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Biomedical subjects

H C Dietz

Publications and source records attributed to H C Dietz.

At least 19 recordsLinked to original sources
PubMed2006

The molecular genetics of Marfan syndrome and related disorders.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

Activin Receptors, Type I↗
PubMed2001

Interaction between krit1 and icap1alpha infers perturbation of integrin beta1-mediated angiogenesis in the pathogenesis of cerebral cavernous malformation.

Cerebral cavernous malformation (CCM) is a common autosomal dominant disorder characterized by venous sinusoids that predispose to intracranial hemorrhage. CCM is genetically heterogeneous, with loci at 7q, 7p and 3q. Mutations in KRIT1 account for all cases linked to 7q (CCM1), but the pathogenesis of CCM is not understood. Krev Interaction Trapped 1 (krit1) was originally identified through its interaction with the Ras-family GTPase krev1/rap1a in a two-hybrid screen, inferring a role in GTPase signaling cascades. We demonstrated additional 5'-coding exons for krit1, extending the N-terminus by 207 amino acids compared to the previously reported protein. Remarkably, by two-hybrid analysis and co-immunoprecipitation, full-length krit1 fails to interact with krev1/rap1a but shows strong interaction with integrin cytoplasmic domain-associated protein-1 (icap1). Icap1 binds to a NPXY motif in the cytoplasmic domain of beta1 integrin and participates in beta1-mediated cell adhesion and migration. The novel N-terminus of krit1 contains a NPXY motif that it is required for icap1 interaction. Like beta1 integrin, krit1 interacts with the 200 amino acid isoform of icap1 (icap1alpha), but not a 150 amino acid form that results from alternative splicing (icap1beta). In a competition assay, induced expression of krit1 diminishes the interaction between icap1alpha and beta1 integrin. Taken together, these data suggest that beta1 integrin and krit1 compete for the same site on icap1alpha, perhaps constituting a regulatory mechanism. Loss-of-function KRIT1 mutations, as observed in CCM1, would shift the balance with predicted consequences for endothelial cell performance during integrin beta1-dependent angiogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Familial thoracic aortic aneurysms and dissections: genetic heterogeneity with a major locus mapping to 5q13-14.

BACKGROUND: Aneurysms and dissections affecting the ascending aorta are associated primarily with degeneration of the aortic media, called medial necrosis. Families identified with dominant inheritance of thoracic aortic aneurysms and dissections (TAA/dissections) indicate that single gene mutations can cause medial necrosis in the absence of an associated syndrome. METHODS AND RESULTS: Fifteen families were identified with multiple members with TAAs/dissections. DNA from affected members from 2 of the families was used for a genome-wide search for the location of the defective gene by use of random polymorphic markers. The data were analyzed by the affected-pedigree-member method of linkage analysis. This analysis revealed 3 chromosomal loci with multiple markers demonstrating evidence of linkage to the phenotype. Linkage analysis using further markers in these regions and DNA from 15 families confirmed linkage of some of the families to 5q13-14. Genetic heterogeneity for the condition was confirmed by a heterogeneity test. Data from 9 families with the highest conditional probability of being linked to 5q were used to calculate the pairwise and multipoint logarithm of the odds (LOD) scores, with a maximum LOD of 4.74, with no recombination being obtained for the marker D5S2029. In 6 families, the phenotype was not linked to the 5q locus. CONCLUSIONS: A major locus for familial TAAs and dissections maps to 5q13-14, with the majority (9 of 15) of the families identified demonstrating evidence of linkage to this locus. The condition is genetically heterogeneous, with 6 families not demonstrating evidence of linkage to any loci previously associated with aneurysm formation.

Aortic Dissection↗
PubMed2001

Characterization of microsatellite markers flanking FBN1: utility in the diagnostic evaluation for Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue with marked interfamilial and intrafamilial variation in phenotype. The primary defect in affected patients resides in the gene for fibrillin-1 (FBN1) on 15q21. Linkage analysis has shown no locus heterogeneity in the classic phenotype, although substantial allelic heterogeneity exists. Recently it has been shown that the size of the gene is approximately 200 kb. These and other factors have precluded routine mutation screening for presymptomatic and prenatal diagnosis. Previously we described four intragenic microsatellite polymorphisms that can be used for haplotype segregation analysis. The utility of this approach is limited because the markers do not fully span the gene and show incomplete informativeness, with 16% homozygosity for the most common haplotype. We have now identified and localized highly polymorphic microsatellite markers that fall within 1 Mb of FBN1. Complete haplotype heterozygosity was observed in a population of 50 unrelated control individuals when the flanking markers and existing intragenic polymorphisms were used in combination. We demonstrate the utility of haplotype segregation analysis in the presymptomatic diagnosis and counseling of families showing atypical or equivocal manifestations of MFS.

