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Biomedical subjects

Gordon K Klintworth

Publications and source records attributed to Gordon K Klintworth.

At least 19 recordsLinked to original sources
PubMed2006

Macular corneal dystrophy types I and II are caused by distinct mutations in the CHST6 gene in Iceland.

PURPOSE: To identify CHST6 mutations in five additional Icelandic cases of macular corneal dystrophy (MCD) type I and in four families with MCD type II from Iceland. METHODS: Genomic DNA was extracted from blood leukocytes of patients with MCD, their healthy family members, and from control individuals. CHST6 mutations were determined by PCR-sequencing. Immunophenotypes of MCD were determined by measuring antigenic keratan sulfate (AgKS) levels in serum and by an immunohistochemical study on corneal tissue. RESULTS: Five additional cases of MCD type I and four families with MCD type II from Iceland were studied. A homozygous p.A128V mutation in the coding region of the CHST6 gene was identified in four of the five MCD type I cases. The other person with MCD type I was a compound heterozygote for p.A128V and a frameshift p.V6fs resulting from a 10-base pair insertion (c.15_16insATGCTGTGCG). Four of five individuals with MCD type II were compound heterozygotes for p.A128V and p.V329L, thus sharing the same p.A128V mutation as MCD type I. One patient with MCD type II was homozygous for p.V329L. The p.V329L mutation was only found in MCD type II patients. An analysis of the upstream region of CHST6 disclosed no upstream deletion or replacements in Icelandic patients with MCD type II. CONCLUSIONS: The findings fit the haplotype analysis that we reported previously in Icelandic MCD families and indicate that different mutations in CHST6 cause MCD type I and type II in Iceland.

Alanine↗
PubMed2006

The human cornea proteome: bioinformatic analyses indicate import of plasma proteins into the cornea.

Increased biochemical knowledge of normal and diseased corneas is essential for the understanding of corneal homeostasis and pathophysiology. In a recent study, we characterized the proteome of the normal human cornea and identified 141 distinct proteins. This dataset represents the most comprehensive protein study of the cornea to date and provides a useful reference for further studies of normal and diseased human corneas. The list of identified proteins is available at the Cornea Protein Database. In the present paper, we review the utilized procedures for extraction and fractionation of corneal proteins and discuss the potential roles of the identified proteins in relation to homeostasis, diseases, and wound-healing of the cornea. In addition, we compare the list of identified proteins with high quality gene expression libraries (cDNA libraries) and Serial Analysis of Gene Expression (SAGE) data. Of the 141 proteins, 86 (61%) were recognized in cDNA libraries from the corneas of dogs and rabbits, or humans with keratoconus, and 98 (69.5%) were recognized in SAGE data of mouse and human corneas. However, the percentages of identified genes in each of the protein functional groups differed markedly. Thus, exceptionally few of the traditional blood/plasma proteins and immune defense proteins that were identified in the human cornea were recognized in the gene expression libraries of the cornea. This observation strongly indicates that these abundant corneal proteins are not expressed in the cornea but originate from the surrounding pericorneal tissue.

Animals↗
PubMed2006

CHST6 mutations in North American subjects with macular corneal dystrophy: a comprehensive molecular genetic review.

PURPOSE: To evaluate mutations in the carbohydrate sulfotransferase-6 (CHST6) gene in American subjects with macular corneal dystrophy (MCD). METHODS: We analyzed CHST6 in 57 patients from 31 families with MCD from the United States, 57 carriers (parents or children), and 27 unaffected blood relatives of affected subjects. We compared the observed nucleotide sequences with those found by numerous investigators in other populations with MCD and in controls. RESULTS: In 24 families, the corneal disorder could be explained by mutations in the coding region of CHST6 or in the region upstream of this gene in both the maternal and paternal chromosome. In most instances of MCD a homozygous or heterozygous missense mutation in exon 3 of CHST6 was found. Six cases resulted from a deletion upstream of CHST6. CONCLUSIONS: Nucleotide changes within the coding region of CHST6 are predicted to alter the encoded protein significantly within evolutionary conserved parts of the encoded sulfotransferase. Our findings support the hypothesis that CHST6 mutations are cardinal to the pathogenesis of MCD. Moreover, the observation that some cases of MCD cannot be explained by mutations in CHST6 suggests that MCD may result from other subtle changes in CHST6 or from genetic heterogeneity.

