the deletion, all of the affected, but no unaffected individuals, were found to carry the deletion. This generated a positive Lod score of 2.6 with the Liped programme.

A large family with Ehlers-Danlos syndrome type IV (EDS IV) has previously been described. Unlike most cases of EDS IV, fibroblasts from affected members secreted near normal amounts of type III collagen. We have localized the mutation in this family to the CB5 peptide of type III collagen, by using both protein and cDNA mapping techniques. Sequence analysis of cDNA revealed a 27-bp deletion within exon 37, a deletion that removed nine amino acids and maintained the Gly-X-Y repeat of the collagen helix. Further sequencing of genomic DNA confirmed its location, and amplification of DNA from family members showed that it was absent in unaffected individuals but present in all the affected individuals tested. This deletion is flanked by two short direct repeats of CTCC; it may have arisen by slipped mispairing, and has subsequently been transmitted to all affected family members.

Fifty-one patients with Ehlers-Danlos syndrome were investigated for abnormalities of platelets and coagulation. Thirty-eight were examined prospectively and 13 retrospectively. A bleeding history was taken from all patients; only four (8%) gave no history of a bruising or bleeding tendency. Nine patients (18%) had significant haemostatic abnormalities of whom four (8%) had a platelet release defect, three (6%) had a factor XI deficiency and two (4%) had a factor XIII deficiency. Additionally 16 patients (31%) had mild abnormalities of uncertain significance of whom four (8%) had prolonged bleeding times (three in association with platelet aggregation abnormalities), 13 (26%) had platelet aggregation abnormalities and two had a positive Hess test. Twenty-four patients (47%) had normal tests for haemostasis of whom 20 (39%) had a bleeding diathesis and four (8%) had no such tendency. Results were analysed according to the type of Ehlers-Danlos syndrome, but there was no pattern to the abnormalities. The high frequency of a bleeding tendency in Ehlers-Danlos patients with normal tests for haemostasis (83%) supports the conventional explanation for this clinical feature, that defects in the structural integrity of skin and blood vessels lead to easy bruising.

We have examined the procollagens and collagens produced by skin fibroblasts from a patient with Ehlers-Danlos syndrome type VII. The patient was heterozygous for an abnormal alpha 2(I) chain migrating with the approximate size of pN alpha 2(I) chains after pepsin digestion. Peptide mapping suggested that the abnormality was located at the amino-terminus of the alpha 2(I) chain. Quantitative analysis of the alpha 2(I) mRNA indicated loss of the exon 6 sequences, and subsequent polymerase chain reaction amplification of cDNA demonstrated a deletion of the 54 bp of exon 6 from some of the alpha 2(I) mRNA. Analysis of genomic DNA from the patient revealed a single base change in one COL1A2 allele, substituting an A for a G as the first base of intron 6. This change mutates the obligate GT-dinulceotide splicing signal to AT and leads to exon skipping with splicing from exon 5 to exon 7. Loss of exon 6 sequences results in the loss of the procollagen-N-propeptidase cleavage site and a lysine residue that normally participates in covalent intermolecular crosslinking within collagen fibres.

Human skin consists of a stratified, cellular epidermis and an underlying dermis of connective tissue [1–5]. The dermal–epidermal junction is undulating in section; ridges of the epidermis, known as rete ridges, project into the dermis. The junction provides mechanical support for the epidermis and acts as a partial barrier against exchange of cells and large molecules. Below the dermis is a fatty layer, the panniculus adiposus, usually designated as ‘subcutaneous’. This is separated from the rest of the body by a vestigial layer of striated muscle, the panniculus carnosus. There are two main kinds of human skin. Glabrous skin (non-hairy skin), found on the palms and soles, is grooved on its surface by continuously alternating ridges and sulci, in individually unique configurations known as dermatoglyphics. It is characterized by a thick epidermis divided into several well-marked layers, including a compact stratum corneum, by the presence of encapsulated sense organs within the dermis, and by a lack of hair follicles and sebaceous glands. Hair-bearing skin (Fig. 3.1), on the other hand, has both hair follicles and sebaceous glands but lacks encapsulated sense organs. There is also wide variation between different body sites. For example, the scalp with its large hair follicles may be contrasted with the forehead, which has only small vellus-producing follicles, albeit associated with large sebaceous glands. The axilla is notable because it has apocrine glands in addition to the eccrine sweat glands, which are found throughout the body. Regional variation is further considered below. Chapter 3

The combined genetic data between the Marfan syndrome and 75 informative loci on 18 autosomes were used to construct an exclusion map for this disorder. Data are also presented for a further two unmapped markers. The most likely location of the Marfan syndrome gene is highlighted and all the unexcluded areas of the genome are displayed in a graphical form. This exclusion map shows that almost 75% of the genome has been excluded as a likely location for the Marfan syndrome gene in the majority of the families studied. Apart from chromosomes 8, 13, 21, and 22, for which no data were available, other regions not excluded yet include 5p, 6p, 9p, 10p, 12p, 15, 17p, 18, and 20p. Future linkage analysis using markers located in the highlighted regions should facilitate the identification of the site of the Marfan syndrome gene.

