Research Perspectives in Heritable Disorders of Connective Tissue

Matrix Vol. 12/1992, pp. 333-342 © 1992 by Gustav Fischer Verlag, Stuttgart

Research Perspectives in Heritable Disorders of Connective Tissue PETER H. BYERS 1 , REED E. PYERITZ2 and JOUNI Uln03 1 2

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Departments of Pathology and Medicine, University of Washington, Seattle, WA 98195; Departments of Medicine and Pediatrics, Johns Hopkins University, Baltimore, MD 21205 and Department of Dermatology and Jefferson Institute for Molecular Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Introduction In response to initiative proposed by the Coalition for Heritable Connective Tissue Disorders, and a directive from Congress, the National Institute of Arthritis, Musculoskeletal and Skin Diseases and the National Institute of Child Health and Human Development sponsored a workshop on "Heritable Disorders of Connective Tissue" in April of 1990 at the National Institutes of Health. Twentyfive discussants and more than forty guests participated. The focus of the workshop was to review the current status of research in heritable disorders of connective tissue and to identify important directions for research in the future. In the ten years since the previous conference on the subject there have been major advances in identifying the molecular components of the extracellular matrix, in isolating and characterizing the genes which encode these proteins, in identifying and characterizing the biosynthetic pathways of most matrix proteins, and in defining interactions among these proteins. In addition, significant work has been done to characterize mutations in genes that produce a limited variety of these disorders. Several common themes emerged from the discussion. These included the need for continued integration of insights gained from molecular biology, biochemistry, physical biochemistry, anatomy and histology and disease processes to the understanding of normal tissues; the need for continued cross-fertilization among basic scientists and clinicians; the need for registeries to recruit patients into clinical trials, to identify individuals with rare disorders for solicit atom of clinically relevant samples and to dissemipate new information concerning disease phenotypes; the recognition that structure-function relationships in connective tissue macromolecules should be pursued and that the manner in which mutations alter molecular structure and function and processing should be examined; finally, the application of insights into specific molecular disorders to

the understanding of common diseases should not go unnoticed.

Clinical perspectives and epidemiology The term "heritable disorders of connective tissue" became embedded in the medical literature with the publication in 1956 of a book by the same name by Victor McKusick (McKusick, 1956). At that time a limited number of disorders including the Marfan syndrome, the Ehlers-Danlos syndrome, osteogenesis imperfecta, pseudoxanthoma elasticum, and the Hurler syndrome were included in this group of disorders. In the intervening 34 years, it has been recognized that mutations in genes that encode matrix proteins or in genes that ultimately affected the structure of the extracellular matrix account for more than 200-distinct disorders. In total, it is estimated that those heritable disorders of connective tissue account for major and, in some cases, lifethreatening illnesses in approximately half a million people in the United States. Further, it is becoming increasingly clear that while some mutations in certain genes give rise to highly deleterious phenotypes, other mutations in the same genes can produce much milder conditions. The milder phenotypes may appear as the more common disorders of osteoporosis, osteoarthritis and, potentially, disorders of vascular integrity such as aortic aneurysm. Because of their contributions to morbidity, the chronic nature of those disorders, and the implications for long term medical care and funding, these disorders have long been recognized as having major impacts on health care in the United States.

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The major disorders of connective tissue The major categories of connective tissue disorders considered at the April 1990 workshop conference were forms of osteogenesis imperfecta (01), chondrodysplasias, forms of epidermolysis bullosa (EB), disorders of the elastic fiber network including pseudoxanthoma elasticum (PXE) and cutis laxa, the Ehlers-Danlos syndromes (EDS), the Marfan syndrome, lysosomal storage disorders, homocystinuria and adult polycystic kidney disease (APKD). Because the focus of the meeting was largely on those disorders due to defects in the synthesis of matrix proteins or in proteins that interact with matrix proteins, we have left the discussion of homocystinuria, of lysosomal storage disorders and of APKD for other.

