Search PubMed⌕ Search

Biomedical subjects

J C Marini

Publications and source records attributed to J C Marini.

At least 37 records · Page 2Linked to original sources
PubMed1999

Extension of phenotype associated with structural mutations in type I collagen: siblings with juvenile osteoporosis have an alpha2(I)Gly436 --> Arg substitution.

Mutations in the type I collagen genes have been identified as the cause of all four types of osteogenesis imperfecta (OI). We now report a mutation that extends the phenotype associated with structural abnormalities in type I collagen. Two siblings presented with a history of back pain and were diagnosed with juvenile osteoporosis, based on clinical and radiological examination. Radiographs showed decreased lumbar bone density and multiple compression fractures throughout the thoracic and lumbar spines of both patients. One child has moderate short stature and mild neurosensory hearing loss. However, neither child has incurred the long bone fractures characteristic of OI. Protein studies demonstrated electrophoretically abnormal type I collagen in samples from both children. Enzymatic cleavage of RNA:RNA hybrids identified a mismatch in type I collagen alpha2 (COL1A2) mRNA. DNA sequencing of COL1A2 cDNA subclones defined the mismatch as a single-base mutation (1715G --> A) in both children. This mutation predicts the substitution of arginine for glycine at position 436 (G436R) in the helical domain of the alpha2(I) chain. Analysis of genomic DNA identified the mutation in the asymptomatic father, who is presumably a germ-line mosaic carrier. The presence of the same heterozygous mutation in two siblings strongly suggests that the probands display the full phenotype. Taken together, the clinical, biochemical, and molecular findings of this study extend the phenotype associated with type I collagen mutations to cases with only spine manifestations and variable short stature into adolescence.

Amino Acid Substitution↗
PubMed1998

New genetic loci that control susceptibility and symptoms of experimental allergic encephalomyelitis in inbred mice.

Experimental allergic encephalomyelitis (EAE), the principal animal model of multiple sclerosis, is a genetically determined phenotype. In this study, analyses of the cumulative disease frequencies in parental, F1 hybrid, and F2 mice, derived from the EAE-susceptible SJL/J strain and the EAE-resistant B10.S/DvTe strain, confirmed that susceptibility to EAE is not inherited as a simple Mendelian trait. Whole genome scanning, using 150 informative microsatellite markers and a panel of 291 affected and 390 unaffected F2 progeny, revealed significant linkage of EAE susceptibility to marker loci on chromosomes 7 (eae4) and 17, distal to H2 (eae5). Quantitative trait loci for EAE severity, duration, and onset were identified on chromosomes 11 (eae6, and eae7), 2 (eae8), 9 (eae9), and 3 (eae10). While each locus reported in this study is important in susceptibility or disease course, interactions between marker loci were not statistically significant in models of genetic control. One locus, eae7, colocalizes to the same region of chromosome II as Orch3 and Idd4, susceptibility loci in autoimmune orchitis and insulin-dependent diabetes mellitus, respectively. Importantly, eae5 and eae7 are syntenic with human chromosomes 6p21 and 17q22, respectively, two regions of potential significance recently identified in human multiple sclerosis genome scans.

Animals↗
PubMed1998

Three novel type I collagen mutations in osteogenesis imperfecta type IV probands are associated with discrepancies between electrophoretic migration of osteoblast and fibroblast collagen.

In three cases of type IV osteogenesis imperfecta (OI), we identified unique point mutations in type I collagen alpha1(I) cDNA. In two cases, the appearance of dimers indicated the presence of cysteine substitutions in the alpha1(I) protein chain. Cyanogen bromide digestion localized these cross-links to CB8 and 3, respectively. In the third case, the overmodification pattern of the CNBr peptides was compatible with a substitution in the aa 123-402 region of either type I collagen chain. We identified a unique point mutation in each proband, which resulted in substitutions for glycine residues in a 300-aa region of the alpha1(I) helix, specifically, Gly to Ala at codon 220 (GGT-->GCT), Gly to Cys at codon 349 (GGT-->TGT) and Gly to Cys at codon 523 (GGT-->TGT). We compared each proband's fibroblast and osteoblast collagen directly, as well as with fibroblast and osteoblast controls. For all cases, the OI osteoblast collagen was more electrophoretically delayed than OI fibroblast collagen. In the patient with G349C, OI fibroblast and osteoblast collagen synthesized in the presence of alpha,alpha'-dipyridyl co-migrated on gels, demonstrating that the electrophoretic discrepancy resulted from differences in post-translational modification. Melting temperature curves for stability of the collagen helix yielded an identical Tm for control fibroblast and osteoblast collagen (41.2 degrees C). By contrast, for collagen with the gly349-->cys substitution, the Tm of the fibroblast collagen was 1 degree C lower than the Tm of the osteoblast collagen. These data indicate that the metabolism of mutant collagen might be cell-specific and has significant implications for understanding the phenotype/genotype correlations and the pathophysiology of OI.