To investigate the role of COL5A1 as a candidate gene for Ehlers-Danlos syndrome (EDS), we have carried out linkage studies in two large British families with EDS type I/II and type II, respectively. Fourteen living, affected individuals were identified by family history, clinical examination and ultrastructural analysis. A polymorphic intragenic simple sequence repeat at the COL5A1 locus showed linkage to EDS without recombination to give a combined lod score of 5.7. We have previously reported linkage to COL5A1 in an EDS type I/II family which brings the total lod score to 9.8 at zero recombination. Taken together, these data implicate COL5A1 as an important cause of EDS and confirm that types I and II are allelic.

A 45 year old man with a 5 year history of primary biliary cirrhosis presented with fatigue, weight loss, bone pains, itch and progressive jaundice. Examination revealed jaundice, hyperpigmantadon, cachexin, extensive xanthelasm a and palmar xanthomata, spider naevi, palmar erythema, moderate hepatosplenomegaly, testicnlar atrophy and a proximal myopathy. Investigations revealed a uholestatie liver enzyme profile, hyperbilirubinaemia (73 umoU1), hypoalbuminaemia and profound hypercholesterolaemia (cholesterol 54.5 mmol/l). Antimitochondrial antibodies(M2) were positive at a titre of 1/ 10,240. A liver biopsy revealed grade III primary biliary cirrhosis, for which he was commenced on ursodeoxycbelie acid. Ultrasound of the abdomen revealed several descrete splenic nodules. CT abdomen noted an enlarged lymph node in the porte hepatis. A splenectomy was performed. Pathology revealed an intermediate grade follicular large cell non-Hodgkin's lympbema. Post-operatively, he received six courses of chemotherapy. The serum cholesterol fell sharply to 10 mmol/l after starting ursodeoxycholie acid. This dramatic fall in cholesterol represents an important beneficial effect of ursodeoxycholic acid therapy. It underlines the potential benefits of ursodeoxycholiu acid as a lipid lowering agent in cases of hypercholesterolemia due to impaired live~ cholesterol homeostasis. A review of the literature suggests that non-Hodgkin's lymphoma complicating primary biliary cirrhosis is very rare. It is suggested that lympbema may complicate autoimmune disease as a result of impaired immunoregulation or chronic antigen stimulation. In summary, this case highlights two important findings; firstly, the effect of ursodeoxycholic acid on hepatic cholesterol metabolism, and secondly, the association between primary biliary cirrhosis and non-Hodgkin's lympfioma.

We report elastosis perforans serpiginosa (EPS) arising in three patients with Ehlers-Danlos syndrome, osteogenesis imperfecta and Down's syndrome. These cases illustrate some of the rare but well-recognized disease associations with EPS. The other causes of EPS are reviewed.

A detailed morphological study was carried out using light and electron microscopy on 36 bone specimens from patients suffering from osteogenesis imperfecta (OI) and 20 age- and site-matched control bone specimens. The findings were grouped into the clinical types of OI according to the Sillence classification. The morphological and ultrastructural alterations observed in OI bone correlate well with clinical severity. Thus, OI type I, the mildest type, showed the least abnormalities in bone ultrastructure. OI type IV closely resembled type I, with only minor abnormalities in the bone cells and osteoid. OI type III showed abnormalities in the structure and distribution of osteoid collagen fibrils, whilst OI type II, the lethal form, revealed many varied abnormalities such as thin cortical bone, sparse trabecular bone, increased numbers of osteoclasts and osteocytes, thin osteoid with thin collagen fibrils, and patchy mineralization.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

Electron microscopy and morphometric measurements of bone osteoid collagen diameter from 42 osteogenesis imperfecta (OI) patients and 25 age- and site-matched controls were carried out. Although the mean diameter did not correlate well with the severity of the disease, it related well with the clinical types and revealed collagen fibrils of reduced diameter in the osteoid of all OI types. Thus, OI type II (the severest type) demonstrated the smallest diameter (45 nm), followed by OI type I (the mildest form) with a mean diameter of 57 nm. The diameter obtained for type III (67 nm) and type IV (64 nm) was lower than the normal control mean diameter (73 nm) but did not show a statistical difference. The thinner fibrils observed in OI bone may be unable to provide nucleating and scaffolding sites for mineral propagation and may play a role in the fragility of bone in this disease.