5' Untranslated Regions↗
PubMed2006

Clinical study of Fuchs corneal endothelial dystrophy leading to penetrating keratoplasty: a 30-year experience.

OBJECTIVE: To review 30 years' clinical experience with Fuchs corneal endothelial dystrophy leading to penetrating keratoplasty (PK). METHODS: We reviewed the clinical records of patients diagnosed histopathologically as having Fuchs corneal endothelial dystrophy who underwent PK at Duke University Medical Center between January 1, 1972, and December 31, 2001. This observational case series included 424 patients (546 eyes). RESULTS: Women represented 77.6% of patients. Mean age at the time of PK for all patients was 69.2 years. Bilateral PK was required in 28.8% of patients, and the mean interval between the 2 PKs was 3.2 years. There was no difference with regard to sex in the likelihood of requiring bilateral PK (P=.59). Among 376 documented family histories, 13.6% were known to be positive for Fuchs dystrophy. Patients with a positive family history underwent PK a mean of 5 years earlier (P<.002) and were more likely to require bilateral PK (P<.003). Patients who underwent bilateral PK were twice as likely to have a positive family history compared with those undergoing unilateral PK (P<.001). Mean visual acuities at corneal thicknesses of 539 to 650 microm, 651 to 750 microm, and greater than 750 microm were 20/60, 20/60, and 20/80, respectively. Patients who underwent bilateral PK and had a preceding cataract extraction on 1 eye required PK of the pseudophakic eye on average 3.2 years earlier than the fellow eye. The mean time from cataract extraction to PK was 2.2 years. CONCLUSIONS: This large study affirms that Fuchs dystrophy is a disorder of aging that predominantly affects women (3.5:1) and is often familial. Pachymetry-determined corneal thickness was a poor predictor of visual acuity until extreme levels of corneal edema were reached. Cataract extraction in an eye with Fuchs dystrophy leads to earlier PK.

Age Distribution↗
PubMed2006

TGFBI gene mutations in corneal dystrophies.

The lattice corneal dystrophies (LCD) and granular corneal dystrophies (GCD) are autosomal dominant disorders of the corneal stroma. They are bilateral, progressive conditions characterized by the formation of opacities arising due to the deposition of insoluble material in the corneal stroma leading to visual impairment. The LCDs and GCDs are distinguished from each other and are divided into subtypes on the basis of the clinical appearance of the opacities, clinical features of the disease, and on histopathological staining properties of the deposits. The GCDs and most types of LCD arise from mutations in the transforming growth factor beta-induced (TGFBI) gene on chromosome 5q31. Over 30 mutations causing LCD and GCD have been identified so far in the TGFBI. There are two mutation hotspots corresponding to arginine residues at positions 124 and 555 of the transforming growth factor beta induced protein (TGFBIp) and they are the most frequent sites of mutation in various populations. Mutations at either of these two hotspots result in specific types of LCD or GCD. The majority of identified mutations involve residues in the fourth fasciclin-like domain of TGFBIp.

Corneal Dystrophies, Hereditary↗
PubMed2005

A dataset of human cornea proteins identified by Peptide mass fingerprinting and tandem mass spectrometry.

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.

Chemical Fractionation↗
PubMed2005

Different mutations in carbohydrate sulfotransferase 6 (CHST6) gene cause macular corneal dystrophy types I and II in a single sibship.

PURPOSE: The aim of this study was to examine the carbohydrate sulfotransferase 6 (CHST6) gene for mutations in a sibship with both macular corneal dystrophy (MCD) types I and II. DESIGN: Clinically relevant laboratory investigation. METHODS: The coding region of the CHST6 gene was examined for mutations. RESULTS: In one sibling, MCD type I was due to a homozygous C1110T (Arg140end) mutation in CHST6. Two MCD type II individuals exhibited three heterozygous nucleotide changes: C1110T, G1360A (Gly223Asp), and G1685T (Gln331His). Analysis of the upstream region was performed on one individual with MCD type II, and no upstream deletion or substitution was found. CONCLUSIONS: These findings fit the haplotype analysis that we reported previously and indicate that the predicted protein that is encoded by CHST6 is more severely affected in the individual with MCD type I than in the siblings with MCD type II.

Codon, Nonsense↗
PubMed2005

Trends in the indications for penetrating keratoplasty, 1980-2001.