Adolescent↗
PubMed1998

An alpha2(I) glycine to aspartate substitution is responsible for the presence of a kink in type I collagen in a lethal case of osteogenesis imperfecta.

Type I collagen synthesized by cultured skin fibroblasts was analyzed biochemically and molecularly to characterize the defect in a patient affected by lethal Osteogenesis Imperfecta. The SDS-Urea-PAGE of procollagen and collagen revealed a broad alpha1(I) band, a normal alpha2(I) and another alpha2(I) band migrating equidistant between alpha1 and alpha2. When synthesized in the presence of alphaalpha'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylation, procollagen and collagen of media and cell layers contained both normal and slower alpha2(I), but only normal alpha1(I). The persistence of the two forms of alpha2(I) chains suggested a mutation in a COL1A2 gene. CNBr cleavage of collagen yielded overmodified alpha1(I) CB3 and CB7 peptides and delayed migration of the alpha2(I) CB3-5 peptide. A delayed CB3-5 was also found after alpha,alpha'-dipyridyl treatment. These data localized the mutation between aa 353 and 551 in alpha2(I) (CB3-5). Sequencing the subcloned alleles in this region revealed a G-->A transition at nt 1671 in one allele, changing Gly 421 to Asp in an alpha2(I) chain. The mutation was demonstrated to occur on the paternally derived allele, using a common C-->A polymorphism at alpha2(I) nt 1585 and by the presence of a rare variant, Arg618-->Gln (Phillips et al., 1990), in the paternal genomic DNA and the proband's mutant allele. Procollagen processing was normal. The Tm of the slow alpha2(I) collagen was 2 degrees C lower than the control, indicating decreased triple helix stability. Mutant collagen was incorporated in the extracellular matrix deposited by cultured fibroblasts. The dramatic delay in alpha2(I) electrophoretic mobility must be induced by the Gly-->Asp substitution, since the Arg-->Gln variant causes only mild electrophoretic delay. Substantial delay in gel mobility even in the absence of overmodification suggested the presence of a kink in the mutated alpha2(I) chains. Rotary shadowing electron microscopy of secreted fibroblast procollagen confirmed the presence of a kink in the region of the helix containing the glycine substitution. The kinking of the collagen helix occurs in the absence of dimer formation. Kinking may interfere with normal helix folding, as well as with the interactions of collagen fibrils with the collagenous and non-collagenous extracellular matrix proteins.

Amino Acid Substitution↗
PubMed1997

Cleavage of collagen RNA transcripts by hammerhead ribozymes in vitro is mutation-specific and shows competitive binding effects.

We report here the in vitro use of hammerhead ribozymes as an approach to the gene therapy of osteogenesis imperfecta (OI). Our strategy for the treatment of this dominant genetic disorder is based on selective reduction of the level of the mRNA transcripts from the mutant allele. We studied the in vitro cleavage activity of five different hammerhead ribozymes targeted against synthetic transcripts of two naturally occurring human collagen mutations and against a point mutation introduced into a construct containing a portion of the mouse COL1A1 gene. This is the first demonstration that ribozyme cleavage is absolutely dependent on the presence of the ribozyme cleavage site introduced by the disease-causing mutation. Cleavage specificity and activity were unchanged when the cleavage site was located in transcripts of progressively longer length. Cleavage efficiency depended directly on the ratio of ribozyme/substrate, as well as on the time and temperature of incubation. We investigated the competitive effects of both total RNA and normal synthetic transcripts on ribozyme cleavage activity. The ribozyme was able to localize and cleave its specific target even in the presence of a vast excess of total RNA. However, cleavage efficiency was linearly inhibited by the presence of a non- cleavable competitor substrate which contained a ribozyme binding site identical to the site present in the cleavable target. Although this competition could be eliminated by introducing a mismatch into one ribozyme binding arm, the presence of the mismatch decreased ribozyme cleavage efficiency. The mutation- specificity of ribozyme cleavage demonstrated in this work provides support for in vivo studies aimed at ribozyme development as a treatment for dominant negative genetic disorders.

Animals↗
PubMed1997

Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: implications for the treatment of multiple sclerosis.

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.

