Biomedical subjects

M K Wirtz

Publications and source records attributed to M K Wirtz.

At least 19 recordsLinked to original sources

PubMed2004

Refining the primary open-angle glaucoma GLC1C region on chromosome 3 by haplotype analysis.

The GLC1C locus for primary open-angle glaucoma (POAG) is inherited as an autosomal dominant trait. This region on chromosome 3 is 11 cM long. DNA samples from members of a Greek and an American GLC1C family were obtained to determine whether additional typing of microsatellite markers in family members might narrow the region. GLC1C family members were evaluated clinically for POAG on the basis of open angles, intraocular pressures, cupping of discs, and visual fields. DNA samples from the Greek and Oregon GLC1C families were used to further refine the GLC1C region using microsatellite markers. A total of 22 affected members were identified in the two families. Common alleles for D3S3637 and D3S3612 were present in the disease haplotype from both families, suggesting that they may have a common founder. A newly diagnosed patient in the American family had a recombination in the distal portion of the GLC1C haplotype. This recombination narrows the GLC1C region from 11 to 4 cM.

Chromosome Mapping↗

PubMed2003

In frame fibrillin-1 gene deletion in autosomal dominant Weill-Marchesani syndrome.

Weill-Marchesani syndrome (WMS) is a connective tissue disorder characterised by short stature, brachydactyly, joint stiffness, and characteristic eye anomalies including microspherophakia, ectopia of the lenses, severe myopia, and glaucoma. Both autosomal recessive (AR) and autosomal dominant (AD) modes of inheritance have been described and a gene for AR WMS has recently been mapped to chromosome 19p13.3-p13.2. Here, we report on the exclusion of chromosome 19p13.3-p13.2 in a large AD WMS family and show that, despite clinical homogeneity, AD and AR WMS are genetically heterogeneous entities. Because two AD WMS families were consistent with linkage to chromosome 15q21.1, the fibrillin-1 gene was sequenced and a 24 nt in frame deletion within a latent transforming growth factor-beta1 binding protein (LTBP) motif of the fibrillin-1 gene was found in a AD WMS family (exon 41, 5074_5097del). This in frame deletion cosegregated with the disease and was not found in 186 controls. This study strongly suggests that AD WMS and Marfan syndrome are allelic conditions at the fibrillin-1 locus and adds to the remarkable clinical heterogeneity of type I fibrillinopathies.

Abnormalities, Multiple↗

PubMed2001

Genetic linkage of autosomal dominant primary open angle glaucoma to chromosome 3q in a Greek pedigree.

A locus for juvenile onset open angle glaucoma (OAG) has been assigned to chromosome 1q in families with autosomal dominant inheritance (GLC1A), due to mutations in the TIGR/MYOC gene. For adult onset OAG, called primary open angle glaucoma or POAG, five loci have so far been mapped to different chromosomes (GLC1B-GLC1F). Except for the GLC1B locus, the other POAG loci have so far been reported only in single large pedigrees. We studied a large family identified in Epirus, Greece, segregating POAG in an autosomal dominant fashion. Clinical findings included increased cup to disc ratio (mean 0.7), characteristic glaucomatous changes in the visual field, and intraocular pressure before treatment more than 21 mmHg (mean 31 mmHg), with age at diagnosis 33 years and older. Linkage analysis was performed between the disease phenotype and microsatellite DNA polymorphisms. Linkage was established with a group of DNA markers located on chromosome 3q, where the GLC1C locus has previously been described in one large Oregon pedigree. A maximal multipoint lod score of 3.88 was obtained at marker D3S1763 (penetrance 80%). This represents the second POAG family linked to the GLC1C locus on chromosome 3q, and haplotype analysis in the two families suggests an independent origin of the genetic defect.

Adult↗

PubMed2000

Identification and expression of a novel type I procollagen C-proteinase enhancer protein gene from the glaucoma candidate region on 3q21-q24.