PURPOSE: To study the leading indications and changing trends for penetrating keratoplasty (PK) over the past 3 decades. METHODS: This is a retrospective review of 696 cases of PK. The indications for PKs performed at the Duke University Eye Center during the years 1980-1981, 1990- 1991, and 2000-2001 were tabulated to determine trends over the past 3 decades. The main outcome measures were indications for PK. RESULTS: During this study, 696 PKs were performed. The leading indications for PK and their respective frequencies during 1980-1981, 1990-1991, and 2000-2001 were failed grafts (10.8%, 19.0%, 27.0%, respectively), pseudophakic bullous keratopathy (PBK)/aphakic bullous keratopathy (ABK) (19.4%, 20.6%, 16.7%, respectively), Fuchs dystrophy (15.6%, 13.0%, 23.8%, respectively), keratoconus (13.4%, 8.2%, 11.8%, respectively), and corneal scar (7.0%, 8.9%, 10.7%, respectively). The number of PKs for failed grafts and Fuchs dystrophy increased over time. CONCLUSIONS: In this study, failed graft has gradually become the leading indication for PK, whereas most other studies have reported PBK as the leading indication. Unlike many other studies, Fuchs dystrophy was a common indication for PK.

Corneal Diseases↗
PubMed2004

Purification and structural characterization of transforming growth factor beta induced protein (TGFBIp) from porcine and human corneas.

Mutations in the TGFBI (BIGH3) gene that encodes for transforming growth factor beta induced protein (TGFBIp) are the cause of several phenotypically different corneal dystrophies. While the genetics of these protein misfolding diseases are well documented, relatively little is known about this extracellular matrix protein itself. In this study, we have purified TGFBIp from normal human and porcine corneas using nondenaturing conditions and standard chromatography techniques. The two homologues were shown to be monomers, and we did not find evidence for posttranslational additions. The C-terminal of both human and porcine TGFBIp is truncated predominantly after the integrin binding sequence Arg(642)-Gly(643)-Asp(644) (RGD). However, using an antibody against the C-terminal fragment (residues 648-683), we also detected a small amount of full-length TGFBIp in corneal extracts. Approximately 60% of TGFBIp was covalently associated with insoluble components of the extracellular matrix in both human and porcine corneas through a disulfide bridge.

Amino Acid Sequence↗
PubMed2004

Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency.

The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.

Adult↗
PubMed2004

Lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in the TGFBI gene.

PURPOSE: To report a phenotypic variant of lattice corneal dystrophy associated with two missense changes, Ala546Asp and Pro551Gln, in the transforming growth factor-beta-induced gene (TGFBI). DESIGN: Experimental study. METHODS: Genomic DNA was obtained from the proband as well as affected and unaffected family members. Exons 4, 11, 12, and 14 of the TGFBI gene were amplified and sequenced. Additionally, a corneal button excised from the proband was examined by light and transmission electron microscopy. Haplotype analysis was performed on the proband's family and members of a previously identified pedigree with the same TGFBI gene missense changes. RESULTS: Bilateral, symmetric, radially arranged, branching refractile lines within and surrounding an area of central anterior stromal haze were noted in the proband. Multiple polymorphic, refractile deposits were noted in the mid and posterior stroma in both the proband and her daughter. Light and electron microscopic analyses demonstrated amyloid and excluded the presence of deposits characteristic of granular corneal dystrophy. Screening of TGFBI exon 12 in the proband and her affected daughter revealed two missense changes, Ala546Asp and Pro551Gln (both absent in 250 control chromosomes). Haplotype analysis suggested that the mutations in this family and in a previously identified pedigree reflect a founder effect, rather than an independent occurrence. CONCLUSIONS: We present a phenotypic variant of lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in exon 12 of the TGFBI gene. A common ancestor appears to account for the missense mutations observed in this pedigree and in a previously reported family.

Adult↗
PubMed2004

Polymorphic corneal amyloidosis: a disorder due to a novel mutation in the transforming growth factor beta-induced (BIGH3) gene.