Osteogenesis imperfecta 01 is a heterogeneous group of disorders of bone fragility that may affect about 1/10000 individuals in the United States (see Sillence et al., 1979 for discussion of the clinical features of 01). The vast majority of individuals affected with 01 have dominant mutations in one of the two genes (COLlA1 and COLlA2) that encode the chains of type I collagen, the major collagen of bone and most other connective tissues (Byers, 1990; Byers et al., 1991; Kuivaniemi et al., 1991). About 10 years ago, on the basis of clinical, genetic and radiographic features, Sillence and colleagues (1979) identified four major osteogenesis imperfecta phenotypes. Individuals with 01 type I, the mildest dominantly inherited form, generally have defects which result in synthesis of approximately half the normal amounts of structurally normal type I procollagen by cultured cells in vitro (Barsh et al., 1982; Rowe et al., 1986; Willing et al., 1990). In contrast individuals with the perinatal lethal form of osteogenesis imperfecta (01 type II), the progressive deformity variety of 01 (01 type III), and a mild to moderate form of 01 (01 type IV), are usually the result of mutations that affect the structure of the proteins (see Byers, 1989; Prockop et al., 1989; Byers, 1990; Byers et al., 1991; and Kuivaniemi et al., 1991 for recent reviews). Multi-exon deletions or insertions are rare events and appear limited to the lethal phenotypes, if they are expressed in the protein. More than 50 mutations in type I collagen genes have been characterized which has allowed the general outline for phenotype and genotype relationships to be developed. In a small number of instances recessive mutations in collagen genes have been identified (Nicholls et al., 1979; Pihlajaniemi et al., 1985); in others there is evidence that mutations are probably in genes other than those that encode the chains of type I collagen (Aitchison et al., 1988; Beighton and Versfeld, 1985) but the nature of the genetic defect and the location of mutation has not been established.

The phenotypic effects of point mutations in collagen genes depend on their location, the nature of the substituting amino acid and the substituted amino acid, and the chain in which they occur. Mutations near the carboxyl terminus of the triple helical domain in the proal (I) chain are generally more deleterious than those near the carboxyl terminus. Mutations in the carboxyl-terminal propeptide have phenotypes that depend on the functional alterations that occur as a consequence of the mutations. Chains that are unincorporated usually result in mild varieties of 01 but if mutant chains are incorporated into molecules the effects of the mutations may be highly deleterious or lethal. Exonskipping mutations are generally lethal if they occur in the proa1(I) chain but may be milder in the proa(I) chain. Abnormal molecules are poorly secreted, have altered thermal stabilities and are poorly incorporated into matrix. Those molecules that are secreted appear to have highly deleterious effects on fibrillogenesis and, consequently, probably affect mineralization and bone formation. However, the precise manner in which mutations give rise to phenotype is not well understood. Early embryonic mutations may lead to very mild or undetectable phenotypes in individuals and yet be passed to future generations in severe or lethal phenotypes (Byers et al., 1988; Cohn et al., 1990; Constantinou et al., 1989; 1991; Wallis et al., 1990). Somatic and germinal mosaicism is an explanation for unexpected recurrence of the lethal 01 phenotype in most families as well as in families in which siblings with other forms of 01 are born to normal parents. The technology of reverse transcription, amplification, and direct sequencing has facilitated the rapid identification of mutations so that the general outline will soon be completed in some detail. There is still a paucity of understanding of how mutations are translated to phenotype and there is virtually no understanding of how mutations affect intracellular processing of proteins. Several bone-specific proteins have been identified including phosphophoryn, osteopontin and other glycoproteins. One disorder, dentinogenesis imperfecta (DI) in the absence of osteogenesis imperfecta (DI type II) is characterized by a paucity of a phosphophoryn in teeth and linkage to a locus on the long arm of chromosome 4 has been demonstrated (Ball et al., 1982) and confirmed (Roulston et al., 1985). This suggests that there maybe important phosphophoryn-collagen interactions and that defects in either protein could give rise to abnormal matrix. Therapy in osteogenesis imperfecta is currently limited to physical attempts to stabilize bone structure by insertion of intramedullary rods or external bracing (Marini, 1988). Those techniques can decrease fracture rate and probably decrease morbidity as a result of recurrent fractures. Linear growth remains a major problem for some children and attempts are now underway to characterize alterations in hormonal response and in structuring bone that may account for defects in linear growth (Marini, 1988).