A novel human Type I procollagen C-proteinase enhancer protein-like gene, PCOLCE2, was identified by sequencing an EST in the primary open-angle glaucoma (POAG) region on 3q21. The total cDNA encoded a 415-amino-acid protein that has 43% identity to the Type I procollagen C-proteinase enhancer protein (PCOLCE1). PCOLCE2 contains two CUB domains, which are thought to be involved in protein-protein interactions, and an NTR module. PCOLCE2 message is expressed in the trabecular meshwork, lungs, heart, brain, liver, skeletal muscle, kidney, pancreas, and placenta as a 2-kb message. PCOLCE2, a 52-kDa protein, is expressed in the trabecular meshwork. A novel gene, PCOLCE2, has been identified and characterized. Based upon its homology with collagen-binding proteins, its expression in the trabecular meshwork, and its chromosome location, PCOLCE2 is a candidate gene for GLC1C. However, no coding sequence mutations were detected in PCOLCE2 in a POAG patient from the GLC1C family.

Animals↗

PubMed1999

GLC1F, a new primary open-angle glaucoma locus, maps to 7q35-q36.

BACKGROUND: A large family with adult-onset primary open-angle glaucoma (POAG) was identified. OBJECTIVE: To initiate a genome-wide scan to map the POAG locus in this family. METHODS: Blood samples or buccal swabs were obtained from 25 members of a large family with POAG after informed consent was obtained. Members and their spouses were evaluated clinically for POAG on the basis of intraocular pressures, cupping of discs, and visual fields. DNA samples were used for a genome-wide screen using microsatellite markers. RESULTS: Ten affected family members in 4 generations showed evidence of POAG including intraocular pressures of 22 mm Hg or more, and/or optic cup-disc ratios of 0.6 or more, and/or visual field defects consistent with glaucomatous damage. Primary open-angle glaucoma segregated as an autosomal dominant trait, with the disease locus mapping to 7q35-q36 between markers D7S2442 and D7S483 with a multipoint lod score of 4.06. CONCLUSION: A sixth gene for POAG (GLC1F) has been mapped to 7q35-q36 in a family with at least 4 generations affected. CLINICAL RELEVANCE: The mapping of this locus further confirms that primary open-angle glaucoma is a heterogeneous group of diseases with at least 6 different loci resulting in a similar phenotype. The eventual ability to classify which major POAG gene an affected person carries could have ramifications for selecting the most effective treatment regimen for that person.

Adult↗

PubMed1998

Age-related macular degeneration. Clinical features in a large family and linkage to chromosome 1q.

OBJECTIVES: To identify the chromosomal location of a disease-causing gene and to describe the clinical characteristics of a large family with age-related macular degeneration (ARMD). METHODS: An ARMD pedigree was identified, and the disease state of family members was documented by stereoscopic fundus photography and was classified using a modified version of the Wisconsin Age-Related Maculopathy Grading System. A genome-wide screen at approximately 6-centimorgan spacing using a DNA-pooling strategy combined with shared-segment analysis was used to identify likely chromosomal regions. The entire family was then screened at each likely locus, and 1 positive locus was refined by screening with markers at an average density of 0.5 centimorgan and subjected to parametric linkage analysis. RESULTS: In the 10 affected family members, ARMD was manifest by the presence of large, soft, confluent drusen accompanied by varying degrees of retinal pigment epithelial degeneration and/or geographic atrophy. Age-related macular degeneration segregated as an autosomal-dominant trait, with the disease locus mapping to chromosome 1q25-q31 between markers D1S466 and D1S413, with a multipoint lod score of 3.00. CONCLUSION: Age-related macular degeneration localized to chromosome 1q25-q31 (gene symbol, ARMD1) as a dominant trait in a large family with a predominantly dry phenotype. CLINICAL RELEVANCE: Identification of ARMD genes will facilitate early diagnosis and aid in understanding the molecular pathophysiological mechanisms of ARMD. This knowledge will contribute to the development of preventive and improved treatment strategies.

Aged↗

PubMed1998

Malattia leventinese: refinement of the genetic locus and phenotypic variability in autosomal dominant macular drusen.