Adult↗
PubMed2001

Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome.

Marfan syndrome is associated with early death due to aortic aneurysm. The condition is caused by mutations in the gene (FBN1) encoding fibrillin-1, a major constituent of extracellular microfibrils. Prior observations suggested that a deficiency of microfibrils causes failure of elastic fiber assembly during late fetal development. Mice homozygous for a targeted hypomorphic allele (mgR) of Fbn1 revealed a predictable sequence of abnormalities in the vessel wall including elastic fiber calcification, excessive deposition of matrix elements, elastolysis, and intimal hyperplasia. Here we describe previously unrecognized concordant findings in elastic vessels from patients with Marfan syndrome. Furthermore, ultrastructural analysis of mgR mice revealed cellular events that initiate destructive changes. The first detectable abnormality was an unusually smooth surface of elastic laminae, manifesting the loss of cell attachments that are normally mediated by fibrillin-1. Adjacent cells adopted alteration in their expression profile accompanied by morphological changes but retained expression of vascular smooth muscle cell markers. The abnormal synthetic repertoire of these morphologically abnormal smooth muscle cells in early vascular lesions included elastin, among other matrix elements, and matrix metalloproteinase 9, a known mediator of elastolysis. Ultimately, cell processes associated with zones of elastic fiber thinning and fragmentation. These data suggest that the loss of cell attachments signals a nonproductive program to synthesize and remodel an elastic matrix. This refined understanding of the pathogenesis of vascular disease in Marfan syndrome will facilitate the development of therapeutic strategies.

Actins↗
PubMed2001

Familial Tetralogy of Fallot caused by mutation in the jagged1 gene.

Tetralogy of Fallot (ToF) is the most common form of complex congenital heart disease, occurring in approximately 1 in 3000 live births. Evaluation of candidate loci in a large kindred segregating autosomal dominant ToF with reduced penetrance culminated in identification of a missense mutation (G274D) in JAG1, the gene encoding jagged1, a Notch ligand expressed in the developing right heart. Nine of eleven mutation carriers manifested cardiac disease, including classic ToF, ventricular septal defect with aortic dextroposition and isolated peripheral pulmonic stenosis (PPS). All forms of ToF were represented, including variants with pulmonic stenosis, pulmonic atresia and absent pulmonary valve. No individual within this family met diagnostic criteria for any previously described clinical syndrome, including Alagille syndrome (AGS), caused by haploinsufficiency for jagged1. All mutation carriers had characteristic but variable facial features, including long, narrow and upslanting palpebral fissures, prominent nasal bridge, square dental arch and broad, prominent chin. This appearance was distinct from that of unaffected family members and typical AGS patients. The glycine corresponding to position 274 is highly conserved in other epidermal growth factor-like domains of jagged1 and in those of other proteins. Its substitution in other proteins has been associated with mild or atypical variants of disease. These data support either a relative loss-of-function or a gain-of-function pathogenetic mechanism in this family and suggest that JAG1 mutations may contribute significantly to common variants of right heart obstructive disease.

Amino Acid Sequence↗
PubMed2001

Rent1, a trans-effector of nonsense-mediated mRNA decay, is essential for mammalian embryonic viability.

The ability to detect and degrade transcripts that lack full coding potential is ubiquitous but non-essential in lower eukaryotes, leaving in question the evolutionary basis for complete maintenance of this function. One hypothesis holds that nonsense-mediated RNA decay (NMD) protects the organism by preventing the translation of truncated peptides with dominant negative or deleterious gain-of-function potential. All organisms studied to date that are competent for NMD express a structural homolog of Saccharomyces cerevisiae Upf1p. We have now explored the consequences of loss of NMD function in vertebrates through targeted disruption of the Rent1 gene in murine embryonic stem cells which encodes a mammalian ortholog of Upf1p. Mice heterozygous for the targeted allele showed no apparent phenotypic abnormalities but homozygosity was never observed, demonstrating that Rent1 is essential for embryonic viability. Homozygous targeted embryos show complete loss of NMD and are viable in the pre-implantation period, but resorb shortly after implantation. Furthermore, Rent1(-/-) blastocysts isolated at 3.5 days post-coitum undergo apoptosis in culture following a brief phase of cellular expansion. These data suggest that NMD is essential for mammalian cellular viability and support a critical role for the pathway in the regulated expression of selected physiologic transcripts.

Animals↗
PubMed2001

A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition.