Heritable Disorders of Connective Tissue

Chondrodysplasias The chondrodysplasias are inherited disorders of linear bone growth that result from defective formation of and transformation to bone of temporary cartilage models of developing bone (Rimoin and Lachman, 1990). Affected individuals are generally characterized by abnormalities of growth, short stature, and skeletal deformities that are unique to each disorder. Only a few mutations in matrix genes have been characterized and the mechanisms by which bone growth is disturbed are not understood. Chondrodysplasias are each uncommon but in total constitute the major reason for short stature and morbidity in humans. The most common of the chondrodysplasias, achondroplasia, affects approximately 1130000 individuals. More than 150 forms of chondrodysplasia are known. Pathologic, genetic and clinical studies suggest that individual disorders can be grouped into a smaller number of disease families (Spranger, 1989) (for example, achondrogenesis, hypochondrogenesis, spondylo-epiphyseal dysplasia, and one form of Stickler syndrome form one such grouping in which there is evidence that mutations in type II collagen genes (COLlA1) (Lee et aI., 1988; Vissing et aI., 1989; Tiller et aI., 1990; Tsipouras et aI., 1990;' Francomano et aI., 1988) give rise to the phenotypes. These mutations have effects on type II collagen synthesis that parallel those on type I collagen in 01. To date other "phenotypic families" have been defined by radiologic evidence of phenotypic and histopathological similarities (see Spranger, 1989). The approaches to identification of molecular abnormalities in chondrodysplasias have stemmed, in large part, from morphologic observations and characterisation of proteins in growth plate cartilage. Genetic linkage studies with the COLlA1 gene have excluded a number of dominantly inherited disorders including achondroplasia (Francomano and Pyeritz, 1988) and some forms of the Stickler syndrome as resulting from mutations in that gene (Francomano et aI., 1987; 1988; Schwartz et aI., 1989). Ultrastructural analysis of cartilaginous tissues have indicated that intracellular storage in the rough endoplasmic reticulum (RER) is a feature of some disorders (Sillence et aI., 1979), notably pseudoachondroplasia, but the nature of the stored material has not yet been definitively identified. Many animal models of chondrodysplasias have been described in mice and are seen occasionally in other mammals and birds. The phenotypes of nanomelia in chickens (Goetinck, 1988) and of cartilage matrix deficiency in mice are both recessively inherited and due to alterations in the structure of the chondroitin sulfate core protein gene of cartilage. An autosomal recessive defect in sulfation in the mouse has been identified an disorder in the brachymorphic mouse (bm/bm) (Melvin and Schwartz, 1988). The

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phenotype is due to defective transfer of sulfate to the glycosaminoglycan chains of proteoglycan molecules. However, few animal models of chondrodysplasia have exact counterparts in human diseases. There is an important need to generate exact animals models, perhaps by transgenic approaches so that management and therapy can be tested in an experimental system. The identification of abnormalities in cartilage genes that result in chondrodysplasias has been hampered by the difficulty in growing target cells which express the mutant proteins and the failure of expression of these genes in surrogate tissues. Culture systems for maintaining welldifferentiated cartilage cells in culture are being developed and should facilitate the identification of additional defects in these disorders. Genetic approaches to the chondrodysplasias has confirmed genetic heterogeneity but has identified relatively few disorders in the candidate genes.

Epidermolysis bullosa: Defects of the cutaneous basement membrane zone Epidermolysis bullosa (EB) is a group of more than a dozen heritable disorders, characterized by fragility of the epidermis, the dermal-epidermal basement membrane, or the upper layers of the dermis. EB is classified by the location of blistering: within the epidermis (simplex), at the dermal-epidermal junction (junctional), or within the papillary dermis (dystrophic): A variety of syndromes exist within each of these major groups and can be differentiated by accompanying features and mode of inheritance (GeddeDahl, 1981; Fine et aI., 1991). The major components within the cutaneous basement membranes are type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. The adjacent basement membrane zone contains complex structures, such as anchoring fibrils (type VII collagen) and hemidesmosomes (which contain the bullous pemphigoid antigens), that provide stability to the dermalepidermal junction (Uitto et aI., 1989). More than twenty-additional proteins are present but currently identified only on the basis of immunohistologic analyses (Fine, 1988). The major genetic disorders which result from mutations in proteins in this region manifest as blistering of the skin and mucous membranes and are grouped under epidermolysis bullosa. The simplex varieties of EB are dominantly inherited, and the blisters and tissue disruption occur in the basal layer of the epidermis. The lesions may be generalized or localized primarily to the hands and feet (Fine et aI., 1991). Because intracellular abnormalities are seen at some stages of the disease, candidate proteins include cystoskeletal elements, as well as membrane and cell attachment proteins. In fact, a close genetic linkage to keratin gene clusters on chromosomes 12q and 17q has been recently demonstrated in families with generalized EBS (Bonifas et aI., 1991; Ryy-