PURPOSE: To study the phenotypic variability in patients inheriting the disease gene for malattia leventinese (dominant macular drusen) and refine the localization of the gene. METHODS: A family with dominant radial drusen was ascertained and studied with clinical examination and DNA linkage analysis. Inheritance of the disease gene was determined by DNA analysis and used to document the variability in phenotypic expression. RESULTS: Fifty family members were studied with fundus photography and genotyping. Linkage analysis showed that the disease in this family was linked to chromosome 2p16-21 with a maximum lod score of 3.72 at D2S2153. An affected patient with obligate recombinations allowed refinement of the disease interval to a 6.2-cM region between D2S2227 and D2S378. The phenotype of older affected patients varied from severe geographic atrophy or subretinal fibrosis to a single druse adjacent to the optic disk. Small and medium-sized, nonradial, and soft macular drusen seen in four older individuals in the family were not specifically associated with the disease haplotype. CONCLUSIONS: Refinement of the localization of the gene for malattia leventinese will facilitate its positional cloning. Genotypic documentation of the variable expression of the disease shows that a single, large, subretinal druse adjacent to the optic disk is consistent with inheritance of the disease gene. Soft macular drusen in low abundance were not specifically associated with inheritance of the disease gene. These results will facilitate the genetic counseling of patients with malattia leventinese. It is unknown what proportion of age-related macular degeneration arises from mutations in disease genes for dominant drusen.

Adolescent↗

PubMed1998

Prospects for genetic intervention in primary open-angle glaucoma.

Recent advances in glaucoma genetics hold potential for dramatically changing the clinical care of glaucoma patients. To date, 5 primary open-angle glaucoma genes and 2 congenital glaucoma genes have been mapped. As more glaucoma genes are identified, earlier diagnosis for glaucoma should become more readily available. Progress in molecular genetics holds considerable promise for both current and future therapy of glaucoma. Glaucoma classification will be tailored to each individual based upon that person's family history, i.e. family glaucoma genotype. In the future, the optimum treatment for a specific glaucoma patient might rely on the knowledge of the phenotype of that person's causal gene, without having to resort to 'trial and error'. At this time, glaucoma treatment is restricted to lowering intraocular pressure. In the near future, with the knowledge of the pathophysiology caused by the defective glaucoma gene, more traditional drug treatments may be used to bypass the gene defect. Ultimately, gene therapy would replace the mutant gene with a normal one before visual loss has occurred as has been done with a model for retinitis pigmentosa, the retinal degeneration mouse.

Forecasting↗

PubMed1998

Insulin-like growth factor binding protein-5 expression by human trabecular meshwork.

PURPOSE: The authors found transcript expression for insulin-like growth factor binding protein-5 (IGFBP-5) while screening for uniquely expressed trabecular meshwork (TM) mRNAs. Because the insulin-like growth factor (IGF) autocrine-paracrine system may provide an important signaling mechanism between TM cells and the outflow pathway, the expression of IGFBP-5 and IGF-I receptor in the TM was characterized. METHODS: Poly(A+) RNA was isolated from cell cultures of human TM, ciliary body, retinal pigment epithelium, and skin fibroblasts and subjected to reverse transcription-polymerase chain reaction (RT-PCR) differential display analysis. A unique 980-bp band present in the TM was cloned and sequenced. Additional PCR and Northern analyses were used to define trabecular IGFBP-5 expression. Western immunoblots and confocal immunohistochemistry were used to evaluate the protein expression patterns of IGFBP-5 and the IGF-I receptor. IGF-I and IGF-II were added to trabecular cells in culture, and matrix metalloproteinase production was evaluated. RESULTS: A unique differential display band was identified in the TM. Sequencing of this band identified it as the 3'-untranslated region of IGFBP-5. RT-PCR, using a variety of specific primers for IGFBP-5, Northern analysis, Western immunoblots, and immunohistochemical analysis, confirmed that IGFBP-5 was expressed in the TM. However, IGFBP-5 was also present at low levels in the ciliary body and skin fibroblasts by Northern and Western analysis, in contrast with the differential display findings. In addition, the IGF-I receptor was expressed by the TM and showed cell-surface staining by immunohistochemistry. Trabecular IGFBP-5 was distributed throughout the meshwork in the extracellular matrix and the cells with more staining in the juxtacanalicular region than in the uveal meshwork. IGF-I, but not IGF-II, modestly increased trabecular stromelysin and gelatinase B but not collagenase, gelatinase A, or tissue inhibitor of metalloproteinases 1 or 2. CONCLUSIONS: IGFBP-5 and IGF-I receptor were expressed at significant levels by TM cells and may serve an important role in trabecular function.