PURPOSE: To characterize the clinicopathologic phenotype as well as the molecular genetic basis of an autosomal dominant form of corneal amyloidosis. DESIGN: Clinicopathologic and molecular genetic study of a family with a form of corneal amyloidosis. PARTICIPANTS: Forty-nine individuals from one family were studied. METHODS: The medical records of affected family members were reviewed, and corneal tissue from those who had undergone penetrating keratoplasty (PK) was examined. Several family members were examined clinically, and corneas were photographed. Deoxyribonucleic acid from blood or buccal swabs was extracted from each consenting family member to determine the status of their transforming growth factor beta-induced (TGFBI) gene. The coding region of the TGFBI gene was analyzed for mutations in the proband's DNA, and compared with the nucleotide sequences of normal individuals. This was performed by amplifying and sequencing all exons of the TGFBI gene. In all other family members, only exons 4, 8, 11, and 12 of the gene were amplified, sequenced, and analyzed for mutations. MAIN OUTCOME MEASURES: Clinicopathologic manifestations in relation to mutational status of the TGFBI gene. RESULTS: Slit-lamp biomicroscopy revealed bilateral multiple polymorphic, polygonal, refractile, chipped ice-appearing gray and white opacities. There were also occasional deep filamentous lines that did not form a distinct lattice pattern. Corneal tissue of affected individuals who underwent PK contained widespread deposits of amyloid within the corneal stroma, particularly in the deep central stroma. Twelve members of the family were found to have a heterozygous single mutation in the TGFBI gene leading to a predicted amino acid substitution of aspartic acid for alanine (A546D). Nine of these individuals had ophthalmologist-documented corneal disease. The remaining 3, who were 11, 14, and 15 years old, were asymptomatic. In addition, 4 inconsequential polymorphisms with the nucleotide changes 387 G/A (R129R), 981 G/A (V327V), 1416 T/C (L472L), and 1620 C/T (F540F) were found. CONCLUSION: A distinct, progressive form of corneal amyloidosis with an autosomal dominant mode of inheritance is characterized clinically by the presence of refractile polymorphic corneal opacities. We have designated this entity, which is caused by an A546D mutation in the TGFBI gene, polymorphic corneal amyloidosis.

Adolescent↗
PubMed2004

Two mutations in the TGFBI (BIGH3) gene associated with lattice corneal dystrophy in an extensively studied family.

PURPOSE: To determine the genetic basis for lattice corneal dystrophy (LCD) in an extensively studied family. METHODS: Ten affected family members were examined clinically, and three individuals were studied with in vivo confocal microscopy and optical coherence tomography (OCT). Corneal tissues from eight affected family members were examined histopathologically. The status of the transforming growth factor beta-induced gene (TGFBI) gene was determined in each consenting family member (six affected, seven nonaffected) by amplifying, sequencing, and analyzing exons 4 and 12 of TGFBI for mutations. All exons from the entire coding region of TGFBI of one affected person were analyzed for mutations. RESULTS: Slit lamp biomicroscopy disclosed the clinical features of LCD in both eyes of affected individuals. In vivo confocal microscopy confirmed the presence of deposits as bright lesions within the corneal stroma. OCT revealed increased reflectivity within the corneal stroma. The corneal stroma in persons undergoing penetrating keratoplasty contained amyloid. Affected members of the family were found to have two heterozygous single-nucleotide mutations in exon 12 of the TGFBI gene (C1637A and C1652A) leading to predicted amino acid substitutions in the encoded TGFbeta-induced protein (A546D and P551Q). Mutations were not detected in exon 4. In addition, an inconsequential single-nucleotide polymorphism T1620C (F540F) was found in some affected and nonaffected family members. CONCLUSIONS: Two mutations in the TGFBI gene (A546D and P551Q) cosegregated with LCD in an extensively studied family that lacked the R124C mutation that frequently accompanies this form of corneal amyloidosis.

Adult↗
PubMed2003

Novel mutations of the carbohydrate sulfotransferase-6 (CHST6) gene causing macular corneal dystrophy in India.

PURPOSE: Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive central haze, confluent punctate opacities and abnormal deposits in the cornea. It is caused by mutations in the carbohydrate sulfotransferase-6 (CHST6) gene, encoding corneal N-acetyl glucosamine-6-O-sulfotransferase (C-GlcNAc-6-ST). We screened the CHST6 gene for mutations in Indian families with MCD, in order to determine the range of pathogenic mutations. METHODS: Genomic DNA was isolated from peripheral blood leukocytes of patients with MCD and normal controls. The coding regions of the CHST6 gene were amplified using three pairs of primers and amplified products were directly sequenced. RESULTS: We identified 22 (5 nonsense, 5 frameshift, 2 insertion, and 10 missense) mutations in 36 patients from 31 families with MCD, supporting the conclusion that loss of function of this gene is responsible for this corneal disease. Seventeen of these mutations are novel. CONCLUSIONS: These data highlight the allelic heterogeneity of macular corneal dystrophy in Indian patients.