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nanen et a1., 1991 c). Also, immunofluorescence of the skin in EBS has suggested abnormal keratin gene expression (Ito et a1., 1991). Finally, transgenic mice expressing truncated keratin 14 developed a blistering disease resembling EBS (Vassar et a1., 1991). Finally, a point mutation in the keratin 14 gene was found to segregate with the EBS phenotype in one family, and in an unrelated family, linkage to the keratin 5 gene on chromosome 12q was demonstrated with a LOD of 7 (Bonifas et aI., 1991). In other forms of EB simplex, linkage of the "Ogna" variety (EBSl) to the GPT locus on chromosome 8 has been noted in a single family (Olaisen and Gedde-Dahl, 1973). The gene at that locus has not, however, been identified. In several additional families with generalized (Koebner) EBS, there is evidence suggestive of linkage with markers in the long arm of chromosome 1 (Mulley et aI., 1984; Humphries et aI., 1990 a), but candidate genes, such as nidogen (lq43), have been excluded (Olsen et a1., 1989; Humphries et aI., 1990 b). The dystrophic forms of EB may be inherited in an autosomal dominant or autosomal recessive fashion (Fine et aI., 1991). These are generally the most severe disorders and the recessive forms may be lethal. Morphologic and immunohistologic studies consistently identify alterations in anchoring fibrils, structures involved in securing stable association of the basal lamina to the dermal structures (Tidman and Eady, 1985). The anchoring fibrils contain type VII collagen as their major component (Keene et aI., 1987). Some anchoring fibrils originate in and then reinsert into the basement membrane zone at the dermal-epidermal junction; while others extend perpendicularly into the dermis and insert into amorphous patches, so-called anchoring plaques, in the dermis. This network can entrap interstitial collagen fibers in the papillary dermis, securing the basement to its underlying dermis. In both the dominant and recessive forms of dystrophic EB type VII collagen staining at the dermal-epidermal junction is diminished, discontinuous or absent (Briggaman, 1985; Bruckner-Tuderman et aI., 1989). In some families with the dystrophic forms EB there is morphologic evidence of retention of type VII collagen in the RER of the basal keratinocytes (Smith and Sybert, 1990; Fine et a1., 1990). There are earlier suggestions that in some forms of the dystrophic EB the absence of type VII collagen might be explained by the destructive effects of increased activity of type I collagenase, but recent evidence excludes the collagenase locus on chromosome 11 as the primary mutation in some families with recessive dystrophic EB (Hovnanian et aI., 1991). Genetic linkage between the type VII collagen locus on human chromosome 3 and dominant dystrophic EB has been documented in a family (Ryynanen et a1., 1991 a). The absence of recombination between the EB phenotype and the type VII collagen gene locus, together with abnormalities in anchoring fibrils, strongly suggested that type VII collagen gene is the mutant locus in this kindred with dominant dystrophic EB. Structural abnor-

malities in other basement membrane and dermal components that bind type VII collagen are also candidates for mutations that could result in the dystropic EB phenotypes (Briggaman, 1985). A number of candidate genes (including those that encode the polypeptide chains of type IV collagen, laminin, nidogen, biglycan, decorin, heparan sulfate core protein, and bullous pemphigoid antigens, a 230-kDa [BPAG1] and a 180-kDa [BPAG2) protein components of hemidesmosomes [Amagai et aI., 1991]) are available fortestingin a genetic fashion in the junctional forms of EB. The genes for the last two proteins have been mapped to chromosomes 6 and 10, respectively, and have been excluded as candidate genes in an EB simplex family (Sawamura et a1., 1990; Sawamura et a1., 1991; Ryynanen et a1., 1991 b; Ditto et a1., 1991 a). The genes for some of the other protein components at the dermal-epidermal junction and in the papillary dermis have not yet been isolated but antibodies to those proteins suggest involvement in some varieties of genetic blistering disorders. For example, defective integrins on the surface of epidermal cells may contribute to the molecular pathology in some of the simplex forms of EB (Peltonen et aI., 1989; Larjava et aI., 1990; Ryynanen et aI., 1991). Animal models of EB simplex, of recessive dystrophic EB in Swiss alpine sheep, and of bovine and equine models of junctional EB are available for study (see Bruckner-Tuderman et aI., 1991).