We describe a 16-year-old patient with Ehlers-Danlos syndrome (EDS) type I and recurrent urinary retention caused by giant bladder diverticulum and review the literature on this association.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders. Recently mutations have been found in the genes for type V collagen in a small number of people with the most common forms of EDS, types I and II. Here we characterise a COL5A2 mutation in an EDS II family. Cultured dermal fibroblasts obtained from an affected subject synthesised abnormal type V collagen. Haplotype analysis excluded COL5A1 but was concordant with COL5A2 as the disease locus. The entire open reading frame of the COL5A2 cDNA was directly sequenced and a single base mutation detected. It substituted a glycine residue within the triple helical domain (G934R) of alpha2(V) collagen, typical of the dominant negative changes in other collagens, which cause various other inherited connective tissue disorders. All three affected family members possessed the single base change, which was absent in 50 normal chromosomes.

To investigate the role of COL5A1 as a candidate gene for Ehlers-Danlos syndrome (EDS), we have carried out linkage studies in two large British families with EDS type I/II and type II, respectively. Fourteen living, affected individuals were identified by family history, clinical examination and ultrastructural analysis. A polymorphic intragenic simple sequence repeat at the COL5A1 locus showed linkage to EDS without recombination to give a combined lod score of 5.7. We have previously reported linkage to COL5A1 in an EDS type I/II family which brings the total lod score to 9.8 at zero recombination. Taken together, these data implicate COL5A1 as an important cause of EDS and confirm that types I and II are allelic.

We report two phenotypically similar patients with primary cutis laxa associated with deficiency of lysyl oxidase, an extracellular copper enzyme the gene for which is located on chromosome 5. Previous reports of this condition have had characteristic occipital projections, abnormality of copper metabolism and X-linked inheritance. The two reported patients have no occipital projections, normal copper metabolism, Wormian bones, and a pattern of inheritance consistent with the autosomal recessive inheritance of the lysyl oxidase gene.

We have recently analysed by histological, protein and molecular DNA techniques 23 mutations of the collagen III gene (COL3A1), most of which cause premature arterial fragility, thin skin and variants of vascular Ehlers-Danlos syndrome. There were 14 glycine substitutions between residues 637 and 1021, eight exon skips between exons 7 and 45 and one small inframe deletion. The glycine substitutions produce a gradient of increasingly abnormal clinical phenotypes from exons 36 to 49 while the clinical severity of exon skips is much more variable. Each mutation is private for the affected family or individual concerned having the potential for early prenatal diagnosis and prevention.

An eight-year-old boy was referred for dental assessment of dentinogenesis imperfecta, a full clinical examination also revealed joint hypermobility and some features of mild osteogenesis imperfecta although he had suffered few fractures. Analysis of the collagens produced by both gingival and skin fibroblast cultures showed the synthesis and intracellular retention of an abnormal alpha 2(I) chain that migrated faster than normal on SDS-PAGE. Cyanogen bromide peptide mapping of this intracellular protein indicated a probable deletion in the N-terminal peptide alpha 2CB4. The denaturation temperature of the mutant protein was only 36 degrees C, some 6 degrees C below normal. At 37 degrees C secretion of abnormal protein was not detectable but a lower temperature (30 degrees C) some was secreted into the medium. RT-PCR amplification of mRNA coding for alpha 2CB4 revealed a heterozygous deletion of the 108 bp exon 21 of COL1A2. Sequencing of PCR amplified genomic DNA identified a G --A transition in the moderately conserved + 5 position of the IVS 21 5' consensus splice site causing the skipping of exon 21. Hybridization with allele-specific oligonucleotides showed no other family member had this base change. Since the cDNA deletion was associated with the (-) allele of a Pvu II polymorphism in exon 25 of COL1A2 we could demonstrate that the mutant pre-mRNA was alternatively spliced yielding both full length and deleted transcripts. Family genotype analysis indicated the mutation had originated in the paternal alpha 2(I) gene.

A young girl presented with severe type III osteogenesis imperfecta; her otherwise healthy mother also had a mild connective tissue disorder with blue sclerae and recurrent joint dislocations. Skin fibroblast cultures from the child produced both normal and post-translationally over-modified type I collagen. The mutant collagen was poorly secreted but had normal thermal stability. Cyanogen bromide peptide maps of the abnormal protein indicated a C-terminal mutation. The mother's cells produced only normal-appearing collagens. Mismatch analysis and extensive sequencing of cDNAs covering the suspect region did not reveal any potentially causal changes in the triple helical domains of either the alpha 1(I) or alpha 2(I) chains. However, examination of the C-propeptide sequences revealed two heterozygous single base changes in the child. One, an A-C changing threonine to proline at residue 29 of the alpha 2(I) C-propeptide was also present in the mother and maternal grandfather but not in 50 unrelated control individuals. The second, a T-C altered the last amino acid residue of the alpha 1(I) C-propeptide from leucine to proline and had occurred de novo in the affected child. This mutation highlights the importance of the C-propeptides in molecular assembly but it is not clear how such an extreme mutation causes the delay in triple helix formation indicated by the extensive over-modification and reduced secretion of the mutant type I collagen. It may inhibit intrachain disulfide bonding or possibly affect the association of the procollagen chain with an intracellular "chaperone" protein that normally assists the assembly of trimeric procollagen molecules.