Animals↗
PubMed1997

Phenotypic comparison of an osteogenesis imperfecta type IV proband with a de novo alpha2(I) Gly922 --> Ser substitution in type I collagen and an unrelated patient with an identical mutation.

We examined the type I collagen synthesized by cultured dermal fibroblasts from a patient affected with osteogenesis imperfecta (OI) type IV. Both normal and abnormal trimers were produced. The mutant collagen molecules were excessively modified intracellularly, had a melting temperature 4 degrees C lower than the control, were secreted at a reduced rate, and underwent delayed processing to mature alpha chains.Molecular investigations identified a G --> A transition in one COL1A2 allele, resulting in a Gly922 --> Ser substitution in the alpha2(I) chain. The proband's mutation was demonstrated to arise "de novo" by the absence of the mutant allele restriction enzyme pattern from parental genomic DNA.We analyzed the insoluble extracellular matrix deposited by long-term cultured fibroblasts from our patient and from a previously described unrelated individual who carries an identical substitution. In both cases, the mutant chain constituted 10-15% of the total alpha chains deposited.We also present here the first detailed comparison of phenotype between unrelated OI patients with an identical collagen mutation. These two patients are both Caucasian females, ages 8 and 9 years, each diagnosed as type IV OI by the Sillence classification. They have a similar phenotype including moderate skeletal fragility with several femur fractures, dentinogenesis imperfecta, wormian bone, and reduced height and weight. We conclude that this phenotype is related both to the location of this mutation and to the similar extent of matrix incorporation by the mutant chains. Molecular and biochemical studies of unrelated individuals with identical amino acid substitutions in type I collagen resulting in either similar or dissimilar clinical outcomes will make a significant contribution to identifying the factors involved in the modulation of the OI phenotype.

Cells, Cultured↗
PubMed1996

Alternative splicing in COL1A1 mRNA leads to a partial null allele and two In-frame forms with structural defects in non-lethal osteogenesis imperfecta.

We have identified a novel multiexon genomic deletion in one COL1A1 collagen allele that results in three alternative forms of mutant mRNA. This mutation occurs in a 9-year-old girl and her father, both affected with severe type III osteogenesis imperfecta (OI). We previously reported detection of a mismatch in their alpha1(I) amino acids 558-861 region by RNA/RNA hybrid analysis (Grange, D. K., Gottesman, G. S., Lewis, M. B., and Marini, J. C. (1990) Nucleic Acids Res. 18, 4227-4236). Single Strand Conformational Polymorphism further localized the mRNA mutation to the amino acids 579-679 coding region. At the gene level, polymerase chain reaction (PCR) amplification of patient leukocyte DNA from the exon 33-38 region yielded the normal 1004-base pair (bp) fragment and an additional 442-bp fragment. Sequencing of the shorter genomic PCR product confirmed the presence of a 562-bp deletion, extending from the last 3 nucleotides (nt) of exon 34 to 156 nt from the 3'-end of intron 36. The genomic deletion was also detected in the clinically normal grandmother, who was confirmed to be a mosaic carrier. PCR amplification and RNase protection experiments were used to investigate the mRNA structure and occurrence of alternative splicing. One form of the mutant cDNA has a deletion with end points that are identical to the genomic deletion. This results in a combination deletion/insertion, with a deletion of amino acids 603-639 followed by an insertion of 156 nt from the 3'-end of intron 36. In addition, we found two alternatively spliced forms. One form uses a cryptic donor site in exon 34 and the exon 37 acceptor. The second form uses the normal exon 32 splice donor and exon 37 acceptor. Use of the cryptic donor results in a coding sequence that is out-of-frame. Both the retained intron form and the use of the exon 32 donor site result in coding sequences that are in-frame. This is the first report of a collagen defect in OI with alternative splicing generating both in-frame and out-of-frame forms of mRNA. Although the in-frame forms constitute more than 60% of the mRNA from the mutant allele, no mutant protein chain was identified. Collagen produced by cultured OI osteoblasts showed a significant increase in the relative amount of type III collagen but no mutant alpha1(I) chain.

Adult↗
PubMed1996

A CD4-CDR3 peptide analog inhibits both primary and secondary autoreactive CD4+ T cell responses in experimental allergic encephalomyelitis.