Premature termination codons (PTCs) have been shown to initiate degradation of mutant transcripts through the nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway. We report a strategy, termed gene identification by NMD inhibition (GINI), to identify genes harboring nonsense codons that underlie human diseases. In this strategy, the NMD pathway is pharmacologically inhibited in cultured patient cells, resulting in stabilization of nonsense transcripts. To distinguish stabilized nonsense transcripts from background transcripts upregulated by drug treatment, drug-induced expression changes are measured in control and disease cell lines with complementary DNA (cDNA) microarrays. Transcripts are ranked by a nonsense enrichment index (NEI), which relates expression changes for a given transcript in NMD-inhibited control and patient cell lines. The most promising candidates can be selected using information such as map location or biological function; however, an important advantage of the GINI strategy is that a priori information is not essential for disease gene identification. GINI was tested on colon cancer and Sandhoff disease cell lines, which contained previously characterized nonsense mutations in the MutL homolog 1 (MLH1) and hexosaminidase B (HEXB) genes, respectively. A list of genes was produced in which the MLH1 and HEXB genes were among the top 1% of candidates, thus validating the strategy.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Cloning of the murine Krit1 cDNA reveals novel mammalian 5' coding exons.

Human KRIT1 (Krev interaction trapped 1), a defective gene product in cerebral cavernous malformation, was cloned from a HeLa cell cDNA library by virtue of its interaction with Krev/rap1A, a small ras-family GTPase. We have now characterized the full-length cDNA for the murine orthologue that encodes a predicted protein of 736 amino acids, 207 amino acids longer than the previously reported human protein. 5' Rapid amplification of cDNA ends analysis of mouse mRNA demonstrated a single transcriptional start site. The putative initiator codon was found within a context that conformed well to the Kozak consensus sequence and was preceded by an in-frame termination codon. BLAST analysis revealed that conceptual translation of a fragment of human genomic DNA upstream of the 5' end of the reported KRIT1 coding sequence predicts extension of the human open reading frame by 207 codons with 95% amino acid identity between the novel putative human and murine amino termini. This block of coding sequence was divided among four exons that are flanked by consensus splice site sequences. The extreme evolutionary conservation of this region, including a putative nuclear localization signal, indicates functional importance. These data have immediate relevance to mutation screening efforts in cerebral cavernous malformation and may contribute to our understanding of the normal biology of KRIT1 and the pathogenesis of this disorder.

Amino Acid Sequence↗
PubMed2000

Enzymatic mutation detection (EMD) of novel mutations (R565X and R1523X) in the FBN1 gene of patients with Marfan syndrome using T4 endonuclease VII.

The Enzymatic Mutation Detection (EMDtrade mark) method is a streamlined and improved version of the original Enzymatic Cleavage of Mismatch (EMC) method. EMD is a fully homogeneous, rapid four step procedure that allows for detection and localization of mismatched or unmatched nucleotides within heteroduplex DNA. To test the utility of EMD for use in the screening of large and complex genes, the fibrillin 1 (FBN1) gene was scanned in a cohort of six patients diagnosed with connective tissue disorders. Four of the six patients were diagnosed with classic Marfan syndrome (MFS). The results were compared with a previous MDEtrade mark scanning of the same patient cohort. Two causative mutations, R565X and R1523X, were detected by EMD that were not detected by MDE. In both cases, the mutation resulted in premature termination of translation. In addition, several polymorphisms were detected by the enzymatic approach that failed detection by heteroduplex analysis. We propose that the EMD method is a sensitive and rapid approach to mutation detection in large genes such as FBN1.

Adult↗
PubMed2000

Mouse models of genetic diseases resulting from mutations in elastic fiber proteins.

The inability to study appropriate human tissues at various stages of development has precluded the elaboration of a thorough understanding of the pathogenic mechanisms leading to diseases linked to mutations in genes for elastic fiber proteins. Recently, new insights have been gained by studying mice harboring targeted mutations in the genes that encode fibrillin-1 and elastin. These genes have been linked to Marfan syndrome (MFS) and supravalvular aortic stenosis (SVAS), respectively. For fibrillin-1, mouse models have revealed that phenotype is determined by the degree of functional impairment. The haploinsufficiency state or the expression of low levels of a product with dominant-negative potential from one allele is associated with mild phenotypes with a predominance of skeletal features. Exuberant expression of a dominant-negative-acting protein leads to the more severe MFS phenotype. Mice harboring targeted deletion of the elastin gene (ELN) show many of the features of SVAS in humans, including abnormalities in the vascular wall and altered hemodynamics associated with changes in wall compliance. The genetically altered mice suggest that SVAS is predominantly a disease of haploinsufficiency. These studies have underscored the prominent role of the elastic matrix in the morphogenesis and homeostasis of the vessel wall.

