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Fibulin-4 is an extracellular matrix glycoprotein essential for elastic fiber formation. Mice deficient in fibulin-4 die perinatally due to severe pulmonary and vascular defects associated with lacking intact elastic fibers. Patients with fibulin-4 mutations demonstrate similar defects and a significant number die shortly after birth or in early childhood owing to cardiopulmonary failure. The patients also demonstrated skeletal and other systemic connective tissue abnormalities, including joint laxity, and flexion contractures of the wrist. A fibulin-4 null mouse strain was generated and used to analyze the roles of fibulin-4 in tendon fibrillogenesis. This mouse model displayed bilateral forelimb contractures, in addition to pulmonary and cardiovascular defects. The forelimb and hindlimb tendons demonstrated a disruption in collagen fibrillogenesis in the absence of fibulin-4 analyzed using transmission electron microscopy. Fewer fibrils were assembled and fibrils were disorganized compared to wild type controls. The organization of developing tenocytes and compartmentalization of the extracellular space also was disrupted. Fibulin-4 was co-localized with fibrillin-1 and fibrillin-2 in limb tendons using immunofluorescence microscopy. Our studies demonstrate that fibulin-4 plays a role in regulating tendon collagen fibrillogenesis, in addition to its essential function in elastogenesis.

Cell-matrix interactions control outgrowth of mammary epithelium during puberty and pregnancy. We demonstrate here that the glycoprotein fibulin-2 (FBLN2) is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth. FBLN2 was specifically localized to the cap cells of the terminal end buds during puberty and to myoepithelial cells during very early pregnancy (days 2-3) even before morphological changes to the epithelium become microscopically visible, but was down-regulated thereafter. Exposure to exogenous oestrogen (E2) or E2 plus progesterone (P) increased Fbln2 mRNA expression in the pubertal gland, indicating hormonal control. FBLN2 was co-expressed and co-localised with the proteoglycan versican (VCAN) and co-localised with laminin (LN), while over-expression of FBLN2 in HC-11 cells increased cell adhesion to several extracellular matrix proteins including LN and fibronectin, but not collagens. Mammary glands from Fbln2 knockout mice showed no obvious phenotype but increased fibulin-1 (FBLN1) staining was detected, suggesting a compensatory mechanism by other fibulin family members. We hypothesise that similar to embryonic aortic smooth muscle development, FBLN2 and VCAN expression alters the cell-matrix interaction to allow mammary ductal outgrowth and development during puberty and to enable epithelial budding during pregnancy.

Type VI collagen (COL6) forms a microfibrillar network often associated with type I collagen and constitutes a major component of the desmoplastic reaction in pancreatic ductal adenocarcinoma (PDA). We have demonstrated recently that the α3 chain of COL6, COL6A3, is highly expressed in PDA tissue and undergoes tumor-specific alternative splicing. In this study, we investigated the diagnostic value and clinical significance of circulating COL6A3 protein and mRNA in PDA. COL6A3 levels in sera from patients with PDA (n = 44), benign lesions (n = 46) and age-matched healthy volunteers (n = 30) were analyzed by enzyme-linked immunosorbent assays (ELISA). Predictive abilities of COL6A3 were examined using receiver operating characteristic (ROC) curves from logistic regression models for PDA versus normal or benign serum levels. Expression levels were correlated with clinicopathological parameters. Real-time PCR was used to analyze the presence of COL6A3 mRNA containing alternative spliced exons E3, E4, and E6. Circulating COL6A3 protein levels were significantly elevated in PDA patients when compared to healthy sera (p = 0.0001) and benign lesions (p = 0.0035). The overall area under the ROC was 0.975. Log(COL6A3) alone provided good discrimination between PDA and benign lesions (area under the curve (AUC) = 0.817), but combined with CA19-9 provided excellent discrimination (AUC = 0.904). Interestingly, high COL6A3 serum levels were significantly associated with perineural invasion and cigarette smoking. Combined E3, E4, and E6 serum RNA values provided good sensitivity but low specificity. Our data demonstrate for the first time the potential clinical significance of circulating COL6A3 in the diagnosis of pancreatic malignancy.

Ullrich congenital muscular dystrophy (UCMD) is a severe disorder caused, in most cases, by a deficiency in collagen VI microfibrils. Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have been identified in eight of the nine UCMD patients reported thus far. A heterozygous COL6A1 gene deletion, resulting in a mutant protein that exerts a dominant negative effect, has recently been described in a severely affected UCMD patient. Here we describe a patient in whom reverse transcription-PCR analysis of fibroblast RNA suggested a heterozygous in-frame deletion of exon 13 in the triple-helical domain of COL6A2, which is predicted to be dominantly acting. However, a homozygous A --G mutation at -10 of intron 12 was found in the genomic DNA. The intron mutation activated numerous cryptic splice acceptor sites, generating normal and exon 13-deleted COL6A2 mRNA, and multiple aberrant transcripts containing frameshifts that were degraded through a nonsense-mediated decay mechanism. Northern analysis indicated diminished COL6A2 mRNA expression as the primary pathogenic mechanism in this UCMD patient. Our results underscore the importance of multifaceted analyses in the accurate molecular diagnosis and interpretation of genotype-phenotype correlations of UCMD.