A structure-based design approach was used to develop a cyclized peptide analog of the murine CD4-CDR3-like region as a potential inhibitor of autoimmune CD4+ T cells responsible for the pathogenesis of experimental allergic encephalomyelitis (EAE). Our results indicate that this peptide, referred to as rD-mPGPtide, is able to significantly inhibit the clinical and pathologic symptoms of EAE in the SJL mouse model when administered on day 12 of induction. The optimum effective dosage range for the peptide, injected i.v., was between 0.125 and 0.5 mg and dosages of as high as 5 mg had no observable toxic effects. Treated mice had normal levels of lymphocytes less than 2 wk later and exhibited normal in vitro primary responses to alloantigen and secondary responses to keyhole limpet hemocyanin Ag. The specificity of the rD-mPGPtide treatment for autoreactive T cells was demonstrated by inhibiting proteolipid protein (p139-151)-induced EAE and finding that the lymph node T cells from these mice had suppressed responses to this Ag, but normal responses to alloantigen or other nominal Ag. Importantly, rD-mPGPtide was found to be effective on secondary T cell responses in an EAE rechallenge situation and was able to establish conditions for long-term resistance to further Ag exposure. Analysis of the cytokine profile of responding T cells during late effector stages of disease revealed that the levels of IFN-gamma and IL-4 are significantly reduced in rD-mPGPtide-treated mice. These results strongly suggest that the administration of a CD4-CDR3 peptide analog is an effective therapeutic approach for the inhibition of the CD4+ T cell-mediated autoimmune response in EAE.

Amino Acid Sequence↗
PubMed1996

Inhibitory effect of a CD4-CDR3 peptide analog on graft-versus-host disease across a major histocompatibility complex-haploidentical barrier.

A structure-based designed peptide has been engineered to exhibit the same molecular surface as a portion of the CDR3-like region in domain 1 of the murine CD4 molecule. Earlier in vitro experiments indicated that this analog, known as rD-mPGPtide, inhibited T-cell proliferation in mixed lymphocyte reactions and blocked activation of both normal CD4+ T cells and T-cell lines after T-cell receptor triggering. In addition, rD-mPGPtide proved to be a potent inhibitor in vivo of CD4+ T-cell-mediated experimental allergic encephalomyelitis disease in the SJL mouse model. In this current report, we have evaluated the potential of rD-mPGPtide for suppressing the development of graft-versus-host disease (GVHD) in an irradiated major histocompatibility complex (MHC)-haploidentical murine bone marrow transplantation (BMT) model [(B6 x DBA/2)F1-->(B6 x CBA)F1 (950 cGy)]. Our results indicated that early administration of rD-mPGPtide was effective in the inhibition of alloreactive responses of the donor T cells against the host and thus delayed or prevented the onset of GVHD. The median survival time of animals treated with rD-mPGPtide was enhanced as much as four-fold with as little as a single dose of peptide at the time of transplant. Decreased alloreactivity was indicated by phenotypic and functional analysis of positively selected thoracic duct lymphocytes 4 days after transplant and by histopathological examination of skin and gastrointestinal tissue samples 4 weeks later. Therefore, the administration of a CD4-CDR3 peptide is an efficacious approach against the development of GVHD during allogeneic BMT.

Acute Disease↗
PubMed1996

Antisense oligodeoxynucleotides selectively suppress expression of the mutant alpha 2(I) collagen allele in type IV osteogenesis imperfecta fibroblasts. A molecular approach to therapeutics of dominant negative disorders.

We are investigating the use of antisense oligodeoxynucleotides to selectively suppress expression of the mutant type I collagen allele in osteogenesis imperfecta (OI). In this report, we target a human collagen mutation in its natural cellular context. We used cultured fibroblasts from a case of type IV OI, in which the mutant alpha 2(I) allele produces mRNA with exon 16 deleted due to a point mutation in the splice donor site. Lipid-mediated transfection was used to deliver antisense, sense and missense phosphorothioates targeted to both the abnormal mRNA exon 15/17 junction and the nuclear level point mutation. Significant suppression of the mutant protein chain and mRNA was achieved with antisense oligonucleotide to both mRNA and nuclear levels. Mutant protein was suppressed to 44-47% and mutant alpha 2(I) mRNA to 37-43% of their levels in control cells, indicating decreased mRNA as the basis for suppression. Selectivity of mutant allele suppression was better with an mRNA target: suppression was sequence specific and normal mRNA was expressed at 79% of its level in untreated cells. With a nuclear target, significant suppression of mutant mRNA occurred not only with antisense and sense, but also with missense oligonucleotide, which suppressed mutant mRNA to 60% of its level in untreated cells. We also investigated the time course of suppression of protein and mRNA in response to a 4 h transfection of antisense oligonucleotide. From 24-72 h after transfection, mutant protein was suppressed to approximately 50% of its untreated level and suppression of mutant message was significantly greater than that of normal message. The suppression achieved in these studies is insufficient for clinical intervention, but our results provide support for further development of antisense therapy as an approach to the treatment of dominant negative disorders.