Disorders of elastin and elastic fiber biogenesis in cutis laxas and pseudoxanthoma elasticum Elastic fibers form a network which contributes to the elasticity and resilience of tissues, such as the skin, the lung and arterial blood vessels (Ditto et aI., 1989; Rosenbloom et aI., 1991). Mature elastic fibers consist of two component elastin and the microfibrils. The human elastin gene has recently been isolated and characterized and mapped to the long arm of chromosome 7 (Bashir et aI., 1989; Fazio et a1., 1991). The primary sequence of elastin, as deduced from complete cDNA sequences (Fazio et a1., 1988), consists of alternating hydrophobic and cross link (hydrophilic) domains which segregate into distinct exons within the gene. In contrast to fibrillar collagens, in which certain elements of sequence are rigorously maintained during evolution, there is considerable variations in elastin primary sequences (Rosenbloom et aI., 1991). These range from relatively small alterations, such as conservative amino acid substitutions, to variation in the length of hydrophobic segments and deletions and insertions within the gene. Furthermore, there is considerable variation in the splicing of several exons within the primary transcript of human elastin which further increases the difficulty in identifying mutant sequences of pathologic consequence (Fazio et aI., 1988). The consequence of multiple biosynthetic

Heritable Disorders of Connective Tissue products generated through alternative splicing for elastic fiber biogenesis is uncertain. The sequence of the entire elastin gene is nearing completion, and a number of interesting features have been identified, including a high number of Alu repeat elements in the introns (Rosenbloom et aI., 1991). In the 5'-flanking region there are several SP-1 and AP2 binding sites, and curiously, the canonical TATA box is missing, probably explaining the presence of multiple transcription initiation sites (Bashir et aI., 1989; Kahari et aI., 1990). Elastin is a relatively late developmental product in most tissues and is initially deposited on a network of microfibrills, of which fibrillin, a glycoprotein with a molecular mass of 320 kDa, is a major component (Sakai et aI., 1986). Cutis laxa is a heterogenous disorder characterized by marked skin laxity which may be accompanied by internal organ abnormalities, such as bladder diverticuli, arterial aneurysms and pulmonary emphysema (Vitto et aI., 1991 b). In a severe form (often inherited in an autosomal recessive fashion) death may occur in childhood. A milder autosomal dominant form has a later onset and organ involvement beyond skin is rare. In the severe form there is often a paucity of elastin, or elastic fibers can be disorganized depicting dissociation of elastin and the microfibrils. Biochemical studies suggest that in the severe congenital forms, there is often a decrease in the amount of elastin synthesized by cultured fibroblasts, accompanied by decreased elastin mRNA levels, but the precise molecular basis of altered synthesis is not known (Olsen et aI., 1988; Sephel et aI., 1989). No studies of gene organization or mRNA structure have been completed and it is uncertain whether deletion or mutations in the elastin gene are responsible for the phenotypes. It is of interest to note that cutis laxa often manifests as premature aging appearance of the affected individuals (Vitto et aI., 1991 b). Consequently, alterations noted in elastic fibers in cutis laxa may provide lesions also applicable to age-associated cutaneous aging (Fazio et aI., 1989). Pseudoxanthoma elasticum (PXE) is a genetically heterogenous disorder characterized by cutaneous, ocular, and arterial abnormalities (Vitto et aI., 1991 b). The affected areas in the skin and arterial blood vessels have marked calcium accumulation in elastic fibers leading to a decrease in skin compliance and arterial rupture. These may be life threatening disorders with early death. The molecular basis of this set of disorders in unknown and no comprehensive studies have been completed.