Fibulins are a newly recognized family of extracellular matrix proteins. The five known members of the family share an elongated structure and many calcium-binding sites, owing to the presence of tandem arrays of epidermal growth factor-like domains. They have overlapping binding sites for several basement-membrane proteins, tropoelastin, fibrillin, fibronectin and proteoglycans, and they participate in diverse supramolecular structures. New insights into their biological roles are now emerging from studies of transgenic mice and of some inherited human diseases.

Fluorescence in situ hybridization (FISH) on mechanically stretched chromosomes (MSCs) and extended DNA fibers enables construction of high-resolution physical maps by accurate ordering and orienting genomic clones as well as by measuring physical lengths of gaps and overlaps between them. These high-resolution FISH targets have hitherto been used mainly in the study of the human genome. Here we have applied both MSCs and extended DNA fibers to the physical mapping of the mouse genome. At first, five mouse collagen genes were localized by metaphase-FISH: Col10a1 to chromosomal bands 10B1-B3; Col13a1 to 10B4; and Col6a1, Col6a2, and Col18a1 to 10B5-C1. The mutual order of the genes, centromere--Col10a1--Col13a1--Col6a2--Col6a1--Col18a1--telomere, was determined by FISH on metaphase chromosomes, MSCs, and extended DNA fibers. To our knowledge, this is the first time mouse metaphase chromosomes have been stretched and used as targets for FISH. We also used MSCs to determine the transcriptional orientations, telomere--5'--3'--centromere, of both Col13a1 and Col18a1. With fiber-FISH, Col18a1, Col6a1, and Col6a2 were shown to be in a head-to-tail configuration with respective intergenic distances of about 350 kb and 90 kb. Comparison of our physical mapping results with the homologous human data reveals both similarities and differences concerning the chromosomal distribution, order, transcriptional orientations, and intergenic distances of the collagen genes studied.

The collagens represent an interesting example of a structurally related but genetically distinct family of proteins. Type I, the most abundant of the vertebrate collagens, comprises two pro alpha 1(I) chains and one pro alpha 2(I) chain, each containing terminal propeptides and a central domain of 338 (Gly, X, Y) repeats. The structure of the chicken pro alpha 2(I) gene shows an intriguing relationship between exon organization and the arrangement of (Gly, X, Y) repeats (see ref. 2 for review). This has led to the suggestion that the collagens evolved from a common ancestral unit of 54 base pairs (bp). Here we present the structure of the entire human pro alpha 1(I) gene and compare this with the chicken pro alpha 2(I). The exon arrangement of the two genes is remarkably similar, although the human pro alpha 1(I) is more compact because of the shorter length of its introns. The data strongly support the notion that the type I genes have evolved from an ancestral multi-exon unit, and that once the gene was translated, a strong evolutionary pressure caused it to maintain this elaborate structure.

A laminin B2 chain cDNA clone was isolated from a human lung cDNA library by screening with antibody against mouse laminin. The authenticity of the human cDNA clone was established by comparison of the nucleotide and deduced amino acid sequences of the cDNA insert with those of the previously reported mouse laminin cDNA clones. The human clone (LC7) contained an insert of 0.75 kb (kilobase pair) that corresponded to the last 232 amino acid residues in the carboxyl terminus of the B2 chain. Northern blot analyses with the LC7 probe detected two mRNA transcripts of 8.2 and 5.6 kb in both normal human skin fibroblasts and three human tumor cell lines. The cDNA probe was also used in Southern blot analysis of DNA from human rodent somatic cell hybrids to localize the gene to human chromosome 1. In situ hybridization of the cDNA with metaphase chromosome spreads confirmed the assignment and further mapped the human laminin B2 chain gene to the long arm of chromosome 1 in the band q31.

Cloned probes specific for unique genes have proven to be powerful tools in defining the nature of genetic diseases such as the thalassaemias and growth hormone deficiencies. A similar approach should be useful in defining heritable diseases of type I collagen, the heterotrimer of two alpha 1(I) chains and one alpha 2(I) chain, which is the most abundant member of the collagen family of proteins. Recently, cloned cDNAs and genomic DNAs for the two polypeptide chains of the type I collagen have become available and have been used to elucidate the chromosomal location of the corresponding genes. Here, we have used several of these cloned DNAs to demonstrate the presence of an internal deletion of about 0.5 kilobases (kb) in one allele for the pro alpha 1(I) chain in a patient with osteogenesis imperfecta (OI), a group of heritable disorders which are characterized by brittle bones but which are highly heterogeneous both phenotypically and biochemically.