Adolescent↗
PubMed2003

Transforming growth factor beta induced protein accumulation in granular corneal dystrophy type III (Reis-Bücklers dystrophy). Identification by mass spectrometry in 15 year old two-dimensional protein gels.

PURPOSE: To investigate the accumulation of TGFBIp in GCDIII and to demonstrate the ability to perform mass spectrometry on old two-dimensional protein gels. METHODS: Proteins were extracted from one cornea with GCDIII from a person with an Arg124Leu mutation in the TGFBI (BIGH3) gene and from one normal human cornea in 1987 and subjected to two-dimensional (2D) gel electrophoresis. After keeping the gels at room temperature for 15 years, protein spots of interest were excised and digested with trypsin. The tryptic-derived peptides were analyzed using mass spectrometry. RESULTS: Four areas of interest were examined and four different proteins (TGFBIp, aldehyde dehydrogenase class 3, actin, and albumin) were identified in the 15-year-old gels. Using image analysis, the amount of TGFBIp was found to be 77 fold higher in the GCDIII affected cornea relative to the normal tissue. In both situations, TGFBIp migrated on the 2D gels as a 63 kDa protein. Mass spectrometry revealed the same nine peptides in TGFBIp from both the normal and the GCDIII affected corneas, including one peptide situated at the amino terminus. Moreover, the cornea with GCDIII contained abundant 40 kDa TGFBIp fragments that were lacking sequences in both the amino and carboxy termini. CONCLUSIONS: Mass spectrometry can be performed on old 2D polyacrylamide gels. In both normal and GCDIII affected corneas, the majority of TGFBIp migrated on 2D gels as a 63 kDa protein with an intact amino terminus. However, the amount of the 63 kDa TGFBIp was 77 fold higher in the GCDIII affected cornea. Furthermore, the GCDIII affected cornea contained abundant 40 kDa fragments that were trunctated in both the amino and carboxy termini.

Actins↗
PubMed2003

The molecular genetics of the corneal dystrophies--current status.

The pertinent literature on inherited corneal diseases is reviewed in terms of the chromosomal localization and identification of the responsible genes. Disorders affecting the cornea have been mapped to human chromosome 1 (central crystalline corneal dystrophy, familial subepithelial corneal amyloidosis, early onset Fuchs dystrophy, posterior polymorphous corneal dystrophy), chromosome 4 (Bietti marginal crystalline dystrophy), chromosome 5 (lattice dystrophy types 1 and IIIA, granular corneal dystrophy types 1, 2 and 3, Thiel-Behnke corneal dystrophy), chromosome 9 (lattice dystrophy type II), chromosome 10 (Thiel-Behnke corneal dystrophy), chromosome 12 (Meesmann dystrophy), chromosome 16 (macular corneal dystrophy, fish eye disease, LCAT disease, tyrosinemia type II), chromosome 17 (Meesmann dystrophy, Stocker-Holt dystrophy), chromosome 20 (congenital hereditary endothelial corneal dystrophy types I and II, posterior polymorphous corneal dystrophy), chromosome 21 (autosomal dominant keratoconus) and the X chromosome (cornea verticillata, cornea farinata, deep filiform corneal dystrophy, keratosis follicularis spinulosa decalvans, Lisch corneal dystrophy). Mutations in nine genes (ARSC1, CHST6, COL8A2, GLA, GSN, KRT3, KRT12, M1S1and TGFBI [BIGH3]) account for some of the corneal diseases and three of them are associated with amyloid deposition in the cornea (GSN, M1S1, TGFBI) including most of the lattice corneal dystrophies (LCDs) [LCD types I, IA, II, IIIA, IIIB, IV, V, VI and VII] recognized by their lattice pattern of linear opacities. Genetic studies on inherited diseases affecting the cornea have provided insight into some of these disorders at a basic molecular level and it has become recognized that distinct clinicopathologic phenotypes can result from specific mutations in a particular gene, as well as some different mutations in the same gene. A molecular genetic understanding of inherited corneal diseases is leading to a better appreciation of the pathogenesis of these conditions and this knowledge has made it imperative to revise the classification of inherited corneal diseases.

Corneal Dystrophies, Hereditary↗