Ehlers-Danlos Syndrome Ehlers-Danlos syndrome is a group of almost a dozen disorders characterized by soft and hyperextensible skin, and joint laxity, with the specific disorders distinguished on genetic, biochemical and clinical grounds (Byers and Hol-

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brook, 1986). The biochemical basis of fewer than half of these have been determined. Although there is morphologic evidence of markedly abnormal collagen fibrillar array in the forms of Ehlers-Danlos syndrome characterized by soft and hyperextensible skin, the molecular basis of this important finding is currently unknown (Holbrook and Byers, 1989). No comprehensive linkage studies have been completed. In a very small group of patients with this phenotype there is evidence that a lack of synthesis of the proa2(1) chains of type I collagen is the underlying defect (Sasaki et aI., 1987). The best characterized form of Ehlers-Danlos syndrome, EDS Type IV, is due to mutations in the COUA1 gene that encodes chains of type III procollagen (see Kuivaniemi et aI., 1991 for recent review of known mutations). In this disorder, life expectancy is about 40years with death resulting from arterial rupture, GI tract rupture and ensuing infection, or uterine rupture during pregnancy. Multiexon deletions, exon-skipping mutations and point mutations can all produce similar phenotypes and there is not yet a clear indication of the relationship between genotype and phenotype. Although type III collagen is a constituent of skin, blood vessels and other hollow organs, it is not clear why the failure to produce normal structures of a relatively minor component in these tissues produces such highly deleterious results. EDS TYPE VII is now known to result from heterozygosity for mutations that affect the correct splicing of exon 6 of the two type I collagen genes (Weil et aI., 1988; 1989; 1990). Splicing of the abnormal exon may be inefficient and contribute to variable expression of the phenotype in different family members. EDS TYPE VI is autosomal recessive disorder due to lysyl hydroxylase deficiency. The molecular basis of this disorder was first identified in the early 1970's and work is now proceeding on identification of mutations in the lysyl hydroxylase gene (Pinnell et aI., 1971). This is a systemic disorder and arterial rupture may complicate the course of this disease (Wenstrup et aI., 1989). EDS TYPE IX (recently classified out of EDS and named the occipital horn syndrome (Beighton et aI., 1988)) is an Xlinked disorder characterized by bladder diverticuli and abnormal bony structures, in addition to abnormalities of skin (Luzzatti et aI., 1977; Byers et aI., 1980). It is due to defective copper transport with high levels of copper accumulating in the cells and low serum copper levels (Kuivaniemi et aI., 1983). It appears that copper cannot be appropriately transferred to its apoenzymes; thus this disorder results in a multi-enzyme deficiency. It may be allelic to Menkes disease or due to a defect in a related X-chromosome encoded copper transport and binding protein.

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The Marfan Syndrome The Marfan syndrome is characterized by dominant inheritance, lens dislocation, cardiovascular abnormalities of mitral valve prolapse, aortic aneurysm and dissection, and musculo-skeletal abnormalities of tall stature, arachnodactyly and scoliosis (Pyeritz and McKusick, 1979). Some affected children develop severe mitral regurgitation and may die from congestive heart failure unless treated aggressively. More often dilatation of the aortic root progresses gradually and asymptomatically. In adulthood aortic regurgitation develops or aortic dissection occurs; the latter often causes sudden death. The Marfan syndrome is one of the more frequent adult lethal mendelian conditions, and more than 25000 Americans are affected. The complex phenotype is caused by different mutations in a single gene expressed in multiple tissues. Medical management remains based on attempts to decrease the severity of aortic involvement. Surgical management of aortic aneurysms prolonge life (Gott et al., 1986; 1991). The effect of medical therapies designed to slow the rate of aortic enlargement is less certain. Attempts to identify the molecular basis of the Marfan syndrome have, until recently, been unrevealing. Biochemical studies had previously suggested that a wide variety of matrix components, including elastin, type I coHagen, other collagens, components of the elastic fiber network, and hyaluronic acid might be abnormal in the Marfan syndrome. Recent genetic studies have excluded the major fibrillar collagen loci as candidate genes. More recently, immunofluoresence studies using monoclonal antibodies to fibrillin, a component of elastic fibers (Sakai et al., 1986), suggested that skin from most individuals with the Marfan syndrome have a defective microfibrillar array and that cells from these people are defective in their ability to lay down a normal microfibrillar network (Godfrey et al., 1990; Hollister et al., 1990). Furthermore, a genetic linkage to the markers in chromosome 15, which also contains the fibrillin locus, has been reported (Peltonen et al., 1991) and confirmed (Dietz et al., 1991). These studies are strongly suggestive of abnormalities in the elastic fiber generating system in the Marfan syndrome; preliminary biochemical studies confirm this suggestion (McGookey et al., 1990). Since the meeting, the sequence of a portion of the fibrillin eDNA has been reported by two groups (Lee et al., 1991; Maslin et al., 1991) and mutations in the gene have been identified in two individuals with the Marfan syndrome (Dietz et aI., 1991). Furthermore, an additional fibrillinlike gene was located on chromosome 5 and linkage to the syndrome of congenital contactural arachnodactyly (CCA) was demonstrated (Lee et al., 10 (' !). These findings set the stage for detailed genotype-phenotype studies.

