Elizabeth A. Putnam

Delineation of the Marfan phenotype associated with mutations in exons 23–32 of the FBN1 gene

Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-based exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 gene in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24-32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling.

CHARACTERIZATION OF CARTILAGE OLIGOMERIC MATRIX PROTEIN (COMP) IN HUMAN NORMAL AND PSEUDOACHONDROPLASIA MUSCULOSKELETAL TISSUES

Cartilage oligomeric matrix protein (COMP), the fifth member of the -thrombospondin gene family, is an extracellular matrix calcium-binding protein. The importance of COMP is underscored by the finding that mutations in COMP cause the human dwarfing condition, pseudoachondroplasia (PSACH). Here, we report the results of human tissue distribution and cell secretion studies of human COMP. COMP is expressed and secreted by cultured monolayer chondrocyte, tendon and ligament cells, and COMP secretion is not restricted to a differentiated chondrocyte phenotype. Whereas COMP is retained in the endoplasmic reticulum that accumulates within PSACH chondrocytes in vivo, COMP is not retained intracellularly in the dedifferentiated PSACH chondrocytes in cultures. These results lend further support to the hypothesis that retention of COMP is related to the terminal PSACH chondrocyte phenotype, processing of proteins related to extracellular matrix formation, and maintenance in cartilage.

Assessment of Autoimmune Responses Associated with Asbestos Exposure in Libby, Montana, USA

Systemic autoimmune responses are associated with certain environmental exposures, including crystalline particles such as silica. Positive antinuclear antibody (ANA) tests have been reported in small cohorts exposed to asbestos, but many questions remain regarding the prevalence, pattern, and significance of autoantibodies associated with asbestos exposures. The population in Libby, Montana, provides a unique opportunity for such a study because of both occupational and environmental exposures that have occurred as a result of the mining of asbestos-contaminated vermiculite near the community. As part of a multifaceted assessment of the impact of asbestos exposures on this population, this study explored the possibility of exacerbated autoimmune responses. Age- and sex-matched sets of 50 serum samples from Libby and Missoula, Montana (unexposed), were tested for ANA on HEp-2 cells using indirect immunofluorescence. Data included frequency of positive tests, ANA titers, staining patterns, and scored fluorescence intensity, all against known controls. Serum immunoglobulin A (IgA), rheumatoid factor, and antibodies to extractable nuclear antigen (ENA) were also tested. The Libby samples showed significantly higher frequency of positive ANA and ENA tests, increased mean fluorescence intensity and titers of the ANAs, and higher serum IgA, compared with Missoula samples. In the Libby samples, positive correlations were found between ANA titers and both lung disease severity and extent of exposure. The results support the hypothesis that asbestos exposure is associated with autoimmune responses and suggests that a relationship exists between those responses and asbestos-related disease processes.

Clustering of FBN2 mutations in patients with congenital contractural arachnodactyly indicates an important role of the domains encoded by exons 24 through 34 during human development

Congenital contractural arachnodactyly (CCA) is an autosomal dominant condition phenotypically related to Marfan syndrome (MFS). CCA is caused by mutations in FBN2, whereas MFS results from mutations in FBN1. FBN2 mRNA extracted from 12 unrelated CCA patient cell strains was screened for mutations, and FBN2 mutations were identified in six of these samples. All of the identified FBN2 mutations cluster in a limited region of the gene, a region where mutations in FBN1 produce the severe, congenital form of MFS (so-called neonatal MFS). Furthermore, three of the identified mutations occur in the FBN2 locations exactly corresponding to FBN1 mutations that have been reported in cases of neonatal MFS. These mutations indicate that this central region of both of the fibrillins plays a critical role in human embryogenesis. The limited region of FBN2 that can be mutated to cause CCA may also help to explain the rarity of CCA compared to MFS.

Profibrillin-1 maturation by human dermal fibroblasts: proteolytic processing and molecular chaperones.

Fibrillin‐1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C‐terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans‐Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin‐1 is processed intracellularly or after secretion. This report provides evidence that profibrillin‐1 processing is not an intracellular event. Bafilomycin A1 and incubation of dermal fibroblasts at 22°C were used to block secretion in the TGN to confirm that profibrillin‐1 processing did not occur in this compartment. Profibrillin‐1 immunoprecipitation studies revealed that two endoplasmic reticulum‐resident molecular chaperones, BiP and GRP94, interacted with profibrillin‐1. To determine the proprotein convertase responsible for processing profibrillin‐1, a specific inhibitor of furin, α‐1‐antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin‐1, providing evidence that furin is the enzyme responsible for profibrillin‐1 processing. These studies delineate the secretion and proteolytic processing of profibrillin‐1, and identify the proteins that interact with profibrillin‐1 in the secretory pathway.

Pharmacogenetics in American Indian populations: analysis of CYP2D6, CYP3A4, CYP3A5, and CYP2C9 in the Confederated Salish and Kootenai Tribes.