Base Sequence↗
PubMed1996

Ribozymes: structure, function, and potential therapy for dominant genetic disorders.

Some dominant genetic disorders, viral processes and neoplastic disorders base their pathogenicity on the production of protein or proteins that negatively affect cellular metabolism or environment. Thus, the inhibition of the synthesis of those proteins should prevent the biological damage. A promising approach to decreasing the level of the abnormal protein(s) is represented by specific interference with gene expression at the level of mRNA. The specific suppression of the expression of an mRNA can be achieved by using ribozymes. Ribozymes are RNA molecules able to break and form covalent bonds within a nucleic acid molecule. These molecules, with even greater potential advantages than antisense oligodeoxynucleotides, are able to bind specifically and cleave an mRNA substrate. There are advantages to using ribozymes instead of antisense oligodeoxynucleotides. Ribozymes can inactivate the target RNA without relying on the host cell's machinery and they have the capacity to cleave more than one copy of the target RNA by dissociating from the cleavage products and binding to another target molecule. Most of the studies performed to date have described the use of ribozymes as therapeutic agents for viral and cancer diseases. However, some dominant genetic disorders may also benefit from this approach. This is the case for some connective tissue disorders such as osteogenesis imperfecta, Marfan syndrome and the craniosynostotic syndromes.

Genetic Diseases, Inborn↗
PubMed1995

A three-hour measurement to evaluate bone calcium turnover.

It is well established that short-term clearance of an intravenous calcium load in vivo reflects bone uptake. Using results from isotope-dilution experiments with 42Ca, a 3-h test has been developed to measure a quantity, gamma, related to bone accretion. This test is proposed as a useful, clinically applicable measure of bone status. For early times, t, after a bolus of 42Ca, plasma tracer dilution was well approximated by t-gamma, where gamma is related to the fractional rate of loss of tracer, q, from blood into bone (1/q)(dq/dt) = -gamma/t). Gamma was evaluated from kinetic measurements on 91 normal female children, adolescents, and adult women in the age range 4-50 years. For t < or = 3 h, all clearance curves were well fit by a power function. Gamma was found to vary from 0.244 +/- 0.031 for adult premenopausal women (N = 22) to 0.392 +/- 0.056 for prepubertal children (N = 29). Using the Spearman rank-order correlation test, gamma was correlated with bone accretion measured from classic calcium kinetic studies with a correlation coefficient of 0.721, significant at p < 0.005. In those cases in which accretion and resorption remain tightly linked, gamma also provides information on the state of calcium loss from bone. Gamma was evaluated in 14 subjects with bone disease characterised by increased resorption (osteoporosis, Paget's disease) and in 27 subjects with decreased accretion (osteogenesis imperfecta, types I, III, IV; steroid-treated juvenile dermatomyositis). All subjects with Paget's disease and with osteoporosis showed increased gamma, consistent with high bone turnover. The osteoporotic patients furthermore exhibited gamma increasing monotonically by approximately 1% per year after age 55.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Neurologic profile in osteogenesis imperfecta.

The clinically important neurlogic complications in 76 patients with OI seen at the NIH included brainstem compression from basilar invagination, skull fracture, and seizure disorders. Neuroimaging studies demonstrated sulcal prominence and ventriculomegaly consistent with communicating hydrocephalus in 17 patients. Basilar invagination was found in 8 individuals, all with clinically severe OI, and caused brain-stem compression in 3 patients. Head circumference growth showed abnormal kinetics with percentile crossing after fontanelle closure in 13 patients and absolute macrocephaly was present in 11 patients. Neurologic evaluation should be part of a team approach in the management of patients with severe OI types. Continued study of the underlying pathophysiology of neurologic features in OI is warranted.

Brain Diseases↗
PubMed1995

Endocrine aspects of growth deficiency in OI.

Growth deficiency is the most common secondary feature of osteogenesis imperfecta. It is unrelated to fracture history and appears to be due to the growth failure of the defective bony matrix. There are characteristic growth curves for different types of OI. We have been investigating the endocrine features of this disorder, in which the skeletal target tissue synthesizes defective matrix. We review the results of our evaluation of the growth hormone axis in 28 children with short stature and OI and of our pilot study to stimulate OI bone to increased growth rates. Our current focus is on the effect of growth hormone treatment on linear growth, bony mineral and bony matrix in OI.

Adolescent↗