Blotting, Northern↗

PubMed1997

Discrete expression and distribution pattern of TIMP-3 in the human retina and choroid.

PURPOSE: Extracellular matrix homeostasis is dependent in part upon a family of matrix metalloproteinases and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Recently, gene defects in TIMP-3 have been identified in the affected individuals of several families with Sorsby's fundus dystrophy (SFD). Very little information is available regarding TIMP-3 function or even its existence in the retina or choroid. METHODS: We used reverse transcription-polymerase chain reaction and Northern analysis to evaluate the expression of TIMP mRNA and Western immunoblots to evaluate TIMP protein produced by select cells of the human retina and choroid. We also used these methods and immunohistochemistry to localize the TIMPs in the retina and choroid. RESULTS: TIMP-3 transcripts are found in cultured human retinal pigment epithelium (RPE), choroidal microcapillary endothelium and pericytes. RPE cells also express and secrete TIMP-3 protein, which is localized to the extracellular matrix and is not found in culture medium; TIMP-1 and -2 are found almost exclusively in the medium. Immunohistochemistry of human retina/choroid sections shows pronounced TIMP-3 immunostaining in Bruch's membrane, particularly near the surface of the RPE and endothelial cells, presumably in their basement membranes, with minimal staining in other portions of the retina. Immunostaining for TIMP-1 is absent and for TIMP-2 is much less prevalent, but detectable in Bruch's membrane. CONCLUSIONS: TIMP-1, -2 and -3 exhibit distinctive expression patterns in the retina and choroid. This distribution and expression pattern partially explains why TIMP-3 mutations result in SFD, rather than other retinal pathologies, such as those associated with proliferative diabetic retinopathy.

Cells, Cultured↗

PubMed1997

Proteoglycan expression by human trabecular meshworks.

PURPOSE: Proteoglycans may serve important roles in trabecular meshwork structure or function. Detailed molecular characterization and identification of specific trabecular proteoglycan core proteins has been limited. METHODS: Radiolabeled proteoglycans were extracted from cultured human trabecular meshworks and subjected to ion exchange and molecular sieve chromatography. Peaks were subjected to glycosaminoglycan content analysis. Reverse transcription with polymerase chain reaction was used to identify trabecular mRNAs of several common proteoglycan core proteins. Western immunoblots of trabecular extracts were also utilized to identify these proteoglycan core proteins. RESULTS: The proteoglycans elute from ion exchange columns at high salt and molecular sieve column profiles, and they exhibit broad peaks typical of the proteoglycan microheterogeneity seen in other tissues. The four common glycosaminoglycan side-chains were identified on these proteoglycans. Trabecular cells in organ or cell culture contain mRNAs coding for decorin, biglycan, versican, perlecan and a basement membrane glycoprotein, SPARC. Syndecan-1 transcripts were present at very low levels, while aggrecan transcripts were not detectable. Decorin, biglycan, versican and perlecan core proteins were also identified by immunoblots of trabecular cell extracts. CONCLUSIONS: Several common proteoglycans are expressed by trabecular cells in organ explant or cell culture. Their general characteristics are not unlike those found in other tissues. These proteoglycans may serve important functions in the trabecular outflow pathway.

Aggrecans↗

PubMed1997

Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q.

Glaucoma is the third-leading cause of blindness in the world, affecting >13.5 million people. Adult-onset primary open-angle glaucoma (POAG) is the most common form of glaucoma in the United States. We present a family in which adult-onset POAG is inherited as an autosomal dominant trait. Twelve affected family members were identified from 44 at-risk individuals. The disease-causing gene was mapped to chromosome 3q21-24, with analysis of recombinant haplotypes suggesting a total inclusion region of 11.1 cM between markers D3S3637 and D3S1744. This is the first report of mapping of an adult-onset POAG gene to chromosome 3q, gene symbol GLC1C.

Adult↗

PubMed1996

Weill-Marchesani syndrome--possible linkage of the autosomal dominant form to 15q21.1.