Animals↗
PubMed2000

Mutations in KRIT1 in familial cerebral cavernous malformations.

OBJECTIVES: The recognition of six unrelated Hispanic-American families in which cerebral cavernous malformations (CCM) segregated as an autosomal dominant trait established a genetic basis for this disease. Linkage analysis subsequently identified locus heterogeneity with disease genes for CCM at chromosomal regions 7q, 7p, and 3q. Recently, mutations in KRIT1, a gene on 7q at the CCM1 locus, were identified in French and Hispanic-American families with CCM. This study confirms the identity the KRIT1 founder mutation in Hispanic-Americans and reports a novel KRIT1 mutation in a Caucasian family. METHODS: Oligonucleotide primers were designed to allow amplification of genomic DNA sequences from four Hispanic-American families and five non-Hispanic families for all 12 exons of the KRIT1 gene using the polymerase chain reaction (PCR). The amplified DNA was then screened using single strand conformation polymorphism analysis (SSCP) and sequencing. The expression pattern of KRIT1 was analyzed by Northern blotting. RESULTS: Analysis of the KRIT1 gene revealed a point mutation in exon 6 that predicts the substitution of a premature termination codon for glutamine at codon 248 in all four Hispanic-American families, confirming previous findings. SSCP analysis and sequencing revealed an 11 base pair duplication in exon 7 leading to a premature termination codon in one Caucasian family. Northern analysis demonstrated widespread expression of this gene, however, the highest level of expression was in the brain. CONCLUSION: The common KRIT1 mutation causing the majority of CCM in Hispanic-Americans has been identified and independently confirmed, allowing efficient presymptomatic molecular diagnosis. In keeping with prior results, both newly identified mutations create a premature termination codon and are predicted to initiate degradation of the mutant mRNA through the nonsense-mediated mRNA decay pathway. These data strongly suggest loss of function as the relevant patho-genetic mechanism.

Brain Neoplasms↗
PubMed2000

Novel Upf2p orthologues suggest a functional link between translation initiation and nonsense surveillance complexes.

Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae, three trans-acting factors (Upf1p to Upf3p) are required for NMD. Orthologues of Upf1p have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution. In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of Upf2p homologues in Schizosaccharomyces pombe and humans (rent2). Disruption of S. pombe UPF2 established that this gene is required for NMD in fission yeast. rent2 was demonstrated to interact directly with rent1, a known trans-effector of NMD in mammalian cells. Additionally, fragments of rent2 were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment. Finally, novel functional domains of Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and other translational regulatory proteins were identified. Directed mutations within these so-called eIF4G homology (4GH) domains were sufficient to abolish the function of S. pombe Upf2p. Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between rent2 and human eIF4AI and Sui1, both components of the translation initiation complex. Based on these findings, a novel model in which Upf2p and rent2 effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with eIF4G-binding partners is proposed.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1.

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory-fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.

Aortic Dissection↗
PubMed1999

Revised genomic organization of FBN1 and significance for regulated gene expression.

FBN1 encodes fibrillin-1, an extracellular matrix protein that is defective in Marfan syndrome. This gene is divided into 65 exons and was previously reported to be approximately 110 kb in length. The existence of 3 exons upstream of the exon containing the putative initiating methionine left open the possibility of alternative fibrillin-1 isoforms that vary at their N-termini. Detailed examination of YACs containing human FBN1 reveal that the gene is 200 kb, almost twice as large as previously thought. Characterization of the porcine FBN1 cDNA and 5' flanking sequence demonstrates extreme conservation between the pig and the human predicted proteins and argues against the possibility of alternative amino-terminal coding sequence. These data further our understanding of the regulatory requirements for gene expression and establish a framework for recombinant expression of fibrillin-1.

Amino Acid Sequence↗
PubMed1999

Nonsense-mediated mRNA decay in health and disease.

All eukaryotes possess the ability to detect and degrade transcripts harboring premature signals for the termination of translation. Despite the ubiquitous nature of nonsense-mediated mRNA decay (NMD) and its demonstrated role in the modulation of phenotypes resulting from selected nonsense alleles, very little is known regarding its basic mechanism or the selective pressure for complete evolutionary conservation of this function. This review will present the current models of NMD that have been generated during the study of model organisms and mammalian cells. The physiological burden of nonsense transcripts and the emerging view that NMD plays a broad and critical role in the regulation of gene expression will also be discussed. Such issues are relevant to the proposal that pharmacological manipulation of NMD will find therapeutic application.

Adaptor Proteins, Signal Transducing↗