Objectives Cytochrome P450 enzymes play a dominant role in drug elimination and variation in these genes is a major source of interindividual differences in drug response. Little is known, however, about pharmacogenetic variation in American Indian and Alaska Native (AI/AN) populations. We have developed a partnership with the Confederated Salish and Kootenai Tribes (CSKT) in northwestern Montana to address this knowledge gap. Methods We resequenced CYP2D6 in 187 CSKT individuals and CYP3A4, CYP3A5, and CYP2C9 in 94 CSKT individuals. Results We identified 67 variants in CYP2D6, 15 in CYP3A4, 10 in CYP3A5, and 41 in CYP2C9. The most common CYP2D6 alleles were CYP2D6*4 and *41 (20.86 and 11.23%, respectively). CYP2D6*3, *5, *6, *9, *10, *17, *28, *33, *35, *49, *1xN, *2xN, and *4xN frequencies were less than 2%. CYP3A5*3, CYP3A4*1G, and *1B were detected with frequencies of 92.47, 26.81, and 2.20%, respectively. Allelic variation in CYP2C9 was low: CYP2C9*2 (5.17%) and *3 (2.69%). In general, allele frequencies in CYP2D6, CYP2C9, and CYP3A5 were similar to those observed in European Americans. There was, however, a marked divergence in CYP3A4 for the CYP3A4*1G allele. We also observed low levels of linkage between CYP3A4*1G and CYP3A5*1 in the CSKT. The combination of nonfunctional CYP3A5*3 and putative reduced function CYP3A4*1G alleles may predict diminished clearance of CYP3A substrates. Conclusion These results highlight the importance of carrying out pharmacogenomic research in AI/AN populations and show that extrapolation from other populations is not appropriate. This information could help optimize drug therapy for the CSKT population.

Mesothelial cell and anti-nuclear autoantibodies associated with pleural abnormalities in an asbestos exposed population of Libby MT

Despite data linking amphibole asbestos exposure with production of autoantibodies, the role of autoantibodies in subsequent disease is unknown. Residents of Libby, Montana have experienced significant exposure to amphibole asbestos due to the mining of asbestos-contaminated vermiculite near the community over several decades. This population predominantly exhibits pleural disease, and an autoimmune-like disorder that has yet to be well defined. This study sought to determine whether autoantibodies from asbestos-exposed subjects were associated with pleural lesions. Serum samples of subjects from Libby were evaluated for anti-nuclear antibodies (ANA) and mesothelial cell autoantibodies (MCAA) using cell based ELISA. The presence of radiographic abnormalities detected during the time frame of serum collection was determined from screening records. In accord with previous studies, 61.3% (76/124) of the Libby samples were ANA positive, a frequency much higher than expected for a healthy population. The odds of having pleural or interstitial abnormalities in Libby was nearly 3.55 times greater for individuals that tested positive for ANA compared with individuals negative for ANA (p=0.004). MCAA were also detected at a strikingly high frequency (18.5%; 23/124) in samples from Libby. Individuals with MCAA had 4.9 times the risk of having pleural abnormalities compared to MCAA-negative subjects (p=0.044). In conclusion, ANA and MCAA were elevated in a study population that was known to have chronic exposure to asbestos, and these autoantibodies were associated with pleural abnormalities, the predominant finding in the asbestos-exposed population of Libby. Additional research is needed to determine the role these autoantibodies may play in pulmonary disease.

Gene expression changes after exposure to six-mix in a mouse model.

The exposure of Libby MT residents to amphibole-contaminated vermiculite is well known. To explore the gene-environment interactions in the development of asbestos-related diseases (ARD), a mouse model of asbestos exposure using Six-mix (a combination of amphibole fibers gathered from six sites at the Libby vermiculite mine), crocidolite asbestos, or saline as a negative control was used to determine both gene expression responses by using mouse 10,000 oligonucleotide array and to visualize these changes histologically. Mice were sacrificed and whole lungs harvested for histology and microarray analysis six months following exposure via intratracheal instillation. Using an arbitrary cutoff of 1.25-fold change, genes whose RNA expression levels were specifically altered in response to the different amphibole exposures were grouped into categories by a gene ontology analysis program, GoMiner. Our hypothesis was that assessment of asbestos-responsive genes would provide a better understanding of response mechanisms. These experiments have provided new candidates for genes involved in the asbestos response pathways.

Collagen accumulation over time in the murine lung after exposure to crocidolite asbestos or Libby amphibole

Libby, MT is the site of a closed vermiculite mine that produced ore contaminated with asbestos‐like amphiboles. Worldwide distribution of the material and the long latency period for manifestation of asbestos‐related diseases (ARDs) has created a significant health threat for many years to come. The composition of the Libby material [termed the Libby amphibole (LA)] differs from other well‐studied types of asbestos in that it is a mixture of several amphibole fibers. The purpose of this study was to determine the fibrotic effects of LA exposure in a mouse model and to compare these effects to those of a well‐characterized amphibole fiber, crocidolite asbestos. We exposed C57Bl/6 mice to LA or crocidolite and analyzed lung RNA, protein, and morphology at 1 week, 1 month, and 3 months post instillation. Our results indicate that both forms of amphibole studied induced increased collagen types I and III mRNA expression and collagen protein deposition in exposed murine lungs compared to the PBS‐instilled control lungs, and that these collagen increases were the most significant at 1 month after exposure. However, crocidolite‐exposed mice demonstrated greater increases in collagen deposition than those exposed to LA, indicating that the fibrotic effects of LA exposure, although not as severe as those of crocidolite in this model system, were still able to induce collagen deposition.

Differences in gene expression profiles from asbestos-treated SPARC-null and wild-type mouse lungs

The role of SPARC in the in vivo lung response to crocidolite asbestos was addressed by instillation of crocidolite asbestos in a series of wild-type or SPARC-null mice. Animals were sacrificed at one week, one month, and three months post-instillation to assess the impact of SPARC on multiple stages in the development of fibrosis. RNA was harvested from 10 animals/time point, pooled, and used to probe a mouse array containing approximately 10,000 probes. Gene expression data were analyzed for fold change, and for broader functional group alterations. As expected, the one-week time point displayed alterations in genes involved in immune recognition, energy utilization, and growth factor production. Later time points showed expression alterations for genes involved in protein degradation, Wnt receptor signaling, membrane protein activity, and transport. Molecules in the Wnt pathway have been implicated in bone growth, mediation of fibroblast activity, and have been directly linked to SPARC regulation.