Recommendations Summary On the basis of the discussion during the conference several areas were identified as research priorities that would further understanding of disease mechanisms of the heritable disorders of connective tissue. These were summarized in the following general recommendations. The more detailed recommendations that are specific for individual disorders are included as Appendix I. 1. Identification and characterization of mutations in all disease phenotypes. 2. Structure-function relationships of normal matrix molecules. 3. Developmental regulation of matrix formation. 4. Multi-disciplinary analysis of disease mechanisms. 5. Clinical studies: natural history, clinical trials in genetic disease. 6. Establishment of disease-based registries. 7. Periodic workshops; inclusion of disease studies in major symposia on matrix biology (Gordon Conferences, FASEB, Cell Biology, American Society of Human Genetics, etc.). 8. Identification and study of animal models; creation of animal models by transgenic technologies to identify phenotypes produced by mutations in specific genes. 9. Application of genetic linkage strategies to heritable disorders of connective tissue. 10. Pursuit of the molecular basis of common disease analogues of specific heritable disorder of connective tissue.

Appendix I: Recommendations in specific disease categories I. Osteogenesis Imperfecta

1. 2. 3. 4.

Continued characterizations of molecular defects Phenotype-genotype correlation Analysis of cellular processing and assembly pathways Analysis of extracellular events and their disturbance by mutations 5. Histopathology of osteogenesis imperfecta using modern immunohistology and electron microscopy 6. Characterization of bone proteins that interact with collagen 7. The molecularbasis of recessively inherited forms of 01 8. The molecular basis of dentinogenesis imperfecta 9. Natural history of OI 10. Identification of genes responsible for variable expression of the phenotype due to a major gene mutation 11. Registry for osteogenesis imperfecta facilitated through 01 Foundation, for example, and modeled on the EB registry

Heritable Disorders of Connective Tissue II. Chondrodysplasias

1. Identification of the candidate genes by direct analysis of gene structure for candidate genes and by linkage in appropriate families. 2. Studies of tissues from affected individuals by immunohistochemical techniques to identify potentially abnormal proteins 3. Isolation and characterization of genes for matrix molecules. 4. Development of transgenic models for chondrodysplasia 5. Characterization of animal models, especially mice, use of chromosome synteny for identification of candidate genes III. Epidermolysis bullosa

1. Identification of candidate proteins in the epidermis, dermalepidermal basement membrane and papillary dermis. 2. Isolation and characterization of complementary and genomic DNAs encoding proteins in the cutaneous basement membrane zone. 3. Genetic linkage analyses with RFLPs in the candidate genes and with chromosome-specific markers. 4. Morphologic correlation by immunohistologic and in situ hybridization studies of tissues from patients. 5. Development of transgenic animals as models of human EB. 6. Definition of "disease groups", based on underlying mutations and phenotypic information derived from EB registry. IV. Cutis laxa 1. Delineation of natural history and inheritance through genetic studies. 2. Identification of elastin mutations through analysis of gene and mRNA structures. 3. Characterization of the microfibrillar proteins and their interactions with elastin during fibrillogenesis of elastic fibers.

V. Pseudoxanthoma elasticum 1. Genetic linkage studies towards identification of candidate genes. 2. Studies of cultured cells for abnormalities in elastin and other candidate proteins. 3. Development of model system to study calcification of elastic fibers.

VI. Marfan Syndrome

1. Definition of the range of mutations in fibrillin that cause MS; continued search for interlocus heterogeneity.

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2. Correlate nuances in phenotype with specific mutations. 3. Structure function studies of fibrillin, expecially incorporating study of mutant proteins. 4. Development of a mouse model of MS by transgeneic methods. 5. Studies of therapy in a mouse model.

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