Weill-Marchesani syndrome comprises short stature, brachydactyly, microspherophakia, glaucoma, and ectopia lentis is regarded as an autosomal recessive trait (McKusick 277600). We present two families each with affected individuals in 3 generations demonstrating autosomal dominant inheritance of Weill-Marchesani syndrome. Linkage analysis in these 2 families suggests a gene for Weill-Marchesani syndrome maps to 15q21.1. The dislocated lenses and connective tissue disorder in these families suggests that fibrillin-1 and microfibril-associated protein 1, which both map to 15q21.1, are candidate genes for Weill-Marchesani syndrome. Immunohistochemistry staining of skin sections from family 1 showed an apparent decrease in fibrillin staining compared to control individuals.

Abnormalities, Multiple↗

PubMed1993

A cysteine for glycine substitution at position 175 in an alpha 1 (I) chain of type I collagen produces a clinically heterogeneous form of osteogenesis imperfecta.

The molecular basis for Osteogenesis Imperfecta in a large kindred with a highly variable phenotype was identified by sequencing the mutant pro alpha 1 (I) protein, cDNA and genomic DNA from the proband. Fibroblasts from different affected individuals all synthesize both normal Type I procollagen molecules and abnormal Type I procollagen molecules in which one or both pro alpha 1 (I) chain(s) contain a cysteine residue within the triple helical domain. Protein studies of the proband localized the mutant cysteine residue to the alpha 1 (I) CB 8 peptide. We now report that cysteine has replaced glycine at triple helical residue 175 disrupting the invariant Gly-X-Y structural motif required for perfect triple helix formation. The consequences include post-translational overmodification, decreased thermal stability, and delayed secretion of mutant molecules. The highly variable phenotype in the present kindred cannot be explained solely on the basis of the cysteine for glycine substitution but will require further exploration.

Amino Acid Sequence↗

PubMed1992

Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies.

Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the N-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller C-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4 degrees C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.

Amino Acid Sequence↗

PubMed1991

Characterization of a COL1A1 splicing defect in a case of Ehlers-Danlos syndrome type VII: further evidence of molecular homogeneity.

A child affected by the type VII form of Ehlers-Danlos syndrome (EDS VII) was shown to have a heterozygous structural defect in the amino-terminus of pro-alpha 1(I) collagen. As a result, type I procollagen trimers containing defective subunits are not converted to mature collagen molecules. To identify the cause of the protein abnormality, specifically primed cDNAs and genomic DNA were PCR amplified and sequenced. This analysis disclosed that the protein structural defect is caused by a single base substitution (A for G) at position -1 of the splice donor site of intron 6 of the pro-alpha 1(I) collagen gene (COL1A1). The affected allele produces (a) transcripts lacking exon 6 sequences and (b), in lesser amount, normally spliced transcripts. Furthermore, the rate of exon 6 skipping is temperature dependent, for it appears to decrease substantially when the patient's fibroblasts are incubated at 31 degrees C. These findings are similar to those we previously reported for other unrelated EDS VII cases and, therefore, reemphasize the molecular homogeneity of this rare connective tissue disorder.

Adult↗

PubMed1990

In vivo and in vitro noncovalent association of excised alpha 1 (I) amino-terminal propeptides with mutant pN alpha 2(I) collagen chains in native mutant collagen in a case of Ehlers-Danlos syndrome, type VII.

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.

Amino Acid Sequence↗

PubMed1989

Temperature-dependent expression of a collagen splicing defect in the fibroblasts of a patient with Ehlers-Danlos syndrome type VII.

In this article we report the characterization of the molecular lesion in a patient with Ehlers-Danlos syndrome Type VII and provide evidence that a de novo substitution of the last nucleotide of exon 6 in one allele of the pro-alpha 2(I) collagen gene produces normally spliced mRNA and transcripts from which exon 6 sequences have been outspliced as well. Unexpectedly, the expression of the alternative splicing was found to be temperature-dependent, for missplicing in cellula is effectively abolished at 31 degrees C and gradually increases to 100% at 39 degrees C. In contrast, in a similar patient harboring a substitution in the obligatory GT dinucleotide of the 5' splice site of intron 6, complete outsplicing of exon 6 sequences was found at all temperatures.

Amino Acid Sequence↗